Neural crest (NC) cells are an embryonic migratory cell population that develops at the junction between the neural plate and the ectoderm. As the neural plate closes to form the neural tube, NC cells emigrate from the epithelium and migrate throughout the embryo. NC cells form a wide range of cell types important in the formation of craniofacial structures, cardiovascular system, endocrine organs, and much of the peripheral nervous system (LeDouarin and Kalcheim, 1999). NC cells arising between the mid-otic and caudal end of the third somite axial level (“cardiac NC cells”) migrate beneath the ectoderm into pharyngeal arches III, IV, and VI, and a subset of these cells invade the outflow tract of the heart and participate in its septation (Kirby et al., 1983; Waldo and Kirby, 1998). Studies show that severe cardiac defects such as persistent truncus arteriosus, double-outlet right ventricle, and tetralogy of Fallot, occur if the migration of these cardiac NC cells is perturbed (Kirby, 1987; Conway et al., 2000).
The involvement of matrix metalloproteinases (MMP) in cell migration is well documented. Many malignant cells synthesize and secrete MMPs or encounter MMP molecules secreted by neighboring stromal cells (Lochter et al., 1998; Hofmann et al., 2000). Natural or synthetic MMP inhibitors reduce tumor cell migration or metastasis in vitro and in vivo (reviewed by, Chambers and Matrisian, 1997). Like tumor cells, NC cells penetrate through basement membranes and invade extracellular matrix (ECM) during their emigration and migration; therefore, NC cells likely use similar invasive mechanisms. In addition, patch mice, which exhibit NC-related craniofacial and cardiac defects (Morrison-Graham et al., 1992; Schatteman et al., 1995), have a deficit in MMP-2 and membrane-type MMP expression, and craniofacial mesenchymal cells from these embryos have a decreased migratory capacity of in vitro (Robbins et al., 1999). The spatial and temporal distribution pattern of MMP-2 also correlates positively with cardiac NC migration in chicken embryos (Cai et al., 2000). However, there is no direct evidence that MMP activity modulates early NC cell migration in vitro or in vivo. Here, we tested the hypothesis that blocking MMP activity inhibits cardiac NC cell migration in vitro and in vivo. We found that the synthetic MMP inhibitor KB8301 significantly decreased the levels of MMP enzymatic activity in embryos and decreased the distance cardiac NC cells migrated both in vitro and in vivo. In addition, KB8301 decreased the rate of cardiac NC cell motility and increased NC cell area and cell perimeter in vitro. These data suggest that MMP enzymatic activity is an important mediator of cardiac NC cell migration in vitro and in vivo.
Before assessing the effect of KB8301 on NC migration, we wanted to verify whether microinjection of KB8301 resulted in decreased MMP activity in vivo. Chick embryos were bilaterally injected at the level of the second somite with dimethyl sulfoxide (DMSO), 46 pmol of KB8301, or 92 pmol of KB8301 in ovo at Hamburger-Hamilton stage 10 (Fig. 1), and the eggs were reincubated for 6 hr. The MMP enzymatic activity in tissue segments isolated from the injected area was then measured by using a fluorometric assay. There was no significant difference between the MMP enzymatic activity found in DMSO-injected embryos and those injected with 46 pmol of KB8301. However, MMP enzymatic activity was significantly reduced in embryos injected with 92 pmol of KB8301 compared with DMSO-treated embryos (Table 1). This finding showed that KB8301 injections effectively reduced MMP activity in vivo when 92 pmol were injected.
Table 1. Effect of KB8301 Injection on MMP Activity In Vivoa
46 pmol Treated Group
92 pmol Treated Group
In each trial, 10 embryos were injected with DMSO or KB8301 and then reincubated for 6 hr. Another 10 embryos were sham operated in each trial. Tissue segments were then collected, and MMP activity was measured as described in the Experimental Procedures section. Data in Table 3 represent the ratio of MMP activity in DMSO-treated to sham embryos and the ratio of KB8301-treated to sham embryos obtained in each trial. An unpaired t-test then was performed. MMP, matrix metalloproteinase; DMSO, dimethyl sulfoxide.
We then evaluated the effect of injecting DMSO and KB8301 on the development of embryos. Approximately 94% (173 of 185) of the embryos injected with DMSO alone were alive, of comparable size, and morphologically indistinguishable from sham-operated embryos. Similar frequency rates were obtained when embryos were injected with 46 pmol (102 of 110; 93%) or 92 pmol of KB8301 (90 of 99; 93%), and embryos were virtually indistinguishable in overall appearance from the DMSO controls. HNK-1 immunostaining revealed that cardiac NC cell emigration and migration patterns in embryos unilaterally injected with DMSO (Fig. 2A) or 46 pmol of KB8301 (not shown) were not noticeably different from the noninjected contralateral side or from sham-operated embryos. However, when embryos were unilaterally injected with 92 pmol of KB8301, the extent of cardiac NC cell migration appeared decreased on both sides (Fig. 2B). Unilateral injection of 184 pmol of KB8301 was devastating in that approximately 60% of treated embryos (43 of 72 treated embryos) died. The remaining 40% exhibited a decreased growth rate, severe curvature defects along the cranial–caudal axis (Fig. 2C), and only a few HNK-1–positive cardiac NC cells were observed in these embryos. The migration of the cranial NC cells in these embryos also appeared perturbed, but the trunk NC migratory pattern looked normal. Tissue sections stained with the DNA-binding fluorescence dye DAPI revealed a pronounced degree of nuclear fragmentation in embryos treated with 184 pmol of KB8301, suggesting large-scale cell death in cardiac NC cells, neural tube, and paraxial mesoderm (Fig. 2F). However, this was not the case for sham-operated embryos or embryos injected with DMSO (Fig. 2D), 46 pmol of KB8301, or 92 pmol of KB8301 (Fig. 2E). Therefore, only 46 and 92 pmol of KB8301 were used in subsequent experiments.
To determine whether KB8301 inhibited cardiac NC cell migration in vivo, the distance cardiac NC cells migrated from their origin (dorsal apex of the neural tube) was measured in cross-sections of embryos at the site of injection (Fig. 2G,H). Cardiac NC cell migratory distance in embryos injected at stages 10-, 10, or 10+ with either DMSO or 46 pmol of KB8301 was not significantly different between the injected side and noninjected side. Nor was there a difference in NC migration distance between the DMSO-injected side and the side injected with 46 pmol of KB8301. However, when embryos were injected with 92 pmol of KB8301, the distance cardiac NC cells migrated in the injected side was significantly decreased compared with that in the noninjected side, whereas there was no such difference observed in DMSO-injected embryos (Fig. 3). In addition, the average migratory distance for cardiac NC cells in the KB8301-injected side of embryos was significantly less than in the DMSO-injected side. We also observed a decrease in NC migratory distance on the noninjected side of KB8301-treated embryos, but it was not quite statistically significantly different from DMSO-treated embryos (P < 0.052). DMSO, the carrier used to deliver KB8301, is very lipophilic and highly diffusible. Given the small size and low tissue density of these young embryos, we attributed the decreased NC migration on the noninjected side to a carryover of KB8301 from the injected side.
Although embryos injected at a particular stage were all incubated for an additional 13 hr, the developmental stage at the end of this period varied. To determine whether KB8301 altered the developmental rate of embryos, we compared the developmental end stage of embryos after treatment with DMSO or KB8301. Based on Hamburger and Hamilton staging, the developmental rate of embryos treated with 46 or 92 pmol of KB8301 was not significantly different from DMSO-treated embryos (Table 2). However, there appeared to be a tendency for embryos treated with 92 pmol of KB8301 to be younger at the time of collection than the DMSO-treated group, based on Hamburger-Hamilton staging. To further evaluate whether differences in the rate of development after injection might be responsible for the differences in migratory distance, we performed a two-way analysis of variance (ANOVA) with the treatment and end stage (stage at time of collection) being the two independent variables. Regardless of the end stage, the migratory distance of cardiac NC cells in KB8301-treated stage 10 embryos was always significantly less than that in DMSO-treated stage 10 embryos (P < 0.001).
Table 2. Effect of KB8301 Injection on Early Embryonic Developmenta
No. of embryos
Stage at collection
Embryos were unilaterally injected with KB8301 or an equivalent amount of DMSO at the second somite level of Hamburger and Hamilton stage 10−, 10, or 10+ embryos, reincubated for 13 hr, and then collected and re-staged. Embryos were classified into three groups based on their developmental stage at the time of collection. The number of KB8301-treated embryos at each end stage was compared with DMSO controls using a chi-squared test. No significant difference with respect to end stage was detected. DMSO, dimethyl sulfoxide.
Although KB8301 injection did not affect the rate of embryonic development based on the end stage, the difference in migratory distance between DMSO- and KB8301-treated embryos could still be attributable to differences in embryo size. To account for possible differences in embryo size, the migratory distance in the injected side and the noninjected side in each embryo was expressed as a ratio and the ratios from DMSO- and the KB9301-treated groups were then compared. Again, KB8301-treated embryos showed a significant reduction in the average distance cardiac NC cells migrated compared with DMSO-treated embryos (Table 3). In addition, inhibition of MMP activity did not change the number of migrating HNK-1–positive NC cells (data not shown).
Table 3. Effect of KB8301 on Migratory Distance When Accounting for Differences in Embryo Sizea
Mean Ratio of Migratory Distance Between Injected and Noninjected Side
Differences in embryo size were accounted for by calculating the ratio between the migratory distance measured on the injected side to the noninjected side for DMSO-treated embryos and KB8301-treated (92 pmol) embryos. The mean of the ratios were then compared using an unpaired t-test. Values represent mean ± SD. DMSO, dimethyl sulfoxide.
We then examined whether inhibiting MMP activity altered NC migratory behavior in vitro. Under these conditions, we could more precisely control and maintain concentrations of KB8301. Analysis of time-lapse images of NC migration (Fig. 4) revealed that KB8301 significantly reduced NC motility rate in a dose-dependent manner compared with controls (Fig. 5). Moreover, the distance NC cells migrated from their original starting point was significantly reduced by KB8301. The decreased motility rate and decrease in distance NC cells migrated from their starting position was accompanied by a significant increase in cell area and cell perimeter, suggesting that NC cell-substrate adhesion was increased in the presence the MMP inhibitor. We also evaluated the effect of another hydroxamate-based MMP inhibitor, GM6001, on NC migratory behavior in vitro. GM6001 also significantly reduced NC motility and migration distance in vitro (Fig. 5), and it also increased cell area and perimeter.
MMPs are not only important in tissue remodeling, wound repair, and pathologies such as cancer metastasis and tumor growth but also may play important roles in embryonic development. Synthetic MMP inhibitors are often used as a tool for investigating the role of MMPs in these processes. Hydroxamate-based MMP inhibitors such as KB8301 and GM6001 use the structural conservation of the catalytic domain among MMPs to block enzymatic activity. KB8301 strongly inhibits MMP activity with a low IC50 (0.3–0.6 nM; Yamamoto et al., 1998). This inhibitor has been used to inhibit MMP activity in explanted mouse pharyngeal arches (Robbins et al., 1999), sheddase activity in shedding of TNF receptors (Dri et al., 2000), and in metalloproteinase-mediated release of Fas ligand (Kayagaki et al., 1995). Previously, we have shown that NC cells encounter ECM containing MMP-2 (Cai et al., 2000); however, we are unaware of any direct evidence showing that MMP enzymatic activity mediates NC cell migration. In this study, microinjection of KB8301 into stage 10 chick embryos caused developmental anomalies, significantly decreased the endogenous levels of MMP activity, reduced cardiac NC migratory distance in vivo and in vitro, and decreased NC motility rate and increased NC surface area in vitro. These results show that MMP activity is an important mediator of early embryonic morphogenesis and NC cell migration.
Injecting 92 pmol of KB8301 before cardiac NC emigration decreased the average distance cardiac NC migrated by approximately 15% on the injected side compared with the noninjected side. This decrease was not attributable to changes in developmental rate or embryo size, showing that KB8301 reduced the local migratory capacity of cardiac NC rather than having a global effect on development at this concentration. KB8301 also decreased MMP activity in embryos, suggesting that the action of KB8301 on cardiac NC migration was mediated through its ability to inhibit MMP activity. Although enzymatic activity and NC migratory distance in embryos decreased only approximately 15–20%, the effectiveness of KB8301 may have been curtailed by molecular diffusion, turnover, and/or growth of the embryo. Moreover, the loss of MMP activity could be partially compensated by a decrease in synthesis of MMP inhibitors, or up regulation of other non-MMP proteinases (e.g., urokinase and plasmin known to play a role in NC migration; Erickson and Isseroff, 1989; Brauer and Yee, 1993). In vitro, where KB8301 levels were more uniform and maintained, NC motility and migratory distance were also reduced by KB8301 in a dose-dependent manner and to a larger degree. Collectively, these results show that MMP activity is an important mediator of cardiac NC migration both in vitro and in vivo.
The decrease in cardiac NC cell migratory distance observed in KB8301-treated embryos could result from a decrease in cardiac NC cell motility, ECM invasiveness, or emigration of NC cells from the neural tube. In vitro, KB8301 decreased not only cardiac NC motility but also increased cardiac NC cell area and cell perimeter, suggesting that there was an increase in cell/substrate adhesion. Cell motility is dependent on the degree of cell adhesion. In general, cells become less mobile when cell adhesion increases beyond an optimal level for cell migration (Izzard and Lochner, 1980; Kolega et al., 1982; Akiyama et al., 1989). When NC cells are plated onto a substrate to which integrin antibody is absorbed, NC cells become extremely flattened and very immobile because of antibody binding to NC fibronectin receptors. However, when plated onto fibronectin substrates, NC cells are almost four times more mobile and more rounded in shape (Duband et al., 1986). By using intact fibronectin and modified fibronectin fragments, Beauvais-Jouneau et al. (1997) also showed an inverse relationship between the degree of spreading and NC motility rate. Compared with intact fibronectin, the degree of NC cell spreading decreases when NC cells are plated on variants of the fibronectin carboxyl-terminal cell-binding domain yet NC motility rate is significantly increased. Therefore, if cell spreading and cell adhesion exceed an optimal degree, the mobility of NC cells decreases.
Several studies suggest that MMP activity is an important mediator of cell adhesion and cell spreading. Tissue inhibitors of metalloproteinase (TIMP) increase cellular adhesion and spreading of human melanoma cells, with a corresponding decrease in proteolytic degradation of cell surface and ECM components (Ray and Stetler-Stevenson, 1995). In transformed fibroblasts, GM6001 and TIMP-1 increase cell spreading and stabilize focal contacts, restoring contact inhibitory characteristics to these cells in vitro (Ho et al., 2001). MMP-9 accumulates opposite type IV collagen in advancing lamellopodia of migrating bronchial epithelial cells. When MMP-9 activation is inhibited or cells are treated with the MMP inhibitor, BB-94, cell motility correspondingly decreases (Legrand et al., 1999). Our observations showing that KB8301 increases NC cell area and cell perimeter with a corresponding decrease in cell motility and migratory distance in vitro suggest the reduction of NC migratory observed in vivo might reflect changes in cell/ECM adhesiveness.
KB8301 could also decrease migratory distance by reducing ECM degradation, thereby reducing invasiveness through ECM barriers. Hydroxamate-based MMP inhibitors reduce degradation of ECM and decrease tissue invasion by cells. The MMP inhibitor BB3103 decreases the degradation of MMP-dependent fibrillar type I collagen and reduces angiogenesis in vivo (Seandel et al., 2001); BB-94 inhibits collagenolysis and invasion of human umbilical vein endothelial cells into collagen gels (Fisher et al., 1994). MMP inhibitors also block ECM degradation and migration of osteoblasts through collagen (Sato et al., 1998). In addition, GM6001 inhibits the invasion of a glioblastoma cell line into Matrigel (Boghaert et al., 1994). Evidence suggests that NC cells degrade ECM as they migrate and that the extracellular distribution of MMP-2 is disrupted at the migratory front of cardiac NC cells (Brauer et al., 1985; Cai et al., 2000). Hence, KB8301 could also restrict cardiac NC migration by reducing MMP-dependent ECM degradation and, hence, cardiac NC invasiveness.
To generate cardiac NC cells, precursor cells must undergo an epithelial-mesenchymal transition necessitating major changes in cell–cell adhesion and/or removal of the underlying basement membrane. Inhibitors of MMPs block similar epithelial-mesenchymal cell transition occurring during tumor metastasis, whereas reducing endogenous MMP inhibitors levels (e.g., TIMPs) can facilitate metastasis (Schultz et al., 1988; Alvarez et al., 1990; Alexander and Werb, 1992; DeClerck et al., 1992; Valente et al., 1998; Prontera et al., 1999). KB8301, by altering cell–cell adhesion or inhibiting basement membrane turnover, could perturb the epithelial–mesenchymal transition process generating NC cells, thereby delaying or reducing the number of emigrating NC cells. However, our observation that there was no measurable change in the number of HNK-1–positive NC cells in KB8301 injected embryos argues against this possibility.
In summary, injecting an MMP synthetic inhibitor into the cardiac NC cell migratory pathway decreased MMP activity and decreased NC cell migration both in vivo and in vitro. In addition, blocking MMP activity increased NC cell spreading and cell perimeter, suggesting that MMP activity mediates NC cell adhesion. Thus, the enzymatic activities of MMPs are likely important mediators of cardiac NC cell migration. Once MMPs are activated, endogenous MMP inhibitors must curtail the enzymatic activity of MMPs so that levels of proteolytic activity are appropriate for NC migration. Future studies will focus on identifying these MMP inhibitors as well as identifying the particular MMPs involved in this process.
MMP Activity Assay
Fertilized White Leghorn chicken eggs were incubated at 38°C in a 60% relative humidity environment until embryos reached Hamburger-Hamilton stage 10 (Hamburger and Hamilton, 1951). Eggs were windowed, and diluted India ink was injected beneath the blastoderm to better visualize the embryos. KB8301 ([4-(N-hydroxyamino)-2R-isobutyl-3S-methylsuccinyl]-L-3-(5,6,7,8,-tetrahydro-1naphyl) alanine-N-methylamide; PharMingen, San Diego, CA) in DMSO or DMSO alone was bilaterally injected beneath the ectoderm at the level of the second somite by using a “Nanoject” automatic oocyte injector (Drummond Scientific Co., Broomall, PA). After injecting the embryos, eggs were then sealed with tape and reincubated for 6 hr. Injected embryos were collected, tissue blocks between mid-otic and third somite level were excised, and splanchnic mesoderm and heart were carefully removed by microdissection from collected tissues. This tissue was then homogenized by vortexing in 0.1% Triton X-100 for 10 min, and supernatants were collected after centrifugation at 12,000 × g for 10 min.
A fluorescent method was used to measure MMP enzymatic activity in the tissue lysates (Murphy and Crabbe, 1995). Briefly, 4 μl of the tissue lysate supernatant was incubated with 12.5 μl of 0.5 mM synthetic MMP substrate, MCA-Pro-Leu-Gly-Leu-[3-DNP-2,3-DAP]-Ala-Arg (American Peptide Co., Sunnyvale, CA), in 250 μl of assay buffer (0.1 M Tris-HCl, 0.1 M NaCl, 10 mM CaCl2, and 0.05% Brij-35, pH 7.5) for 20 min at 37°C. After cleavage of the Gly-Leu bond, this substrate fluorescences at 390 nm when excited at a wavelength of 326 nm. Fluorescence was read on a TD-700 Fluorometer (Turner Designs, Sunnyvale, CA). The enzymatic activity of the samples was calculated based on a standard curve using known amounts of active human MMP-2 (Alexis Biochemicals, San Diego, CA). MMP enzymatic activity was normalized to levels of DNA in the lysates measured by using a H33258/TKO 100 DNA assay method on a minifluorometer (Hoefer Scientific Instruments, San Francisco, CA; Brunk et al., 1979). The ratio of the enzymatic activity (μU of MMP-2 activity/μg DNA/time) in KB8301-injected to sham-control embryos was compared with the ratio of enzymatic activity in DMSO-injected to sham-control embryos by using a nonpaired t test. The test was considered statistically significant when the P values were less than 0.05.
In Vitro Migration Assays
Eggs were incubated until they reached stage 10 to 11- and the neural tube segments from the cardiac NC axial level (mid-otic placode to somite level 3) were removed and freed of associated tissue by microdissection. Neural tubes were incubated on fibronectin-coated tissue culture dishes (25 μg/ml) containing Medium 199 with 1% heat-inactivated and plasminogen-depleted fetal bovine serum to which either DMSO (the carrier), DMSO containing KB8301, or DMSO containing GM6001 (N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide; Calbiochem, San Diego, CA) was added. GM6001 is a hydroxamic acid–based MMP inhibitor that inhibits MMP activity both in vitro and in vivo (Galardy et al., 1994; Jones et al., 1997; Solorzano et al., 1997). For time-lapse videomicrography, neural tubes were maintained in a 5% CO2 environment at 37°C in a micro-incubation chamber located on an inverted microscope. After 1 hr, the migratory behavior of cardiac NC cells emerging from the neural tube explants was accessed by capturing an image every 5 min for the next 5 hr by using a CCD camera-equipped computer and the images were stored. To evaluate NC cell migratory behavior, the cell position, cell area, and cell perimeter of randomly selected cells were measured on each frame by using image analysis software (IPLab, Scanalytics, Inc., Fairfax, VA). From these data, the average rate of cell motility, the average distance each cell migrated from its initial starting position, and the average cell area and perimeter were calculated over the 5-hr period. All measurements were made without prior knowledge as to which treatment group was being analyzed. Statistical analysis was performed by using a Mann-Whitney nonparametric test or a Kruskal-Wallis nonparametric ANOVA coupled with Dunn's multiple-comparison posthoc tests by using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA).
KB8301 Injection and In Vivo Cardiac NC Migration Assessment
KB8301 (46, 92, or 184 pmol in 9.2 nl) was microinjected unilaterally into the cell-free space adjacent to the premigratory cardiac NC cells at the second somite level of chicken embryos at stages 10-, 10, and 10+ (Fig. 1). Control embryos received an equivalent volume of the KB8301 vehicle (DMSO). Eggs were sealed and returned to incubators for 13 hr, by which time the embryos reached stage 13–14. Embryos were collected, fixed in 4% paraformaldehyde, and permeabilized with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM ethylenediaminetetraacetic acid, 50 mM Tris-HCl, pH 8.0) for 3 hr. Embryos were then incubated at 4°C overnight with a 1:3 dilution of hybridoma-conditioned culture medium containing the HNK-1 immunoglobulin M (IgM) antibody (American Type Culture Collection, Rockville, MD), which recognizes an epitope on NC cells. The embryos were washed five times with PBS-0.1% Tween-20 and then incubated with donkey anti-mouse IgM-TRITC (Jackson ImmunoResearch, West Grove, PA) at 4°C overnight. The embryos were washed in PBS-0.1% Tween-20 and then refixed in 4% paraformaldehyde, dehydrated, and embedded in JB-4 plastic (Electron Microscopy Sciences, Fort Washington, PA). Four-micron-thick serial cross-sections were prepared by using a Leica RM2135 microtome (Leica Microsystems, Inc., Bannockburn, IL). Serial sections at the second somite level were then stained with 0.5 μg/ml of DAPI (Sigma Chemical Co., St. Louis, MO) to label the nuclei, the sections were examined, and images were captured by using an epifluorescence microscope equipped with a color CCD camera. HNK-1 immunostaining and DAPI images of the same tissue section were then combined by using image analysis software. The distance between the dorsal midline of the neural tube to the leading edge of cardiac NC cells was measured along the basal surface of the ectoderm (Fig. 1). Five adjacent serial tissue sections, beginning with the most cranial section containing the second somite, were measured and averaged to represent the migratory distance of the leading cardiac NC cells on each side for each embryo. The number of HNK-1–positive NC cells was also counted by using the DAPI nuclear stain in these sections. Again, all measurements were made without prior knowledge as to which treatment group was being analyzed. The migratory distance between the injected and noninjected side of KB8301- or DMSO-treated embryos was then compared by using a paired t-test. The migratory distance in the injected side of KB8301- and DMSO-treated embryos was also compared by using a nonpaired t-test. To account for possible effects on embryo size, the migratory distance in the injected side (d) and the noninjected side (d′) was expressed as a ratio for each embryo and the ratios then compared between the DMSO- and KB8301-treated embryos (Fig. 1). Finally, a two-way ANOVA was used to analyze whether differences in migration distance between DMSO- and KB8301-injected embryos were independent of the rate of development (embryonic stage reached at the time of collection) by using GraphPad Prism software. Tests were considered statistically significant at P values < 0.05.
P.R.B. received funding from the American Heart Association, Heartland Affiliate.