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. Author manuscript; available in PMC: 2011 Jul 23.
Published in final edited form as: Circ Res. 2010 Jun 3;107(2):242–251. doi: 10.1161/CIRCRESAHA.109.210229

INCREASED ENDOTHELIAL EXOCYTOSIS AND GENERATION OF ENDOTHELIN-1 CONTRIBUTES TO CONSTRICTION OF AGED ARTERIES

Aditya Goel 1, Baogen Su 2, Sheila Flavahan 3, Charles J Lowenstein 4, Dan E Berkowitz 5, Nicholas A Flavahan 6
PMCID: PMC2909353  NIHMSID: NIHMS213415  PMID: 20522806

Abstract

Rationale

Circulating levels of endothelin-1 (ET-1) and endogenous ETA-mediated constriction are increased in human aging. The mechanisms responsible are not known.

Objective

Investigate the storage, release and activity of ET-1 system in arteries from young and aged Fischer-344 rats.

Methods and Results

After NO synthase inhibition (L-NAME), thrombin contracted aged arteries, which was inhibited by endothelial-denudation, ETA-receptor antagonism (BQ123), ECE inhibition (phosphoramidon, SM19712) or by inhibiting exocytosis (TAT-NSF,N-ethylmaleimide-sensitive-factor inhibitor). Thrombin did not cause endothelium-dependent contraction of young arteries. In aged but not young arteries, thrombin rapidly increased ET-1 release, which was abolished by endothelium-denudation or TAT-NSF. L-NAME did not affect ET-1 release. ET-1 immunofluorescent staining was punctate and distinct from Von Willebrand factor (VWF). VWF and ET-1 immunofluorescent intensity was similar in young and aged quiescent arteries. Thrombin rapidly increased ET-1 staining and decreased VWF staining in aged but had no effect in young aortas. After L-NAME, thrombin decreased VWF staining in young aortas. NO donor DEA-NONOate (1–100nmol/L) reversed thrombin-induced exocytosis in young (VWF) but not aged L-NAME-treated aortas (VWF,ET-1). Expression of preproET-1 mRNA and ECE-1 mRNA were increased in aged compared to young endothelium. BigET-1 levels and contraction to exogenous BigET-1 (but not ET-1) were also increased in aged compared to young arteries.

Conclusions

The stimulated exocytotic release of ET-1 is dramatically increased in aged endothelium. This reflects increased reactivity of exocytosis, increased expression and storage of ET-1 precursor peptides, and increased expression of ECE-1. Altered endothelial exocytosis of ET-1 and other mediators may contribute to cardiovascular pathology in aging.

Keywords: aorta, mesenteric arteries, Weibel-Palade bodies, thrombin, von Willebrand factor

INTRODUCTION

Human aging is associated with an increase in baseline systemic vasoconstriction and increased circulating levels of endothelin-1 (ET-1)1–4. Indeed, inhibition of ETA-receptors causes powerful vasodilation in aged but not young individuals5,6 and normalizes the reduced blood flow and increased vascular resistance in elderly subjects5. Constriction to exogenous ET-1 appears diminished in the elderly suggesting that the increased activity of ET-1 reflects the increased levels of the mediator6. Increased ET-1 production can cause oxidant stress, endothelial dysfunction, vascular inflammation and vascular remodeling, and likely contributes to age-related cardiovascular dysfunction7–9. Indeed, ET-1 contributes to stiffening and calcification of central arteries10–14, which is a prominent characteristic of the aging vascular system and is associated with increased risk for stroke, myocardial infarction, heart failure and mortality in the elderly15.

ET-1 is the major vascular ET isoform and is produced predominantly by the endothelium9,16. In cultured endothelial cells, ET-1 is released continuously through a constitutive pathway that is regulated principally through gene transcription and translation9,16. However, stimulation of cultured endothelial cells also causes a rapid increase in ET-1, which suggests storage of ET-1 or its precursors and regulated exocytosis17. Indeed, ET-1 has been localized to Weibel-Palade bodies (WPBs), the most recognizable endothelial storage granule, although the primary localization in cultured and native endothelium may be in distinct storage granules17–22. This pattern of partial localization in WPBs is similar to other endothelial mediators, including tissue plasminogen activator23–26. ET-1 is formed from precursor peptides by proteolytic processing. PreproET-1 mRNA is translated, stripped of its signal sequence and further cleaved by a furin-like peptidase to generate BigET-19,16. Further processing to biologically active ETs is achieved predominantly by endothelin-converting enzyme (ECE)9,16. Despite the evidence for increased activity of ET-1 in the aging vascular system, no previous studies have directly analyzed the exocytotic release of ET-1 from aging endothelial cells. Therefore, the present experiments were performed to investigate the ET-1 signaling system in aging native arterial endothelium.

METHODS

Young (3–4 months) and aged (18–20 months) Fischer-344 rats were used in the study, which was approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University and complied with the NIH Guide for the Care and Use of Laboratory Animals. Thoracic aorta and mesenteric arteries were rapidly and carefully removed from anesthetized rats into cold physiological buffer solution. Tissues were processed for measurement of vasomotor activity or endothelial mediator expression and release by ELISA, laser scanning microscopy and real time-PCR. Methods and analyses are described in the Online Data Supplement.

RESULTS

Endothelium, ET-1 and Thrombin-Induced Contractions

AORTAS

In aged aortas, thrombin (1U/ml) caused a transient dilation that peaked at 37.7 ± 5.9% (n = 6) of the contraction to phenylephrine (Figure 1). After NO synthase (NOS) inhibition (L-NAME, 100 μmol/L), the relaxation to thrombin (1U/ml) was abolished and converted to rapid contraction (Figure 1). In endothelium-denuded arteries (with or without L-NAME), thrombin (1U/ml) caused only a small contractile response suggesting that the transient dilation and the major component of the contraction to thrombin were mediated by the endothelium (Figure 1, Supplemental Figure I). Although the selective ETA-receptor antagonist BQ123 (1μmol/L) did not affect the transient dilation to thrombin, it markedly reduced the contraction to thrombin in L-NAME-treated aged aortas (Figure 1). The inhibitory effect of BQ123 in L-NAME-treated aged aortas was similar to the inhibitory effect of endothelial denudation (Figure 1). The ETB-receptor antagonist BQ788 (1μmol/L, alone or in presence of BQ123) did not affect contractions to thrombin in aged L-NAME-treated aortas (Supplemental Figure II).

Figure 1.

Figure 1

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Thrombin-induced vasomotor responses in aged and young rat aortas. Responses to thrombin (1U/ml) were assessed in aortic rings contracted with phenylephrine in the absence or presence of the ETA antagonist BQ123 (1μmol/L) and/or the NOS inhibitor L-NAME (100μmol/L), and in endothelium-denuded rings (E-). Upper trace is a representative experiment in aged aortas, whereas the lower graphs present combined data for aged (top) or young aortas (bottom). Responses to thrombin were determined after 5 mins, expressed as a percentage change in the contraction to phenylephrine, and presented as means ± SEM for n = 3 to 6. *, significant difference from Control (**P < 0.01;***P < 0.001); #, significant difference from L-NAME-treated aortas (# P < 0.05; ## P < 0.01).

In young aortas, thrombin (1 U/ml) caused relaxation that was greater in magnitude and was more sustained than in aged blood vessels (69.9 ± 6.0% of the contraction to phenylephrine, n = 5, P < 0.01 compared to aged arteries) (Figure 1). As in aged aortas, NOS inhibition (L-NAME, 100μmol/L) abolished the relaxant response to thrombin and converted it to contraction (Figure 1). However, the contractile response to thrombin in young aortas was not affected by ET-receptor antagonism (BQ123 1μmol/L and/or BQ788 1μmol/L) or by endothelial denudation (Figure 1, Supplemental Figure II).

Exogenous ET-1 (in the presence of L-NAME, 100 μmol/L), caused similar concentration-dependent contractions of young and aged aortas that were virtually abolished by the ETA-receptor antagonist BQ123 (1μmol/L) (Supplemental Figure III)

MESENTERIC ARTERIES

In control quiescent arteries, thrombin (1U/ml) did not affect the baseline diameter of young or aged mesenteric arteries. However, in the presence of L-NAME (100 μmol/L), thrombin (1U/ml) caused constriction that was significantly increased in aged compared to young arteries (Supplemental Figure IV).

In aged L-NAME-treated arteries, thrombin-induced constriction was markedly reduced by endothelial denudation or by antagonism of ETA and ETB receptors (BQ123 1μmol/L, BQ788 1μmol/L) (Supplemental Figure IV). Constriction to phenylephrine was not significantly affected by these interventions (Supplemental Figure IV).

The small constriction to thrombin in young L-NAME-treated arteries was not affected by endothelial denudation or by antagonism of ETA and ETB receptors (BQ123 1μmol/L, BQ788 1μmol/L) (Supplemental Figure IV).

Exogenous ET-1 caused similar concentration-dependent constrictions of young and aged L-NAME-treated mesenteric arteries that were virtually abolished by antagonism of ETA and ETB receptors (BQ123 1μmol/L, BQ788 1μmol/L) (Supplemental Figure V)

Endothelial Exocytosis and Thrombin-Induced Contractions

TAT-NSF is a cell-permeable inhibitor of endothelial exocytosis that acts by blocking the activity of NSF (N-ethylmaleimide-sensitive factor), a critical component of the exocytotic machinery27,28. In aged rat aortas treated with L-NAME (100μmol/L), the endothelium-dependent contraction to thrombin (1U/ml) was significantly reduced by TAT-NSF (1μmol/L) but not by a control scrambled peptide (TAT-CON, 1μmol/L) (Figure 2).

Figure 2.

Figure 2

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Effect of TAT-NSF, a cell-permeable inhibitor of endothelial exocytosis on endothelium-dependent contractions to thrombin (1U/ml) in aged rat aorta. Thrombin-induced contraction was assessed in aortic rings, which were treated with the NOS inhibitor L-NAME (100μmol/L) and contracted with phenylephrine, in the absence or presence of TAT-NSF or a control scrambled peptide TAT-CON (each at 1μmol/L). Responses to thrombin were determined after 5 mins, expressed as a percentage change in the contraction to phenylephrine, and presented as means ± SEM for n = 5. #, significant difference from L-NAME-treated aortas (P < 0.05).

ET-1 Release and Aging Endothelium

In aged aortas, thrombin (1U/ml, 5 min) caused a rapid 3.7-fold increase in ET-1 release that was prevented by endothelial denudation or by the exocytosis inhibitor TAT-NSF (1μmol/L), but not affected by the control peptide (TAT-CON) (Figure 3). Inhibition of NOS (L-NAME, 100 μmol/L) did not significantly affect ET-1 release under basal conditions or in response to thrombin in aged aortas (Figure 3).

Figure 3.

Figure 3

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Analysis of the release of ET-1 from aged and young rat aortas. Release of ET-1 from aortic segments (upper: aged, lower: young) was determined under basal conditions and following stimulation with thrombin (1U/ml, 5 mins) in the absence or presence of the NOS inhibitor L-NAME (100μmol/L), the inhibitor of exocytosis (TAT-NSF) or a control scrambled peptide (TAT-CON) (each at 1μmol/L), and in endothelium-denuded rings (E-). ET-1 levels were determined by ELISA, expressed relative to basal unstimulated conditions, and presented as means ± SEM for n = 4 to 5. *, significant difference from Control (C) (*P < 0.05,**P < 0.01;***P < 0.001); #, significant difference from thrombin (THR) treated aortas P < 0.05.

In young aortas, thrombin did not affect ET-1 release in the presence or absence of L-NAME (Figure 3). The basal release of ET-1 was not significantly different between young and aged aortas (111.4 ± 23.7 and 87.1 ± 20.1 pg/g dry tissue, respectively, n = 5, P = NS).

Imaging ET-1 and WPBs in Native Endothelium

VWF immunofluorescent staining identified WPBs in the native endothelium of young and aged aortas (Figure 4 and 5, Supplemental Figure VI). ET-1 immunofluorescence revealed punctate staining in the endothelium that was distinct from WPBs (Figure 4). Under control unstimulated conditions, there was no significant difference in the intensity of VWF or of ET-1 staining between young and aged endothelium (Figure 5). Thrombin (1U/ml, 5 min) did not significantly affect ET-1 or VWF staining in young control aortas (Figure 5). However, thrombin (1U/ml, 5 min) significantly increased ET-1 immunofluorescence and decreased VWF immunofluorescence associated with the endothelium of aged control aortas (Figure 5). Acute inhibition of ECE (SM19712, 200μmol/L 30 mins prior to thrombin) prevented the thrombin-induced increase in ET-1 immunofluorescence but not the thrombin-induced decrease in VWF immunofluorescence in aged aortas (Figure 6).

Figure 4.

Figure 4

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Localization of ET-1 and VWF in endothelial cells of aged rat aorta. Representative LSM images of endothelial cells lining the lumen of aged rat aorta, which was stained for VWF (green), ET-1 (red) and nuclei (blue). Individual images and the overlay are presented. Bar: 25μm. To enhance visualization of ET-1, this image was acquired using image accumulation (2 images).

Figure 5.

Figure 5

Figure 5

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Effect of thrombin (1U/ml, 5 mins) on immunofluorescent staining for VWF and ET-1 in endothelial cells lining young and aged rat aortas. TOP: Representative LSM images of native endothelial cells, which were stained for VWF (green), ET-1 (red) and nuclei (blue). Aortas from young (upper) and aged rats (lower) were processed under control conditions (left) and following stimulation with thrombin (1U/ml, 5 mins) (right). Bar: 10μm. BOTTOM: Quantification of immunofluorescent images. Fluorescence intensity is expressed as a percentage of the signal in young control aortas, and is presented as means ± SEM (N = 11–19 scanned images for individual groups; n = 3 animals). *, statistically significant difference (*P < 0.05;***P < 0.001); NS, not significant.

Figure 6.

Figure 6

Figure 6

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Effect of ECE inhibitor SM19712 (200μmol/L, 30 mins) on thrombin (1U/ml, 5 mins)-induced changes in immunofluorescent staining for VWF and ET-1 in endothelial cells lining aged rat aortas. TOP: Representative LSM images of native endothelial cells, which were stained for VWF (green), ET-1 (red) and nuclei (blue). Aortic segments from aged rats were incubated in the presence and absence of SM19712 followed by incubation in the presence and absence of thrombin (1U/ml, 5 mins). Bar:10μm. BOTTOM: Quantification of immunofluorescent images. Fluorescence intensity is expressed as a percentage of the signal in control aortas, and presented as means ± SEM (N = 16–23 scanned images for individual groups; n = 3 animals). *, significant difference from Control (***P < 0.001); #, significant difference from thrombin treated aortas (### P < 0.001).

Although thrombin had no significant effect on WPBs in young control aortas, in the presence of L-NAME (100μmol/L) thrombin caused a dramatic mobilization of these endothelial storage granules, evidenced by the presence of VWF immunofluorescent aggregates and strands (Supplemental Figure VII) and by a significant decrease in intensity of VWF fluorescence (figure 7). In aged arteries, the effect of thrombin was not influenced by the presence of L-NAME (Supplemental Figure VII, Figure 5 and 7).

Figure 7.

Figure 7

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Effects of the NO donor DEA-NONOate on smooth muscle and endothelium of young and aged aortas treated with the NOS inhibitor L-NAME (100μmol/L). A: Effect of DEA-NONOate (0.1–100 nmol/L) to cause dilation of phenylephrine-contracted young and aged aortas. Responses are expressed as a percentage change in the contraction to phenylephrine, and presented as means ± SEM for n = 6. B and C: Effect of DEA-NONOate (1,10,100 nmol/L; 10 mins) on thrombin-induced exocytosis of ET-1 (B) or VWF (C) from native endothelium of aged and young aortas, determined by quantification of immunofluorescent staining. Fluorescence intensity was expressed as a percentage of the signal in control aortas, and presented as means ± SEM (N = 23–54 scanned images for individual groups; n = 5 (B) or 3 (C) animals). a, significantly different from corresponding control arteries (a, P < 0.001; a′, P < 0.01; a″, P < 0.05); b, significantly different from corresponding young aortas (b, P < 0.001; b′, P < 0.01).

The effects of the NO donor DEA-NONOate were analyzed in L-NAME-treated aortas. DEA-NONOate (0.1 to 100 nmol/L) caused similar relaxation of aged and young aortas contracted with phenylephrine (Figure 7A). However, in aged arteries, DEA-NONOate (1 to 100nmol/L) did not significantly affect the stimulation of endothelial exocytosis by thrombin (1U/ml), which was characterized by increased fluorescent staining for ET-1 (Figure 7B) and decreased fluorescent staining for VWF (Figure 7C). In contrast, DEA-NONOate (1 to 100nmol/L) significantly reversed the thrombin-induced decrease in VWF staining in young arteries (Figure 7C). Thrombin (1U/ml) did not affect ET-1 immunofluorescence in young L-NAME-treated aortas (Figure 7B).

Processing of ET-1 in Young and Aging Arteries

In aged L-NAME-treated aortas, the rapid contraction to thrombin (1U/ml) was inhibited by acute treatment with ECE inhibitors SM19712 (200μmol/L) or phosphoramidon (30μmol/L) (each 30 mins prior to thrombin), which caused similar inhibition as the ETA-receptor antagonist BQ123 (Supplemental Figure VIII). The ECE inhibitors did not affect the contractile response to thrombin in young aortas (Supplemental Figure VIII).

Analysis of aortic lysates revealed a 3.2-fold increase in the ET-1 precursor Big ET-1 in aged compared to young blood vessels (Figure 8A), which was associated with increased expression of preproET-1 mRNA in aged aortas (Figure 8C). Endothelial-denudation reduced the aortic expression of preproET-1 mRNA and abolished the difference in expression between young and aged blood vessels.

Figure 8.

Figure 8

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Processing of ET-1 in aortas of young and aged rats. A: Quantification of BigET-1 content of aged and young aortas determined by ELISA of aortic lysates. Data is presented as pg/g of aortic protein and is presented as means ± SEM, n = 4. **, significant difference from aged aortas (P < 0.01). B: Contractions to BigET-1 (30 nmol/L) in aged (left) and young aortas (panel) in the absence and presence of the ECE inhibitor phosphoramidon (30μmol/L, Phos). Aortic rings were treated with the NOS inhibitor L-NAME (100μmol/L) and contracted with phenylephrine. Responses to BigET-1 were assessed after 15 mins, expressed as a percentage change in contraction to phenylephrine, and presented as means ± SEM for n = 6–7. **, significant difference from corresponding group in aged aortas (P < 0.01); #, significant difference from aortas not treated with phosphoramidon (##, P < 0.01; ###, P < 0.001). C: Expression of mRNA for preproET-1 (ppET-1), ECE-1 and ECE-2 in endothelium-containing (+) and endothelium-denuded (−) aortas from young and aged rats. mRNA was determined using realtime PCR, expressed relative to levels in aged endothelium-containing aortas, and presented as means ± SEM for n = 5 or 7. *, significant difference from corresponding aged aortas (endothelium + or −) (**P < 0.01; ***P < 0.001); #, significant difference from endothelium + (# P < 0.05; ### P < 0.001).

Expression of ECE-1 mRNA was also increased in aged compared to young aorta (Figure 8C). Endothelial-denudation reduced the expression of ECE-1 mRNA in aged aortas and abolished the difference in expression between young and aged blood vessels (Figure 8C). ECE-2 expression was similar in aged and young aortas (Figure 8C). Consistent with increased ECE-1 expression, the ET-1 precursor, BigET-1 (30 nmol/L) caused contraction that was significantly greater in aged compared to young aortas (Figure 8B). The ECE inhibitor phosphoramidon (30μmol/L) decreased the contraction to BigET-1 in young and aged arteries, and after phosphoramidon, there was no longer any significant difference between young and aged aortas (Figure 8B).

DISCUSSION

The results of the present study demonstrate that ET-1 is generated during stimulated endothelial exocytosis in aged but not in young arteries and that this ET-1 contributes to constriction of the aged vascular system. ET-1 was formed predominantly and rapidly during the exocytotic process. An aging-associated specific increase in the stimulated generation of ET-1 reflects increased expression and storage of ET-1 precursors and increased expression of ECE-1 in aged endothelium. Stimulated exocytosis from aged endothelium was also less sensitive to inhibition by NO compared to young arteries, and so an important restraint on endothelial exocytosis is diminished. The increased ability of aged endothelium to generate ET-1, combined with an increased excitability of the exocytotic process may contribute to the cardiovascular pathology of aging.

Endothelial secretagogues such as thrombin can cause rapid release of mediators from distinct storage granules in cultured endothelial cells as well as de novo synthesis of endothelial vasodilators including NO24,29,30. Indeed, thrombin caused NOS and endothelium-dependent relaxation, which was decreased in aged compared to young aortas. This is consistent with the known effect of aging to decrease endothelium-dependent relaxation and NO activity31–33. After NOS inhibition, the thrombin response converted to rapid contraction, which was increased in aged compared to young arteries. The aging-dependent increase in thrombin-induced contraction was entirely dependent on the endothelium (absent in endothelium-denuded arteries) and was prevented by blocking ET receptors. Studies on cultured endothelial cells have suggested that ET-1 release can occur through two processes: a constitutive pathway regulated principally through gene transcription and synthesis of ET-1 precursors, and a stimulated pathway that results from rapid exocytosis of stored peptides9,16,17. Thrombin rapidly increased the release of ET-1 from aged but not young aortas, consistent with the thrombin-induced contractile responses. The stimulated release of ET-1 from aged aortas was abolished by endothelial-denudation or by TAT-NSF, a cell-permeable inhibitor of NSF and endothelial exocytosis27,28,34,35. TAT-NSF also inhibited the endothelium-dependent contraction to thrombin in aged aorta. The scrambled control peptide TAT-CON27,28 did not significantly affect the thrombin-induced release of ET-1 or the resulting endothelium-dependent contraction occurring in aged aortas. In contrast to the stimulated release of ET-1, the basal release of the peptide, which may reflect the constitutive pathway, was not significantly different between young and aged aortas. Therefore, aged arterial endothelial cells have an increased ability to rapidly generate ET-1 through regulated exocytosis, which subsequently causes vasoconstriction of the aging vascular system.

Endothelium-derived NO is an important and powerful endogenous inhibitor of endothelial exocytosis34–37. However, in aged arteries, NOS inhibition did not significantly affect the release of ET-1 occurring under basal conditions or following stimulation with thrombin. An altered role of endogenous NO activity in regulating endothelial exocytosis of young and aged aortas was evident when imaging exocytosis of WPBs. VWF is the major component of WPBs and is stored as large multimers reaching an ultimate size of 20,000kD (ultra large or ULVWF)38,39. Stimulated release of ULVWF does not occur from individual WPBs: multiple granules fuse prior to release and exocytosis is associated with formation of craters on the endothelial surface38,40. After exocytosis, ULVWF can be unfurled by flow revealing a pearls-on-a-string appearance with adherent platelets41,42. In control aged arteries, thrombin caused a significant decrease in intensity of VWF staining concomitant with the appearance of ULVWF strands and aggregates, all consistent with stimulated exocytosis of the protein. In contrast, thrombin did not alter the intensity or pattern of VWF staining in young control arteries. However, after NOS inhibition, thrombin caused the same pattern of ULVWF aggregation and release, and a decrease in VWF staining in young and aged arteries. These results suggest that endogenous NO normally restrains endothelial exocytosis in young arteries, but this restraint is diminished in aged arteries. This could reflect a generalized aging-induced decrease in endothelial NO activity31–33. However, although NOS inhibition did not influence thrombin-induced endothelial exocytosis in aged arteries, it was an absolute requirement to observe the resulting thrombin-induced ET-1-mediated constriction of these blood vessels. Although the NOS-dependent endothelium-dependent relaxation to thrombin was partly reduced in aging arteries, NOS activity was still associated with marked inhibition of contractions to phenylephrine or thrombin in aging blood vessels. This suggests that aging arteries may retain sufficient endothelial NOS activity to relax smooth muscle but not to inhibit exocytosis. Aging can cause uncoupling of NOS resulting in increased generation of reactive oxygen species (ROS) and decreased production of NO by the enzyme43. Altered generation of NOS-derived mediators could then contribute to differential regulation of endothelial and smooth muscle function. However, ROS such as H2O2 not only mediate dilation to uncoupled NOS44, but also inhibit endothelial exocytosis45. Furthermore, catalase (1200 U/ml), which degrades H2O2, did not act like L-NAME and had no effect on constriction to phenylephrine or dilation to thrombin in aged aortas (data not shown). Therefore, the most likely explanation is that exocytosis in aging endothelial cells has a reduced sensitivity to endogenous NO. Indeed, exogenous NO (DEA-NONOate) had a markedly decreased ability to inhibit endothelial exocytosis in aged compared to young arteries. Interestingly, there was no difference in the ability of exogenous NO to cause smooth muscle relaxation in young and aged arteries. These results therefore highlight a novel form of aging-induced endothelial dysfunction, namely a selective insensitivity of endothelial exocytosis to the inhibitory effects of NO. This may reflect an altered pattern of S-nitrosylation or reduced cyclic GMP signaling, which can mediate the modulatory effects of NO on endothelial exocytosis27,34,35,46,47.

Enhanced activity of endothelial exocytosis cannot solely explain the increased ET-1 activity in aging arteries. Although NO synthase inhibition enabled exuberant thrombin-induced exocytosis in young aortas, it did not uncover thrombin-induced release of ET-1 in these arteries. This indicates that in addition to enhanced exocytosis, aging also caused specific changes in endothelial ET-1 processing.

ET-1 immunofluorescence revealed diffuse punctate staining distinct from VWF suggesting that the primary storage site in native aortic endothelium is in small granules and not WPBs, which is consistent with previous studies in cultured and native endothelium17–22. Under quiescent conditions, there was no significant difference in ET-1 staining between the endothelium of young and aged aortas. However, thrombin caused a dramatic and rapid increase in ET-1 staining associated with the endothelium of aged but not young arteries. These results suggest that the increased activity of ET-1 in aging arteries does not reflect increased storage of the active peptide, and that the dramatic thrombin-induced generation of ET-1 in aged arteries reflects rapid formation of ET-1. Indeed, the thrombin-induced ET-1 mediated contraction and increase in ET-1 immunofluorescent staining were markedly reduced by acute treatment of the aortas with ECE inhibitors, which would not affect ET-1 that had already been formed and stored in endothelial granules. ECE inhibition did not affect the ability of thrombin to cause exocytosis of WPBs and release of VWF. These results suggest that ET-1 is stored as a precursor and that it is formed rapidly during the exocytotic process; a process that is dramatically increased in aged compared to young endothelium. Indeed, there was increased expression of preproET-1 mRNA and also of ECE-1 mRNA in aged compared to young aortas. In each case, the increased expression was abolished by endothelium-denudation indicating that it reflected a selective increase in endothelial expression. The increase in preproET-1 mRNA expression was paralleled by increased concentrations of BigET-1 in aortic lysates of aged compared to young aortas. Likewise, the functional significance of the increase in ECE-1 mRNA was demonstrated by the marked increase in phosphoramidon-sensitive contractions to exogenous BigET-1 in aged compared to young arteries. In contrast to BigET-1, exogenous ET-1 caused contraction that was similar in young and aged arteries. Therefore, the increased generation of ET-1 in aged arteries results from increased expression of endothelin precursors and converting enzyme and an increased sensitivity of the exocytotic process, compared to young arteries. Increased expression of preproET-1 and ECE-1 may result from (or be amplified by) chronic exposure to altered hemodynamics, including arterial stiffness, or altered mediator activity within the aging blood vessel wall, including diminished NO activity, increased oxidant stress and inflammatory activation of the endothelium. Altered activity of the ET-1 signaling pathway may be an important early component of the aging endothelial phenotype. Although endothelial senescence in cultured systems is associated with a loss of WPBs and ET-1 immunofluorescence, pre-senescent aging of cultured endothelial cells increases ET-1 expression48,49. Likewise, ECE-1 expression and circulating levels of ET-1 are increased in third generation telomerase-deficient mice50.

Human aging is associated with increased circulating levels of ET-1 and VWF1–4,51. Increased ET-1 production can cause oxidant stress, endothelial dysfunction, vascular inflammation and vascular remodeling7,8, and likely contributes to age-related cardiovascular dysfunction including arterial stiffening and atherosclerosis9–15. Likewise, VWF is an important thrombotic mediator and increased VWF levels are associated with myocardial infarction and mortality in individuals with vascular disease52. Increased expression of ET-1 precursors and converting enzyme and increased excitability of the exocytotic process likely contributes to increased circulating levels of these mediators and to the cardiovascular pathology of aging.

Novelty and Significance.

What is known?

  • Studies in cultured endothelial cells have demonstrated that endothelin-1(ET-1) is released both continuously via a constitutive pathway and rapidly in response to stimulated exocytosis, suggesting storage of ET-1 or its precursors in endothelial storage granules such as Weibel-Palade Bodies.

  • ET-1, which can cause oxidant stress, endothelial dysfunction, vascular inflammation, vascular constriction and vascular remodeling, has increased activity in aging individuals and likely contributes to age-related cardiovascular dysfunction.

  • No previous studies have directly assessed the stimulated endothelial exocytosis of ET-1 or other mediators from native arteries or its potential modulation in the aging vasculature.

What new information does this article contribute?

  • Stimulation of endothelium caused rapid exocytotic release of ET-1 from aged but not young arteries, and the released ET-1 contributed to constriction of aged arteries.

  • The generation of ET-1 in aging arteries occurred during the exocytotic process and reflected rapid conversion of its precursor Big ET-1 by endothelin converting enzyme-1 (ECE-1), both of which had increased expression in aged endothelium.

  • Aging also increased the excitability of the exocytotic process due to a decreased sensitivity of aging endothelium to the inhibitory effects of nitric oxide, resulting in increased stimulated exocytosis of stored mediators from aging endothelium.

Summary.

Despite evidence for increased ET-1 activity in the aging vascular system, no previous studies have directly analyzed the endothelial exocytotic release of ET-1 from aging arteries. We report that stimulation of native arteries with a secretogogue (thrombin) can cause a rapid exocytotic release of ET-1 from the endothelium of aged but not young arteries, resulting in constriction of aged arteries. Interestingly, there was no difference in storage of ET-1 between young and aged endothelial cells, and the increased activity of ET-1 in aged arteries became evident only during the stimulated exocytotic process. Compared to young endothelial cells, aging cells stored increased levels of the ET-1 precursor, Big ET-1, and had increased expression of preproET-1 and ECE-1, which cleaves BigET-1. Indeed, acute inhibition of ECE, which would not inhibit stored ET-1, abolished ET-1 release and the associated constriction in aged arteries. Aging arteries also displayed a novel type of endothelial dysfunction, namely a diminished ability of endothelium-derived NO to inhibit the stimulated exocytotic release of endothelial mediators, including ET-1 and von Willebrand factor. The increased ability of aged endothelium to generate ET-1, combined with an increased excitability of the exocytotic process may contribute to the cardiovascular pathology of aging.

Supplementary Material

Acknowledgments

SOURCES OF FUNDING

NIH (HL080119,HL102715,OH008531) to NAF

Non-standard Abbreviations and Acronyms

BQ123

Cyclo(D-Asp-Pro-D-Val-Leu-D-Trp)

BQ788

N-[(cis-2,6-Dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl-1-(methoxycarbonyl)-D-tryptophyl-D-norleucine sodium salt

DEA-NONOate

Diethylamine NONOate

ECE

Endothelin Converting Enzyme

ET

Endothelin

L-NAME

Nω-Nitro-L-arginine methyl ester hydrochloride

LSM

Laser Scanning Microscope

NOS

Nitric oxide synthase

SM19712

4-Chloro-N-[[(4-cyano-3-methyl-1-phenyl-1H-pyrazol-5-yl)amino]carbonyl]benzenesulfonamide sodium

TAT-NSF

human immunodeficiency virus transactivator of transcription (TAT) protein transduction domain fused to a N-ethylmaleimide-sensitive factor (NSF) homohexamerization domain

TAT-CON

human immunodeficiency virus transactivator of transcription (TAT) protein transduction domain fused to scrambled amino acid sequence of N-ethylmaleimide-sensitive factor (NSF) homohexamerization domain

VWF

Von Willebrand Factor

WPB

Weibel Palade body

Footnotes

DISCLOSURES

None

Contributor Information

Aditya Goel, Department of Anesthesiology, Johns Hopkins University, Baltimore, MD 21205.

Baogen Su, Department of Anesthesiology, Johns Hopkins University, Baltimore, MD 21205.

Sheila Flavahan, Department of Anesthesiology, Johns Hopkins University, Baltimore, MD 21205.

Charles J. Lowenstein, Department of Medicine, University of Rochester, Rochester, NY 14642

Dan E. Berkowitz, Department of Anesthesiology, Johns Hopkins University, Baltimore, MD 21205

Nicholas A. Flavahan, Department of Anesthesiology, Johns Hopkins University, Baltimore, MD 21205

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Supplementary Materials

NIHMS213415-supplement-supplement_1.pdf (4.5MB, pdf)

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