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The Notch ligand Delta-like 1 is elevated and associated with mortality in patients with symptomatic aortic stenosis
International Journal of Cardiology 180 (2015) 18–20
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
The Notch ligand Delta-like 1 is elevated and associated with mortality in patients with symptomatic aortic stenosis Aurelija Abraityte a,d,e,f,⁎, Lars Gullestad b,d,e, Erik Tandberg Askevold a,b,e,i, Ståle Nymo a,d, Christen P. Dahl a,b,e, Sven Aakhus b,d,e, Pål Aukrust a,c,d,g, Thor Ueland a,d,f,g,h a
Research Institute of Internal Medicine, Oslo University Hospital, Rikshospitalet, Oslo, Norway Department of Cardiology, Oslo University Hospital, Rikshospitalet, Oslo, Norway Section of Clinical Immunology and Infectious Diseases, Oslo University Hospital, Rikshospitalet, Oslo, Norway d Faculty of Medicine, University of Oslo, Oslo, Norway e Center for Heart Failure Research, University of Oslo, Oslo, Norway f K.G. Jebsen Cardiac Research Center, University of Oslo, Oslo, Norway g K. G. Jebsen Inflammation Research Center, University of Oslo, Oslo, Norway h K. G. Jebsen Thrombosis Research and Expertise Center, University of Tromsø, Tromsø, Norway i Department of Medicine, Lovisenberg Diakonale Hospital, Oslo, Norway b c
a r t i c l e
i n f o
Article history: Received 15 September 2014 Received in revised form 10 November 2014 Accepted 16 November 2014 Available online 18 November 2014 Keywords: Aortic stenosis Notch signaling DLL1
Aortic valve stenosis (AS) is the most common cardiac valve disease in developed countries and its prevalence increases markedly with age [1]. The pathogenesis of AS is still not fully understood, but during disease progression, myofibroblast-like cells acquire osteoblastic features, including spontaneous calcification and bone formation that take place in the valves [2]. Therapeutic strategies to delay the disease progression are lacking, and a better understanding of relevant signaling pathways could reveal new prognostic biomarkers and novel therapeutic targets in AS. Notch is an evolutionary conserved signaling pathway that regulates cell-fate determination, proliferation, and tissue patterning. Notch pathway may be reactivated from its quiescent state during myocardial remodeling in heart failure [3,4]. This signaling also contributes to cardiovascular calcification [5]. Mutations in Notch1 receptor have been linked to valve defects and severe valve calcification [6]. There are two distinct families of Notch ligands known as Delta-like (DLL) and Jagged ligands, and of these, DLL1 has been shown to promote osteogenic differentiation and calcification [7]. We hypothesized that circulating ⁎ Corresponding author at: Research Institute of Internal Medicine, Oslo University Hospital, Rikshospitalet, P.B. 4950 Nydalen, 0424 Oslo, Norway. E-mail address: [email protected] (A. Abraityte).
http://dx.doi.org/10.1016/j.ijcard.2014.11.111 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
DLL1 levels could represent a link between valvular calcification and myocardial dysfunction and provide prognostic information in patients with symptomatic AS. A total of 136 patients with confirmed symptomatic AS assessed by echocardiography and evaluated for aortic valve replacement (AVR) were consecutively enrolled in the study [8]. All patients underwent echocardiographic, angiographic examinations and blood sampling. All patients were clinically stable and none had severe comorbidities. Aortic valve specimens were obtained from 14 patients undergoing AVR (mean ± SD: 70 ± 12 years, 7 women). Serum levels of DLL1 were determined by enzyme immunoassay (R&D Systems). DLL1 mRNA was quantified on an ABI Prism 7500 qPCR instrument using Power SYBR Green Master Mix (Applied Biosystems), sequence-specific primers and normalized to the expression of GAPDH. Variables which were not normally distributed were log-transformed. Differences between groups were analyzed with Student's t-test or Mann–Whitney U test, as appropriate. Relationships between variables were assessed by Spearman's correlation. Restricted cubic spline interpolation was used to evaluate nonlinear relationships between DLL1 levels and all-cause mortality. Kaplan–Meier analysis with log-rank test was done to compare the number of events in relation to tertiles of DLL1 levels (paired for each stratum due to non-linearity). Cox proportional hazard analysis was performed to estimate adjusted hazard ratios (covariates in figure legend). Follow-up time for all-cause mortality was calculated from time of inclusion to death from any cause. p values (two-sided) were considered significant when b 0.05. The study protocol conforms to the ethical guidelines of the Declaration of Helsinki. Signed informed consent was obtained from all study objects. The authors of the manuscript certify that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. Baseline characteristics according to DLL1 levels comparing the tertiles are shown in Table 1. Patients with symptomatic AS (n = 136) had upregulated serum levels of DLL1 compared to healthy controls (n = 95) (Fig. 1A). Patients with the highest DLL1 levels were older, had higher creatinine and lower LDL levels. There was no association
A. Abraityte et al. / International Journal of Cardiology 180 (2015) 18–20
19
Table 1 Patient characteristics in relation to DLL1 levels. Total population N = 136
DLL1 (≤5.30)
DLL1 (5.31–6.92)
DLL1 (≥6.93)
p-Value
Age (years) Male (%) BMI (kg m−2) NYHA functional class (II/III/IV) (%) Coronary artery disease (%) Current smokers (%) DM2 (%) Hypertension (%) Atrial fibrillation AVR
77 (69, 81) 79 (58) 26.3 ± 4.2 3/33/63/1 64 (47) 45 (34) 14 (10) 36 (27) 43 (32) 108 (79)
74 (65, 78) 24 (53) 25.6 ± 4.0 5/34/61/0 19 (42) 11 (25) 6 (13) 11 (24) 34 (76) 40 (38)
78 (70, 81) 28 (62) 26.7 ± 3.8 5/30/63/2 22 (49) 17 (40) 4 (9) 13 (29) 33 (73) 33 (31)
79 (74, 82) 26 (59) 27.0 ± 4.7 0/35/65/0 23 (52) 17 (40) 4 (9) 12 (27) 24 (55) 33 (31)
0.01 0.69 0.24 0.64 0.63 0.36 0.74 0.89 0.07 0.14
Hemodynamics LVEF (%) CO (l/min) Aortic valve area (cm2) Mean aortic gradient (mm Hg)
63 (56, 70) 4.8 (4.3, 5.5) 0.62 (0.50, 0.80) 54.4 ± 19.1
63 (56, 70) 4.7 (4.3, 5.4) 0.65 (0.53, 0.78) 53.1 ± 15.7
66 (57, 70) 4.8 (4.3, 5.3) 0.70 (0.50, 0.83) 53.7 ± 21.9
62 (54, 70) 4.9 (4.1, 5.6) 0.60 (0.50, 0.80) 54.3 ± 18.0
0.47 0.98 0.56 0.96
Biochemistry eGFR (ml min−1 1.73 m−2) CRP (mg L−1) HDL-Ch (mmol L−1) LDL-Ch (mmol L−1) Nt-proBNP [pmol L−1] Cholesterol (mmol L−1) TnT (ng L−1)
66 (52, 88) 1.9 (0.9, 4.3) 1.6 (1.3, 1.9) 3.2 ± 1.1 98 (42, 270) 5.0 (4.3, 5.7) 14.1 (8.3, 25.0)
75 (58, 103) 1.4 (1.0, 3.6) 1.6 (1.4, 1.9) 3.7 ± 1.2 62 (37, 159) 5.3 (4.8, 6.1) 10.1 (6.0, 17.1)
68 (55, 89) 1.3 (0.5, 3.6) 1.7 (1.3, 1.9) 3.2 ± 0.9 99 (51, 154) 4.7 (4.3, 5.5) 13.6 (8.8, 19.2)
56 (43, 71) 3.7 (1.7, 7.7) 1.5 (1.2, 1.9) 2.9 ± 1.2 174 (41, 512) 4.5 (3.7, 5.4) 19.5 (12.0, 31.4)
0.001 0.008 0.39 0.005 0.03 0.002 0.003
Medication (%) ACE inhibitor ARB β-Blocker Statins Warfarin Aspirin
18 (13) 28 (21) 65 (48) 68 (50) 27 (20) 65 (48)
4 (9) 5 (11) 19 (42) 21 (47) 6 (13) 20 (44)
6 (13) 14 (31) 20 (44) 28 (62) 8 (18) 26 (58)
7 (16) 9 (21) 25 (57) 19 (43) 13 (30) 18 (41)
0.60 0.07 0.33 0.16 0.14 0.24
Continuous data as mean ± SD or median and interquartile range (Q1, Q3). DLL1, delta-like 1; BMI, body mass index; NYHA, New York Heart Association; DM2, type 2 diabetes mellitus; AVR, aortic valve replacement; LVEF, left ventricular ejection fraction; CO, cardiac output; eGFR, estimated glomerular filtration rate; CRP, C-reactive protein; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NT-proBNP, N-terminal probrain natriuretic peptide; TnT, troponin T, ACE, angiotensin-converting enzyme; ARB, angiotensin receptor blocker.
Fig. 1. DLL1 in patients with symptomatic AS. (A) Tukey plots showing that serum DLL1 levels are elevated in patients with symptomatic AS (n = 136) compared to healthy controls (n = 95), p-value age-adjusted. (B) Association between serum DLL1 and DLL1 mRNA expression in aortic valves (n = 14). (C) Restricted cubic spline interpolation showing a U shaped association between DLL1 levels (red and blue lines, mean and 95% CI) and all-cause mortality. (D) Kaplan–Meier curves according to tertiles of circulating DLL1 demonstrating a higher rate of mortality in patients with low (i.e. T1) and high (i.e. T3) DLL1. (E) Unadjusted (UNI) and adjusted hazard ratios (Model [M] 1: adjustment for AVR, age, gender, mean aortic gradient, LVEF and eGFR; M2 adjusted for M1 + CRP and NT-proBNP) showing association between DLL1 tertile 2 vs. the combined tertiles 1 and 3 for mortality. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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A. Abraityte et al. / International Journal of Cardiology 180 (2015) 18–20
between DLL1 levels and previous comorbidities (cancer, n = 8; stroke, n = 7; asthma, n = 6). There was a correlation between elevated DLL1 levels and well-characterized biomarkers of cardiovascular disease, i.e., NT-proBNP (r = 0.28, p b 0.001), TnT (r = 0.33, p b 0.001) and CRP (r = 0.30, p = 0 b 001). DLL1 levels did not correlate with echocardiographic parameters of valve stenosis or cardiac function, clinical history or the use of cardiovascular medications. DLL1 mRNA expression was determined in 14 excised aortic valves. Notably, mRNA levels correlated with serum DLL1 levels from the same patients (Fig. 1B). During a mean follow-up of 4.6 [±2.3 (S.D.)] years, 35 patients died. A restricted cubic spline interpolation model shows a U shaped association between DLL1 levels and all-cause mortality (Fig. 1C). A Kaplan–Meier plot illustrates this association further, revealing that patients in the first (p = 0.05) and third (p = 0.029) tertiles of DLL1 had lower survival rate, comparing to the second tertile (Fig. 1D). Fig. 1E shows the unadjusted and adjusted hazard ratios for tertile 2 vs. the combined tertiles 1 and 3 for mortality. Intermediate (i.e. tertile 2) DLL1 levels were associated with decreased mortality rate in unadjusted and also after multivariable adjustment for demographic features, mean aortic gradient, LVEF, NTproBNP, TnT, CRP and eGFR. We show for the first time a relationship between serum levels of the Notch ligand DLL1 and symptomatic AS. Our main and novel findings were that a) circulating DLL1 levels were elevated in patients with symptomatic AS, b) DLL1 was expressed in calcified aortic valves and correlated with systemic DLL1 concentrations, c) there was an association between DLL1 levels and established biomarkers of cardiovascular disease, and d) DLL1 levels were independently associated with long-term mortality. DLL1 has been shown to enhance mineral deposition and stimulate osteoblast differentiation [7]. Our findings may suggest that similar, DLL1-induced signaling could be operating in AS, thus promoting calcification and AS progression. The correlation between DLL1 mRNA expression in calcified valves and circulating levels suggests that valvular derived DLL1 contributes to the systemic pool. Furthermore, the positive association between DLL1 and TnT and NT-proBNP, as a reflection of myocardial damage and neurohormonal activation, respectively, may support DLL1 role in the development of heart failure accompanying symptomatic
AS. Finally, the U shaped association between serum DLL1 and mortality may suggest that intermediate levels of DLL1 are beneficial. One could speculate that low levels of DLL1 could reflect inadequate response to pathological Notch activity, while high levels might represent an overactivation of repair mechanisms in the presence of increased wall stress. Our findings support involvement of DLL1 and Notch pathways in the pathophysiology of AS and suggest that manipulation of Notch signaling could offer new therapeutic approaches for this disease. Future studies should also examine the ability of DLL1 to predict prognosis as well as prosthetic valve failure in these patients. Conflict of interest The authors report no relationships that could be construed as a conflict of interest. Acknowledgments The study was funded by grant # 39587 from the Southern and Eastern Norway Regional Health Authority. References [1] B.A. Carabello, Introduction to aortic stenosis, Circ. Res. 113 (2013) 179–185. [2] N.M. Rajamannan, Calcific aortic stenosis: lessons learned from experimental and clinical studies, Arterioscler. Thromb. Vasc. Biol. 29 (2009) 162–168. [3] J.L. de la Pompa, J.A. Epstein, Coordinating tissue interactions: notch signaling in cardiac development and disease, Dev. Cell 22 (2012) 244–254. [4] G. Aquila, M. Pannella, M.B. Morelli, C. Caliceti, C. Fortini, P. Rizzo, et al., The role of notch pathway in cardiovascular diseases, Glob. Cardiol. Sci. Pract. 2013 (2013) 364–371. [5] G. Rusanescu, R. Weissleder, E. Aikawa, Notch signaling in cardiovascular disease and calcification, Curr. Cardiol. Rev. 4 (2008) 148–156. [6] V. Garg, A.N. Muth, J.F. Ransom, M.K. Schluterman, R. Barnes, I.N. King, et al., Mutations in NOTCH1 cause aortic valve disease, Nature 437 (2005) 270–274. [7] M. Nobta, T. Tsukazaki, Y. Shibata, C. Xin, T. Moriishi, S. Sakano, et al., Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Delta1/ Jagged1-activated Notch1 signaling, J. Biol. Chem. 280 (2005) 15842–15848. [8] T. Ueland, L. Gullestad, C.P. Dahl, P. Aukrust, S. Aakhus, O.G. Solberg, et al., Undercarboxylated matrix Gla protein is associated with indices of heart failure and mortality in symptomatic aortic stenosis, J. Intern. Med. 268 (2010) 483–492.
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
The Notch ligand Delta-like 1 is elevated and associated with mortality in patients with symptomatic aortic stenosis Aurelija Abraityte a,d,e,f,⁎, Lars Gullestad b,d,e, Erik Tandberg Askevold a,b,e,i, Ståle Nymo a,d, Christen P. Dahl a,b,e, Sven Aakhus b,d,e, Pål Aukrust a,c,d,g, Thor Ueland a,d,f,g,h a
Research Institute of Internal Medicine, Oslo University Hospital, Rikshospitalet, Oslo, Norway Department of Cardiology, Oslo University Hospital, Rikshospitalet, Oslo, Norway Section of Clinical Immunology and Infectious Diseases, Oslo University Hospital, Rikshospitalet, Oslo, Norway d Faculty of Medicine, University of Oslo, Oslo, Norway e Center for Heart Failure Research, University of Oslo, Oslo, Norway f K.G. Jebsen Cardiac Research Center, University of Oslo, Oslo, Norway g K. G. Jebsen Inflammation Research Center, University of Oslo, Oslo, Norway h K. G. Jebsen Thrombosis Research and Expertise Center, University of Tromsø, Tromsø, Norway i Department of Medicine, Lovisenberg Diakonale Hospital, Oslo, Norway b c
a r t i c l e
i n f o
Article history: Received 15 September 2014 Received in revised form 10 November 2014 Accepted 16 November 2014 Available online 18 November 2014 Keywords: Aortic stenosis Notch signaling DLL1
Aortic valve stenosis (AS) is the most common cardiac valve disease in developed countries and its prevalence increases markedly with age [1]. The pathogenesis of AS is still not fully understood, but during disease progression, myofibroblast-like cells acquire osteoblastic features, including spontaneous calcification and bone formation that take place in the valves [2]. Therapeutic strategies to delay the disease progression are lacking, and a better understanding of relevant signaling pathways could reveal new prognostic biomarkers and novel therapeutic targets in AS. Notch is an evolutionary conserved signaling pathway that regulates cell-fate determination, proliferation, and tissue patterning. Notch pathway may be reactivated from its quiescent state during myocardial remodeling in heart failure [3,4]. This signaling also contributes to cardiovascular calcification [5]. Mutations in Notch1 receptor have been linked to valve defects and severe valve calcification [6]. There are two distinct families of Notch ligands known as Delta-like (DLL) and Jagged ligands, and of these, DLL1 has been shown to promote osteogenic differentiation and calcification [7]. We hypothesized that circulating ⁎ Corresponding author at: Research Institute of Internal Medicine, Oslo University Hospital, Rikshospitalet, P.B. 4950 Nydalen, 0424 Oslo, Norway. E-mail address: [email protected] (A. Abraityte).
http://dx.doi.org/10.1016/j.ijcard.2014.11.111 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
DLL1 levels could represent a link between valvular calcification and myocardial dysfunction and provide prognostic information in patients with symptomatic AS. A total of 136 patients with confirmed symptomatic AS assessed by echocardiography and evaluated for aortic valve replacement (AVR) were consecutively enrolled in the study [8]. All patients underwent echocardiographic, angiographic examinations and blood sampling. All patients were clinically stable and none had severe comorbidities. Aortic valve specimens were obtained from 14 patients undergoing AVR (mean ± SD: 70 ± 12 years, 7 women). Serum levels of DLL1 were determined by enzyme immunoassay (R&D Systems). DLL1 mRNA was quantified on an ABI Prism 7500 qPCR instrument using Power SYBR Green Master Mix (Applied Biosystems), sequence-specific primers and normalized to the expression of GAPDH. Variables which were not normally distributed were log-transformed. Differences between groups were analyzed with Student's t-test or Mann–Whitney U test, as appropriate. Relationships between variables were assessed by Spearman's correlation. Restricted cubic spline interpolation was used to evaluate nonlinear relationships between DLL1 levels and all-cause mortality. Kaplan–Meier analysis with log-rank test was done to compare the number of events in relation to tertiles of DLL1 levels (paired for each stratum due to non-linearity). Cox proportional hazard analysis was performed to estimate adjusted hazard ratios (covariates in figure legend). Follow-up time for all-cause mortality was calculated from time of inclusion to death from any cause. p values (two-sided) were considered significant when b 0.05. The study protocol conforms to the ethical guidelines of the Declaration of Helsinki. Signed informed consent was obtained from all study objects. The authors of the manuscript certify that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. Baseline characteristics according to DLL1 levels comparing the tertiles are shown in Table 1. Patients with symptomatic AS (n = 136) had upregulated serum levels of DLL1 compared to healthy controls (n = 95) (Fig. 1A). Patients with the highest DLL1 levels were older, had higher creatinine and lower LDL levels. There was no association
A. Abraityte et al. / International Journal of Cardiology 180 (2015) 18–20
19
Table 1 Patient characteristics in relation to DLL1 levels. Total population N = 136
DLL1 (≤5.30)
DLL1 (5.31–6.92)
DLL1 (≥6.93)
p-Value
Age (years) Male (%) BMI (kg m−2) NYHA functional class (II/III/IV) (%) Coronary artery disease (%) Current smokers (%) DM2 (%) Hypertension (%) Atrial fibrillation AVR
77 (69, 81) 79 (58) 26.3 ± 4.2 3/33/63/1 64 (47) 45 (34) 14 (10) 36 (27) 43 (32) 108 (79)
74 (65, 78) 24 (53) 25.6 ± 4.0 5/34/61/0 19 (42) 11 (25) 6 (13) 11 (24) 34 (76) 40 (38)
78 (70, 81) 28 (62) 26.7 ± 3.8 5/30/63/2 22 (49) 17 (40) 4 (9) 13 (29) 33 (73) 33 (31)
79 (74, 82) 26 (59) 27.0 ± 4.7 0/35/65/0 23 (52) 17 (40) 4 (9) 12 (27) 24 (55) 33 (31)
0.01 0.69 0.24 0.64 0.63 0.36 0.74 0.89 0.07 0.14
Hemodynamics LVEF (%) CO (l/min) Aortic valve area (cm2) Mean aortic gradient (mm Hg)
63 (56, 70) 4.8 (4.3, 5.5) 0.62 (0.50, 0.80) 54.4 ± 19.1
63 (56, 70) 4.7 (4.3, 5.4) 0.65 (0.53, 0.78) 53.1 ± 15.7
66 (57, 70) 4.8 (4.3, 5.3) 0.70 (0.50, 0.83) 53.7 ± 21.9
62 (54, 70) 4.9 (4.1, 5.6) 0.60 (0.50, 0.80) 54.3 ± 18.0
0.47 0.98 0.56 0.96
Biochemistry eGFR (ml min−1 1.73 m−2) CRP (mg L−1) HDL-Ch (mmol L−1) LDL-Ch (mmol L−1) Nt-proBNP [pmol L−1] Cholesterol (mmol L−1) TnT (ng L−1)
66 (52, 88) 1.9 (0.9, 4.3) 1.6 (1.3, 1.9) 3.2 ± 1.1 98 (42, 270) 5.0 (4.3, 5.7) 14.1 (8.3, 25.0)
75 (58, 103) 1.4 (1.0, 3.6) 1.6 (1.4, 1.9) 3.7 ± 1.2 62 (37, 159) 5.3 (4.8, 6.1) 10.1 (6.0, 17.1)
68 (55, 89) 1.3 (0.5, 3.6) 1.7 (1.3, 1.9) 3.2 ± 0.9 99 (51, 154) 4.7 (4.3, 5.5) 13.6 (8.8, 19.2)
56 (43, 71) 3.7 (1.7, 7.7) 1.5 (1.2, 1.9) 2.9 ± 1.2 174 (41, 512) 4.5 (3.7, 5.4) 19.5 (12.0, 31.4)
0.001 0.008 0.39 0.005 0.03 0.002 0.003
Medication (%) ACE inhibitor ARB β-Blocker Statins Warfarin Aspirin
18 (13) 28 (21) 65 (48) 68 (50) 27 (20) 65 (48)
4 (9) 5 (11) 19 (42) 21 (47) 6 (13) 20 (44)
6 (13) 14 (31) 20 (44) 28 (62) 8 (18) 26 (58)
7 (16) 9 (21) 25 (57) 19 (43) 13 (30) 18 (41)
0.60 0.07 0.33 0.16 0.14 0.24
Continuous data as mean ± SD or median and interquartile range (Q1, Q3). DLL1, delta-like 1; BMI, body mass index; NYHA, New York Heart Association; DM2, type 2 diabetes mellitus; AVR, aortic valve replacement; LVEF, left ventricular ejection fraction; CO, cardiac output; eGFR, estimated glomerular filtration rate; CRP, C-reactive protein; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NT-proBNP, N-terminal probrain natriuretic peptide; TnT, troponin T, ACE, angiotensin-converting enzyme; ARB, angiotensin receptor blocker.
Fig. 1. DLL1 in patients with symptomatic AS. (A) Tukey plots showing that serum DLL1 levels are elevated in patients with symptomatic AS (n = 136) compared to healthy controls (n = 95), p-value age-adjusted. (B) Association between serum DLL1 and DLL1 mRNA expression in aortic valves (n = 14). (C) Restricted cubic spline interpolation showing a U shaped association between DLL1 levels (red and blue lines, mean and 95% CI) and all-cause mortality. (D) Kaplan–Meier curves according to tertiles of circulating DLL1 demonstrating a higher rate of mortality in patients with low (i.e. T1) and high (i.e. T3) DLL1. (E) Unadjusted (UNI) and adjusted hazard ratios (Model [M] 1: adjustment for AVR, age, gender, mean aortic gradient, LVEF and eGFR; M2 adjusted for M1 + CRP and NT-proBNP) showing association between DLL1 tertile 2 vs. the combined tertiles 1 and 3 for mortality. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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A. Abraityte et al. / International Journal of Cardiology 180 (2015) 18–20
between DLL1 levels and previous comorbidities (cancer, n = 8; stroke, n = 7; asthma, n = 6). There was a correlation between elevated DLL1 levels and well-characterized biomarkers of cardiovascular disease, i.e., NT-proBNP (r = 0.28, p b 0.001), TnT (r = 0.33, p b 0.001) and CRP (r = 0.30, p = 0 b 001). DLL1 levels did not correlate with echocardiographic parameters of valve stenosis or cardiac function, clinical history or the use of cardiovascular medications. DLL1 mRNA expression was determined in 14 excised aortic valves. Notably, mRNA levels correlated with serum DLL1 levels from the same patients (Fig. 1B). During a mean follow-up of 4.6 [±2.3 (S.D.)] years, 35 patients died. A restricted cubic spline interpolation model shows a U shaped association between DLL1 levels and all-cause mortality (Fig. 1C). A Kaplan–Meier plot illustrates this association further, revealing that patients in the first (p = 0.05) and third (p = 0.029) tertiles of DLL1 had lower survival rate, comparing to the second tertile (Fig. 1D). Fig. 1E shows the unadjusted and adjusted hazard ratios for tertile 2 vs. the combined tertiles 1 and 3 for mortality. Intermediate (i.e. tertile 2) DLL1 levels were associated with decreased mortality rate in unadjusted and also after multivariable adjustment for demographic features, mean aortic gradient, LVEF, NTproBNP, TnT, CRP and eGFR. We show for the first time a relationship between serum levels of the Notch ligand DLL1 and symptomatic AS. Our main and novel findings were that a) circulating DLL1 levels were elevated in patients with symptomatic AS, b) DLL1 was expressed in calcified aortic valves and correlated with systemic DLL1 concentrations, c) there was an association between DLL1 levels and established biomarkers of cardiovascular disease, and d) DLL1 levels were independently associated with long-term mortality. DLL1 has been shown to enhance mineral deposition and stimulate osteoblast differentiation [7]. Our findings may suggest that similar, DLL1-induced signaling could be operating in AS, thus promoting calcification and AS progression. The correlation between DLL1 mRNA expression in calcified valves and circulating levels suggests that valvular derived DLL1 contributes to the systemic pool. Furthermore, the positive association between DLL1 and TnT and NT-proBNP, as a reflection of myocardial damage and neurohormonal activation, respectively, may support DLL1 role in the development of heart failure accompanying symptomatic
AS. Finally, the U shaped association between serum DLL1 and mortality may suggest that intermediate levels of DLL1 are beneficial. One could speculate that low levels of DLL1 could reflect inadequate response to pathological Notch activity, while high levels might represent an overactivation of repair mechanisms in the presence of increased wall stress. Our findings support involvement of DLL1 and Notch pathways in the pathophysiology of AS and suggest that manipulation of Notch signaling could offer new therapeutic approaches for this disease. Future studies should also examine the ability of DLL1 to predict prognosis as well as prosthetic valve failure in these patients. Conflict of interest The authors report no relationships that could be construed as a conflict of interest. Acknowledgments The study was funded by grant # 39587 from the Southern and Eastern Norway Regional Health Authority. References [1] B.A. Carabello, Introduction to aortic stenosis, Circ. Res. 113 (2013) 179–185. [2] N.M. Rajamannan, Calcific aortic stenosis: lessons learned from experimental and clinical studies, Arterioscler. Thromb. Vasc. Biol. 29 (2009) 162–168. [3] J.L. de la Pompa, J.A. Epstein, Coordinating tissue interactions: notch signaling in cardiac development and disease, Dev. Cell 22 (2012) 244–254. [4] G. Aquila, M. Pannella, M.B. Morelli, C. Caliceti, C. Fortini, P. Rizzo, et al., The role of notch pathway in cardiovascular diseases, Glob. Cardiol. Sci. Pract. 2013 (2013) 364–371. [5] G. Rusanescu, R. Weissleder, E. Aikawa, Notch signaling in cardiovascular disease and calcification, Curr. Cardiol. Rev. 4 (2008) 148–156. [6] V. Garg, A.N. Muth, J.F. Ransom, M.K. Schluterman, R. Barnes, I.N. King, et al., Mutations in NOTCH1 cause aortic valve disease, Nature 437 (2005) 270–274. [7] M. Nobta, T. Tsukazaki, Y. Shibata, C. Xin, T. Moriishi, S. Sakano, et al., Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Delta1/ Jagged1-activated Notch1 signaling, J. Biol. Chem. 280 (2005) 15842–15848. [8] T. Ueland, L. Gullestad, C.P. Dahl, P. Aukrust, S. Aakhus, O.G. Solberg, et al., Undercarboxylated matrix Gla protein is associated with indices of heart failure and mortality in symptomatic aortic stenosis, J. Intern. Med. 268 (2010) 483–492.