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Adaptation of uterine artery thick- and thin-filament regulatory pathways to pregnancy

Center for Perinatal Biology, Department of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, California

Submitted 2 July 2004 ; accepted in final form 18 August 2004

	ABSTRACT

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Little is known about the adaptation of uterine artery smooth muscle contractile mechanisms to pregnancy. The present study tested the hypothesis that pregnancy differentially regulates thick- and thin-filament regulatory pathways in uterine arteries. Isometric tension, intracellular free Ca²⁺ concentration, and phosphorylation of 20-kDa myosin light chain (MLC₂₀) were measured simultaneously in uterine arteries isolated from nonpregnant and near-term (140 days gestation) pregnant sheep. Phenylephrine-mediated intracellular free Ca²⁺ concentration, MLC₂₀ phosphorylation, and contraction tension were significantly increased in uterine arteries of pregnant compared with nonpregnant animals. In contrast, phenylephrine-mediated Ca²⁺ sensitivity of MLC₂₀ phosphorylation was decreased in the uterine arteries of pregnant sheep. Simultaneous measurement of phenylephrine-stimulated tension and MLC₂₀ phosphorylation in the same tissue indicated a decrease in MLC₂₀ phosphorylation-independent contractions in the uterine arteries of pregnant sheep. In addition, activation of PKC produced significantly lower sustained contractions in uterine arteries of pregnant compared with nonpregnant animals in the absence of changes in MLC₂₀ phosphorylation levels in either vessels. In uterine arteries of nonpregnant sheep, the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase inhibitor PD-098059 significantly increased phenylephrine-mediated, MLC₂₀ phosphorylation-independent contractions. The results suggest that in uterine arteries, pregnancy upregulates ₁-adrenoceptor-mediated Ca²⁺ mobilization and MLC₂₀ phosphorylation. In contrast, pregnancy downregulates the Ca²⁺ sensitivity of myofilaments, which is mediated by both thick- and thin-filament pathways.

₁-adrenoceptor; protein kinase C; calcium; myosin light chain phosphorylation; extracellular signal regulated kinase

Smooth muscle contraction is regulated through both thick- and thin-filament regulatory pathways. Thick-filament regulation is mediated by both Ca²⁺-dependent mechanisms that lead to activation of myosin light chain kinase (MLCK) and phosphorylation of 20-kDa regulatory light chain of myosin (MLC₂₀) and Ca²⁺-independent mechanisms that involve inactivation of myosin light chain phosphatase (MLCP) and decreased MLC₂₀ dephosphorylation (20, 21, 35). In addition to thick-filament regulation, many studies have demonstrated dissociation between MLC₂₀ phosphorylation and cross-bridge cycling rates and/or tension development, which suggests a thin-filament regulatory pathway (5, 28).

Our recent studies on pregnant ovine uterine arteries have shown that ₁-adrenoceptor-mediated contractions are regulated through both thick- and thin-filament pathways with the thick-filament regulatory pathway, i.e., MLC₂₀ phosphorylation, predominating (49). In contrast, activation of PKC induced contractions without changing MLC₂₀ phosphorylation levels (49). These studies have also shown that extracellular signal-regulated kinase (ERK) modulates contractile force in uterine arteries during pregnancy by dual regulation of thick- and thin-filament pathways. ERK potentiated ₁-adrenoceptor-stimulated MLC₂₀ phosphorylation via increases in intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) and Ca²⁺ sensitivity of MLC₂₀ phosphorylation. In contrast, ERK attenuated the thin-filament regulatory pathway by suppressing the contractions independent of changes in MLC₂₀ phosphorylation levels.

In the present study, we tested the hypothesis that pregnancy differentially regulates thick- and thin-filament regulatory pathways in uterine arteries. Isometric tension, [Ca²⁺]_i, and MLC₂₀ phosphorylation levels were measured simultaneously in uterine arteries isolated from nonpregnant and near-term (140 days gestation) pregnant sheep. The present study provides evidence that pregnancy upregulates ₁-adrenoceptor-mediated intracellular Ca²⁺ mobilization and MLC₂₀ phosphorylation but downregulates the Ca²⁺ sensitivity of MLC₂₀ phosphorylation. In addition, pregnancy significantly downregulates MLC₂₀ phosphorylation-independent contractions in uterine arteries.

	METHODS

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Tissue preparation. Nonpregnant and pregnant (140 days gestation) sheep were anesthetized with thiamylal (10 mg/kg) administered via the external left jugular vein. The ewes were then intubated, and anesthesia was maintained with 1.5–2.0% halothane in O₂ throughout the surgery. An incision in the abdomen was made, and the uterus was exposed. The uterine arteries were isolated and removed without stretching and were placed into a modified Krebs solution (pH 7.4) of the following composition (in mM): 115.2 NaCl, 4.7 KCl, 1.80 CaCl₂, 1.16 MgSO₄, 1.18 KH₂PO₄, 22.14 NaHCO₃, 0.03 EDTA, and 7.88 dextrose. The Krebs solution was oxygenated with a mixture of 95% O₂-5% CO₂. After the tissues were removed, animals were killed with T-61 euthanasia solution (Hoechst-Roussel; Somerville, NJ). All procedures and protocols used in the present study were approved by the Animal Research Committee of Loma Linda University and followed the guidelines in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Contraction studies. The third (in nonpregnant sheep) and fourth (in pregnant sheep) branches of the main uterine arteries that had similar external diameter were separated from the surrounding tissue and were cut into 2-mm ring segments. Isometric tension was measured in the Krebs solution in a tissue bath at 37°C as described previously (49). After 60 min of equilibration, each ring was stretched to the optimal resting tension as determined by the tension developed in response to 120 mM KCl added at each stretch level. Tissues were then stimulated with phenylephrine or phorbol 12,13-dibutyrate (PDBu; Sigma; St. Louis, MO), and contractile tensions and MLC₂₀ phosphorylation levels were measured simultaneously in the same tissues. Tensions developed were continuously recorded with an online computer. To measure phosphorylated MLC₂₀ (MLC₂₀-P) simultaneously in the same tissue, arterial rings were snap frozen with liquid N₂-cooled clamps at the indicated times and were rapidly immersed in a dry ice-acetone slurry that contained a 10% trichloroacetic acid (TCA) and 10 mM DTT mixture. Tissues were then stored at –80°C until analysis of MLC₂₀ phosphorylation. In certain experiments, tissues were pretreated with 30 µM PD-098059 (Sigma) or DMSO vehicle for 30 min before stimulation with phenylephrine.

Measurements of MLC₂₀ phosphorylation. Tissue MLC₂₀ phosphorylation levels were measured as described previously (49). Briefly, tissues were brought to room temperature in a dry ice-acetone-TCA-DTT mixture and washed three times with ether to remove the TCA. Tissues were then extracted in 100 µl of sample buffer that contained 20 mM Tris base and 23 mM glycine (pH 8.6), 8.0 M urea, 10 mM DTT, 10% glycerol, and 0.04% bromophenol blue as previously described. Samples (20 µl) were electrophoresed at 12 mA for 2.5 h after a 30-min prerun in 1.0-mm mini-polyacrylamide gels that contained 10% arcelamide-0.27% bisacrylamide, 40% glycerol, and 20 mM Tris base (pH 8.8). Proteins were transferred to nitrocellulose membranes and subjected to immunoblot with a specific MLC₂₀ antibody (1:500 dilution; Sigma). Goat anti-mouse IgG conjugated with horseradish peroxidase was used as a secondary antibody (1:2,000 dilution; Calbiochem). Bands were detected using enhanced chemiluminescence, visualized on films, and analyzed using Kodak 1D image-analysis software. Moles of phosphate per mole of MLC₂₀ (fraction of MLC₂₀ phosphorylated) were calculated by dividing the density of the phosphorylated band by the sum of the densities of the phosphorylated plus the unphosphorylated bands. In some experiments, MLC₂₀-P was corrected for protein-content differences between uterine arteries of nonpregnant and pregnant animals and was expressed as milligrams of MLC₂₀-P per gram of tissue wet weight as previously described for ovine uterine arteries by Annibale et al. (4).

Simultaneous measurement of [Ca²⁺]_i and tension. Simultaneous recordings of contraction tension and [Ca²⁺]_i in the same tissue were conducted as described previously (53). Briefly, the arterial ring was attached to an isometric force transducer in a 5-ml tissue bath mounted on a CAF-110 intracellular Ca²⁺ analyzer (Jasco; Tokyo, Japan). The tissue was equilibrated in Krebs buffer under a resting tension of 0.5 g for 40 min. The ring was then loaded with 5 µM fura 2-AM (Molecular Probes; Eugene, OR) for 3 h in the presence of 0.02% Cremophor EL at room temperature (25°C). After the tissue was loaded, it was washed with Krebs solution at 37°C for 30 min to allow for hydrolysis of fura 2 ester groups by endogenous esterase. Contractile force and fura 2 fluorescence were measured simultaneously at 37°C in the same tissue. The tissue was illuminated alternatively (125 Hz) at excitation wavelengths of 340 and 380 nm, respectively, by means of two monochromators in the light path of a 75-W xenon lamp. Fluorescence emission from the tissue was measured at 510 nm by a photomultiplier tube. The fluorescence intensity at each excitation wavelength (F₃₄₀ and F₃₈₀, respectively) and the ratio of these two fluorescence values (R_f340/380) were recorded with a time constant of 250 ms and stored with the force signal on a computer.

Data analysis. Data were analyzed by computer-assisted linear or nonlinear regression to fit the data using GraphPad Prism (GraphPad; San Diego, CA). Results were expressed as means ± SE. Differences were evaluated for statistical significance (P < 0.05) by one-way ANOVA and Student’s t-test.

	RESULTS

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Phenylephrine-induced [Ca²⁺]_i and tension. Phenylephrine produced dose-dependent increases in [Ca²⁺]_i and contractions of uterine arteries from both nonpregnant and pregnant animals (Fig. 1). As shown in Fig. 1A, the phenylephrine-induced concentration-contraction curve for the uterine artery of the pregnant sheep was shifted to the left compared with that of the nonpregnant sheep as indicated by the –log of the EC₅₀ values obtained (pD₂; 5.86 ± 0.13 vs. 5.15 ± 0.18, respectively; P < 0.05). The maximal responses were 2.51 ± 0.14 and 1.79 ± 0.19 g for the uterine arteries of pregnant and nonpregnant animals, respectively (P < 0.05). Accordingly, phenylephrine produced a dose-dependent increase in [Ca²⁺]_i (measured via fura 2, R_f340/380) in uterine arteries of both nonpregnant and pregnant sheep (pD₂, 5.6 ± 0.14 vs. 6.0 ± 0.12, respectively; P = 0.07; Fig. 1B). The maximal response was significantly increased in uterine arteries of pregnant (0.103 ± 0.009) compared with nonpregnant (0.032 ± 0.004) animals (P < 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Phenylephrine-induced contractions (A) and intracellular free Ca2+ concentrations ([Ca2+]i; B) in uterine arteries. Cumulative concentration-response curves of phenylephrine-induced increases in contractions and [Ca2+]i were measured simultaneously in the same tissues of uterine arteries obtained from nonpregnant and pregnant ovine. Data are means ± SE of tissues from 4 animals. Values for pD2 and the maximal responses are presented in the text.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Phenylephrine-induced time courses of 20-kDa myosin light chain (MLC20) phosphorylation in uterine arteries. Arterial rings of uterine arteries from nonpregnant (NP) and pregnant (P) ewes were stimulated with 3 µM phenylephrine. Phosphorylation of MLC20 was detected by Western immunoblot (as described in METHODS). Representative immunoblot (top) shows unphosphorylated MLC20 and phosphorylated MLC20 (MLC20-P, expressed as mg phosphorylated MLC20/g tissue wet weight) induced by phenylephrine at 0–300 s (bottom). Data are means ± SE of tissues from 4 animals.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Phenylephrine-induced, dose-dependent increases in MLC20 phosphorylation in uterine arteries. Arterial rings of uterine arteries from nonpregnant and pregnant ewes were stimulated with increasing doses of phenylephrine. Phosphorylation of MLC20 was detected by Western immunoblot. Data are means ± SE of tissues from 4 animals; *P < 0.05, pregnant vs. nonpregnant.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Phenylephrine-induced MLC20 phosphorylation-[Ca2+]i relation in uterine arteries. Arterial rings of uterine arteries from nonpregnant and pregnant ewes were stimulated with increasing doses of phenylephrine (0.3–30 µM). Increased levels of MLC20-P stimulated by phenylephrine were plotted to show responses as a function of [Ca2+]i [fura 2 signal and ratio of fluorescence intensities at each excitation wavelength, F340 and F380, respectively (Rf340/380)] at each corresponding concentration. Data are means ± SE of tissues from 4 animals.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Phenylephrine-induced force-MLC20-P relation in uterine arteries. Arterial rings of uterine arteries from nonpregnant and pregnant ewes were stimulated with increasing doses of phenylephrine (0.3–30 µM). Increased tension stimulated by phenylephrine was plotted to show responses as a function of MLC20-P measured simultaneously in the same tissue at each corresponding concentration. Data are means ± SE of tissues from 4 animals.

View larger version (14K): [in this window] [in a new window]   Fig. 6. Effects of PD-098059 on phenylephrine-induced contractions and MLC20 phosphorylation in uterine arteries from nonpregnant animals. Arterial rings were pretreated with 30 µM PD-098059 or DMSO vehicle (control) for 30 min before stimulation with 3 µM phenylephrine. Contractions (A) and MLC20 phosphorylation (B) were measured simultaneously in uterine arteries. Increased tension stimulated by phenylephrine was plotted to show responses as a function of MLC20-P measured simultaneously in the same tissue at each corresponding time point (C). Data are means ± SE of tissues from 5 animals.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Phorbol 12,13-dibutyrate (PDBu)-induced contractions and MLC20 phosphorylation in uterine arteries. Arterial rings of uterine arteries from nonpregnant and pregnant ewes were stimulated with 5 µM PDBu. PDBu-induced contractions and MLC20 phosphorylation were measured simultaneously in the same tissues. Data are means ± SE of tissues from 4–8 animals; *P < 0.05, pregnant vs. nonpregnant.

	DISCUSSION

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The present finding that ₁-adrenoceptor-mediated contractions of isolated uterine arteries were increased in the pregnant animals is in agreement with previous studies (3, 4, 11, 12, 33, 47, 51). This is probably due in part to a transient and reversible sympathetic denervation of the uterine artery during pregnancy (23, 30, 31) that results in a sensitization of postsynaptic ₁-adrenoceptor-mediated responses. In contrast, pregnancy did not result in changes in postsynaptic ₁-adrenoceptor-mediated contractions in systemic vessels (3, 19). In addition, 5-HT- and KCl-stimulated contractions were the same between uterine arteries of nonpregnant and pregnant animals (52). These findings suggest a specific effect of pregnancy on ₁-adrenoceptors in uterine arteries and are consistent with the notion of sympathetic denervation-mediated sensitization of postsynaptic ₁-adrenoceptor-mediated responses. Previous studies demonstrated that the increased ₁-adrenoceptor-mediated contractions in pregnant ovine uterine arteries were not the result of increased cellularity in the arterial cross-section but rather were related to properties of the vascular smooth muscles themselves (4). In addition, uterine arterial wall thickness and actin-to-myosin ratio were found to be unaltered during pregnancy (4).

Consistent with the sensitization of postsynaptic ₁-adrenoceptor-mediated Ca²⁺ mobilization and contractions, we have demonstrated that the density of ₁-adrenoceptors and the norepinephrine binding affinity to ₁-adrenoceptors are significantly increased in pregnant compared with nonpregnant ovine uterine arteries and result in increased inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] synthesis (46, 47). The mechanisms for pregnancy-mediated increase in agonist binding affinity of ₁-adrenoceptors in uterine arteries are not clear at present. The role of Ins(1,4,5)P₃ as the messenger of agonist-mediated Ca²⁺ mobilization in smooth muscle has been firmly established (40). In addition to the possible sensitization of postsynaptic ₁-adrenoceptors due to sympathetic denervation in uterine arteries during pregnancy, pregnancy and steroid hormones may also have direct effects on ₁-adrenoceptor-mediated responses in uterine arteries. In isolated female rat aorta, estrogen pretreatment increased the number of ₁-adrenoceptors and the amount of intracellular Ca²⁺ available for contraction (6). In myometrial smooth muscle, progesterone had no effect on Ins(1,4,5)P₃-dependent or -independent Ca²⁺ release from intracellular Ca²⁺ stores (15) but significantly increased ₁-adrenoceptor-mediated synthesis of inositol phosphates (37). It has been demonstrated (16) that progesterone of endogenous or exogenous origin increases ₁-adrenoceptor numbers on uterine arterial smooth muscle membranes.

The increased Ca²⁺ mobilization is likely to play an important role in the increased MLC₂₀ phosphorylation by activation of ₁-adrenoceptors in the uterine arteries of pregnant sheep in the present study. This is consistent with the previous finding with ovine uterine arteries that the phenylephrine-stimulated total amount of MLC₂₀-P was increased in uterine arteries of pregnant compared with nonpregnant animals (4). In contrast, studies on human myometrial smooth muscle demonstrated that KCl-mediated MLC₂₀ phosphorylation was significantly reduced in tissues from pregnant compared with nonpregnant individuals (45). This also suggests that increased MLC₂₀ phosphorylation observed in the uterine artery during pregnancy is specific to the ₁-adrenoceptor-mediated pathway. As discussed above, this selective sensitization occurred upstream of the signal transduction pathway at the receptor levels, i.e., ₁-adrenoceptor density and endogenous agonist binding affinity. Unlike ₁-adrenoceptors, KCl-mediated MLC₂₀ phosphorylation depends on Ca²⁺ influx through voltage-sensitive Ca²⁺ channels. Both estrogen and progesterone have been demonstrated to inhibit Ca²⁺ influx in smooth muscle (15, 38).

In addition to [Ca²⁺]_i, phosphorylation of MLC₂₀ is also regulated by activities of MLCK and MLCP. Although it is unknown at present whether MLCK activity is altered in uterine arteries during pregnancy, the specific activity of MLCK was not different in myometria of nonpregnant and pregnant individuals (45). In the present study, we have demonstrated that pregnancy is associated with a significant decrease in the Ca²⁺ sensitivity of MLC₂₀ phosphorylation in response to phenylephrine, i.e., less MLC₂₀ phosphorylation occurs at a given [Ca²⁺]_i in uterine arteries of pregnant animals, which suggests an increase in MLCP activity in these vessels. It has been demonstrated that steroid hormones regulate the expression of MLCP catalytic (PP1-) and large regulatory (MYPT) subunits and increase the levels of PP1- and MYPT in myometria of pregnant individuals (43). In addition, estrogen and progesterone treatment inhibited agonist- and GTPS-induced Ca²⁺ sensitization of smooth muscle by increasing the expression of Rnd1 and Rnd3, which inhibited the RhoA-dependent pathways (9, 26).

The present finding that ₁-adrenoceptor-mediated contractions at given levels of MLC₂₀ phosphorylation were significantly deceased in uterine arteries of pregnant compared with nonpregnant sheep suggests that pregnancy enhances the thin-filament-mediated inhibition of contractions in uterine arteries. This is further supported by the results of PKC-mediated contractions in uterine arteries. In this study, we demonstrated that PKC-induced contractions are independent of changes in MLC₂₀ phosphorylation in uterine arteries of both nonpregnant and pregnant animals, which indicates that in uterine arteries, PKC-induced contractions were mediated predominately through thin-filament regulatory pathways. Although many studies have demonstrated that PKC mediates Ca²⁺-independent contractions by increasing myofilament Ca²⁺ sensitivity in vascular smooth muscle, the significance of MLC₂₀ phosphorylation in PKC-induced contractions remains controversial and may be tissue dependent. Consistent with the present findings, the dissociation between MLC₂₀ phosphorylation and tension development in response to phorbol esters has been demonstrated previously (17, 41). In addition to thick-filament regulation, i.e., MLC₂₀ phosphorylation through MLCP, the Ca²⁺ sensitivity of myofilaments is also regulated through smooth muscle thin-filament actin-binding proteins such as caldesmon that inhibit myosin ATPase activity. Studies using the antagonist peptide GS17C and the corresponding antisense (28) strongly suggest an important and physiologically relevant role for caldesmon in suppressing smooth muscle tone. It has been demonstrated (42, 44, 49) that PKC induces phosphorylation of caldesmon, which reverses its inhibitory effect on myosin ATPase. The finding that PKC-mediated contractions were attenuated in uterine arteries of pregnant ewes in the present study further suggests an adaptation of the thin-filament regulatory pathway to pregnancy. Indeed, it has been reported (25, 45) that during pregnancy, caldesmon levels increase in both human and rat myometria, which contributes to suppression of contractility.

In agreement with our previous findings with uterine arteries of pregnant animals, the present study demonstrates that the ERK inhibitor PD-098059 causes a significant leftward shift in the force-MLC₂₀ phosphorylation relation in uterine arteries of nonpregnant sheep. This suggests that ERK plays a key role in maintaining the thin-filament-mediated inhibitory effect on contractions of uterine arteries. We previously demonstrated (51) that pregnancy selectively enhances the role of ERK in ₁-adrenoceptor-mediated contractions and its effect in suppressing PKC-mediated contractions in uterine arteries. In vascular smooth muscle, ERK has been demonstrated to be a physiologically relevant caldesmon kinase that phosphorylates caldesmon at Ser⁷⁸⁹ (1, 2). However, whether caldesmon phosphorylation at ERK-specific sites induces smooth muscle contraction remains controversial (10, 24, 32). Our recent studies with uterine arteries of pregnant sheep suggest that ERK-mediated phosphorylation of caldesmon may stabilize the inhibitory effect of caldesmon on actin-activated myosin ATPase (49). Pregnancy significantly upregulates ERK-signaling pathways in both endothelium and vascular smooth muscle in uterine artery, although different mechanisms may be involved (7, 14, 51). Taken together, ERK is likely to play an important role in the adaptation of uterine artery contractility to pregnancy by increasing the thin-filament-mediated inhibitory effect on uterine artery contractions.

From the physiological perspective, during pregnancy, the uterine vasculature acts as a low-resistance shunt to accommodate the large increase in uteroplacental blood flow that is required for normal fetal development. In addition to increased endothelial nitric oxide synthesis and release, pregnancy is associated with a transient and reversible sympathetic denervation of the uterus and uterine artery that is associated with a profound decrease in contractions of smooth muscle in response to electric field stimulation (23, 31). Although decreased sympathetic innervation combined with increased nitric oxide release maintain low uterine vascular tone during pregnancy, sympathetic denervation is likely to sensitize the postsynaptic -adrenergic pathway and increase the ability of nonsynaptic -adrenergic-mediated contractions in uterine artery. This may be important for a mother to protect herself under stress and may allow a redistribution of blood by contracting the uterine artery in response to circulating catecholamines. Indeed, uterine blood flow was found to be significantly reduced under the stress of short-term exercise in pregnant women and sheep (27). This may be achieved in part by enhanced intracellular Ca²⁺ mobilization through activation of ₁-adrenoceptors that result in activation of MLCK and phosphorylation of MLC₂₀. On the other hand, the decreased Ca²⁺ sensitivity of myofilaments contributes to maintaining low basal vascular tone of uterine arteries during pregnancy and helps to maintain low vascular tone in response to increased blood flow through uterine arteries during pregnancy. Although it is reasonable to suggest that steroid hormones play a key role in the adaptation to pregnancy of both thick- and thin-filament regulatory pathways in uterine arteries, additional investigation is needed to establish the cause-and-effect relationship in an ovariectomized ovine model as described previously (18, 36).

	GRANTS

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This work was supported in part by National Institutes of Health Grants HL-57787, HL-67745, and HD-31226 and by the Loma Linda University School of Medicine. D. Xiao is a recipient of American Heart Association Predoctoral Fellowship AHA-0215040Y.

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. Zhang, Center for Perinatal Biology, Dept. of Physiology and Pharmacology, Loma Linda Univ. School of Medicine, Loma Linda, CA 92350 (E-mail: lzhang{at}som.llu.edu)

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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