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. Author manuscript; available in PMC: 2016 May 4.
Published in final edited form as: Clin Chem. 2011 Jul 18;57(9):1272–1278. doi: 10.1373/clinchem.2011.169151

ProBNP1–108 Is Resistant to Degradation and Activates Guanylyl Cyclase-A with Reduced Potency

Deborah M Dickey 1, Lincoln R Potter 1,2,*
PMCID: PMC4855511  NIHMSID: NIHMS781592  PMID: 21768217

Abstract

BACKGROUND

B-type natriuretic peptide (BNP) compensates for the failing heart and is synthesized as a 108-residue prohormone that is cleaved to a 32-residue C-terminal maximally active peptide. During heart failure, serum concentrations of proBNP1–108 exceed concentrations of BNP1–32. The aim of this study was to determine why the proBNP1–108/BNP1–32 ratio increases and whether proBNP1–108 is bioactive.

METHODS

Using cGMP elevation and 125I-ANP binding assays, we measured binding and activation of individual human natriuretic peptide receptor populations by recombinant human proBNP1–108 and human synthetic BNP1–32. Using receptor bioassays, we measured degradation of recombinant proBNP1–108 and BNP1–32 by human kidney membranes.

RESULTS

ProBNP1–108 stimulated guanylyl cyclase-A (GC-A) to near-maximum activities but was 13-fold less potent than BNP1–32. ProBNP1–108 bound human GC-A 35-fold less tightly than BNP1–32. Neither proBNP1–108 nor BNP1–32 activated GC-B. The natriuretic peptide clearance receptor bound proBNP1–108 3-fold less tightly than BNP1–32. The half time for degradation of proBNP1–108 by human kidney membranes was 2.7-fold longer than for BNP1–32, and the time required for complete degradation was 6-fold longer. BNP1–32 and proBNP1–108 were best fitted by first- and second-order exponential decay models, respectively.

CONCLUSIONS

ProBNP1–108 activates GC-A with reduced potency and is resistant to degradation. Reduced degradation of proBNP1–108 may contribute to the increased ratio of serum proBNP1–108 to BNP1–32 observed in patients with congestive heart failure.

Secretion of atrial natriuretic peptide (ANP)3 and B-type natriuretic peptide (BNP) from cardiomyocytes is markedly increased during heart failure (1). Both ANP and BNP compensate for the failing heart by reducing blood pressure, blood volume, and cardiac remodeling. The signaling receptor for ANP and BNP is guanylyl cyclase-A (GC-A), which catalyzes the synthesis of cGMP (2). C-type natriuretic peptide (CNP) also possesses cardioprotective properties (3) but activates the homologous GC-B receptor (4). Circulating concentrations of all natriuretic peptides are reduced by natriuretic peptide receptor C (NPR-C)–mediated endocytosis and cell-surface proteases (5).

Natriuretic peptides are synthesized as preprohormones that are proteolytically cleaved to bioactive C-terminal peptides. ProBNP1–108 is processed to an inactive amino-terminal fragment and a C-terminal 32 residue peptide (BNP1–32) that binds GC-A with high potency and maximal activity. Multiple BNP species are independently associated with heart failure progression (6). In heart failure, the majority of BNP is secreted from ventricular myocytes. The protease that cleaves ProBNP1–108 to BNP1–32 is not definitively known. Corin performs this function in vitro, and inactivating mutations in corin correlate with reduced ratios of BNP1–32 to ProBNP1–108 in African Americans (7, 8). Furin inhibitors also block proBNP processing, however, and a recent study reported that furin and corin cleavage of human ProBNP1–108 produced BNP1–32 and BNP4–32, respectively (9, 10).

Both high and low molecular weight species of BNP are found in human plasma (11, 12). The high molecular weight species is the most prevalent form (12–15) and appears to be an O-glycosylated version of ProBNP1–108 (16), but about 30% of human ProBNP1–108 was suggested to exist in a nonglycosylated form (10). The low molecular weight species is likely a mixture of BNP1–32 and N- and C-terminally degraded forms of BNP1–32, such as BNP3–32, BNP1–30, BNP3–30, BNP3–29, BNP1–29, and BNP4–31, which were observed when BNP1–32 was incubated with whole blood or with purified dipeptidyl-peptidase IV (17, 18).

Why the ratio of ProBNP1–108 to BNP1–32 is increased in plasma is not known. One possibility is that the enzyme that converts ProBNP1–108 to BNP1–32 is overwhelmed and fails to completely cleave ProBNP1–108 to BNP1–32. Another possibility is that BNP1–32 is degraded faster than proBNP1–108 in the circulation. The aims of this study were to examine the ability of the 2 known natriuretic peptide– degrading pathways to degrade BNP1–108 and determine whether BNP1–108 is biologically active. We found significant decreases in the affinity of NPR-C for ProBNP1–108 compared with BNP1–32 and that ProBNP1–108 was more resistant to proteolytic inactivation by human kidney membranes than BNP1–32. We also determined that ProBNP1–108 increases cGMP concentrations in cells expressing GC-A. These results provide 1 mechanistic explanation for why circulating proBNP1–108–to–BNP1–32 ratios are increased in heart failure patients. They also indicate that ProBNP1–108 contains reduced but significant bioactivity and may activate GC-A during congestive heart failure.

Methods

REAGENTS

Human ProBNP1–108 containing an N-terminal His6-tag followed by a rTEV protease cleavage site (MHHHHHNYNIPTTENLYFQG) was expressed and purified from the Rosetta DE3 Escherichia coli to >95% purity by immobilized metal affinity chromatography followed by strong cation-exchange chromatography as described (15). The affinity tag was not removed. This preparation is not glycosylated and was a kind gift from Dr. Ute Schellenberger (Scios, Inc). We purchased human BNP (BNP1–32) from Sigma-Aldrich and Phoenix Pharmaceutical; it was >95% pure. Protease inhibitors were from Roche (Complete Roche protease inhibitor cocktail tablet), MP Biomedicals (phosphoramidon), and Sigma-Aldrich (actinonin). We purchased 125I-ANP and the cGMP RIA kit from PerkinElmer.

MEMBRANES

We obtained human kidneys from the Tissue Procurement Program at the University of Minnesota and prepared them as described (19). Briefly, kidney tissue was homogenized in 3 mL ice-cold buffer lacking protease inhibitors (50 mmol/L Tris-HCl, pH 7.5, 20% glycerol, 50 mmol/L NaF) then centrifuged at 10000g for 10 min at 4 °C. The supernatant was removed, and the pellet was washed 2 times in 2 mL buffer by resuspension and centrifugation. The membranes were resuspended in 2–3 volumes of ice-cold buffer, aliquoted, and stored at −80 °C.

CELLS

Human embryonic kidney 293 cells stably expressing human GC-A, GC-B, or NPR-C were maintained as described (20).

CELLULAR cGMP ELEVATIONS

Cells in 48-well plates were incubated 4–12 h in serum-free media until 75%–90% confluent. The medium was aspirated and replaced with 0.15 mL Dulbecco modified Eagle medium (DMEM) containing 1 mmol/L 1-methyl-3-isobutylxanthine (IBMX) and 25 mmol/L HEPES pH 7.4 for 10 min at 37 °C. The medium was aspirated, and cells were incubated with DMEM containing 1 mmol/L IBMX and 25 mmol/L HEPES pH 7.4 with or without natriuretic peptide for 3 min. Treatment medium was aspirated and the reaction was stopped with 0.2 mL ice-cold 80% ethanol. An aliquot of the resulting supernatant was dried in a centrifugal vacuum concentrator and analyzed for cGMP content by RIA.

ANP BINDING ASSAYS

We added cells to 24-well plates precoated with poly-d-lysine. When 75%–90% confluent, the cells were washed with DMEM and incubated with DMEM containing 0.2% BSA at 37 °C for 1–2 h. Medium was aspirated and replaced with 0.2 mL binding medium containing 75 pmol/L 125I-ANP and 1% BSA in the presence or absence of increasing concentrations of unlabeled ligand as described (19). The plates were incubated at 4 °C for 1 h before the binding medium was aspirated and the cells washed twice with 0.5 mL ice-cold PBS. The cells were solubilized in 0.5 mL of 1 mol/L NaOH, transferred to glass tubes, and bound radioactivity was determined in a γ counter. Data were plotted as cpm specifically bound to cells divided by cpm added to wells, which is defined as B/Bo, vs the log of the competing peptide concentration.

PROTEOLYSIS ASSAYS

We performed proteolysis assays using 0.02 mL crude membranes in a 0.1-mL reaction volume as described (21). An aliquot of membranes was thawed and diluted to 2 g/L with ice-cold proteolysis buffer. The reaction was started with the addition of 0.04 mg crude membranes to a solution containing 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 5 mmol/L Mg2Cl, 0.1% BSA, and 1 μmol/L natriuretic peptide and incubated for the indicated period of time at 37 °C. The assay was terminated with 0.1 mL of 0.5 N perchloric acid. The samples were neutralized with 0.005 mL of 10 mol/L NaOH before centrifugation for 5 min at 20000g at 4 °C. An aliquot of the supernatant was mixed with DMEM containing 1 mmol/L IBMX and 25 mmol/L HEPES and then added to cells stably expressing human GC-A for 3 min at 37 °C to determine peptide bioactivity. We added 0.3 mL of 0.1 N HCl to stop the reaction and assayed aliquots of the acid extracts for cGMP content. We determined the amount of starting peptide remaining by converting the cGMP to nano-moles of peptide using a peptide standard concentration response curve performed with each bioassay and then graphing that value as a percent of the starting peptide. In some assays, membranes were preincubated with inhibitors on ice for 10 min before membrane aliquots were added to 1 μmol/L peptide and incubated at 37 °C for 20 min. The reactions were stopped and assayed as above. Final inhibitor concentrations were leupeptin, 0.0002 g/L; phosphoramidon, 0.01 mmol/L; actinonin, 0.01 mmol/L; and 1×concentrations of Roche complete protease inhibitor cocktail.

STATISTICAL ANALYSIS

We performed the cGMP elevation and ANP binding experiments in triplicate in 3 separate assays and determined P values using paired 2-tailed Student t-test. The data were graphed as the mean of all combined assays with error bars representing the standard error. We performed the proteolysis experiments in duplicate in 5 separate assays. The individual data were analyzed as previously reported (21) and then combined and graphed. The formulas used for 1- and 2-phase exponential decay models were Y = Span*exp(−K*X) + Plateau and Y = Span1*exp(−K1*X) + Span2*exp(−K2*X) + plateau, respectively, where X is time, Span is the maximal Y value (100%) minus the plateau, and the plateau is where the curve levels off. For these analyses, the plateau was set to zero. We then calculated the half-lives in Prism from the equation t1/2 = 0.69/K. P values for proteolysis were determined using an unpaired t-test.

Results

ProBNP1–108 ACTIVATED GC-A WITH REDUCED POTENCY

We investigated the ability of proBNP1–108 and BNP1–32 to activate GC-A by measuring cGMP elevations in 293 cells stably expressing GC-A and no other natriuretic peptide receptor (20). Neither BNP1–32 nor proBNP1–108 increased cGMP concentrations in the parental 293 cells that were used to make the stable cell lines, because they lack GC-A expression (data not shown). Thus, the cGMP increases observed in the 293T-GC-A cells were solely due to activation of human GC-A. ProBNP1–108 activated human GC-A in a concentration-dependent manner, but more proBNP1–108 than BNP1–32 was required to achieve a given level of activation (Fig. 1). The half-maximal effective concentration (EC50) for BNP1–32 was 27 nmol/L (95% CI 16–46 nmol/L), and the EC50 for proBNP1–108 was 372 nmol/L (214–649 nmol/L), which is approximately 13-fold greater than the EC50 for BNP1–32.

Fig. 1. ProBNP1–108 activates GC-A with reduced potency.

Fig. 1

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Confluent HEK293 cells stably expressing human GC-A were incubated with increasing concentrations of ligand for 3 min. Intracellular cGMP concentrations were measured and plotted as a function of peptide concentration. Data points represent the mean and SEM from 3 separate experiments assayed in triplicate where P = 0.004.

ProBNP1–108 FAILED TO ACTIVATE GC-B

Previous studies investigated effects of proBNP1–108 in primary cells that often express both GC-A and GC-B (15, 22). To directly measure the ability of the BNP molecules to activate human GC-B, we performed whole-cell stimulation assays in 293 cells expressing only GC-B. CNP potently increased cGMP in these cells. BNP1–32 was a poor activator of human GC-B, however, and proBNP1–108 was an even worse activator (Fig. 2). Thus, the cGMP increases in response to proBNP1–108 in primary cells were due solely to activation of GC-A.

Fig. 2. ProBNP1–108 does not activate GC-B.

Fig. 2

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Confluent HEK293 cells stably expressing human GC-B were incubated with increasing concentrations of ligand for 3 min. Cellular cGMP concentrations were measured and plotted as a function of peptide concentration. Data points represent the mean and SEM from 2 separate experiments assayed in triplicate.

REDUCED BINDING OF ProBNP1–108 TO GC-A

To determine if the reduced activation of GC-A by proBNP1–108 was due to decreased affinity to the receptor as opposed to decreased receptor activation, we performed whole-cell binding assays by measuring the ability of BNP1–32 and proBNP1–108 to compete for 125I-ANP binding to cells expressing human GC-A. The calculated half-maximal inhibitory concentration (IC50) for proBNP1–108 was 295 nmol/L (95% CI 189–460 nmol/L), which was approximately 35-fold higher than the IC50 of 8.3 nmol/L (7–10 nmol/L) determined for BNP1–32 (Fig. 3). Thus, the reduced ability to activate GC-A correlated with the reduced ability of proBNP1–108 to bind the receptor.

Fig. 3. Human GC-A binds proBNP1–108 with a lower affinity than BNP1–32.

Fig. 3

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293-GC-A cells were incubated for 1 h with 50 pmol/L 125I-ANP at 4 °C with or without unlabeled ligand. Bound 125I-ANP was plotted as a function of competing peptide concentration. Symbols represent the mean and SEM from 3 experiments assayed in triplicate where P = 0.03.

NPR-C BOUND ProBNP1–108 LESS TIGHTLY THAN IT BOUND BNP1–32

All natriuretic peptides are cleared from the circulation by NPR-C–mediated endocytosis, but BNP has been reported to bind NPR-C less tightly than ANP (23). We determined the affinity of BNP1–32 and proBNP1–108 for NPR-C by competition binding to 125I-ANP. The IC50 for proBNP1–108 was increased approximately 2.8-fold from 2.6 (95% CI 2.1–3.2 nmol/L) to 7.4 nmol/L (5.8–9.5 nmol/L) compared to BNP1–32 (Fig. 4). Thus, ProBNP1–108 binds NPR-C less tightly than BNP1–32.

Fig. 4. ProBNP1–108 binds human NPR-C with reduced affinity.

Fig. 4

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293-NPR-C cells were incubated with125I-ANP in the presence or absence of unlabeled ligand. Bound 125I-ANP was plotted as a function of competing peptide concentration. Symbols represent mean and SEM of 3 experiments assayed in triplicate where P = 0.007.

ProBNP1–108 WAS RESISTANT TO PROTEOLYTIC DEGRADATION BY HUMAN KIDNEY MEMBRANES

The kidney is a major target organ for BNP (2). Therefore, we investigated the ability of human kidney membranes to proteolytically inactivate 1-μmol/L concentrations of BNP1–32 and proBNP1–108 as a function of time. BNP1–32 was inactivated after approximately 20 min, whereas 30% of the initial bioactivity of proBNP1–108 remained after 20 min (Fig. 5). Complete inactivation of proBNP1–108 was observed only after 2 h, which was 6-fold longer than the time required to completely inactivate BNP1–32. The best fit for the degradation of BNP1–32 was a single-site model where the half-life was 4.2 min. The best fit for BNP1–108 degradation was a second-order exponential model where the calculated half-life for the first phase was very similar to the single site model for BNP1–32 but also had a longer second component. The second-order decay model did not improve the fit for BNP1–32.

Fig. 5. ProBNP1–108 is resistant to proteolytic inactivation.

Fig. 5

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BNP1–32 or proBNP1–108 was incubated with kidney membranes at 37 °C for the indicated times, and the ability of the extracts to increase cGMP in 293-GC-A cells was determined. BNP1–32 and proBNP1–108 were fitted to 1- and 2-phase exponential decay models, respectively. Symbols represent mean and SEM where n = 10. t, time.

The vast majority of the inactivation was abolished in boiled membranes, consistent with a proteolytic-dependent mechanism, and neither the neutral endopeptidase inhibitor, phosphoramidon, nor the meprin inhibitor, actinonin, blocked inactivation (Fig. 6). However, the general serine threonine protease inhibitor, leupeptin, and a broad-range protease inhibitor cocktail completely blocked inactivation of BNP1–32 and proBNP1–108, consistent with both peptides being degraded by a common pathway (Fig. 6).

Fig. 6. ProBNP1–108 and BNP1–32 are degraded similarly.

Fig. 6

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Peptides were incubated with kidney membranes for 20 min at 37 °C with or without the indicated protease inhibitors (leupeptin, 0.2 μg/mL; phosphoramidon, 0.01 mmol/L; actinonin, 0.01 mmol/L; 1× Roche complete protease inhibitor cocktail). cGMP elevating activity in extracts was determined as for Fig. 5. Symbols represent the mean and SEM where n = 4. ***P < 0.0001.

Discussion

BNP isolated from the serum of heart failure patients indicated increased concentrations of high molecular weight species (11–15). The assumption was that much of the BNP measured in plasma by commercially available immunoassays was unprocessed proBNP1–108, which was assumed to be biologically inactive (24). Subsequent measurements on the biological activity of proBNP1–108 produced conflicting results. One group reported that proBNP1–108 does not activate GC-A in cultured cardiac fibroblasts or cardiomyocytes, whereas another group reported that proBNP1–108 elevates cGMP in human endothelial and vascular smooth muscle cells but with reduced potency (15, 22). We found that proBNP1–108 stimulates GC-A to near maximal activities but has a 13-fold reduced potency, similar to the 6- to 8-fold reduced potency reported by Liang and colleagues (15). In a second set of experiments, we used in vitro assays to show that both major natriuretic peptide degradation pathways are less effective at inactivating proBNP1–108 compared with BNP1–32.

Our studies determined that proBNP1–108 exhibits both reduced binding and activation of human GC-A in 293 cells. This cell line allowed a definitive comparison of the activity of the peptides on a single natriuretic peptide receptor. Interestingly, whereas the EC50 of proBNP1–108 for GC-A was only 13-fold less than that of BNP1–32, the dissociation constant (Kd) was reduced 35-fold. The explanation for this inconsistency is not definitively known but is not due to competition for binding between GC-A and NPR-C, since the cells express only a single transfected receptor. It most likely relates to the previously described discrepancy between binding and activation constants observed for trans-membrane guanylyl cyclases (25).

Theoretically, the ratio of proBNP1–108 to BNP1–32 can increase as a result of incomplete proteolytic processing of proBNP1–108 to BNP1–32 or as a result of reduced degradation of proBNP1–108 compared to BNP1–32, or both. Shimizu and colleagues suggested that BNP1–32 is more rapidly cleared from the circulation than BNP1–108, but they did not measure degradation rates of the peptides (11, 12). Our results provide biochemical evidence in support of the differential degradation of BNP1–32 and proBNP1–108 by both elimination processes. The best fit for the proteolytic inactivation of BNP1–32 followed a first-order exponential decay model, but a second-order decay model better fitted the inactivation of proBNP1–108. One hypothesis that is consistent with these data is that proBNP1–108 and BNP1–32 are initially cleaved by the same protease but that the proBNP1–108 cleavage product is a poor substrate for a subsequent inactivating cleavage and maintains a low level of biological activity, whereas BNP1–32 is more susceptible to the subsequent inactivating cleavage. Similar rate constants (0.166 and 0.160), half-lives (4.2 and 4.3 min), and protease inhibitor profiles support this idea.

A recent article by Semenov and colleagues reported that human forms of BNP1–32 and proBNP1–108 have similar clearance rates when injected into the circulation of rats and that nonglycosylated proBNP1–108 was processed to BNP1–32 or smaller fragments in the rat circulation (26). The latter observation is exciting because it suggests that circulating proBNP1–108 could serve as a reservoir for a signaling form of BNP if the tissue processes the appropriate processing enzymes. Because we failed to observe an increase in bioactivity corresponding to an increase in the more potent smaller forms of BNP when we used proBNP1–108 as a substrate in our proteolysis assay, it seems unlikely that our human kidney samples contain detectable concentrations of the processing enzyme required to yield smaller, more potent forms of BNP. Alternatively, the degrading enzyme is much more active than the processing enzyme in human kidney membranes.

Semenov et al. also measured the clearance of nonglycosylated and glycosylated version of proBNP1–108 and found that the latter had a longer clearance time and concluded that increased serum proBNP1–108 observed in heart failure results from an increased secretion of unprocessed proBNP1–108, not reduced clearance of proBNP1–108 (26). However, the relevance of this study to BNP degradation in humans is unclear because the sequences of human and rat BNP are radically different. Human and rat BNP contain 32 and 45 residues, respectively, and 18 of the 32 residues differ between the 2 species (21). Furthermore, the half-life of BNP is 20 min in humans but only 1 min in rats (27, 28), the proteases that degrade human BNP in human and rat kidney membranes are different (21), and the half-life of fully glycosylated proBNP1–108 is longer than less glycosylated forms of proBNP1–108 (29).

Studies by Liang et al. demonstrated no significant difference in the potencies of glycosylated versed nonglycosylated versions of proBNP1–108 on the activation of GC-A (15). Thus, our studies using nonglycosylated proBNP1–108 likely reflect physio logic activations of GC-A by proBNP1–108. A relative limitation of our studies is that the nonglycosylated version of proBNP1–108 may be degraded differently than the glycosylated version of proBNP1–108. We did not measure the degradation of glycosylated human proBNP1–108, but since glycosylated proBNP1–108 had a longer half-life than the nonglycosylated proBNP1–108 in rats (26), glycosylated proBNP1–108 may be more resistant to degradation. Future studies comparing the glycosylated versed nonglycosylated forms of proBNP1–108 will be required to determine the role of glycosylation on the proteolytic degradation of this peptide. Another limitation is that the N-terminal His6 tag used for purification was not removed from our proBNP1–108 sample, which could impede binding to receptors and proteases. Thus, it is possible that the EC50 of proBNP1–108 was overestimated.

Recent studies have shown that C-terminal extensions of 12 or 15 amino acids to ANP or CNP, respectively, protect these peptides from proteolytic degradation (19, 30). Here, a large N-terminal extension was shown to decrease degradation, but it also reduced the affinity and potency of the peptide. From a drug development prospective, it will be interesting to test whether shorter N-terminal extensions can be created that reduce degradation without affecting receptor activation. Regarding degrading and processing enzymes, Ralat et al. recently reported that insulin-degrading enzyme cleaves terminal residues from BNP that modify the receptor preference of the peptide (31). Although the increased size of proBNP1–108 is likely to reduce access to the catalytic chamber of insulin-degrading enzyme, the ability of insulin-degrading enzyme to process proBNP1–108 in the circulation requires further study.

Finally, it has been suggested that congestive heart failure represents a BNP-deficient state, where immunologically reactive, but biologically inactive, BNP species are increased in the blood of heart failure patients (22, 32). Our data do not challenge the idea of a BNP-deficient state. However, they are not consistent with an increased ratio of proBNP1–108 to BNP1–32 solely explaining the deficiency, because although the EC50 for proBNP1–108 was reduced 13-fold in our study and 6- to 8-fold in the study by Liang et al. (15), circulating concentrations of immunoreactive BNP (BNP1–32 and proBNP1–108) are increased at least this much during advanced heart failure (33). It could be argued that proBNP1–108 antagonizes binding of more active species like BNP1–32, BNP3–32, or ANP to GC-A, but the reduced Kd for proBNP1–108 indicates that it would not be an effective competitor. Thus, we conclude that although the increased ratio of proBNP1–108 to BNP1–32 contributes to the BNP-deficient state, additional factors or processes also contribute to the diminished GC-A response observed during heart failure.

Acknowledgments

We thank Dr. Ute Schellenberger and her colleagues at Scios, Inc. for the kind gift of proBNP1–108 and for helpful discussions.

Research Funding: NIH grant R021HL093402 and a grant from Scios, Inc.

Role of Sponsor: The funding organizations played a direct role in the design of study, review and interpretation of data, and preparation or approval of manuscript.

Footnotes

3

Nonstandard abbreviations: ANP, atrial natriuretic peptide; BNP, B-type natriuretic peptide; CNP, C-type natriuretic peptide; GC, guanylyl cyclase; NPR-C, natriuretic peptide receptor C; DMEM, Dulbecco modified Eagle medium; IBMX, 1-methyl-3-isobutylxanthine; EC50, half-maximal effective concentration; IC50, half-maximal inhibitory concentration.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: L.R. Potter, Medtronic and Eli Lilly.

Expert Testimony: None declared.

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