Effect of acute heparin administration on glycocalyx thickness and endothelial function in healthy younger adults

Abstract

The endothelial glycocalyx is a dynamic, gel-like layer that is critical to normal vascular endothelial function. Heparin impairs the endothelial glycocalyx and reduces vascular endothelial function in a murine model; however, this has yet to be tested in healthy humans. We hypothesized that a single bolus dose of heparin would increase circulating glycocalyx components and decrease endothelial glycocalyx thickness resulting in blunted brachial artery vasodilation in healthy younger adults. Healthy adults (n = 19, aged 18–39 yr, 53% female) underwent measurements of the endothelial glycocalyx and vascular endothelial function at baseline and after a single bolus 5,000 U dose of heparin. The glycocalyx components syndecan-1 and heparan sulfate were measured from plasma samples using enzyme-linked immunosorbent assays. Glycocalyx thickness was determined as perfused boundary region (PBR) in sublingual microvessels using the GlycoCheck. Endothelial function was measured via ultrasonography and quantified as brachial artery flow-mediated dilation (FMD). Following acute heparin administration, there was no increase in syndecan-1 or heparan sulfate (P = 0.90 and P = 0.49, respectively). In addition, there was no change in PBR 4–7 µm (P = 0.55), PBR 10–25 µm (P = 0.63), or 4–25 µm (P = 0.49) after heparin treatment. Furthermore, we did not observe a change in FMDmm (P = 0.23), FMD% (P = 0.35), or plasma nitrite concentrations (P = 0.10) in response to heparin. Finally, time to peak dilation and peak FMD normalized to shear stress were unchanged following heparin (P = 0.59 and P = 0.21, respectively). Our pilot study suggests that a single bolus intravenous dose of heparin does not result in endothelial glycocalyx degradation or vascular endothelial dysfunction in healthy younger adults.

NEW & NOTEWORTHY The endothelial glycocalyx’s role in modulating vascular endothelial dysfunction with aging and disease is becoming increasingly recognized. This study presents novel findings that acute heparin administration is not a feasible method to experimentally degrade the endothelial glycocalyx and measure concurrent changes in vascular endothelial function in healthy humans. Alternative approaches will be needed to translate findings from preclinical studies and test the effects of acute endothelial glycocalyx degradation on vascular endothelial function in humans.

INTRODUCTION

Cardiovascular disease (CVD) is the leading cause of death worldwide, in part, because of age-related vascular endothelial dysfunction (1, 2). One potential mechanism contributing to age-related vascular endothelial dysfunction is degradation of the endothelial glycocalyx. The glycocalyx is a dynamic, gel-like layer that lines the luminal surface of endothelial cells and is crucial for the maintenance of normal endothelial function (3, 4). Importantly, glycocalyx thickness declines with advancing age (5) and is predictive of major adverse CVD events among healthy middle-aged and older adults, independent of traditional CVD risk factors (6). Therefore, it is plausible that the age-related reduction in endothelial glycocalyx thickness may contribute to vascular endothelial dysfunction and elevated CVD with aging. Consistent with this, a recent cross-sectional study of middle-aged adults revealed that lower glycocalyx thickness was associated with impaired vascular endothelial function (7). In addition, preclinical investigations have utilized enzymes to selectively degrade specific glycocalyx components and quantify concurrent changes in endothelial function. Indeed, acute enzymatic degradation of endothelial glycocalyx components decreases vascular endothelial function in cultured endothelial cells and isolated vessels obtained from multiple animal species (8–14). However, glycocalyx-degrading enzymes used in preclinical studies have not been tested in human trials and no studies have investigated the effect of acute endothelial glycocalyx degradation on vascular endothelial function in healthy humans.

Heparin is a widely used anticoagulant drug indicated to prevent and treat blood clots in patients at risk for thrombotic events and during certain medical procedures (15). After injection, heparin binds to the endothelium and disrupts the glycocalyx layer (16). For this reason, acute heparin administration could be a useful experimental approach to translate preclinical findings and elucidate the effect of acute endothelial glycocalyx degradation on vascular endothelial function in humans. However, results from investigations examining the effects of heparin on endothelial glycocalyx degradation and vascular endothelial function are conflicting (17). Heparin inhibits the shedding of endothelial glycocalyx components into the circulation in disease-free rats (18) and improves brachial artery flow-mediated dilation (FMD) in high-risk humans (19–21). Conversely, heparin impairs endothelial glycocalyx barrier properties and reduces arteriolar vasodilation in disease-free mice (22), and releases proteins bound to the endothelial glycocalyx into the circulation in healthy humans (23–25). Taken together, it is unclear whether acute heparin administration alters endothelial glycocalyx thickness and reduces vascular endothelial function in humans. Thus, the purpose of our study was to determine the effect of acute experimental heparin administration on endothelial glycocalyx integrity and vascular endothelial function in healthy younger adults. We hypothesized that a single bolus intravenous dose of heparin would increase circulating glycocalyx components and decrease endothelial glycocalyx thickness resulting in blunted brachial artery vasodilation in healthy younger adults.

METHODS

Study Population

Healthy adults, aged 18 to 39 yr, were recruited from Iowa City and the surrounding communities to complete an intervention testing the effect of acute heparin administration on glycocalyx integrity and endothelial function. Exclusion criteria included body mass index (BMI) greater than 40 kg/m², resting systolic blood pressure of greater than 130 mmHg and/or diastolic blood pressure greater than 89 mmHg, current pregnancy or plans to become pregnant, having given birth within the past 6 mo, currently breastfeeding, postmenopausal, clinically abnormal thyroid function, platelet count of less than 150,000/mm³, use of aspirin, nonsteroidal anti-inflammatory drugs, or omega-3 fatty acids within the past 30 days, surgery within the past 30 days, plasma donation within the past 2 wk, current tobacco use or history of tobacco use within the past 3 mo, self-reported history of pregnancy related disorder, blood clots, stroke, dementia, diabetes mellitus, pulmonary/renal/neurological/hepatic disease, history of major hemorrhagic event (hemorrhagic stroke, gastrointestinal bleed, bleeding in urine, or severe nose bleed requiring emergency department visit), previous CVD event (myocardial infarction, stent, bypass surgery, heart failure), current CVD or evidence of CVD during a resting 12-lead electrocardiogram, and hypersensitivity or allergy to pork.

Study Procedures

On the day of vascular testing, participants were instructed to arrive following an overnight fast for a minimum of 8-h and abstain from caffeine intake the morning of the study. In addition, participants were instructed to abstain from exercise and alcohol consumption at least 24-h before testing. Nonpregnancy status was confirmed in female participants using a urine pregnancy test on the day of vascular testing.

Following baseline measurements, participants were administered a single intravenous bolus dose of unfractionated heparin (5,000 U). A heparin dose of 5,000 U was chosen based on established clinical recommendations for initial bolus dosing of unfractionated heparin (26). Blood pressure, plasma blood samples, glycocalyx integrity, and vascular endothelial function were assessed at baseline and 30-min post-heparin administration. In the event of hemorrhage, a safety protocol was put in place to administer slow infusion protamine sulfate, a drug that quickly neutralizes heparin. All procedures and the informed consent document were approved by the University of Iowa Institutional Review Board and conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent before participating in the research study.

Clinical Characteristics

Race, ethnicity, menopause status, smoking status, and health history were evaluated by questionnaire. BMI was computed after measurement of weight and height in kg/m². Supine brachial blood pressure was obtained in triplicate using an inflatable cuff with a built-in microphone and determined via auscultation, where the 1st and 5th Korotkoff sounds denoted systolic and diastolic blood pressure respectively (Noninvasive Hemodynamics workstation; Cardiovascular Engineering, Inc., Norwood, MA). Clinical laboratories were measured at the University of Iowa Diagnostics laboratories and included fasting lipid panel and fasting glucose.

Glycocalyx Thickness

Glycocalyx thickness was determined by non-invasively imaging the sublingual microcirculation using a sidestream dark field imaging camera (KK Technology) and automated acquisition software (GlycoCheck, Microvascular Health Solutions LLC), as previously described (27). Briefly, five trials were performed, with each trial lasting 2–3 min and consisting of at least ten 2-s video recordings 40 frames in length. Values from the five trials were averaged to obtain a single value per measurement. In each recording, microvessels 4–25 µm in lumen diameter were identified using contrast between red blood cells (RBCs) and the background and were divided into 10 µm long microvessel segments. Video recordings were repeated until a total of 3,000 microvessel segments were identified. Microvessels ranging from 4 to 7 µm in diameter were considered capillaries and microvessels ≥ 10 µm in diameter were considered feed vessels (27).

Perfused boundary region (PBR) reflects the depth at which RBCs penetrate into the glycocalyx, with a larger PBR indicative of decreased glycocalyx thickness (28). To calculate PBR, the GlycoCheck software identifies the lateral movement of RBCs in each valid segment to determine the median RBC column width (RBCW), as well as the outer edge of the RBC perfused lumen (Dperf). PBR was reported in vessels 4–25 µm in diameter and calculated using the following equation (27):

$$\mathrm{P}\mathrm{B}\mathrm{R}=(\mathrm{D}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{f}-\mathrm{R}\mathrm{B}\mathrm{C}\mathrm{W})/2$$

Circulating Glycocalyx Components

Venous blood samples were obtained at baseline and 30 min after heparin infusion for the determination of heparan sulfate and syndecan-1 in plasma. Heparan sulfate (Lifeome Biolabs, Catalog No. E09585h) and syndecan-1 (Sigma-Aldritch, Catalog No. RAB0736) were quantified using commercially available enzyme-linked immunosorbent assays performed according to assay instructions.

Endothelial Function

Endothelial function was determined using brachial artery FMD and assessed by high-resolution ultrasonography (Logiq 7, GE Healthcare), as previously described (29). Following baseline diameter measurements, a cuff placed on the upper forearm was inflated to suprasystolic pressure (250 mmHg) for 5 min. After 5 min, the cuff was rapidly deflated, and measurements were continued for an additional 2 min. Offline software (Vascular Analysis Tools 5.5, Medical Imaging Applications, LLC, Coralville, IA) with automatic edge detection was used to determine changes in brachial artery diameter and mean blood velocity (V_mean) in response to reactive hyperemia during baseline and deflation images. FMD was calculated as the percent increase in peak diameter (D_peak) from baseline diameter (D_base) using the following equation: FMD = [(D_peak – D_base)/D_base × 100]. Brachial artery shear stress (SR) was calculated as: SR = 8 × V_mean/diameter and shear rate area under the curve (SR_AUC) was quantified as the area from the time of cuff deflation to the time of D_peak and calculated using the trapezoidal rule, as previously described (30). Normalization of FMD to shear rate was expressed as the peak FMD:SR_AUC ratio (31). Reactive hyperemia, an established index of microvascular function (32), was determined as the difference in blood flow area under the curve (BF_AUC) between baseline and 120 s post-occlusion and calculated using the trapezoidal rule equation as was used for SR_AUC.

Plasma Nitrite

Nitric oxide is rapidly converted to nitrite in plasma (33), therefore plasma nitrite was used to estimate nitric oxide production. Venous blood samples obtained at baseline and 30 min after heparin infusion were used to determine plasma nitrite, as previously described (34). Quantification of plasma nitrite was performed using a Sievers chemiluminescence nitric oxide analyzer (NOA 280i, Sievers Instruments, Boulder, CO), according to previously established recommendations (35).

Statistical Analysis

Normally distributed data are presented as means ± SD, nonnormally distributed data as median (interquartile range), and categorical data as number (percentage of participants). Shapiro–Wilk tests were used to assess whether the change in outcome variables following heparan infusion was normally distributed. Paired student’s t tests were used to test outcome variables with a normally distributed change from baseline and paired Wilcoxon rank sum tests were used to test outcome variables with a nonnormally distributed change from baseline. Pearson correlation tests were performed to assess the relation between PBR and circulating glycocalyx components at baseline. Significance was set at an α level of 0.05. All analyses were performed in R, version 4.2.3 (R Foundation for Statistical Computing, Vienna, Austria).

RESULTS

A total of 22 participants free of overt CVD and metabolic disease were screened and consented. Of those, 19 (86%) completed the experimental intervention, whereas the remaining participants were excluded because of elevated blood pressure (n = 1), a scheduling conflict (n = 1), or withdrawn because of a change in health status (n = 1) (Fig. 1). Baseline clinical characteristics are presented in Table 1. On average, participants were normal weight, younger adults with optimal blood pressure, blood glucose, and blood lipids.

Figure 1.

Study schematic.

Table 1.

Participant characteristics (n = 19)

Variable	Value
Age, yr	23 (21, 27)
Sex, no. (%)
Female	10 (53)
Male	9 (47)
Race, no. (%)
White	17 (89)
Black	1 (5)
Asian	2 (11)
Ethnicity, no. (%)
Hispanic	2 (11)
Body mass index, kg/m2	23.7 ± 2.7
Glucose, mg/dL	81 ± 6
Total cholesterol, mg/dL	156 ± 27
Triglycerides, mg/dL	59 (46, 86)
LDL-C, mg/dL	90 ± 20
HDL-C, mg/dL	54 ± 13
Systolic blood pressure, mmHg	117 ± 8
Diastolic blood pressure, mmHg	60 ± 6
Brachial artery FMD, %	5.67 (3.98, 9.41)
PBR 4-25, µm	2.03 (1.95, 2.11)

Variables are presented as means ± SD, median (interquartile range), or number (percentage). LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; FMD, flow-mediated dilation; PBR, perfused boundary region.

Glycocalyx thickness, determined from PBR, was unchanged following the heparin infusion across all diameter ranges (Table 2). In addition, there was no change in both circulating syndecan-1 and heparan sulfate plasma concentrations following heparin infusion (Table 2). Cross-sectional analyses performed at baseline revealed that circulating syndecan-1 was not associated with PBR 4–25 (r = −0.20, P = 0.49), PBR 4–7 (r = −0.43, P = 0.11), or PBR 10–25 (r = −0.30, P = 0.27). However, circulating heparan sulfate was associated with PBR 4–7 (r = 0.56, P = 0.04), but not PBR 4–25 (r = 0.24, P = 0.40), or PBR 10–25 (r = 0.36, P = 0.21).

Table 2.

Change in endothelial glycocalyx perfused boundary region and circulating glycocalyx concentrations after a single dose of heparin

Variable	Baseline	Post-Heparin	Change from baseline	P Value
PBR 4–25, µm	2.04 ± 0.18	2.02 ± 0.20	−0.02 ± 0.11	0.49
PBR 4–7, µm	0.94 (0.89, 0.96)	0.93 (0.91, 0.99)	−0.01 (−0.03, 0.05)	0.55
PBR 10–25, µm	2.52 ± 0.23	2.50 ± 0.25	−0.02 ± 0.15	0.63
Syndecan-1, pg/mL	2910 (1824, 12581)	2539 (1758, 16426)	−121.1 (−707.9, 387.7)	0.90
Heparan sulfate, ng/mL	2980 ± 871	3215 ± 921	206 ± 1036	0.49

Data are means ± SD or median (interquartile range). Paired Student's t tests were used when the change from baseline was normally distributed variables and paired Wilcoxon rank sum tests were used when the change from baseline nonnormally distributed variables. PBR, perfused boundary region.

Changes in endothelial function following heparin infusion are shown in Table 3. There was no change in brachial artery FMD% or FMDmm following heparin infusion (Table 3). In addition, no changes were observed in baseline diameter, peak diameter, and plasma nitrite after heparin (Table 3). Moreover, there were no changes in time to peak dilation, peak SR, SR_AUC, and FMD:SR_AUC (Table 3). Furthermore, microvascular function, determined from brachial artery post-inflation hyperemic BF_AUC, was unchanged following heparin infusion (Table 3). Finally, there was no observed change in systolic or diastolic blood pressure after heparin (P = 0.42 and P = 0.63, respectively).

Table 3.

Change in circulating nitrite concentrations and vascular endothelial function after a single dose of heparin

Variable	Baseline	Post-heparin	Change from baseline	P Value
Nitrite, µM	447 ± 149	410 ± 129	−37 ± 93	0.10
Baseline diameter, mm	3.44 (3.13, 3.87)	3.44 (3.11, 3.80)	−0.03 (−0.11, 0.11)	0.70
Peak diameter, mm	3.62 (3.32, 4.22)	3.54 (3.33, 3.97)	−0.07 (−0.20, 0.12)	0.26
Brachial artery FMD, %	6.45 ± 3.14	5.42 ± 3.09	−1.03 ± 4.65	0.35
Brachial artery FMD, mm	0.23 ± 0.12	0.19 ± 0.11	−0.47 ± 1.64	0.23
Time to peak dilation, s	45.9 ± 18.6	42.5 ± 19.7	−3.3 ± 26.3	0.59
Peak SR, 1/s	1230 ± 434	1147 ± 375	−84 ± 258	0.54
SRAUC, au	32339 ± 20083	32039 ± 15712	−300 ± 12589	0.92
FMD:SRAUC, au	0.18 (0.13, 0.34)	0.15 (0.09, 0.28)	−0.06 (−0.12, 0.05)	0.21
BFAUC, au	14797 ± 9959	13722 ± 8711	−1075 ± 3603	0.22

Data are means ± SD or median (interquartile range). Paired Student’s t tests were used when the change from baseline was normally distributed variables and paired Wilcoxon rank sum tests were used when the change from baseline nonnormally distributed variables. FMD, flow-mediated dilation; SR, shear rate; BF, blood flow.

DISCUSSION

The purpose of our study was to determine whether a single experimental bolus dose of heparin would increase circulating glycocalyx components and lower endothelial glycocalyx thickness, thereby reducing brachial artery vasodilation in healthy younger adults. Our primary finding was that heparin did not alter the endothelial glycocalyx, as demonstrated by no change in PBR, and no change in circulating syndecan-1 and heparan sulfate plasma concentrations. In addition, there was no change in endothelial function following acute intravenous heparin administration. Taken together, these data demonstrate that a single bolus dose of heparin (5,000 U) is not sufficient to reduce endothelial glycocalyx thickness or vascular endothelial function in healthy younger adults.

Shear stress is a tangential force generated by the friction of laminar blood flowing adjacent to the endothelium of the vascular wall. The endothelial glycocalyx is well-positioned to sense shear stress because of its location on the surface of endothelial cells and protrusion into the blood vessel lumen (36). This positioning enables the endothelial glycocalyx to participate in mechanotransduction, the process of converting a mechanical force (shear stress) into a biochemical signal (nitric oxide) (12). Heparan sulfate is a primary component of the endothelial glycocalyx (37). Importantly, enzymatic degradation of heparan sulfate with heparinase blunts FMD and nitric oxide production in preclinical studies (10, 12–14). Notably, endothelial cells exposed to heparin may lose their heparan sulfate coating (38). Therefore, it is plausible that heparin could degrade heparan sulfate and impair vascular endothelial function in humans. In our study, we observed that a single bolus dose of heparin (5,000 U) did not increase syndecan-1 or heparan sulfate circulating in plasma or decrease glycocalyx thickness, FMD, and nitrite production in healthy young adults. Furthermore, time to peak dilation and FMD:SR_AUC were unchanged after heparin, suggesting that there was not a significant difference in the amount of shear stress required to elicit peak vasodilation. Finally, reactive hyperemia, quantified as BF_AUC, was unchanged following heparin, indicating that heparin did not impair microvascular function in our study.

The results of our study indicate that acute intravenous heparin administration is not a suitable method to study the effects of acute endothelial glycocalyx degradation on vascular endothelial function in healthy humans. However, our findings differ from a previous study by VanTeeffelen et al. (22) that revealed heparin, administered at a similar dose per body weight as our study, impairs the endothelial glycocalyx and reduces vascular endothelial function in disease-free mice. Briefly, the researchers administered heparin to mice and then estimated endothelial glycocalyx integrity from the systemic clearance of fluorescein isothiocyanate-labeled dextrans and shear-induced arteriolar vasodilation in the cremaster muscle (22). The researchers observed a significant elevation in dextran clearance over time following a single bolus injection of heparin, indicating degradation of the endothelial glycocalyx. In addition, heparin reduced flow-mediated arteriolar vasodilation in response to prolonged occlusion (22). Furthermore, treating the arterioles with the nitric oxide inhibitor N^ω-nitro-l-arginine reduced arteriolar vasodilation similar to heparin (22), suggesting that heparin potentially impaired arteriolar vasodilation via reduced nitric oxide production. However, this study did not directly measure circulating glycocalyx components, rather the researchers used fluorescein isothiocyanate-labeled dextran clearance to estimate glycocalyx properties (22); therefore, it is unknown if heparin degraded heparan sulfate and other specific glycocalyx components. Additional studies performed in healthy humans found that heparin releases glycocalyx-bound proteins into the circulation (23–25), further suggesting that heparin disrupts the endothelial glycocalyx. However, these studies also did not measure glycocalyx components, thus it is difficult to determine whether the release of endothelial glycocalyx-bound proteins was accompanied by the degradation of endothelial glycocalyx components. Since heparin is widely used in clinical settings, future studies could measure changes in circulating glycocalyx components in response to prolonged heparin treatment to corroborate our findings.

Conversely, heparin’s ability to release proteins bound to the endothelial glycocalyx appears to have beneficial effects in high-risk populations. Myeloperoxidase is a heme-enzyme released from immune cells, at sites along the vascular tree with elevated inflammation and oxidative stress, that degrades the endothelial glycocalyx and reduces nitric oxide bioavailability (39, 40). Heparin treatment liberates vessel-immobilized myeloperoxidase into plasma and increases FMD in patients undergoing coronary angiography, patients with stable coronary artery disease, and pregnant women with a heightened risk of preeclampsia (19–21). Thus, it is plausible that heparin induces the release of harmful proteins bound to the endothelial glycocalyx and has promise as a therapeutic intervention in individuals with chronic inflammation and oxidative stress. In our study, it is likely that we did not observe an increase in FMD after heparin administration because our participants were healthy younger adults with likely no inflammation or oxidative stress.

There were several limitations in our study. First, we measured endothelial glycocalyx thickness and circulating glycocalyx components at only one time point (30-min) after administering a single dose of heparin. However, the effective half-life of heparin is 60 to 90 min and previous human studies observed elevated glycocalyx-bound proteins in circulation within 15-min after heparin infusion (25, 41). Second, imaging sublingual microvessels with the GlycoCheck may not reflect changes in endothelial glycocalyx thickness throughout the arterial tree. However, sublingual endothelial glycocalyx thickness has been well validated in older adults (5) and our findings regarding the change in circulating glycocalyx components after heparin administration reflect our observations in the sublingual microvessels. Third, we did not utilize a saline infusion to serve as a control against the heparin infusion, which may have strengthened our findings. Instead, each participant’s baseline values served as their own control. A strength of our study was the measurement of endothelial glycocalyx integrity using both the GlycoCheck and plasma glycocalyx components. We observed that plasma heparan sulfate, but not syndecan-1, was associated with endothelial glycocalyx thickness and this association was only observed in vessels 4–7 microns in diameter. Our study expands upon findings from a recent meta-analysis that revealed structural endothelial glycocalyx thickness was associated with plasma glycocalyx components in only 60% of studies included in the analysis (42). An additional strength of our study was that we examined young, healthy adults without cardiovascular risk factors. This enabled us to study the direct effect of heparin on endothelial glycocalyx degradation without the influence of additional glycocalyx-degrading stimuli such as hypertension and hyperglycemia (43, 44).

In conclusion, a single bolus dose of heparin does not increase circulating glycocalyx components, decrease endothelial glycocalyx thickness, or reduce shear stress-mediated brachial artery vasodilation in healthy younger adults. These data suggest that, to replicate preclinical models, future studies should utilize other methodological techniques to investigate the effects of acute endothelial glycocalyx degradation on vascular endothelial function in humans.

GRANTS

Colin J. Gimblet is supported by the American Heart Association predoctoral fellowship grant (23PRE1012593). Gary L. Pierce is supported by the Russell B. Day and Florence D. Day Endowed Chair in Liberal Arts and Sciences at the University of Iowa. The research reported in this publication was supported by the National Center For Advancing Translational Sciences of the National Institutes of Health (UM1TR004403) grant awarded to the University of Iowa.

DISCLAIMERS

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

C.J.G., K.D.B., A.K.S., A.J.D., D.I.J., and G.L.P. conceived and designed research; C.J.G. performed experiments; C.J.G. and J.W.E. analyzed data; C.J.G. and G.L.P. interpreted results of experiments; C.J.G. prepared figures; C.J.G. drafted manuscript; C.J.G., J.W.E., A.J.D., D.I.J., and G.L.P. edited and revised manuscript; C.J.G., J.W.E., K.D.B., A.K.S., A.J.D., D.I.J., and G.L.P. approved final version of manuscript.