Baseline Aortic Valve Calcium Score and Use of Anticoagulants are associated with Rapid Progression of Aortic Valve Calcification

Annapoorna Singh^*, John T Saxon, Kevin F Kennedy and Randall C Thompson
^*Correspondence: Annapoorna Singh, School of Medicine, Saint Luke’s Mid America Heart Institute and The University of Missouri, Kansas City, USA, Tel: 816-844-1042, Email:

Keywords

Aortic valve calcification • X-Ray computed tomography • Anticoagulation

Introduction

Calcific aortic valve disease (CAVD) is a common disorder affecting up to 13% of the United States population [1] and which is anticipated to double over the next 50 year [2]. Progressive cardiovascular disease (CAVD) results in hemodynamically significant aortic stenosis (AS), which is associated with heart failure and reduced survival. The association between traditional cardiovascular disease risk factors and the development of aortic stenosis (AS) in the general population remains insufficiently addressed, despite the increased risk of AS with cumulative age [3-5]. CAVD is characterized by progressive fibro-calcific remodeling and thickening of the aortic valve leaflets that evolves over years to cause severe obstruction to cardiac outflow [6]. Until recently, CAVD and vascular calcification were considered degenerative and unregulated processes, but it has become increasingly apparent that CVAD is not a passive process of calcium deposition, but rather a complex process that involves genetic factors, lipoprotein deposition, and oxidation, chronic inflammation, as well as osteoblastic transition of cardiac valve interstitial cells and active leaflet calcification [6]. Calcification in the aortic valve can be measured non-invasively on gated x-ray computed tomography [1,7,8]. As the calcium largely causes the obstruction, the amount of calcium roughly

Research Methodology

This is a single-center retrospective longitudinal study involving the chart review of patients at Saint Luke’s Mid America Heart Institution (MAHI), Kansas City, Missouri. Subjects who had undergone screening gated x-ray non-contrast CT scanning of the coronary arteries for coronary calcium scoring were eligible for inclusion. The MAHI coronary calcium scoring database was queried (total n=112,665, 1/2005-12/31/2019) and individuals who had aortic valve calcium identified on a coronary calcium score study (aortic valve calcium score >0) and had at least one other gated CT calcium score at another point in time between 2005 to 2019 were included (n=360). Baseline demographics including age, gender, history of hypertension, hyperlipidemia, diabetes mellitus, chronic kidney disease, coronary artery disease (CAD), myocardial infarction (MI), stroke, peripheral vascular disease, history of the bicuspid aortic valve (BAV), and medications use such as angiotensin-converting enzyme inhibitors (ACE), angiotensin receptor blockers (ARB’s), statin use, and anticoagulation use were obtained from electronic health records (EHR). Saint Luke's Hospital Institutional Review Board granted a waiver of informed consent for this retrospective study.

Aortic valve calcium (AVC) scoring was obtained as has been previously described [1,7,8]. AVC was defined as any calcified lesion localized to the AV leaflets and aortic annulus. Calcification in the ascending aorta, subaortic annulus space or coronary arteries was excluded. AVC was quantified using Agatston methodology [10], which accounts for both lesion area and calcium density (using Hounsfield brightness). If AVC was not detected, the Agatston score was recorded as zero. All CT scans were scored on the same workstation (Singo. via coronary calcium scoring program, Siemens Healthineers, Erlangen Germany) by one of the authors (AS) and reviewed and confirmed by the senior author (RCT). The study participants were categorized into four groups based on Agatston score of their first CT scan as follows: <250 AU, 250-500 AU, 500-1000 AU, and those with scores >1000 AU.

Continuous variables are expressed as mean +/- standard deviation and categorical variables as proportions. Patients were divided into 4 groups based on their baseline AV calcium scores as mentioned above. Continuous variables were analyzed with a one-way ANOVA test, and categorical variables were compared with Chi-square tests. We then performed a multivariable linear regression model predicting the rate of AVCS using the following variables: age, sex, smoking, AC use, HTN, DM, FH of CAD, statin use, bicuspid aortic valve, creatinine, baseline CACS, and baseline AVCS. Output from this model is shown graphically with the linear beta weights and 95% confidence intervals (Figure 1). Statistical analyses were performed using SAS 9.4 (Cary, NC). Statistical significance was defined as p <0.05.

Results

A total of 360 patients with at least two separate gated coronary calcium scoring chest CT scans between 2005 and 2019 were identified from the MAHI cardiovascular CT scan database. The mean between-scan days was 2381 +/- 1274 sd. Demographic and clinical characteristics are presented in Table 1. The mean baseline age was 73.4 +/- 8.4 years sd with 62.8% being male. Hypertension was present in 80.6% of the cases, diabetes in 33.0% and 50.7% of cases had an abnormal coronary artery calcium score. About 50.3% of individuals had a family history of CAD. The prevalence of chronic kidney disease (CKD) stage I, II, III, IV and V was 17.0%, 46.1%, 16.4%, 3.1%, and 2.8% respectively. About 50% of the cohort was on ACE/ARBs, 73.6% on statins, and 20.3% on anticoagulants.

Predictors of higher baseline aortic valve calcium and predictors of progression of aortic valve calcium

Higher baseline aortic valve calcium score was associated with older age (Table 2). AVCS mean age was 71.9 ± 8.1 in individuals < 250 AU and 77.0 ± 8.9 in individuals AVCS > 1000 AU (p-value <0.001). The mean rate of increase in AVCS per year was incrementally higher according to baseline AVCS (Figures 2 and 3). For patients with baseline AVCS <250 AU, the mean ACVS rate per year was 15.8± 26.8; for 250-500 AU, the mean rate was 39.9 ± 36.9, for 500-1000 AU, the mean rate was 96.8 ± 76.0, and for > 1000 AU, the mean rate was 280.3 ± 284.2 (p-value <0.001). The difference between the old and new scan AVCS was also significant across all the groups – a mean difference of 80.9 +/- 55.6 in group 1 to 1326 +/- 1170.8 in group 4. Use of anticoagulation (33.3% in group 4 vs 12.0% in group 1, p<0.05) was found to be significantly associated with a higher rate of AVCS score at baseline and associated with faster rate of AVCS increase. In our full multivariable model, only two variables were noted to have an increased rate of AVCS - baseline AVCS and oral anticoagulant use (both p <0.05).

With regards to CACS, the initial CAC scores were noted to be higher in individuals with higher baseline AVCS; 182.3±319.0 in individuals with AVCS <250, and 689.2±734.5 in individuals with ACVS >1000 (p-value of <0.001). However, the mean rate of increase in CAC score was not found to be statistically significantly related to aortic valve calcium. The median rate of change in AVCS per year is higher in patients with higher baseline calcium score and the rate of increase seems to level off between baseline scores of 1000-2000 (Figure 3). Only baseline aortic valve calcium score and use of warfarin predicted the progression of AVCS (Figure 1). The rate of AVCS change per year rises with an increased baseline AVCS reaching the peak at ~1100 baseline score (Figure 3).

Discussion

Our study aimed to determine the rate of progression of AV calcium using CT and to evaluate and identify the risk factors associated with the progression of AV calcification. Since valvular calcification is the intrinsic mechanism leading to AS development, and in the light of the fact that it may be precisely estimated by computed tomography (CT) [11], aortic valve calcification load assessment has been of significant interest. Prior studies have demonstrated that the prevalence of AVC and the degree of valve calcification depend on age [12,13]. In the study by Walsh, the incidence of AVC was 3% for subjects <50 years of age, 6% for subjects 50–59 years of age, 17% for subjects 60–69 years of age, and 34% for subjects ≥70 years of age (p <0.0001) [14]. However, AV calcification is not simply a consequence of aging, but rather a complex mechanism involving deposition of lipoproteins, chronic inflammation and the calcification cascade [15]. In our study, increasing age was associated with increasing prevalence of AVCS in univariable analysis, but not in multivariable analysis. Our study also did not reveal a significant correlation between gender and the prevalence and degree of AVC, similar to the study by Koos et al. analyzing AVC in 402 individuals [16].

It is known that AS hemodynamically progresses over time, with valve area declining approximately by 0.1 cm²/year [17]. The processes and determinants leading to this progression are poorly known. While prior epidemiologic investigations have reported cross-sectional associations between AVC and traditional cardiovascular risk factors, including male gender, smoking, hyperlipidemia, diabetes, metabolic syndrome [18-20] and hypertension [18,21,22], as well as bicuspid aortic valves [23], our study did not find any significant correlation between these factors and the progression of AVCS, after correcting for age.

Despite the favorable effects of statins observed in some studies [24,25], conflicting and predominantly no significant association has been reported in the two largest trials [26]. Similar somewhat conflicting results were observed in our study. For example, we found that the participants with higher baseline AVCS also had higher baseline coronary artery calcium score (CACS) supporting the theory that the CVD risk factors are perhaps responsible for the initiation of AVC. However, baseline CACS was not associated with more rapid progression of AVCS. These findings could be explained if the risk factors associated with initial aortic stenosis differ from those associated with disease progression due to pathophysiological differences in the initiation and the progression of the process [27]. This early phase fits well with the atherosclerotic concepts of aortic calcification with hyperlipidemia, inflammation, and rapid coronary calcification, consistent with previous studies on early aortic lesion pathology [28], characterized by inflammation and oxidized lipoproteins deposition [29,30], and colocalized with early calcium deposit [28,30]. The secondary phase of calcium accumulation with ultimately ossification [31], is unrelated to vascular risk factors and AVC grows faster with calcification load [32], thus reflecting the biological concept of centripetal expansion of calcific nodules, which are surrounded by osteoblast-like cells [33], and fits the observed independence of AS progression from lipid profile [17,34].

We found that for any given aortic valve calcium score severity, a larger baseline calcific load leads to faster calcification, emphasizing that as the disease progresses, there is near exponential calcium deposition and the rate of calcification is independent of conventional cardiovascular risk factors. Lipoprotein a (Lp(a)) increases expression of key osteogenic genes in human valvular interstitial cells (VIC), committing them to an osteoblastic-like cell type [35]. Although our study did not evaluate the Lp(a), prior studies have shown that in AS, patients with elevated Lp(a) and OxPL-apoB plasma levels demonstrate increased valvular calcification activity, faster disease progression, and an increased risk of AV replacement (AVR) or death than subjects with lower levels. The pro-osteogenic effects of Lp(a) and OxPL on VIC are potentially reversible with targeted treatment inactivating OxPL [2]. These findings provide a rationale for clinical studies aiming to reduce elevated Lp(a) and OxPL levels in patients with AS as a means of slowing disease progression and delaying the need for AVR. Lp(a) is unaffected by statin therapy, but levels can now be reduced with novel compounds [36], making Lp(a) a potential therapeutic target in AS.

Our study also demonstrates a strong association of the level of AVC with the use of anticoagulants (3.3% in group 1 vs 12.0% in group 4, p <0.05), especially warfarin (18.9% in group 3 and 14.6% in group 4 vs 4.0% in group 1, p <0.05). Oral anticoagulants were also independently associated with a more rapid progression of aortic valve calcium over time (beta weight 71.06 ci 28.47-113.66). Vitamin K antagonist (VKA) have been previously implicated in the progression of AS and our results confirm and expand upon this finding [37,38]. This observation is theoretically supported by the fact that VKA inactivates matrix γ-carboxyglutamic acid protein (MGP), through its incomplete γ-carboxylation by antagonization of vitamin K [39]. MGP is a potent inhibitor of vascular calcification. Only the carboxylated form of MGP (c-MGP) is protective towards calcium deposition whereas the uncarboxylated (uc- MGP) is ineffective in protecting the vascular tissue from mineralization [40,41] and the carboxylation of MGP is dependent on vitamin K. MGP prevents both the crystal complexes nucleation and the Bone Morphogenic Protein-2 (BMP- 2) induced osteogenic differentiation of vascular cells [40].

Koos et al. used CT imaging to quantify coronary and AV calcification in patients on long-term oral anticoagulation with VKA and compared it to those not on anticoagulation [40]. The patients on VKA had higher coronary calcium (p=0.024) as well as AV calcium (p=0.002) [38]. Similar results have been reported in a series of 430 subjects where a significantly greater incidence of AVC was found to be associated with the use of warfarin (18.0% vs 6.9% in warfarin vs non-warfarin-treated subjects; p=0.014) [37], findings similar to our study. Direct oral (DOAC) anticoagulants that do not act via the vitamin K pathway conceivably would solve this problem, and this possibility is supported in several studies.

Tastet et al. assessed the advancement of AS by measuring progression of peak aortic jet velocity (Vpeak) on Doppler echocardiography and AVCS on MDCT in participants who had at least mild aortic stenosis and were taking warfarin versus DOAC versus no anticoagulant group. The median annualized increase in Vpeak was larger in the warfarin group compared with the DOAC and no anticoagulant therapy groups. The median follow-up time between the first and last MDCT examinations was 2.0 years (25^th to 75^th percentile: 1.5 to 3.9 years). In the subset of patients who also underwent MDCT, the annualized increase in AVC score was at least 2-fold larger in the warfarin compared with the DOAC and no anticoagulation therapy groups [42]. Our study extends the findings by Tastet et al. and magnifies their observation to include individuals who have milder levels of aortic valve disease – those who have lower levels of aortic valve calcification likely prior to hemodynamic aortic stenosis.

AVC as measured by MDCT is low risk, noninvasive, and highly reproducible [8]. MDCT is not indispensable in patients with an obvious surgical indication of AVI but is helpful in considering transcatheter AVI. Thus, the measurement of AVC by MDCT should be considered for not only diagnosis, but also riskstratification purposes in the evaluation of and therapeutic decision making in some patients with AS [8]. For example, in patients with echocardiographic findings which could be possibly be explained by either low-gradient- severe AS or moderate aortic stenosis, heavy AVC load is helpful as it is consistent with severe calcified aortic valve disease [7]. Higher AVC load is also associated with worse outcome [11,43].

Limitations

This was a single center, retrospective, non-randomized study and has the limitations associated with such a design. Also, not all characteristics may be captured and some baseline characteristics such as kidney function and use of anticoagulation can change over time, limiting the accuracy of a predictive model. Likewise, while to a certain extent the reference ranges of AVCA are dependent on the imaging protocol and CT equipment used [44], we performed aortic calcium scoring in a consistent manner on all our patients.

Conclusion

Our study showed that a high baseline AV calcium score and the use of warfarin oral anticoagulation are associated with a faster rate of progression of aortic valve calcium. We did not find any statistically significant association with the conventional CAD risk factors, emphasizing that progression of AVC seems to be unrelated to conventional CVD risk factors. The faster rise in individuals with high baseline AVC load suggest a positive feedback of calcium deposition with accelerated calcification growth over the years. The study confirmed, in a population of mostly pre-clinical aortic valve calcification, the rapid progression of AVCS previously described with warfarin use in patients with aortic stenosis, implying that alternative anticoagulation strategies should be considered as an alternative in individuals with CAVD. These data also could help determine appropriate follow up intervals for patients with aortic valve calcifications.

Funding Source

This study was supported in part by Saint Luke's Hospital of Kansas City Foundation.

Author Contributions

Annapoorna Singh: Data curation, writing-original draft preparation; John Saxon: conceptualization, visualization, supervision, reviewing and editing; Kevin Kennedy: Methodology, software and validation; Randall Thompson: Conceptualization, visualization, supervision, writing-reviewing and editing.

References

1. Nasir Khurram, Ronit Katz, Junichiro Takasu, David Shavelle, et al. "Ethnic differences between extra-coronary measures on cardiac computed tomography: multi-ethnic study of atherosclerosis (MESA)." Atherosclerosis 198(2008): 104-114.
2. Zheng Kang, Sotirios Tsimikas, Tania Pawade, Jeffrey Kroon, et al. "Lipoprotein (a) and oxidized phospholipids promote valve calcification in patients with aortic stenosis." J Am Coll Cardiol 73(2019): 2150-2162.
3. Otto Catherine and Bernard Prendergast. "Aortic-valve stenosis-from patients at risk to severe valve obstruction." N Engl J Med 371(2014): 744-756.
4. Nishimura Rick, Catherine Otto, Robert Bonow, Blase Carabello, et al. "2014 AHA/ACC guideline for the management of patients with valvular heart disease: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines." Journal of the American College of Cardiology 63(2014): 2438-2488.
5. Vahanian A. "Task force on the management of valvular hearth disease of the European Society of Cardiology: ESC Committee for Practice Guidelines. Guidelines on the management of valvular heart disease: the Task Force on the Management of Valvular Heart Disease of the European Society of Cardiology." Eur Heart J 28(2007): 230-268.
6. Lindman Brian, Marie-Annick Clavel, Patrick Mathieu, Bernard Iung, et al. "Calcific aortic stenosis." Nat Rev Dis Primer 2(2016): 1-28.
7. Clavel Marie-Annick, David Messika-Zeitoun, Philippe Pibarot, Shivani Aggarwal, et al. "The complex nature of discordant severe calcified aortic valve disease grading: new insights from combined Doppler echocardiographic and computed tomographic study." J Am Coll Cardiol 62(2013): 2329-2338.
8. Clavel Marie-Annick, Philippe Pibarot, David Messika-Zeitoun, Romain Capoulade, et al. "Impact of aortic valve calcification, as measured by MDCT, on survival in patients with aortic stenosis: results of an international registry study." J Am Coll Cardiol 64(2014): 1202-1213.
9. Helmut Baumgartner, Philipp Bonhoeffer, Natasja De Groot, Fokko de Haan, et al. "ESC Guidelines for the management of grown-up congenital heart disease (new version 2010) The Task Force on the Management of Grown-up Congenital Heart Disease of the European Society of Cardiology (ESC)." Eur Heart J 31(2010): 2915-2957.
10. Agatston Arthur, Warren Janowitz, Frank Hildner, Noel Zusmer, et al. "Quantification of coronary artery calcium using ultrafast computed tomography." J Am Coll Cardiol.15(1990): 827-832.
11. Messika-Zeitoun David, Marie-Christine Aubry, Delphine Detaint, Lawrence Bielak, et al. "Evaluation and clinical implications of aortic valve calcification measured by electron-beam computed tomography." Circulation 110(2004): 356-362.
12. Boon Arthur, Emile Cheriex, Jan Lodder and Fons Kessels. "Cardiac valve calcification: characteristics of patients with calcification of the mitral annulus or aortic valve." Heart Br Card Soc 78(1997): 472-474.
13. O’Brien Kevin. "Pathogenesis of calcific aortic valve disease: a disease process comes of age (and a good deal more)." Arterioscler Thromb Vasc Biol 26(2006): 1721-1728.
14. Walsh Craig, Martin Larson, Michelle Kupka, Daniel Levy, et al. "Association of aortic valve calcium detected by electron beam computed tomography with echocardiographic aortic valve disease and with calcium deposits in the coronary arteries and thoracic aorta." Am J Cardiol 93(2004): 421-425.
15. Dweck Marc, Nicholas Boon, David Newby. "Calcific aortic stenosis: a disease of the valve and the myocardium." J Am Coll Cardiol 60(2012): 1854-1863.
16. Koos Ralf, Harald Kühl, Georg Mühlenbruch, Joachim Wildberger, et al. "Prevalence and clinical importance of aortic valve calcification detected incidentally on CT scans: comparison with echocardiography." Radiology 241(2006): 76-82.
17. Bellamy Michael, Patricia Pellikka, Kyle Klarich, Jamil Tajik, et al. "Association of cholesterol levels, hydroxymethylglutaryl coenzyme-A reductase inhibitor treatment, and progression of aortic stenosis in the community." J Am Coll Cardiol 40(2002): 1723-1730.
18. Stewart Fendley, David Siscovick, Bonnie Lind, Julius Gardin, et al. "Clinical Factors Associated With Calcific Aortic Valve Disease fn1fn1This study was supported in part by Contracts NO1-HC85079 through HC-850086 from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland." J Am Coll Cardiol 3(1997): 630-634.
19. Aronow Wilbert, Kenneth Schwartz, Mordecai Koenigsberg. "Correlation of serum lipids, calcium, and phosphorus, diabetes, mellitus and history of systemic hypertension with presence or absence of calcified or thickened aortic cusps or root in elderly patients." Am J Cardiol 59(1987): 998-999.
20. Katz Ronit, Nathan Wong, Richard Kronmal, Junichiro Takasu, et al. "Features of the metabolic syndrome and diabetes mellitus as predictors of aortic valve calcification in the Multi-Ethnic Study of Atherosclerosis." Circulation 113(2006): 2113-2119.
21. Yan Andrew, Maria Koh, Kelvin Chan, Helen Guo, et al. "Association between cardiovascular risk factors and aortic stenosis: the CANHEART Aortic Stenosis Study." J Am Coll Cardiol 69(2017): 1523-1532.
22. Messika-Zeitoun David, Lawrence Bielak, Patricia Peyser, Patrick Sheedy, et al. "Aortic valve calcification: determinants and progression in the population." Arterioscler Thromb Vasc Biol 27(2007): 642-648.
23. Roberts Clifford and Jong Mi Ko. "Weights of operatively-excised stenotic unicuspid, bicuspid, and tricuspid aortic valves and their relation to age, sex, body mass index, and presence or absence of concomitant coronary artery bypass grafting." Am J Cardiol 92(2003): 1057-1065.
24. Novaro Gian, Irving Tiong, Gregory Pearce, Michael Lauer, et al. "Effect of hydroxymethylglutaryl coenzyme a reductase inhibitors on the progression of calcific aortic stenosis." Circulation 104(2001): 2205-2209.
25. Rosenhek Raphael, Florian Rader, Nicole Loho, Harald Gabriel, et al. "Statins but not angiotensin-converting enzyme inhibitors delay progression of aortic stenosis." Circulation 110(2004): 1291-1295.
26. Rossebø Anne, Terje Pedersen, Kurt Boman, Philippe Brudi, et al. "Intensive lipid lowering with simvastatin and ezetimibe in aortic stenosis." N Engl J Med 359(2008): 1343-1356.
27. Pawade Tania, David Newby and Marc Dweck. "Calcification in aortic stenosis: the skeleton key." J Am Coll Cardiol 66(2015): 561-577.
28. Otto Catherine, Johanna Kuusisto, Dennis Reichenbach, Allen Gown, et al. "Characterization of the early lesion of'degenerative'valvular aortic stenosis. Histological and immunohistochemical studies." Circulation 90(1994): 844-853.
29. O’Brien Kevin, Dennis Reichenbach, Santica Marcovina, Johanna Kuusisto, et al. "Apolipoproteins B,(a), and E accumulate in the morphologically early lesion of ‘degenerative’valvular aortic stenosis." Arterioscler Thromb Vasc Biol 16(1996): 523-532.
30. Olsson Margareta, Johan Thyberg and Jan Nilsson. "Presence of oxidized low density lipoprotein in nonrheumatic stenotic aortic valves." Arterioscler Thromb Vasc Biol 19(1999): 1218-1222.
31. Mohler Emile, Francis Gannon, Carol Reynolds, Robert Zimmerman, et al. "Bone formation and inflammation in cardiac valves." Circulation 103(2001): 1522-1528.
32. Yoon Hyo-Chun, Aletha Emerick, Jennifer Hill, David Gjertson, et al. "Calcium begets calcium: progression of coronary artery calcification in asymptomatic subjects." Radiology 224(2002): 236-241.
33. Mohler ER, Chawla MK, Chang AW, Vyavahare N, et al. "Identification and characterization of calcifying valve cells from human and canine aortic valves." J Heart Valve Dis 8(1999): 254-260.
34. Cowell Joanna, David Newby, Robin Prescott, Peter Bloomfield, et al. "A randomized trial of intensive lipid-lowering therapy in calcific aortic stenosis." N Engl J Med 352(2005): 2389-2397.
35. Rutkovskiy Arkady, Anna Malashicheva, Gareth Sullivan, Maria Bogdanova, et al. "Valve interstitial cells: the key to understanding the pathophysiology of heart valve calcification." J Am Heart Assoc 6(2017): e006339.
36. Viney Nicholas, Julian van Capelleveen, Richard Geary, Shuting Xia, et al. "Antisense oligonucleotides targeting apolipoprotein (a) in people with raised lipoprotein (a): two randomised, double-blind, placebo-controlled, dose-ranging trials." The Lancet 388(2016): 2239-2253.
37. Yamamoto Kazuhiro, Yukihiro Koretsune, Takashi Akasaka, Akira Kisanuki, et al. "Effects of vitamin K antagonist on aortic valve degeneration in non-valvular atrial fibrillation patients: Prospective 4-year observational study." Thromb Res 160(2017): 69-75.
38. Koos Ralf, Andreas Mahnken, Georg Mühlenbruch, Vincent Brandenburg, et al. "Relation of oral anticoagulation to cardiac valvular and coronary calcium assessed by multislice spiral computed tomography." The American journal of cardiology 96(2005): 747-749.
39. Vermeer Cees. "Gamma-carboxyglutamate-containing proteins and the vitamin K-dependent carboxylase." Am J Cardiol 266(1990): 625.
40. Schurgers Leon, Jouni Uitto and Chris Reutelingsperger. "Vitamin K-dependent carboxylation of matrix Gla-protein: a crucial switch to control ectopic mineralization." Trends Mol Med 19(2013): 217-226.
41. Nigwekar Sagar, Donald Bloch, Rosalynn Nazarian, Cees Vermeer, et al. "Vitamin K–dependent carboxylation of matrix Gla protein influences the risk of calciphylaxis." J Am Soc Nephrol 28(2017): 1717-1722.
42. Tastet Lionel, Philippe Pibarot, Mylène Shen, Marine Clisson, et al. "Oral anticoagulation therapy and progression of calcific aortic valve stenosis." J Am Coll Cardiol 73(2019): 1869-1871.
43. Rosenhek Raphael, Thomas Binder, Gerold Porenta, Irene Lang, et al. "Predictors of outcome in severe, asymptomatic aortic stenosis." N Engl J Med 343(2000): 611-617.
44. Koos Ralf, Andreas Mahnken, Anil Sinha, Joachim Wildberger, et al. "Aortic valve calcification as a marker for aortic stenosis severity: assessment on 16-MDCT." American Journal of Roentgenology 183(2004): 1813-1818.