Placental growth factor reverses decreased vascular and uteroplacental MMP-2 and MMP-9 and increased MMP-1 and MMP-7 and collagen types I and IV in hypertensive pregnancy

Abstract

Preeclampsia is a complication of pregnancy manifested as maternal hypertension (HTN) and fetal intrauterine growth restriction, with unclear mechanisms. Placental ischemia increases antiangiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) relative to angiogenic placental growth factor (PlGF); however, the molecular targets are unclear. To test the hypothesis that placental ischemia-induced changes in sFlt-1 and PlGF target vascular and uteroplacental matrix metalloproteinases (MMPs), we tested whether raising the sFlt-1-to-PlGF ratio by infusing sFlt-1 (10 µg·kg⁻¹·day⁻¹) in pregnant (Preg) rats increases blood pressure (BP) and alters MMPs and whether correcting sFlt-1/PlGF by infusing PlGF (20 µg·kg⁻¹·day⁻¹) in Preg rats with reduced uterine perfusion pressure (RUPP) improves BP and reverses the changes in MMPs. On gestational day 19, BP was higher and the litter size and uterine, placenta, and pup weight were less in Preg + sFlt-1 and RUPP than Preg rats and restored in RUPP + PlGF versus RUPP rats. Gelatin and casein zymography and Western blots revealed decreases in MMP-2 and MMP-9 and increases in MMP-1 and MMP-7 in the aorta, uterine artery, uterus, and placenta of Preg + sFlt-1 and RUPP versus Preg rats, which were reversed in RUPP + PlGF versus RUPP rats. Collagen types I and IV were more abundant in Preg + sFlt-1 and RUPP versus Preg rats and were reversed in RUPP + PlGF versus RUPP rats. Thus, PlGF reverses decreased vascular and uteroplacental MMP-2 and MMP-9 and increased MMP-1, MMP-7, and collagen types I and IV induced by placental ischemia and sFlt-1 in HTN in pregnancy. Angiogenic factors and MMP modulators could rectify changes in MMPs and collagen, restore vascular and uteroplacental remodeling, and improve HTN and intrauterine growth restriction in preeclampsia.

NEW & NOTEWORTHY Understanding the mechanisms of preeclampsia could help in its prevention and management. This study shows that correcting soluble fms-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PlGF) imbalance by infusing PlGF reverses the decreases in vascular and uteroplacental matrix metalloproteinase (MMP)-2 and MMP-9 and the increases in MMP-1, MMP-7, and collagen types I and IV induced by placental ischemia and antiangiogenic sFlt-1 in hypertension in pregnancy. Angiogenic factors and MMP modulators could rectify changes in vascular and uteroplacental MMPs and collagen content and ameliorate hypertension and intrauterine growth restriction in preeclampsia.

INTRODUCTION

Normal pregnancy is associated with hemodynamic and uteroplacental adaptations. Increases in maternal plasma volume, heart rate, and cardiac output and decreases in vascular resistance maintain blood flow to different tissues with little change in blood pressure (BP). The uterus undergoes substantial expansion to accommodate the growing fetus. The placenta also undergoes marked development, and extravillous trophoblasts invade the spiral arteries in the decidua, replacing the endothelium and muscular wall and creating dilated low-resistance vessels to maintain nutrient requirements of the developing fetus (51, 65). These hemodynamic and uteroplacental adaptations involve significant structural remodeling and functional changes in the maternal vasculature, uterus, and placenta (52, 92).

Preeclampsia (PE) is a major disorder affecting 5–8% of pregnancies in the United States and ~8 million pregnancies worldwide (91). PE is manifested as hypertension (HTN) in pregnancy (HTN-Preg) with occasional proteinuria (84). PE could progress to eclampsia, with severe HTN, cerebral edema, and seizures (37, 67), accounting for 15–20% of maternal deaths (10, 71). PE is also often associated with fetal intrauterine growth restriction (IUGR), accounting for 10–15% of preterm pregnancy and premature childbirth (80, 91).

Although PE is a major cause of maternal and fetal morbidity and mortality, it remains enigmatic with unclear pathophysiological mechanisms. Inadequate placentation and placental ischemia/hypoxia could be initiating events. In PE, trophoblasts only partially invade the superficial decidual vessels, whereas the deeper spiral arteries do not lose their endothelial lining and musculoelastic tissue and dilate to only half the size of those in normal pregnancy. In support, induction of placental ischemia by reducing uterine perfusion pressure (RUPP) in late pregnant (Preg) rats and mice shows some of the features of PE, including HTN-Preg and IUGR (4, 29, 101). PE-like characteristics have also been observed in rat models of gestational hypoxia (100), supporting a role of placental ischemia/hypoxia in HTN-Preg. However, the intermediary factors and tissue targets linking placental ischemia to the vascular and uteroplacental alterations in HTN-Preg have not been clearly identified.

Placental ischemia/hypoxia trigger the release of bioactive factors that could affect angiogenesis and vascular and uteroplacental remodeling (29, 42, 95). Angiogenic factors include VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF) (35). VEGF-A, VEGF-B, and PlGF activate tyrosine kinase receptor Flt-1 [VEGF receptor (VEGFR)-1], and VEGF-A activates VEGFR-2 (Flk-1 or KDR) to promote placental vascularization (35). However, measurements of circulating VEGF showed decreases (68), no change (54, 55), and even increases in women with PE (9, 34, 89). PlGF has 1/10th of the affinity of VEGF for VEGFR-1, but its levels are ~40 times higher than VEGF during normal pregnancy (40) and are consistently decreased in PE (8, 89). On the other hand, soluble fms-like tyrosine kinase-1 (sFlt-1 or sVEGFR-1) is an antiangiogenic alternatively spliced variant of VEGFR-1 that lacks both the transmembrane and cytoplasmic domains and binds circulating VEGF and PlGF and prevents them from activating their cell surface VEGFR-1 (43). Studies have shown that adenovirus-mediated overexpression of sFlt-1 in non-Preg rats causes increases in BP (46). Also, circulating levels of sFlt-1 and the sFlt-1-to-PlGF ratio are greater in PE than normal Preg women (8, 35, 53), suggesting that angiogenic imbalance could be an intermediary factor linking RUPP to HTN-Preg and PE. However, the vascular and uteroplacental targets underlying the relationship between angiogenic imbalance and the increased BP and IUGR in HTN-Preg are unclear.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade different components of the extracellular matrix (ECM) and play a role in remodeling of various tissues (45, 59). MMPs are produced as pro-MMPs, which are cleaved into active MMPs (73, 93). MMPs include collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other MMPs (73). MMP-2 (gelatinase A) and MMP-9 (gelatinase B) play a role in endometrial tissue remodeling during the estrous and menstrual cycles and during pregnancy (58, 90, 99). We have shown that MMP-2 and MMP-9 are upregulated in rat aorta and uterus during pregnancy, supporting their role in pregnancy-related vascular and uterine remodeling (14, 96). Some studies have showed increases in circulating MMP-2 and MMP-9 (20), whereas other studies have shown a decrease in MMP-9 in PE versus normal Preg women (59). We have shown that MMP-2 and MMP-9 are reduced in the aorta, uterus, and placenta of RUPP rats, which would favor collagen accumulation and interfere with trophoblast invasion and spiral artery remodeling (45). However, gelatinases mainly degrade collagen type IV and partially degrade collagen type I, suggesting that other MMPs are involved in vascular and uteroplacental remodeling. Consistent with this paradigm, MMP-1 is expressed in cytotrophoblasts of the placenta and decidua and may play a role in trophoblast invasion (15). Also, MMP-7 is expressed in the uterus and could play a role in endometrial tissue remodeling during estrous and menstrual cycles and during pregnancy (81). We have recently shown that MMP-1 and MMP-7 are increased in the aorta, uterus, and placenta of RUPP rat model of placental ischemia (44). Although changes in sFlt-1 and the sFlt-1-to-PlGF ratio could link placental ischemia to HTN-Preg, the role of MMPs and their tissue substrates as potential targets of sFlt-1 and PlGF is less clear.

The present study was designed to test the hypothesis that vascular and uteroplacental gelatinases MMP-2 and MMP-9, collagenase MMP-1, matrilysin MMP-7, and their protein substrates collagen types I and IV are molecular targets of angiogenic factors in HTN-Preg. We used Preg, Preg + sFlt-1, RUPP, and RUPP + PlGF rats to investigate whether 1) increasing the sFlt-1-to-PlGF ratio by infusing sFlt-1 in Preg rats increases BP and alters vascular and uteroplacental MMPs, 2) correcting the sFlt-1-to-PlGF ratio by infusing PlGF in RUPP rats reduces BP and reverses the changes in vascular and uteroplacental MMPs, and 3) the angiogenic factor-induced changes in vascular and uteroplacental MMPs affect their tissue substrate collagen.

METHODS

Animals.

Timed Preg (day 11) Sprague-Dawley rats (12 wk of age, Charles River Laboratories, Wilmington, MA) were housed in the animal facility and maintained on ad libitum standard rat chow and tap water in a 12:12-h light-dark cycle. On gestational day 14, some of the rats were anesthetized with isoflurane inhalation (3.0% induction, 1.5% for maintenance using a ventilator and a vaporizer) and infused intravenously via a jugular vein catheter (PE-50) and osmotic minipump with murine recombinant sFlt-1 (VEGF-R1/Flt-1 Fc chimera, 471-F1-100, R&D Systems, Minneapolis, MN) at 10 µg·kg⁻¹·day⁻¹ for 5 days (Preg + sFlt-1), a dose sufficient to cause HTN, proteinuria, glomerular endotheliosis, and vascular dysfunction (55, 101). Previous studies projected a high homology between murine and rat VEGFR-1 (7, 47) and did not report any immune response or secondary effects of murine recombinant sFlt-1 in similar rat models of HTN-Preg (33, 55). Other day 14 Preg rats were anesthetized with isoflurane inhalation (3.0% induction, 1.5% for maintenance using a ventilator and a vaporizer) and underwent a surgical procedure, RUPP by banding the lower abdominal aorta above the iliac bifurcation and main uterine branches of the ovarian arteries as previously described (16, 29, 101). Briefly, the abdominal aorta near the iliac bifurcation was dissected free of perivascular fat and separated from the vena cava. A blunt plastic rod (outer diameter: 0.3 mm) was placed parallel to the aorta, and a 4-0 silk braided ligature was knotted twice around the aorta and adjacent plastic rod. Once taut, the rod was removed from the knotted ligature, thus creating a constrictive band (inner diamter: 0.3 mm) and reducing blood flow through the aorta. This procedure reduces uterine perfusion pressure in the gravid rat by ∼40% (19). Because compensation of blood flow to the placenta occurs through adaptive increase in ovarian blood flow (62), a blunt plastic rod (outer diameter: 0.1 mm) was used to place a ligature band (inner diameter: 0.1 mm) on the main uterine branches of both the right and left ovarian arteries, specifically avoiding the main ovarian artery. RUPP rats received postoperative analgesia in the form of subcutaneous buprenorphine (0.05 mg/kg) every 12 h for 48 h plus meloxicam (2 mg/kg) every 24 h for 48 h. RUPP rats in which the banding procedure resulted in maternal death or total reabsorption of the pups were excluded from data analyses, and the RUPP procedure success rate was ~85%. Normal Preg rats were sham operated.

Some RUPP rats were simultaneously infused intravenously via a jugular vein catheter and osmotic pump with recombinant PlGF-1 (MBS696135, MyBiosource, San Diego, CA) at 20 µg·kg⁻¹·day⁻¹ for 5 days (RUPP + PlGF) (101). A lower dose of PlGF (10 µg·kg⁻¹·day⁻¹) was not sufficient to decrease BP in RUPP rats. A previous study infused recombinant VEGF at 90 or 180 µg·kg⁻¹·day⁻¹ to restore the angiogenic balance and showed that it lowered BP and improved renal function in RUPP rats (30). We selected PlGF over VEGF because most studies have shown decreased PlGF levels in PE (8, 53, 89), whereas measurements of VEGF have not been consistent, with studies showing decreased (68), no change (54, 55), or even increased levels (9, 34, 89). Also, PlGF is specific for VEGFR-1 and its soluble form sFlt-1, whereas VEGF also binds to VEGFR-2 and could increase vascular permeability and edema (49) and promote cancer (77). VEGF levels are also controlled at the maternal-fetal interface, partly through feedback modulation of sFlt-1, to prevent damage to the placenta or fetus by excess VEGF (21), and dysregulation of this feedback mechanism could complicate the measurement of angiogenic factors. A recent study also showed that infusion of recombinant human PlGF at 180 µg·kg⁻¹·day⁻¹ abolished placental ischemia-induced HTN-Preg in rats (87). We avoided using higher doses of PlGF, as they may cause microvascular abnormalities (39). Also, because the angiogenic/antiangiogenic balance is tightly controlled by feedback mechanisms, excess PlGF could drive a feedback increase in sFlt-1 to maintain the sFlt-1-to-PlGF ratio. We used a smaller dose of 20 µg·kg⁻¹·day⁻¹ PlGF based on our guiding measurements of sFlt-1 and PlGF levels in Preg, RUPP, and Preg + sFlt-1 rats, and the dose of PlGF was sufficient to counterbalance sFlt-1 and decrease the sFlt-1-to-PlGF ratio in RUPP + PlGF versus RUPP rats to levels close to those in control Preg rats. All procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the guidelines of the American Physiological Society and were approved by the Institutional Animal Care and Use Committee of the Brigham and Women Hospital.

Blood pressure.

On day 19 of pregnancy, rats were anaesthetized with isoflurane, and a PE-50 catheter was inserted in the carotid artery and exteriorized at the back of the neck. Rats were allowed to recover from anesthesia for at least 1 h. The carotid arterial catheter was connected to a pressure transducer attached to an amplifier and pressure recorder (Living System Instrumentation, Burlington, VT). Animals were not tethered and did not receive analgesia during measurement of BP. BP in conscious rats was monitored over a 30-min period, and the average BP was measured as previously described (56).

Plasma sFlt-1 and PlGF.

After BP had been measured, blood samples were collected via the arterial catheter into sterile heparin tubes (Tyco Healthcare, Mansfield, MA), and plasma was separated by centrifugation at 2,000 g for 10 min and stored at −80°C. Plasma sFlt-1 levels were measured using rat sFlt-1 ELISA microplate kit (MBS2602003, MyBiosource), with 0.05 ng/ml sFlt-1 sensitivity, intra-assay and interassay precision, and a coefficient of variability (CV) of <12%. The ELISA kit detects complexed sFlt-1 bound to PlGF and VEGF in rat plasma. Plasma PlGF levels were measured using rat PlGF ELISA microplate kit (MBS703282, MyBiosource), with <0.78 pg/ml PlGF sensitivity, intra-assay and interassay precision, and a CV of <10%. The ELISA kit detects both free PlGF and complexed PlGF bound to sFlt-1 in rat plasma. Absorbance was measured at 450 nm on a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA) as previously described (101).

Tissue preparation.

On gestational day 19, rats were euthanized by inhalation of CO₂, and the thoracic aorta and gravid uterus were excised and placed in Krebs solution. The uterine artery was carefully dissected under microscopic visualization. The gravid uterus was cut open, the pups and placentae were separated and gently blotted between filter papers, and the litter size and individual pup and placenta weight were recorded. The aorta, uterine artery, uterus, and placenta were cut into 3 × 3-mm segments to facilitate tissue homogenization and protein extraction. Placental segments were obtained from the entire placenta, and uterine segments containing the decidua were used. Experiments were performed on 8−12 tissue segments from each rat, 8−11 rats/group.

Gelatin zymography.

Aortic, uterine artery, uterine, and placental segments were homogenized using a 2-ml tight-fitting homogenizer (Kontes Glass, Vineland, NJ) and homogenization (no dithiothreitol) buffer containing 20.00 mM MOPS, 4.00% SDS, 10.00% glycerol, 1.20 mM EDTA, 0.02% BSA, 5.50 μM leupeptin, 5.50 μM pepstatin, 2.15 μM aprotinin, and 20.00 μM 4-(2-aminoethyl)-benzenesulfonyl fluoride. The homogenate was centrifuged at 10,000 g for 10 min, the supernatant was collected, and protein concentration was determined using a protein assay kit (Bio-Rad, Hercules, CA).

Our preliminary gelatin zymography analysis in uterine tissue homogenate from Preg or RUPP rats showed concentration-dependent increases in the intensity of pro-MMP-2 and MMP-2 bands at loading protein amounts from 0.1 and 0.2 to 0.5 μg and clearly discernible bands at 1.0 and 2.0 μg protein. Further increases in loaded protein to 5.0 and 10.0 μg showed further increases in gelatinolytic activity, and MMP-2 bands became almost saturated (14, 45); therefore, all gelatin zymography experiments for MMP-2 and MMP-9 were performed using 1.0 μg loading protein. Also, careful examination of the zymograms revealed additional bands at ~45 and 29 kDa corresponding to MMP-1 and MMP-7, respectively. The intensity of MMP-1 and MMP-7 bands was dependent on the amount of loading protein being undetectable at 0.1, 0.2, and 0.5 μg, barely detectable at 1.0 and 2.0 μg, and clearly discernible at 5.0 μg protein. Further increases in loading protein to 10.0 μg showed insignificant increases in gelatinolytic activity for MMP-1 or MMP-7. Because 5.0 μg loading protein produced clearly discernible MMP-1 and MMP-7 bands, all gelatin zymography experiments comparing MMP-1 and MMP-7 in different rat groups were performed using 5.0 μg loading protein (44). To control for intergel variations, an equal amount of loading protein from different groups was used in different gels.

Tissue homogenates were subjected to electrophoresis on a 8.0% SDS-polyacrylamide gel containing 0.1% gelatin (Sigma, St. Louis, MO). The gel was incubated in a zymogram renaturing buffer containing 2.5% Triton X-100 (Sigma) with gentle agitation for 30 min at room temperature. The gel was then incubated in a zymogram developing buffer (pH 6.7) containing 50.00 mM Tris base, 0.20 M NaCl, 5.00 mM CaCl₂, 0.02% Brij35 (Fisher Scientific, Pittsburgh, PA), and 1.00 μM ZnCl₂ (Sigma) at room temperature for 30 min and then at 37°C for 16 h. The gel was stained with 0.5% Coomassie blue R-250 (Sigma) for 30 min and then destained with an appropriate destaining solution [methanol-acetic acid-water (50:10:40)]. Areas corresponding to MMP gelatinolytic activity appeared as clear bands against a dark blue background. The clear bands were analyzed by optical densitometry and ImageJ software (National Institutes of Health, Bethesda, MD), and the integrated protease activity was measured as pixel intensity × mm² normalized to actin intensity to correct for loading (14, 45).

Casein zymography.

Because gelatin may not be an ideal substrate for MMP-1 and MMP-7, we repeated the zymography experiments using the MMP substrate casein (86). In preliminary casein zymography experiments, we used increasing concentrations of casein in the gel (0.2%, 0.4%, 0.6%, 0.8%, 1.0%, and 2.0%) and increasing amounts of loading protein (0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 µg) from uterine tissue homogenates of Preg rats (44). The intensity of the caseinolytic bands corresponding to MMP-1 and MMP-7 increased with increasing casein concentrations in the gel and reached a maximum at 1.0% casein. Further increases in casein concentration to 2.0% did not cause further increases in the intensity of the caseinolytic bands. The MMP-1 and MMP-7 caseinolytic bands were also dependent on the amount of loading protein being undetectable at 0.1, 0.2, 0.5, and 1.0 µg and clearly discernible at 2.0 µg protein, and further increases in protein amount to 5.0, 10.0 and 20.0 µg did not further increase the intensity of the caseinolytic bands. Because 1.0% casein and 2.0 μg protein produced detectable MMP-1 bands and clearly discernible MMP-7 bands, all casein zymography experiments were performed using 1.0% casein in the gel and 2.0 μg loading protein. Aortic, uterine artery, uterine, and placental tissue homogenate was subjected to electrophoresis on an 8.0% SDS-polyacrylamide gel containing 1.0% casein (Sigma). The zymograms were then developed, stained, destained, and analyzed as previously described for gelatin zymography (44).

Western blot analysis.

Aortic, uterine artery, uterine, and placental segments were homogenized using a 2-ml tight-fitting homogenizer and homogenization buffer containing 20.00 mM MOPS, 4.00% SDS, 10.00% glycerol, 2.30 mg dithiothreitol, 1.20 mM EDTA, 0.02% BSA, 5.50 μM leupeptin, 5.50 μM pepstatin, 2.15 μM aprotinin, and 20.00 μM 4-(2-aminoethyl)-benzenesulfonyl fluoride. The tissue homogenate was centrifuged at 10,000 g for 10 min. If the supernatant contained floating debris, centrifugation was repeated at least two times to obtain a clear supernatant. Protein concentration in the supernatant was determined using a protein assay kit (Bio-Rad). Protein extracts (20 μg) were combined with an equal volume of 2× Laemmli loading buffer, boiled for 5 min, and size fractionated by electrophoresis on 8% SDS-polyacrylamide gels. Proteins were transferred from the gel to a nitrocellulose membrane by electroblotting. Membranes were incubated in 5% nonfat dry milk in PBS-Tween for 1 h and then overnight at 4°C with polyclonal rabbit antibody to MMP-2 (sc-10736, 1:1,000), MMP-9 (sc-10737, 1:1,000), MMP-1 (2583988, 1:1,000), MMP-7 (sc-30071, 1:1,000), collagen type I (sc-28657, 1:1,000), or collagen type IV (sc-70246, 1:1,000) (Santa Cruz Biotechnology, Dallas, TX). The antibodies were recommended by the vendor for the detection of MMPs of rat and human origin. The authenticity of the antibodies was validated by immunoreactivity with recombinant MMPs. Negative control experiments were performed with the omission of primary antibody or with heat-inactivated primary antibody (heating at 75°C for 30 s and cooling at 4°C for 1 min, repeated 10 times) (38) and showed no detectable immunoreactive bands. Membranes were washed 5 × 15 min in PBS-Tween then incubated with horseradish peroxidase-conjugated secondary antibody (1:3,000) for 2 h. Membranes were washed another 5 × 15 min in PBS-Tween, and the immunoreactive bands were detected using enhanced chemiluminescence (ECL) Western blotting detection reagent (GE Healthcare Bio-Sciences, Piscataway, NJ). Membranes were stripped in stripping buffer and subsequently reprobed with monoclonal β-actin antibody (A1978, 1:3,000, Sigma). Equal amounts of sample protein were loaded onto the gels, and equal exposure times were used for the processing and development of Western blots. The reactive bands were analyzed by optical densitometry and ImageJ software. The densitometry values represented the pixel intensity normalized to β-actin to correct for loading (14, 44, 45).

Solutions and drugs.

Krebs solution was used for tissue dissection and contained the following (in mM): 120.0 NaCl, 5.9 KCl, 25.0 NaHCO₃, 1.2 NaH₂PO₄, 11.5 dextrose, 2.5 CaCl₂, and 1.2 MgCl₂ and bubbled with 95% O₂-5% CO₂, pH 7.4. PBS contained the following (in mM): 137.0 NaCl, 2.7 KCl, 8.0 Na₂HPO₄, and 2.0 KH₂PO₄ at pH 7.4. All other chemicals were of reagent grade or better.

Statistical analysis.

Experiments were conducted on the aorta, uterine artery, uterus, and placenta isolated from 8–11 rats/group, and cumulative data are presented as means ± SE, with the n value representing the number of rats per group. Data were analyzed using Prism (v.5.01, GraphPad Software, San Diego, CA). Data were first analyzed using ANOVA. When a statistical difference was observed, data were further analyzed using Bonferroni’s post hoc correction for multiple comparisons. Student’s unpaired t-test was used for comparison of two means. Differences were statistically significant when P < 0.05.

RESULTS

Effect of sFlt-1/PlGF on maternal and fetal parameters.

On gestational day 19, maternal body weight was not significantly different in Preg, Preg + sFlt-1, RUPP, and RUPP + PlGF rats (Table 1). BP was increased in Preg + sFlt-1 and RUPP versus Preg rats and reduced in RUPP + PlGF versus RUPP rats to levels not significantly different from control Preg rats (Table 1).

Table 1.

Maternal and fetal parameters in Preg, Preg + sFlt-1, RUPP, and RUPP + PlGF rats

Parameter	Preg	Preg + sFlt-1	RUPP	RUPP + PlGF
Maternal body weight, g	331.4 ± 9.2	311.5 ± 7.0	313.2 ± 4.2	322.9 ± 5.4
Blood pressure, mmHg	97.6 ± 3.4	125.4 ± 1.7*	128.6 ± 1.8*	102.5 ± 3.2†
Plasma sFlt-1, pg/ml	1588.2 ± 36.6	1846.7 ± 64.8*	1921.1 ± 121.3*	1818.8 ± 69.7*
Plasma PlGF, pg/ml	2.69 ± 0.33	1.62 ± 0.18*	1.47 ± 0.23*	2.38 ± 0.34†
Plasma sFlt-1-to-PlGF ratio	682.1 ± 83.7	1273.5 ± 156.7*	1553.0 ± 220.1*	894.7 ± 141.6†
Uterus weight without placentae or pups, g	4.65 ± 0.10	2.66 ± 0.08*	2.96 ± 0.23*	4.32 ± 0.15†
Litter size, number of pups	11.91 ± 0.48	9.67 ± 0.75*	8.33 ± 0.73*	11.38 ± 0.42†
Pup weight, g	2.09 ± 0.12	1.42 ± 0.23*	1.47 ± 0.19*	2.15 ± 0.25†
Placenta weight, mg	0.45 ± 0.02	0.38 ± 0.01*	0.37 ± 0.02*	0.44 ± 0.01†
Placental efficiency (pup/placenta weight), g/g	4.77 ± 0.34	3.70 ± 0.59	4.00 ± 0.50*	4.94 ± 0.61

Data are expressed as means ± SE. Rats were divided into the following four groups: pregnant (Preg; n = 11), Preg + soluble fms-like tyrosine kinase-1 (sFlt-1; n = 9), reduced uterine perfusion pressure (RUPP; n = 9), and RUPP + placental growth factor (PlGF; n = 8).

^*Significantly different (P < 0.05) vs. Preg rats;

^†significantly different (P < 0.05) vs. RUPP rats.

Plasma sFlt-1 levels were higher in Preg + sFlt-1, RUPP, and RUPP + PlGF than Preg rats and not different in RUPP + PlGF versus RUPP rats. Plasma PlGF was deceased in Preg + sFlt-1 and RUPP versus Preg rats and increased in RUPP + PlGF versus RUPP rats to levels not different from Preg rats. The sFlt-1-to-PlGF ratio was increased in Preg + sFlt-1 and RUPP versus Preg rats and restored in RUPP + PlGF rats to levels lower than RUPP rats and not different from Preg rats (Table 1).

The uterus weight without placentae and pups was reduced in Preg + sFlt-1 and RUPP versus Preg rats and increased in RUPP + PlGF versus RUPP rats to levels not different from control Preg rats. The litter size (number of pups) and average pup weight were reduced in Preg + sFlt-1 and RUPP versus Preg rats and increased in RUPP + PlGF versus RUPP rats to levels not different from Preg rats. The average placenta weight was significantly reduced in Preg + sFlt-1 and RUPP versus Preg rats and increased in RUPP + PlGF versus RUPP rats to levels not different from Preg rats (Table 1). Placental efficiency defined as individual pup weight/placenta weight (24, 94) showed an insignificant reduction in Preg + sFlt-1 and RUPP versus Preg rats and was not significantly different in RUPP + PlGF versus control Preg rats (Table 1).

Effect of sFlt-1/PlGF on vascular and uteroplacental MMP-2 and MMP-9.

Gelatin zymography of aortic, uterine artery, uterine, and placental tissue homogenates from Preg rats revealed proteolytic bands corresponding to pro-MMP-2, MMP-2, pro-MMP-9, and MMP-9 (Fig. 1). Because 1 μg protein produced clearly discernible bands for MMP-2 and MMP-9 (14, 45), we compared the intensity of the gelatinolytic bands using 1 μg loading protein from tissues of the different groups. Gelatin zymography revealed that pro-MMP-2, MMP-2, pro-MMP-9, and MMP-9 were reduced in the aorta, uterine artery, uterus, and placenta of Preg + sFlt-1 and RUPP versus Preg rats and enhanced in RUPP + PlGF versus RUPP rats to levels not different from control Preg rats. The zymograms also showed a gelatinolytic band at ~74 kDa adjacent to the pro-MMP-2 band. The nature of this band is unclear, but its intensity followed that of MMP-2 particularly in the uterine artery and placenta, suggesting MMP-2 complexation with another MMP-2 molecule (homodimer) or other MMP subspecies (heterodimer).

Fig. 1.

Aortic, uterine artery, uterine, and placental matrix metalloproteinase (MMP)-2 and MMP-9 gelatinase activity in pregnant (Preg), Preg + soluble fms-like tyrosine kinase-1 (Preg + sFlt-1), reduced uterine perfusion pressure (RUPP), and RUPP + placental growth factor (RUPP + PlGF) rats. Equal protein amounts (1 μg) of tissue homogenates from the aorta (A), uterine artery (B), uterus (C), and placenta (D) of Preg, Preg + sFlt-1, RUPP, and RUPP + PlGF rats were prepared for gelatin zymography analysis. The densitometry values of the proteolytic bands corresponding to pro-MMP-2, MMP-2, pro-MMP-9, and MMP-9 are presented as pixel intensity × mm² and were normalized to actin to correct for loading. Bar graphs represent means ± SE; n = 4 rats/group. *Significantly different (P < 0.05) vs. Preg rats; #significantly different (P < 0.05) vs. RUPP rats.

Western blots revealed immunoreactive bands corresponding to pro-MMP-2 (72 kDa), MMP-2 (63 kDa), pro-MMP-9 (92 kDa), and MMP-9 (82 kDa) in the aorta, uterine artery, uterus, and placenta that were reduced in Preg + sFlt-1 and RUPP versus Preg rats and enhanced in RUPP + PlGF versus RUPP rats to levels not different from control Preg rats (Fig. 2). The Western blot data also showed a band at ~74 kDa adjacent to pro-MMP-2, supporting possible dimerization or complexation with another MMP-2 molecule or other MMP subspecies.

Fig. 2.

Protein levels of aortic, uterine artery, uterine, and placental matrix metalloproteinase (MMP)-2 and MMP-9 in pregnant (Preg), Preg + soluble fms-like tyrosine kinase-1 (Preg + sFlt-1), reduced uterine perfusion pressure (RUPP), and RUPP + placental growth factor (RUPP + PlGF) rats. Tissue homogenates of the aorta (A), uterine artery (B), uterus (C), and placenta (D) of Preg, Preg + sFlt-1, RUPP, and RUPP + PlGF rats were prepared for Western blot analysis using antibodies to MMP-2 (1:1,000) and MMP-9 (1:1,000). Immunoreactive bands corresponding to pro-MMP-2, MMP-2, pro-MMP-9, and MMP-9 were analyzed by optical densitometry and normalized to β-actin to correct for loading. Bar graphs represent means ± SE; n = 4 rats/group. *Significantly different (P < 0.05) vs. Preg rats; #significantly different (P < 0.05) vs. RUPP rats.

Effect of sFlt-1/PlGF on vascular and uteroplacental MMP-1 and MMP-7.

Because 5 μg loading protein produced clearly discernible MMP-1 and MMP-7 bands (44, 45), all gelatin zymography experiments comparing MMP-1 and MMP-7 in different rat groups were performed using 5 μg loading protein. At 5 μg loading protein, gelatinase activity corresponding to pro-MMP-2 and MMP-2 was almost saturated, and no difference in the intensity of MMP-2 could be observed in the aorta, uterine artery, uterus, or placenta of the different rat groups. Consistent with our experiments using 1 μg loading protein, experiments with 5 μg loading protein showed that pro-MMP-9 and MMP-9 bands were not completely saturated and confirmed that they were reduced in tissues of Preg + sFlt-1 and RUPP versus Preg rats and restored in RUPP + PlGF versus RUPP rats. In comparison, MMP-1 and MMP-7 bands were significantly greater in the aorta, uterine artery, and uterus of Preg + sFlt-1 and RUPP versus Preg rats and reduced in RUPP + PlGF versus RUPP rats to levels not different from control Preg rats (Fig. 3). MMP-1 and MMP-7 bands were less prominent in the placenta but generally showed a similar pattern to that in the aorta, uterine artery, and uterus of the different rat groups.

Fig. 3.

Matrix metalloproteinase (MMP)-1 and MMP-7 gelatinase activity in pregnant (Preg), Preg + soluble fms-like tyrosine kinase-1 (Preg + sFlt-1), reduced uterine perfusion pressure (RUPP), and RUPP + placental growth factor (RUPP + PlGF) rats. Equal protein amounts (5 μg) of tissue homogenates from the aorta (A), uterine artery (B), uterus (C), and placenta (D) of Preg, Preg + sFlt-1, RUPP, and RUPP + PlGF rats were prepared for gelatin zymography analysis. At 5 µg loading protein, pro-MMP-2 and MMP-2 proteolytic bands were saturated and not different between groups. In line with the gelatin zymography data using 1 µg loading protein (see Fig. 1), pro-MMP-9 and MMP-9 proteolytic bands appeared to be reduced in Preg + sFlt-1 and RUPP versus Preg rats and enhanced in RUPP + PlGF versus RUPP rats. The densitometry values of the proteolytic bands corresponding to MMP-1 and MMP-7 are presented as pixel intensity × mm² and were normalized to actin to correct for loading. Bar graphs represent means ± SE; n = 4–5 rats/group. *Significantly different (P < 0.05) vs. Preg rats; #significantly different (P < 0.05) vs. RUPP rats.

Because gelatin may not be an ideal substrate for MMP-1 and MMP-7, we performed zymography experiments using the MMP substrate casein (86). Casein zymography revealed that MMP-1 and MMP-7 were increased in the aorta, uterine artery, and uterus of Preg + sFlt-1 and RUPP versus Preg rats and decreased in RUPP + PlGF versus RUPP rats to levels not different from Preg rats (Fig. 4). MMP-1 and MMP-7 caseinolytic bands were not easily detectable in the placenta compared with the aorta, uterine artery, and uterus and were not different in the different rat groups (Fig. 4), supporting a less prominent role in the placenta.

Fig. 4.

Matrix metalloproteinase (MMP)-1 and MMP-7 caseinase activity in pregnant (Preg), Preg + soluble fms-like tyrosine kinase-1 (Preg + sFlt-1), reduced uterine perfusion pressure (RUPP), and RUPP + placental growth factor (RUPP + PlGF) rats. Equal protein amounts (2 μg) of tissue homogenates from the aorta (A), uterine artery (B), uterus (C), and placenta (D) of Preg, Preg + sFlt-1, RUPP, and RUPP + PlGF rats were prepared 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 were normalized to actin to correct for loading. Bar graphs represent means ± SE; n = 5–6 rats/group. *Significantly different (P < 0.05) vs. Preg rats; #significantly different (P < 0.05) vs. RUPP rats.

Western blot showed detectable bands corresponding to MMP-1 at 45 kDa and MMP-7 at 29 kDa in the aorta, uterine artery, and uterus of Preg rats. MMP-1 and MMP-7 levels were significantly increased in the aorta, uterine artery, and uterus of Preg + sFlt-1 and RUPP versus Preg rats and decreased in RUPP + PlGF versus RUPP rats, to levels not different from control Preg rats (Fig. 5). In contrast with zymography data, Western blots showed MMP-1 and MMP-7 bands in the placenta that were increased in Preg + sFlt-1 and RUPP versus Preg rats and decreased in RUPP + PlGF versus RUPP rats to levels not different from control Preg rats (Fig. 5). This could be due to greater sensitivity of Western blots versus gel zymography in detecting placental proteins or the possibility that MMP-1 and MMP-7 have less gelatinolytic or caseinolytic activity or perform other functions in the placenta.

Fig. 5.

Protein amount of matrix metalloproteinase (MMP)-1 and MMP-7 in pregnant (Preg), Preg + soluble fms-like tyrosine kinase-1 (Preg + sFlt-1), reduced uterine perfusion pressure (RUPP), and RUPP + placental growth factor (RUPP + PlGF) rats. Tissue homogenates of the aorta (A), uterine artery (B), uterus (C), and placenta (D) of Preg, Preg + sFlt-1, RUPP, and RUPP + PlGF 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 rats/group. *Significantly different (P < 0.05) vs. Preg rats; #significantly different (P < 0.05) vs. RUPP rats.

Effect of sFlt-1/PlGF on vascular and uteroplacental collagen types I and IV.

We examined the potential downstream target substrates of MMPs. Western blots revealed that both collagen types I and IV were increased in the aorta, uterine artery, uterus, and placenta of Preg + sFlt-1 and RUPP versus Preg rats and decreased in RUPP + PlGF versus RUPP rats to levels not significantly different from control Preg rats (Fig. 6).

Fig. 6.

Protein levels of collagen types I and IV in pregnant (Preg), Preg + soluble fms-like tyrosine kinase-1 (Preg + sFlt-1), reduced uterine perfusion pressure (RUPP), and RUPP + placental growth factor (RUPP + PlGF) rats. Tissue homogenates of the aorta (A), uterine artery (B), uterus (C), and placenta (D) of Preg, Preg + sFlt-1, RUPP, and RUPP + PlGF rats were prepared for Western blot analysis using antibodies to collagen type I (1:1,000) and collagen type IV (1:1,000). Immunoreactive bands corresponding to collagen types I and IV were analyzed by optical densitometry and normalized to β-actin to correct for loading. Bar graphs represent means ± SE; n = 4–7 rats/group. *Significantly different (P < 0.05) vs. Preg rats; #significantly different (P < 0.05) vs. RUPP rats.

DISCUSSION

We assessed the sFlt-1/PlGF imbalance in RUPP rats and found that inducing a comparable sFlt-1/PlGF imbalance by infusing sFlt-1 in Preg rats was sufficient to increase BP, decrease vascular and uteroplacental MMP-2 and MMP-9, and increase MMP-1, MMP-7, and collagen types I and IV to levels similar to those in RUPP rats. We then tested the reverse hypothesis and demonstrated that restoring the sFlt-1/PlGF balance by infusing PlGF in RUPP rats reversed the increase in BP, decreases in vascular and uteroplacental MMP-2 and MMP-9, and increases in MMP-1, MMP-7, and collagen types I and IV to levels similar to those in control Preg rats, highlighting a potential relationship between angiogenic imbalance, vascular and uteroplacental MMPs, and collagen deposition in the setting of HTN-Preg.

Angiogenic imbalance is associated with HTN-Preg, placental inefficiency, and IUGR.

Because of the difficulty of performing a mechanistic study in Preg women, animal models of HTN-Preg have provided mechanistic insights on the pathophysiological changes in PE (5, 37). Late Preg sheep, dog, rabbit, and rat with RUPP show characteristics of PE, including HTN-Preg and IUGR (5, 13, 48), supporting the concept that inadequate trophoblast invasion of spiral arteries and the ensuing uteroplacental ischemia/hypoxia are important events in the pathogenesis of PE (28, 100). Placental ischemia could cause the release of antiangiogenic factors such as sFlt-1 and lead to an angiogenic imbalance (29, 42, 43, 55). The observed increase in plasma sFlt-1 levels, decreases in PlGF, and increases in sFlt-1-to-PlGF ratio in RUPP rats are consistent with similar reports in PE women (8, 53, 89) and in RUPP rats, Dahl salt-sensitive rats, and other animal models of HTN-Preg (16, 29, 31, 75, 85, 101).

We assessed the effects of inducing a sFlt-1/PlGF imbalance and found that infusing sFlt-1 in Preg rats was associated with increases in BP. This is consistent with reports that infusion of sFlt-1 in Preg rats is associated with HTN, proteinuria, and glomerular endotheliosis (33, 55). The present study also showed that infusing PlGF in RUPP rats improved the sFlt-1-to-PlGF ratio and reduced BP, providing evidence for a role of PlGF in regulating the sFlt-1/PlGF balance and BP in HTN-Preg. This is consistent with reports that infusion of recombinant VEGF or PlGF ameliorates HTN, IUGR, and renal and vascular dysfunction in RUPP rats (30, 87, 101). Also, consistent with previous reports (16, 87, 101), the litter size and individual pup and placenta weight were reduced in Preg + sFlt-1 and RUPP versus Preg rats and restored in RUPP + PlGF versus RUPP rats to levels not different from control Preg rats. Changes in placental efficiency occur as a result of alterations in the weight of the fetus, placenta, or both (24, 94). Variations in placental efficiency occur naturally, and changes in the fetal-to-placental weight ratio may occur during manipulation of uterine blood flow in RUPP rats (24). Placental efficiency showed an insignificant reduction in RUPP and Preg + sFlt-1 rats, suggesting reduced placental development relative to fetal size and the nutrient demand. Also, we have previously detected measureable amounts of sFlt-1 and PlGF in placental and uterine extracts of Preg rats and observed increases in sFlt-1 and decreases in PlGF in placental and uterine extracts of RUPP rats (16). These findings are consistent with reports showing that sFlt-1 levels are greater in villous explants from PE than normal Preg women (2) and that sFlt-1 increases, whereas PlGF decreases, in twin versus singleton pregnancies (22) and in twin pregnancies with suspected PE (18, 76). These findings support that uteroplacental cells coexpress sFlt-1 and PlGF during normal pregnancy and that their levels are different during HTN-Preg. Together, these observations suggest a role of the angiogenic imbalance in the increased BP and IUGR in HTN-Preg and support a role of angiogenic balance in regulating BP, placental development, and fetal growth and thus made it important to examine the mechanisms via which angiogenic imbalance could alter vascular and uteroplacental remodeling in the setting of HTN-Preg and IUGR.

Angiogenic imbalance is associated with decreased vascular and uteroplacental gelatinases.

MMPs are major regulators of tissue remodeling during pregnancy, and the gelatinases MMP-2 and MMP-9 are involved in ECM degradation and trophoblast invasion of the spiral arteries. MMP-2 is the main collagenolytic enzyme in the umbilical cord artery (6), and serum MMP-9 levels are elevated in normal Preg women (59). Also, we have shown increases in MMP-2 and MMP-9 in the aorta and uterus during the course of pregnancy in rats (14). These observations are consistent with the concept that MMP-2 and MMP-9 play a role in the vascular and uteroplacental remodeling during healthy pregnancy (14, 36, 82, 96). Although some studies have shown an increase in circulating MMP-2 and MMP-9 in PE versus normal Preg women (20), other studies have shown a decrease in serum MMP-9 in PE (59). Also, in cultured trophoblasts, suppression of MMP-9 expression inhibits their invasive capability, supporting a role of MMP-9 in modulating trophoblast invasion (98). In the present study, we tested whether MMP-2 and MMP-9 are potential targets of placental ischemia and angiogenic imbalance in HTN-Preg. We examined the aorta as representative of the vascular changes in the maternal systemic circulation, the uterine artery as representative of the specific vascular changes in the uteroplacental circulation, the uterus, which undergoes expansive remodeling to accommodate the growing fetus, and the placenta, which provides blood and nutrient supply to the developing fetus. Consistent with our previous report (45), MMP-2 and MMP-9 levels were reduced in the aorta, uterine artery, uterus, and placenta of RUPP versus Preg rats. Also, we have previously shown histological evidence supporting that the decreases in MMP-2 and MMP-9 as detected by Western blot analysis and gelatin zymography coincide with significant decreases in immunohistochemical staining of MMP-2 and MMP-8 in aortic, uterine, and placental tissue sections of RUPP versus normal Preg rats (45). We have also shown prominent MMP-2 and MMP-9 immunostaining in the aortic media (45), consistent with reports that vascular smooth muscle cells are a major source of MMPs (83). These observations are in agreement with other reports that MMP levels are decreased in the umbilical cord artery and microvascular endothelial cells of PE women (25, 26) and that pharmacological inhibition of MMPs is associated with decreased placenta weight and IUGR in both normal Preg and HTN-Preg rats and reduces trophoblast invasion and placental perfusion in HTN-Preg rats (66, 78, 97). In search for the cause of reduced MMPs during placental ischemia, the present study provides evidence that the decreases in MMP-2 and MMP-9 in RUPP rats are likely due to a sFlt-1/PlGF imbalance because infusing sFlt-1 in Preg rats caused decreases in MMP-2 and MMP-9 similar to those in RUPP rats, and infusing PlGF in RUPP rats restored MMP-2 and MMP-9 to the levels observed in control Preg rats. Although these observations support a role of the angiogenic imbalance in decreasing vascular and uteroplacental MMP-2 and MMP-9 in the setting of HTN-Preg and IUGR, they raise questions regarding whether other MMPs could be modulated during HTN-Preg, the target substrates of MMPs, and the role of angiogenic factors in modulating these MMPs and their substrates.

Angiogenic imbalance is associated with increased collagenase type 1 and matrilysin.

In addition to gelatinases, the MMP family includes collagenases, stromelysins, matrilysins, membrane-type-MMPs, and other MMPs that could influence tissue remodeling and other cellular functions (50, 73). In the present study, careful examination of the gelatin zymograms showed additional gelatinolytic activities corresponding to MMP-1 and MMP-7 because, first, the gelatinolytic bands ran at 45 and 29 kDa, which correspond to reported molecular weights of MMP-1 and MMP-7, respectively (50, 73). Second, MMP-1 and MMP-7 gelatinolytic activity was detected only at high amounts of loading protein, consistent with reports showing that gelatin may not be an ideal substrate for MMP-1 (50, 86). Third, zymography using the other MMP substrate casein demonstrated caseinolytic bands at molecular weights corresponding to MMP-1 and MMP-7. Finally, Western blots showed specific immunoreactive bands with MMP-1 and MMP-7 antibodies. These observations are consistent with reports showing that MMP-1 is expressed in cytotrophoblasts of the placenta and decidua (15) and that MMP-7 could be involved in uterine tissue remodeling during pregnancy (81). We have recently shown immunohistochemistry data suggesting that MMP-1 and MMP-7 are localized in uterine trophoblasts and aortic smooth muscle and supporting that these cells represent a major source of MMP-1 and MMP-7 (44). Also, consistent with our previous report (44), the present gelatin zymography, casein zymography, and Western blots suggest that the levels of MMP-1 and MMP-7 are increased in the aorta, uterine artery, uterus, and placenta of RUPP versus Preg rats. The increases in MMP-1 and MMP-7 in RUPP rat model of placental ischemia are likely due to changes in sFlt-1/PlGF because infusing sFlt-1 in Preg rats caused increases in MMP-1 and MMP-7 similar to those observed in RUPP rats, and infusing PlGF in RUPP rats restored the levels of MMP-1 and MMP-7 to those observed in control Preg rats. The MMP-1 and MMP-7 gelatinolytic and caseinolytic activities were less clear in the placenta compared with the aorta, uterine artery, or uterus, suggesting specific changes in MMP activity in the placenta compared with other maternal tissues. This is supported by our previous report of tissue-specific changes in MMP activity in the placenta compared with the aorta and uterus of late Preg versus mid-Preg rats (14). Nevertheless, the increases in MMP-1 and MMP-7 in blood vessels and the uterus of Preg + sFlt-1 and RUPP rats and their reversal in RUPP + PlGF rats support a role of angiogenic factors in the regulation of vascular and uterine MMP-1 and MMP-7 and tissue remodeling during HTN-Preg and IUGR.

Angiogenic imbalance is associated with increased MMP substrates collagen types I and IV.

In search of the MMP downstream targets that could influence tissue remodeling, we investigated possible changes in MMP substrates. MMPs degrade different substrates, including gelatin, collagen, and other proteins (41, 73, 93). We have previously shown an increase in total collagen content with no change in elastin content in the aorta, uterus, and placenta of RUPP versus Preg rats (45). However, collagen has 18 types and different subtypes (27). Gelatinases mainly degrade collagen type IV, although MMP-2 can also degrade collagen types I, II, III, V, VII, X, and XI and MMP-9 can degrade collagen types V, VII, X, and XIV (41, 73, 93). Thus, although MMP-2 is a gelatinase, it can also function as interstitial collagenase, acting much like MMP-1 but in a weaker manner (60). MMP-2 can degrade collagen in two stages, interstitial collagenase-like degradation followed by gelatinolysis promoted by the fibronectin-like domain (3). MMP-9 can also function as a collagenase, where it binds the α₂-chains of collagen type IV with high affinity even when it is inactive and makes the substrate readily available, then as a gelatinase (64). We have previously shown increases in collagen type I in the aorta, uterus, and placenta of RUPP versus Preg rats (44). The present study showed increases not only in collagen type I but also collagen type IV in the aorta, uterine artery, uterus, and placenta of RUPP versus Preg rats. The increases in collagen types I and IV in RUPP rats are likely due to changes in sFlt-1/PlGF because infusing sFlt-1 in Preg rats caused increases in collagen types I and IV similar to those in RUPP rats, and infusing PlGF in RUPP rats restored the collagen levels to those observed in control Preg rats. Because MMPs facilitate cell growth and migration by promoting proteolysis of the ECM, we suggest that the decreased MMP-2 and MMP-9 and consequently the increased collagen deposition in Preg + sFlt-1 and RUPP tissues could impede cell growth, proliferation, and migration and in turn interfere with uteroplacental tissue invasion and remodeling of spiral arteries. The increased collagen deposition in blood vessels of Preg + sFlt-1 and RUPP rats could also decrease vascular plasticity and in turn contribute to increased vascular resistance and HTN (45).

MMPs also promote angiogenesis by detaching pericytes from the vessels undergoing angiogenesis, releasing ECM-bound angiogenic growth factors, exposing cryptic proangiogenic integrin-binding sites in the ECM, generating promigratory ECM component fragments, and cleaving endothelial cell-cell adhesions (70). Thus, downregulation of MMP-2 and MMP-9 by sFlt-1 could decrease angiogenesis, trophoblast invasion of spiral arteries, uteroplacental vascularization, and fetoplacental growth during HTN-Preg. On the other hand, PlGF could act in an autocrine or paracrine fashion to accelerate angiogenesis, trophoblast invasion of spiral arteries, uteroplacental vascularization and fetoplacental growth, and MMP-2 and MMP-9 may mediate these effects by virtue of their proteolytic activity and degradation of collagen type IV in the ECM. The increases in collagen type I could also contribute to collagen deposition, vascular rigidity, and uteroplacental restrictive remodeling in Preg + sFlt-1 and RUPP rats, and decreases in collagen type I could reverse growth restriction and improve vascular and uteroplacental remodeling in RUPP + PlGF rats. However, if collagen type I is a substrate of MMP-1 and possibly MMP-7 (50, 73), one would expect that an increase in MMP-1 and MMP-7 would lead to a decrease in collagen type I in Preg + sFlt-1 and RUPP rats and that a decrease in MMP-1 and MMP-7 in RUPP + PlGF rats would increase collagen type I, which is opposite from the present results. One possibility is that collagen turnover is a dynamic process, and an increase in the collagenolytic activity of MMP-1 and MMP-7 could trigger a feedforward compensatory mechanism to upregulate and provide additional supply of the substrate collagen type I. Another possibility is that collagen type I turnover is controlled by other MMPs and proteases, and, although an increase in MMP-1 and MMP-7 would decrease collagen type I, a decrease in MMP-2 and MMP-9 may increase collagen type I. Another possibility is that collagen type I may not be the ideal substrate for MMP-1 and MMP-7, and, therefore, other collagen subtypes should be examined in vascular and uteroplacental tissues of HTN-Preg rats. RT-PCR experiments should also determine whether any increases in a collagen subtype are due to decreased degradation or also involve decreased de novo mRNA expression and protein biosynthesis.

In addition to their proteolytic effects on the ECM, MMPs may affect vascular and uterine function and the mechanisms of smooth muscle contraction. We have previously shown that MMP-2 and MMP-9 cause relaxation of the precontracted rat aorta (12) and uterus (96) and induce vasodilation in rat inferior vena cava via hyperpolarization and activation of K⁺ channels (72, 74). Other studies have shown that MMP-2 and MMP-9 may increase the production of vasoconstrictor peptides such as endothelin-1 and decrease vasodilator peptides such as adrenomedullin (1, 23, 61), leading to endothelial dysfunction and imbalance between vasodilator and constrictor factors. Studies have also shown that MMP-1 enhances vascular contraction to angiotensin II via an endothelium-dependent protease-activated receptor and the endothelin-1 pathway in omental vessels of normal Preg women (63). Thus, changes in vascular MMP-2, MMP-9, MMP-1, and MMP-7 could affect vascular contraction and promote HTN-Preg, whereas changes in uterine MMPs could affect uterine contraction and trigger premature labor (45, 96). We and other groups have shown that vascular contraction is enhanced in Preg + sFlt-1 and RUPP rats and reduced in RUPP + PlGF rats (13, 56, 101), and the observed changes in vascular MMPs may contribute to the increased vasoconstriction and BP in Preg + sFlt-1 and RUPP rat models of HTN-Preg and their reversal in RUPP + PlGF rats.

Other observations/considerations.

There are other observations and considerations. First, some studies have shown that low levels of MMP-1 in the umbilical cord blood, placenta, and decidua of PE versus normal Preg women and that the low MMP-1 levels are correlated with the severity of PE (15). Also, cultured human decidual microvascular endothelial cells from PE pregnancies express lower levels of MMP-1 than those from normal pregnancies (26). However, MMP-1 causes vasoconstriction and enhances reactivity to angiotensin II in studies in omental vessels of Preg women, supporting a role of MMP-1 in the increased vasoconstriction in PE (63). Also, whereas MMP-7 may play a role in endometrial tissue remodeling during the menstrual cycle and pregnancy (81), the expression of MMP-7 by extravillous trophoblast cells, especially those close to the spiral arteries, is reduced in PE women (79). Therefore, the measurements of MMP-1 and MMP-7 levels should be carefully interpreted in the context of differences in species (human vs. animals), the specimens examined (plasma vs. placenta, uterus, and aorta), and nature of tissues or cells examined (native vs. cultured). Second, MMP is a large family of at least 28 proteolytic enzymes (41, 73, 93). Although we examined MMP-2, MMP-9, MMP-1, and MMP-7, other MMPs have been detected in the aorta, uterus, and placenta, and the changes in these MMPs in HTN-Preg need to be examined. For instance, trophoblast- and vascular smooth muscle-derived MMP-12 mediate proteolysis and uterine spiral artery remodeling during pregnancy (32). Also, MMP activity could be influenced by other MMP activators and inhibitors. In effect, some MMPs may cleave other pro-MMPs, and membrane-type 1 MMP is a key activator of pro-MMP-2 (69, 73, 93). Also, tissue inhibitors of metalloproteinase (TIMPs) are endogenous modulators of MMPs (59, 69, 93), and the changes in vascular and uteroplacental TIMPs in HTN-Preg need to be examined. Our previous reverse zymography experiments revealed detectable bands corresponding to TIMP-1 and TIMP-2 in the uterine artery, uterus, and placenta of Preg rats, and TIMP-1 levels were enhanced in RUPP versus Preg rats (11), which may explain the observed decrease in MMP-2 and MMP-9 in RUPP rats. Third, in the present study, MMPs were measured on gestational day 19, and the progressive changes in MMPs during the course of pregnancy and their reversal in the postpartum period need to be examined. Also, while plasma levels of MMPs may show increase during pregnancy (57), MMPs are released from different maternal tissues, and plasma MMP levels reflect global changes in MMPs in multiple tissues. In the present study, we showed decreases in aortic, uterine artery, uterine, and placental MMP-2 and MMP-9 and increases in MMP-1 and MMP-7 in HTN-Preg rats. The changes in MMP expression/activity and their effects on the structure and function of other tissues, e.g., the small resistance vessels that significantly affect BP, need to be examined. Finally, the present study supports a role of sFlt-1/PlGF as a potential upstream mechanism linking placental ischemia to the changes in vascular and uteroplacental MMPs and collagen deposition in HTN-Preg.

These observations make it important to determine the mechanistic connection between sFlt-1/PlGF and MMPs. An important question is whether the infusion of sFlt-1 and PlGF in vivo affects vascular and uteroplacental MMPs directly or indirectly via other cytoactive factors. We have previously tested a direct mechanistic relationship between angiogenic factors and MMPs and have shown that treatment of uterine and placental explants from Preg rats with sFlt-1 (0.1 μg/ml) in tissue culture for 48 h causes decreases in MMP-2 and MMP-9 and that treatment of uterine and placental explants from RUPP rats with VEGF or PlGF (0.1 μg/ml) in tissue culture for 48 h increases MMP-2 and MMP-9 in tissues of RUPP rats (16, 45), supporting direct effects of angiogenic factors on MMP-2 and MMP-9 levels. Whether sFlt-1/PlGF directly affects the levels of MMP-1 and MMP-7 or acts indirectly via other cytoactive factors should be examined in future studies. Another question is whether MMP inhibition prevents the effects of PlGF. We have performed preliminary experiments and found that intravenous infusion of the MMP inhibitor doxycycline at 30 mg·kg⁻¹·day⁻¹ in normal Preg rats, where plasma PlGF levels are naturally elevated and both MMP-2 and MMP-9 are overexpressed in the vascular and uteroplacental tissues, was associated with increased BP (108.1 ± 0.2 vs. 96.0 ± 2.8 mmHg in control) and reduced pup weight (1.57 ± 0.02 vs. 2.59 ± 0.04 g in control), supporting a role of MMPs in maintaining BP and fetoplacental growth during pregnancy.

Perspectives

The present study showed that, similar to RUPP rats, inducing a sFlt-1/PlGF imbalance by infusing sFlt-1 in Preg rats increases BP and causes changes in MMPs and collagen, supporting changes in MMPs as a central mechanism in HTN-Preg and that sFlt-1 is an important intermediary factor linking placental ischemia to changes in MMPs in HTN-Preg. In this respect, counterbalancing sFlt-1 by infusing PlGF improved MMP levels, collagen deposition, BP, and IUGR in RUPP rats. Although delivery of the baby and placenta is the only effective measure to reverse PE, the present findings could be useful in designing new approaches for the management of HTN-Preg and IUGR. Extracorporeal removal of circulating sFlt-1 from patients with PE decreases the sFlt-1-to-PlGF ratio and could improve PE symptoms and prolong pregnancy (88) but may require advanced apheresis equipment and highly trained clinical staff that may only be feasible in large medical centers. Infusion of PlGF could be an alternative approach to restore the sFlt-1/PlGF balance and improve MMP profile and vascular remodeling and in turn reverse the increases in BP in PE. Preterm birth is another undesirable outcome of PE and could be the only measure to prevent the progress to eclampsia. Our data in RUPP and Preg + sFlt-1 rats support that a sFlt-1/PlGF imbalance is associated with decreased litter size and pup weight. On the other hand, infusion of PlGF in HTN-Preg rats improved litter size and pup weight, suggesting that PlGF could promote fetal growth and development and prolong pregnancy in PE. Although PlGF may have potentially limiting side effects, localized administration of PlGF in the vicinity of the fetoplacental interface using direct injection of PlGF or incorporation of PlGF into implantable scaffolds or matrixes could reverse the changes in MMPs induced by placental ischemia and sFlt-1 and restore the activity and remodeling capacity of growth-permissive MMPs, thus maintaining fetal growth and prolonging gestation in PE. MMP modulators that increase MMP-2 and MMP-9 and decrease MMP-1 and MMP-7 could provide alternative approaches to improve vascular and uteroplacental remodeling and in turn BP and IUGR in the management of PE.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-65998 and HL-111775.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Z.R., N.C., M.Z., and R.A.K. conceived and designed research; Z.R., N.C., and M.Z. performed experiments; Z.R., N.C., M.Z., and R.A.K. analyzed data; Z.R., N.C., M.Z., and R.A.K. interpreted results of experiments; Z.R. and R.A.K. prepared figures; Z.R., N.C., M.Z., and R.A.K. edited and revised manuscript; Z.R., N.C., M.Z., and R.A.K. approved final version of manuscript; R.A.K. drafted manuscript.