Review For reprint orders, please contact: [email protected]
Emerging biomarkers for heart failure: an update
A growing array of biological pathways underpins the syndrome we recognize as heart failure. These include both deleterious pathways promoting its development and progression, as well as compensatory cardioprotective pathways. Components of these pathways can be utilized as biomarkers of this condition to aid diagnosis, prognostication and potentially guide management. As our understanding of the pathophysiology of heart failure deepens further candidate biomarkers are being identified. We provide an overview of the more recently emerging biomarkers displaying potential promise for future clinical use. Keywords: biomarkers • emerging • heart failure • novel
Heart failure is associated with significant mortality, morbidity and economic cost [1] . Over the past 30 years, our understanding of the pathophysiology of heart failure has advanced greatly. It is now recognized as a systemic syndrome characterized by maladaptive neurohormonal, metabolic and inflammatory processes. Recognition of these pathways has prompted the evaluation of some of their components as biomarkers in both chronic heart failure and acutely decompensated heart failure. We have previously summarized the more novel additions to this growing portfolio of candidate biomarkers [2] . Many of the biomarkers previously discussed have progressed to the forefront of clinical heart failure research with much expectation surrounding them, particularly the midregional peptides, ST2 and galectin-3. In this paper, we review only the more recently identified plasma biomarkers that have emerged as potential additions to the heart failure biomarker portfolio since our last review in 2009 [2] . Tenascin-C Tenascin-C (TN-C) is an extracellular glycoprotein that is released in response to inflammatory tissue remodeling under various path-
10.2217/BMM.14.51 © 2014 Future Medicine Ltd
ological conditions such as acute myocardial infarction [3,4] , chronic kidney disease [5] , myocarditis [6,7] as well as idiopathic dilated [8] and hypertrophic cardiomyopathy [9] . TN-C loosens the attachment of cardiomyocytes to connective tissue and upregulates expression and activity of matrix metalloproteinases, which may promote degradation of connective tissue and contribute to left ventricular (LV) dilatation. Serum levels of TN-C independently predict LV remodeling and all-cause mortality post-MI [4,10] . Circulating serum TN-C was measured in 107 patients with heart failure secondary to dilated cardiomyopathy (DCM) [11] . Levels in DCM patients were higher than those in normal controls (p < 0.001), showed a significantly positive correlation with New York Heart Association (NYHA) functional class (p < 0.001), B-type natriuretic peptide (p < 0.001), cardiothoracic ratio on chest x-ray (p < 0.01) and a significantly negative correlation with left ventricular ejection fraction (p < 0.01) (Table 1) [11] . Similar results were noted in a study of 110 consecutive DCM patients hospitalized with acute decompensated heart failure [12] . Serum TN-C and BNP levels were checked at discharge and at a mean of 22.4 months follow-
Biomarkers Med. (2014) 8(6), 833–840
Jonathan R Dalzell*,1, Jane A Cannon2, Colette E Jackson1, Ninian N Lang1 & Roy S Gardner1 Scottish Advanced Heart Failure Unit, Golden Jubilee National Hospital, Glasgow, G81 4DY, UK 2 British Heart Foundation Cardiovascular Research Centre, University of Glasgow, Glasgow, G12 8TA, UK *Author for correspondence: Tel.: +44 141 330 2237 Fax: +44 141 330 6955 j.dalzell@ nhs.net 1
part of
ISSN 1752-0363
833
Review Dalzell, Cannon, Jackson, Lang & Gardner
Table 1. Summary of novel biomarkers in acute and chronic heart failure. Biomarker
Plasma levels in Independent decompensated HF prognostic information?
Levels Plasma levels altered with in CHF therapy?
Independent prognostic information?
Levels altered with therapy?
Tenascin-C
↑
U
U
FL1
U
U
U
↑
Yes
Yes
↑
No
U
Syndecan-4
U
U
U
↑
No
U
Midkine
U
CCL21
U
U
U
↑
Yes
U
U
U
↑
Yes
Yes
S100A8/A9
U
ACE2
↑
U
U
↑
Yes
U
U
Yes
↑
α-defensins
U
Yes
U
U
U
↑
Yes
U
PAP
U
U
U
↑
U
U
U
U
U
Yes
LRG
U
U
U
↑
sEng
U
U
U
↑
↑: Raised; CHF: Chronic heart failure; HF: Heart failure; U: Unknown.
up. The optimal cutoff value for serum TN-C levels predicting the combined end-point of rehospitalization and cardiac death (due to heart failure) was ≥78.4 ng/ml and ≥219 pg/ml for BNP. Patients with serum TN-C levels ≥78.4 ng/Ml at discharge had higher NYHA functional class, larger LV end-diastolic and end-systolic diameters, and higher plasma BNP levels [12] . Furthermore, combined serum TN-C levels with plasma BNP levels at discharge were a stronger predictor of cardiac events for heart failure than either single biomarker alone. Circulating levels were measured in 83 chronic heart failure patients and 30 healthy controls. TN-C concentrations were significantly raised in heart failure patients in whom they correlated significantly with BNP worsening EF. TN-C was an independent predictor of a composite of all-cause mortality or hospitalization over a 12-month follow-up period [13] . When considering markers indicative of reverse cardiac remodeling, 64 patients scheduled for cardiac resynchronization therapy (CRT) had serum biomarkers (including TN-C) checked at baseline and 6 months postimplant [14] . In total, 72% of patients were classified as ‘responders’ to device therapy (i.e., showed a >10% reduction in LV end-systolic volume at followup) and among these patients a significant decrease in circulating levels of TN-C (from 60 ± 40 ng/ml to 40 ± 30 ng/ml; p < 0.01) was observed. This suggests an important role of extracellular matrix modulation in the process of reverse ventricular remodeling in patients responding to CRT [14] . Follistatin Like-1 Follistatin like-1 (FL1) is an extracellular glycoprotein belonging to the BM-40/Osteonectin family. It
834
Biomarkers Med. (2014) 8(6)
was first identified as a TGF-β-induced protein from a mouse osteoblast cell line and is thought to work by neutralizing activins (members of the TGF-β family) that are implicated in diverse biological processes such as wound healing, inflammation and fibrosis [15,16] . It has since been found to be upregulated in cardio vascular injury models (where it protects cardiac myocytes from hypoxia and reperfusion injury) [17] and in cardiac myocytes in the failing heart, returning to normal after myocardial recovery. Myocardial FL1 levels are significantly raised in samples obtained from patients with advanced heart failure undergoing LVAD implantation [18] . Plasma concentrations were assessed in 83 patients with stable chronic heart failure and were found to be significantly raised compared with healthy controls. FL1 levels correlated with LV dimensions and mass, left atrial size and BNP concentrations [18] . FL1 was a predictor of mortality on univariate but not multivariate analysis [18] . Syndecan-4 Components of the extra cellular matrix, of which collagen is the best characterized to date, are now recognized to play significant role in myocardial remodeling. Proteoglycans represent a major component of the extracellular matrix. Syndecan-4 is a transmembrane proteoglycan and preclinical studies have suggested it plays a crucial role in the development of compensatory myocardial hypertrophy following experimental myocardial infarction or pressure overload [19–23] and circulating concentrations have previously been shown to be elevated in patients after acute myocardial infarction [24] . Plasma syndecan-4 was measured in 45 patients, predominantly (76%) NYHA II or better. Syndecan-4
future science group
Emerging biomarkers for heart failure: an update
concentrations were significantly raised in HF patients compared with healthy controls. Although this study is too small to derive meaningful conclusions, a negative correlation was seen between syndecan-4 levels and LVEF. Syndecan-4 levels predicted death or HF-related hospital admission over a 3-year follow-up period on univariate analysis only [25] . A prospective study of 68 patients with LV systolic dysfunction secondary to dilated cardiomyopathy similarly demonstrated an increase in circulating syndecan-4 concentrations with an identical correlation with LVEF [26] . A positive correlation was noted with LV end systolic and diastolic diameters. Midkine Midkine is a multifunctional growth factor with roles in angiogenesis, cell growth and inflammation. It is predominantly expressed in the kidney, GI tract and lung in humans but expression can also be induced by ischemia in the cerebral cortex, vascular endothelium and myocardium. Experimental data suggest that midkine plays a crucial role in the development of LV hypertrophy and adverse remodeling [27] . Plasma concentrations were assessed in 216 patients with chronic heart failure and 60 healthy controls [28] . Circulating midkine levels were significantly raised in chronic heart failure patients in whom they correlated with NYHA class but not with LV ejection fraction or plasma BNP. In a multivariable analysis that included age, NYHA class BNP and LVEF, midkine was an independent predictor of cardiovascular death, heart failure hospitalization over a mean follow-up of 653 ± 375 days. Plasma midkine is also significantly raised in stable patients (n = 134) post cardiac transplantation. Similar correlations were noted with NY-HA class, NT-proBNP, creatinine and cystatin C [29] . CCL21 CCL21 is one of a family of so called homeostatic chemokines playing a crucial role in inflammatory responses, particularly T cell migration in nonlymphoid tissue. Expression of CCL21 has been shown to be upregulated in the myocardium of patients (n = 29) with advanced heart failure undergoing cardiac transplantation [30] . Myocardial samples were also taken from nine patients with advanced heart failure at the time of left ventricular assist device implantation and again at subsequent transplantation. In these patients, the raised CCL21 expression seen at LVAD implantation was significantly attenuated following a period of LV assistance (mean 8 ± 1.7 months) at the time of transplantation [30] . In 150 patients with stable chronic heart failure, circulating CCL21 was significantly elevated compared
future science group
Review
with healthy controls and independently predicted allcause mortality, in a multivariable model that included NT-proBNP, over a mean follow-up period of 24 (±12) months [30] . Plasma concentrations were significantly positively correlated with NT-proBNP and age while a negative correlation with cardiac index was noted [30] . Interestingly, CCL21 levels were higher in the subgroup of patients with an etiology of coronary artery disease as compared with those with idiopathic dilated cardiomyopathy [30] . CCL21 concentrations were also studied in the CORONA (n = 1456) and GISSI-HF (n = 1145) trials [31] . Plasma CCL21 correlated positively with NTproBNP, age and CRP while a negative correlation was noted with estimated glomerular filtration rate. CCL21 was noted to be an independent predictor of all-cause and cardiovascular mortality in both trial cohorts in multivariable analyses that included NT-proBNP. S100A8/A9 S100A8/A9 is a proinflammatory extracellular heterodimic protein secreted by neutrophils, monocytes and macrophages, and exerts its effects via receptor of advanced glycation end products and toll-like receptors. Myocardial levels are raised in a murine model of post-MI heart failure and S100A8/A9 supplementation exacerbates myocardial ischemia-reperfusion injury [32] . Plasma concentrations were measured in 54 patients with chronic heart failure, 31 hypertensive patients without heart failure and 23 healthy controls [33] . Levels were significantly higher in the heart failure patients compared with the other two groups. Plasma levels correlated significantly with IL-2, IL-8 and creatinine. In a multivariable model that included BNP, S100A8/A9 was an independent predictor of all-cause mortality at 1 year, but not at 2 years, of follow-up. ACE2 ACE2 is an 805-amino acid transmembrane peptidase that negatively regulates the RAS by counterbalancing the actions of ACE. In humans, ACE2 is most abundant in the heart, kidney and testes [34] where it is predominantly membrane bound. It displays 42% sequence homology with ACE in its extracellular (catalytic) domain but is resistant to contemporary ACE inhibitors. ACE2 hydrolyses angiotensin II, for which it has the greatest affinity [35] , to angiotensin-(1–7) by cleaving the C-terminal amino acid [36] and has also been shown to hydrolyze numerous peptides in vitro via the same mechanism. ACE2 can undergo cleavage of its extracellular catalytic domain by the enzyme TACE with subsequent systemic release [37] . ACE2 knockout mice demonstrate a progressive, severe reduction in LV systolic function by 6 months of
www.futuremedicine.com
835
Review Dalzell, Cannon, Jackson, Lang & Gardner age along with an increase in myocardial angiotensin-II levels. These effects are abolished by additional deletion of the ACE gene [38] . A significantly quicker development of CHF following aortic constriction [38] and myocardial infarction compared with wild-type controls is also observed [39–42] . Overexpression of ACE2 in the rat heart significantly attenuates the deleterious myocardial effects of angiotensin II infusion [43] and also significantly reduces infarct size and preserves LV function in a rat model of myocardial infarction [44] . Infusion of soluble ACE2 improves right ventricular function in a murine model of right ventricular pressure overload [45] . ACE2 is upregulated in the myocardium of CHF patients on standard therapy [46,47] and ACE inhibitor or angiotensin receptor blocker therapy upregulates myocardial ACE2 levels in normal [48] and CHF [49,50] rats. Circulating ACE2 activity has been shown to be significantly raised in human chronic heart failure [51] . Positive correlations were seen with worsening NYHA class, worsening LVEF and plasma BNP concentrations. Plasma ACE2 activity independently predicted outcome in 113 patients with chronic heart failure over a median follow-up of 34 ± 17 months in a multivariable analysis which included NT-proBNP and LVEF [52] . Similar correlations and impressive prognostic power was noted in 111 patients with chronic heart failure secondary to Chagas’ disease [53] . Plasma ACE2 activity was measured within 12 h of admission and again 48–72 h after intensive contemporary treatment in 70 patients (of which 57 were followed up) with acute decompensated heart failure [54] . In this cohort ACE2 activity correlated with BNP; however, no correlation was noted with LV systolic or diastolic function. Somewhat paradoxically, when compared with the above data in chronic heart failure, a 50% increase from baseline ACE2 levels following treatment was associated with a significant reduction in the subsequent risk of death or requirement for transplantation or subsequent rehospitalization [54] . Clearly, larger studies are required to ascertain the significance of ACE2 activity levels and any potential confounders. Perhaps more importantly, the potential of therapeutic manipulation of this putative cardioprotective arm of the renin angiotensin system merits investigation. α-defensins α-defensins are peptides secreted by neutrophils as part of the innate inflammatory response and have been implicated in the progression of atherosclerosis [55] . Circulating levels were assessed in 194 patients with stable chronic heart failure and compared with 98 age-matched healthy controls [56] . Concentrations were significantly raised in patients with heart fail-
836
Biomarkers Med. (2014) 8(6)
ure in whom significant correlations were noted with NYHA status and less strongly with NT-proBNP. No correlation was noted with LVEF. In a multivariable model which included age, sex, NYHA class, LVEF and NT-proBNP, α-defensins concentrations were an independent predictor of all-cause mortality over a median follow-up of 2.6 years. The combination of high NT-proBNP and α-defensins levels provided incremental prognostic information compared with either biomarker alone. Pancreatitis-associated protein Pancreatitis-associated peptide (PAP) is a novel cytokine with putative anti-inflammatory properties [57] . It was initially discovered in pancreatic secretions from rats with experimental acute pancreatitis [58] . Myocardial PAP expression is markedly increased in a rat model of autoimmune myocarditis [59] . Plasma PAP was measured in 70 stable chronic heart failure patients and compared with 17 healthy controls. PAP concentrations were significantly raised in patients with heart failure in whom significant correlations were noted with BNP, NYHA class and pulmonary artery pressure [60] . Leucine-rich alpha-2-glycoprotein The trace protein leucine-rich alpha-2-glycoprotein (LRG) has a putative role in the inflammatory response and neutrophilic differentiation [61–64] . It has been postulated that LRG may have a role in the exaggerated inflammatory responses leading to myocardial fibrosis. Leading on from this, Watson et al. sampled coronary sinus serum from 11 stable patients undergoing cardiac catheterization and analyzed for LRG, TNF-α and IL-6 [65] . They also sampled peripheral venous blood from 40 asymptomatic hypertensive patients and 105 patients with a range of ventricular dysfunction. They found LRG levels were significantly correlated with BNP in hypertensive, asymptomatic left ventricular diastolic dysfunction, diastolic HF and systolic HF patient groups (p ≤ 0.05). LRG levels correlated with coronary sinus serum levels of TNF-α (p = 0.009) and IL-6 (p = 0.021). They concluded that LRG levels can accurately identify patients with HF and multivariable analyses confirmed LRG was a stronger identifier of heart failure than BNP, independent of age, sex, creatinine and BNP. There are no prognostic data available for LRG in heart failure as yet. Soluble endoglin The glycoprotein endoglin is a TGF-β1 coreceptor that is released into the circulation as soluble endoglin (sEng). TGF-β1 is a profibrogenic cytokine that
future science group
Emerging biomarkers for heart failure: an update
contributes to multiple fibroproliferative disorders including myocardial fibrosis associated with heart failure. In cardiac tissue, endoglin is expressed by endothelial cells, fibroblasts and fibroblast-like stromal cells of valve leaflets. sEng is also used as a marker of endothelial function and is known to regulate the expression and activity of endothelial NO synthase, reducing NO production and making it an important modulator of endothelial vascular tone. Recently the difference in levels of sEng between smokers and nonsmokers with HF has been explored [66] . Brachial artery flow-mediated dilatation (FMD) was recorded, as a measure of conduit vessel endothelial function, as well as sEng in 33 smokers and nonsmokers with HF versus controls. They found that smokers with HF had higher brachial FMD (p < 0.05) and lower sEng than nonsmokers with HF (p < 0.05), and values were comparable to nonsmokers without HF. It was concluded that HF appears to modulate the relationship between smoking exposure and endothelial biology with HF patients who smoke having preserved endothelial function and lower sEng compared with non-smokers with HF. A study of 82 consecutive patients referred for evaluation of suspected left ventricular dysfunction including right and left heart catheterization measured serum sEng levels and compared the results with calculated left ventricular end diastolic pressure (LVEDP) [67] . Patients with an LVEDP of ≥16mmHg had significantly greater serum sEng concentrations, compared with those with an LVEDP of ≤16 mmHg (p < 0.001) even after adjusting for age, sex, renal function and diuretic use. A reduction in pulmonary capillary wedge pressure correlated with a reduction in sEng following diuretic therapy (p = 0.008) suggesting a potential application as a marker of response to therapy [66] .
the targeting of advanced device therapy to appropriate patients. Mortality and morbidity remain high and further improvement is therefore necessary. Many of the biomarkers discussed here have shown promise in one or more of these categories. However, further work is required if we are to ascertain their relevance to contemporary heart failure management. The incremental impact on current clinical acumen arising from the prognostic information provided by this myriad of emerging biomarkers is not yet clear. Even the role of the more established heart failure biomarkers in guiding therapy is unclear with conflicting results in large randomized trials [68,69] . It would seem that our ability to simply correlate components of neurohormonal, inflammatory and other pathways with clinical outcomes has far exceeded progress in using the accrued information to improve care and outcomes in heart failure. The critical question remains: what clinically meaningful information is provided by these novel biomarkers that is not already provided by other established clinical parameters (e.g., NT-proBNP/BNP, peak VO2, serum sodium, 6-min walk test, invasive hemodynamic data, LVEF, QRS duration, among others)? It remains to be seen whether the apparent increase in prognostic power reported by many of these studies translates into a clinically meaningful benefit to patients. It is therefore crucial to comprehensively explore whether or not the incremental information provided by this expanding array of potential biomarkers has any translatable clinical impact on contemporary chronic heart failure management incremental to the aforementioned variables. It is also important that research focuses on the potential therapeutic manipulation of these pathways, which may allow the development of new disease-modifying therapies, and perhaps help to improve prognosis as well as effectively predict it.
Conclusion The use of biomarkers in heart failure has improved diagnosis and prognostication, as well as improving
Future perspective The ability to tailor specific therapy to the individual patient based upon a multibiomarker approach is an
Review
Executive summary Background • Heart failure is a complex systemic syndrome characterized by abnormalities of a spectrum of biological systems and pathways, components of which are potential biomarkers of the syndrome. • Established biomarkers, such as BNP, have improved the diagnosis and prognostication of heart failure. • The introduction of further biomarkers may further improve the management of these patients.
Conclusion • Any novel biomarkers should add to contemporary diagnostic and prognostic acumen whilst being cost effective. • Much work is required to ascertain which, if any, of these candidate biomarkers have a place in contemporary clinical practice and, if so, how they will be best utilized. • The improvement in the understanding of the pathophysiology of CHF should also prompt investigation into the development of novel disease modifying therapies.
future science group
www.futuremedicine.com
837
Review Dalzell, Cannon, Jackson, Lang & Gardner exciting prospect. Whether a multimarker approach will allow us to identify if particular pathways, be it neurohormonal or inflammatory, require more intensive inhibition from patient to patient remains to be seen. The rapidly evolving fields of genomics and proteomics will likely revolutionize the appliance of biomarkers not only in heart failure but medicine as a whole and may ultimately lead to a personalized approach to heart failure management.
Financial & competing interests disclosure
References
13
Yao HC, Han QF, Zhao AP, Yao DK, Wang LX. Prognostic values of serum tenascin-C in patients with ischaemic heart disease and heart failure. Heart Lung Circ. 22, 184–187 (2013).
14
Hessel MH, Bleeker GB, Bax JJ et al. Reverse ventricular remodelling after cardiac resynchronization therapy is associated with a reduction in serum tenascin-C and plasma matrix metalloproteinase-9 levels. Eur. J. Heart Fail. 9, 1058–1063 (2007).
15
Shibanuma M, Mashimo J, Mita A, Kuroki T, Nose K. Cloning from a mouse osteoblastic cell line of a set of transforming-growth-factor-beta 1-regulated genes, one of which seems to encode a follistatin-related polypeptide. Eur. J. Biochem. 217, 13–19 (1993).
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Papers of special note have been highlighted as: • of interest; •• of considerable interest 1
Mosterd A, Hoes AW. Clinical epidemiology of heart failure. Heart 93, 1137–1146 (2007).
2
Dalzell JR, Jackson CE, McDonagh TA, Gardner RS. Novel biomarkers in heart failure: an overview. Biomark. Med. 3, 453–463 (2009).
3
Nishioka T, Onishi K, Shimojo N et al. Tenascin-C may aggravate left ventricular remodeling and function after myocardial infarction in mice. Am. J. Physiol. Heart Circ. Physiol. 298, H1072–H1078 (2010).
4
Sato A, Aonuma K, Imanaka-Yoshida K et al. Serum tenascin-C might be a novel predictor of left ventricular remodeling and prognosis after acute myocardial infarction. J. Am. Coll. Cardiol. 47, 2319–2325 (2006).
16
Liabeuf S, Barreto DV, Kretschmer A et al. High circulating levels of large splice variants of tenascin-C is associated with mortality and cardiovascular disease in chronic kidney disease patients. Atherosclerosis 215, 116–124 (2011).
Kawabata D, Tanaka M, Fujii T et al. Ameliorative effects of follistatin-related protein/TSC-36/FSTL1 on joint inflammation in a mouse model of arthritis. Arthritis Rheum. 50, 660–668 (2004).
17
Oshima Y, Ouchi N, Sato K, Izumiya Y, Pimentel DR, Walsh K. Follistatin-like 1 is an Akt-regulated cardioprotective factor that is secreted by the heart. Circulation 117, 3099–3108 (2008).
18
El-Armouche A, Ouchi N, Tanaka K et al. Follistatin-like 1 in chronic systolic heart failure: a marker of left ventricular remodeling. Circ. Heart Fail. 4, 621–627 (2011).
5
6
7
838
Imanaka-Yoshida K, Hiroe M, Yasutomi Y et al. Tenascin-C is a useful marker for disease activity in myocarditis. J. Pathol. 197, 388–394 (2002). Morimoto S, Imanaka-Yoshida K, Hiramitsu S et al. Diagnostic utility of tenascin-C for evaluation of the activity of human acute myocarditis. J. Pathol. 205, 460–467 (2005).
8
Aso N, Tamura A, Nasu M. Circulating tenascin-C levels in patients with idiopathic dilated cardiomyopathy. Am. J. Cardiol. 94, 1468–1470 (2004).
9
Kitaoka H, Kubo T, Baba Y et al. Serum tenascin-C levels as a prognostic biomarker of heart failure events in patients with hypertrophic cardiomyopathy. J. Cardiol. 59, 209–214 (2012).
10
Sato A, Hiroe M, Akiyama D et al. Prognostic value of serum tenascin-C levels on long-term outcome after acute myocardial infarction. J. Card. Fail. 18, 480–486 (2012).
11
Terasaki F, Okamoto H, Onishi K et al. Higher serum tenascin-C levels reflect the severity of heart failure, left ventricular dysfunction and remodeling in patients with dilated cardiomyopathy. Circ. J. 71, 327–330 (2007).
12
Fujimoto N, Onishi K, Sato A et al. Incremental prognostic values of serum tenascin-C levels with blood B-type natriuretic peptide testing at discharge in patients with dilated cardiomyopathy and decompensated heart failure. J. Card. Fail. 15, 898–905 (2009).
Biomarkers Med. (2014) 8(6)
•
First description of chronic heart failure.
19
Finsen AV, Woldbaek PR, Li J et al. Increased syndecan expression following myocardial infarction indicates a role in cardiac remodeling. Physiol. Genomics 16, 301–308 (2004).
20
Finsen AV, Lunde IG, Sjaastad I et al. Syndecan-4 is essential for development of concentric myocardial hypertrophy via stretch-induced activation of the calcineurin-NFAT pathway. PLoS ONE 6, e28302 (2011).
21
Matsui Y, Ikesue M, Danzaki K et al. Syndecan-4 prevents cardiac rupture and dysfunction after myocardial infarction. Circ. Res. 108, 1328–1339 (2011).
22
Echtermeyer F, Harendza T, Hubrich S et al. Syndecan-4 signalling inhibits apoptosis and controls NFAT activity during myocardial damage and remodelling. Cardiovasc. Res. 92, 123–131 (2011).
23
Strand ME, Herum KM, Rana ZA et al. Innate immune signaling induces expression and shedding of the heparan sulfate proteoglycan syndecan-4 in cardiac fibroblasts and myocytes, affecting inflammation in the pressure-overloaded heart. FEBS J. 280, 2228–2247 (2013).
future science group
Emerging biomarkers for heart failure: an update
24
Kojima T, Takagi A, Maeda M et al. Plasma levels of syndecan-4 (ryudocan) are elevated in patients with acute myocardial infarction. Thromb. Haemost. 85, 793–799 (2001).
25
Takahashi R, Negishi K, Watanabe A et al. Serum syndecan-4 is a novel biomarker for patients with chronic heart failure. J. Cardiol. 57, 325–332 (2011).
26
Bielecka-Dabrowa A, von Haehling S, Aronow WS, Ahmed MI, Rysz J, Banach M. Heart failure biomarkers in patients with dilated cardiomyopathy. Int. J. Cardiol. 168, 2404–2410 (2013).
27
Netsu S, Shishido T, Kitahara T et al. Midkine exacerbates pressure overload-induced cardiac remodeling. Biochem. Biophys. Res. Commun. 443, 205–210 (2014).
28
Kitahara T, Shishido T, Suzuki S et al. Serum midkine as a predictor of cardiac events in patients with chronic heart failure. J. Card. Fail. 16, 308–313 (2010).
•
First description in chronic heart failure.
29
Przybylowski P, Malyszko J, Malyszko JS. Serum midkine is related to NYHA class and cystatin C in heart transplant recipients. Transplant Proc. 42, 3704–3707 (2010).
30
Yndestad A, Finsen AV, Ueland T et al. The homeostatic chemokine CCL21 predicts mortality and may play a pathogenic role in heart failure. PLoS ONE 7, e33038 (2012).
31
Ueland T, Nymo SH, Latini R et al. Investigators of the Controlled Rosuvastatin Multinational Study in Heart Failure (CORONA) trial; Investigators of the GISSI-Heart Failure (GISSI-HF) trial. CCL21 is associated with fatal outcomes in chronic heart failure: data from CORONA and GISSI-HF trials. Eur. J. Heart Fail. 15, 747–755 (2013).
••
Demonstrates the prognostic power of CCL21 in two large heart failure clinical trial cohorts.
32
Volz HC, Laohachewin D, Seidel C et al. S100A8/A9 aggravates post-ischemic heart failure through activation of RAGE-dependent NF-κB signaling. Basic Res. Cardiol. 107, 250 (2012).
33
Ma LP, Haugen E, Ikemoto M, Fujita M, Terasaki F, Fu M. S100A8/A9 complex as a new biomarker in prediction of mortality in elderly patients with severe heart failure. Int. J. Cardiol. 155, 26–32 (2012).
•
First description of chronic heart failure.
34
Donoghue M, Hsieh F, Baronas E et al. A novel angiotensinconverting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1–9. Circ. Res. 87, e1–e9 (2000).
35
Vickers C, Hales P, Kaushik V et al. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J. Biol. Chem. 277, 14838–14843 (2002).
36
Danilczyk U, Penninger JM. Angiotensin-converting enzyme II in the heart and the kidney. Circ. Res. 98, 463–471 (2006).
37
Lambert DW, Yarski M, Warner FJ et al. Tumor necrosis factor-alpha convertase (ADAM17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (SARS-CoV) receptor, angiotensinconverting enzyme-2 (ACE2). J. Biol. Chem. 280, 30113–30119 (2005).
future science group
38
Crackower MA, Sarao R, Oudit GY et al. Angiotensinconverting enzyme 2 is an essential regulator of heart function. Nature 417, 822–828 (2002).
39
Nakamura K, Koibuchi N, Nishimatsu H et al. Candesartan ameliorates cardiac dysfunction observed in angiotensinconverting enzyme 2-deficient mice. Hypertens. Res. 31, 1953–1961 (2008).
40
Gurley SB, Allred A, Le TH et al. Altered blood pressure responses and normal cardiac phenotype in ACE2-null mice. J. Clin. Invest. 116, 2218–2225 (2006).
41
Kassiri Z, Zhong J, Guo D et al. Loss of angiotensinconverting enzyme 2 accelerates maladaptive left ventricular remodeling in response to myocardial infarction. Circ. Heart Fail. 2, 446–455 (2009).
42
Yamamoto K, Ohishi M, Katsuya T et al. Deletion of angiotensin-converting enzyme 2 accelerates pressure overload-induced cardiac dysfunction by increasing local angiotensin II. Hypertension 47, 718–726 (2006).
43
Huentelman MJ, Grobe JL, Vazquez J et al. Protection from angiotensin II-induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats. Exp. Physiol. 90, 783–790 (2005).
44
Der Sarkissian S, Grobe JL, Yuan L et al. Cardiac overexpression of angiotensin converting enzyme 2 protects the heart from ischemia-induced pathophysiology. Hypertension 51, 712–718 (2008).
45
Johnson JA, West J, Maynard KB, Hemnes AR. ACE2 improves right ventricular function in a pressure overload model. PLoS ONE 6, e20828 (2011).
46
Burrell LM, Risvanis J, Kubota E et al. Myocardial infarction increases ACE2 expression in rat and humans. Eur. Heart J. 26, 369–375 (2005).
47
Goulter AB, Goddard MJ, Allen JC, Clark KL. ACE2 gene expression is up-regulated in the human failing heart. BMC Med. 2, 19 (2004).
48
Ferrario CM, Jessup J, Chappell MC et al. Effect of angiotensin-converting enzyme inhibition and angiotensin II receptor blockers on cardiac angiotensin-converting enzyme 2. Circulation 111, 2605–2610 (2005).
49
Ishiyama Y, Gallagher PE, Averill DB, Tallant EA, Brosnihan KB, Ferrario CM. Upregulation of angiotensinconverting enzyme 2 after myocardial infarction by blockade of angiotensin II receptors. Hypertension 43, 970–976 (2004).
50
Kaiqiang Ji, Minakawa M, Fukui K, Suzuki Y, Fukuda I. Olmesartan improves left ventricular function in pressureoverload hypertrophied rat heart by blocking angiotensin II receptor with synergic effects of upregulation of angiotensin converting enzyme 2. Ther. Adv. Cardiovasc. Dis. 3, 103–111 (2009).
51
Epelman S, Tang WH, Chen SY, Van Lente F, Francis GS, Sen S. Detection of soluble angiotensin-converting enzyme 2 in heart failure: insights into the endogenous counterregulatory pathway of the renin–angiotensin–aldosterone system. J. Am. Coll. Cardiol. 52, 750–754 (2008).
52
Epelman S, Shrestha K, Troughton RW et al. Soluble angiotensin-converting enzyme 2 in human heart failure:
www.futuremedicine.com
Review
839
Review Dalzell, Cannon, Jackson, Lang & Gardner relation with myocardial function and clinical outcomes. J. Card. Fail. 15, 565–571 (2009). •
840
signaling pathways downstream of PU.1 and C/EBPepsilon that modulate neutrophil activation. J. Leukoc. Biol. 83, 1277–1285 (2008).
First description of prognostic power in chronic heart failure.
62
53
Wang Y, Moreira Mda C, Heringer-Walther S et al. Plasma ACE2 activity is an independent prognostic marker in Chagas’ disease and equally potent as BNP. J. Card. Fail. 16, 157–163 (2010).
Codina R, Vanasse A, Kelekar A, Vezys V, Jemmerson R. Cytochrome c-induced lymphocyte death from the outside in: inhibition by serum leucine-rich alpha-2-glycoprotein-1. Apoptosis 15, 139–152 (2010).
63
54
Shao Z, Shrestha K, Borowski AG et al. Increasing serum soluble angiotensin-converting enzyme 2 activity after intensive medical therapy is associated with better prognosis in acute decompensated heart failure. J. Card. Fail. 19, 605–610 (2013).
O’Donnell LC, Druhan LJ, Avalos BR. Molecular characterization and expression analysis of leucine-rich alpha2-glycoprotein, a novel marker of granulocytic differentiation. J. Leukoc. Biol. 72, 478–485 (2002).
64
Shirai R, Hirano F, Ohkura N, Ikeda K, Inoue S. Upregulation of the expression of leucine-rich alpha(2)glycoprotein in hepatocytes by the mediators of acute-phase response. Biochem. Biophys. Res. Commun. 382, 776–779 (2009).
65
Watson CJ, Ledwidge MT, Phelan D et al. Proteomic analysis of coronary sinus serum reveals leucine-rich alpha2glycoprotein as a novel biomarker of ventricular dysfunction and heart failure. Circ. Heart Fail. 4, 188–197 (2011).
66
Heffernan KS, Kuvin JT, Patel AR, Karas RH, Kapur NK. Endothelial function and soluble endoglin in smokers with heart failure. Clin. Cardiol. 34, 729–733 (2011).
67
Kapur NK, Heffernan KS, Yunis AA et al. Usefulness of soluble endoglin as a noninvasive measure of left ventricular filling pressure in heart failure. Am. J. Cardiol. 106, 1770–1776 (2010).
68
Pfisterer M, Buser P, Rickli H et al. TIME-CHF Investigators. BNP-guided vs symptom-guided heart failure therapy: the Trial of Intensified vs Standard Medical Therapy in Elderly Patients With Congestive Heart Failure (TIMECHF) randomized trial. JAMA 301, 383–392 (2009).
69
Lainchbury JG, Troughton RW, Strangman KM et al. N-terminal pro-B-type natriuretic peptide-guided treatment for chronic heart failure: results from the BATTLESCARRED (NT-proBNP-Assisted Treatment To Lessen Serial Cardiac Readmissions and Death) trial. J. Am. Coll. Cardiol. 55, 53–60 (2009).
55
Bdeir K, Cane W, Canziani G et al. Defensin promotes the binding of lipoprotein(a) to vascular matrix. Blood 94, 2007–2019 (1999).
56
Christensen HM, Frystyk J, Faber J et al. α-defensins and outcome in patients with chronic heart failure. Eur. J. Heart Fail. 14, 387–394 (2012).
•
First description of chronic heart failure.
57
Closa D, Motoo Y, Iovanna JL. Pancreatitis-associated protein: from a lectin to an anti-inflammatory cytokine. World J. Gastroenterol. 13, 170–174 (2007).
58
Rouquier S, Verdier JM, Iovanna J, Dagorn JC, Giorgi D. Rat pancreatic stone protein messenger RNA. Abundant expression in mature exocrine cells, regulation by food content, and sequence identity with the endocrine reg transcript. J. Biol. Chem. 266, 786–791 (1991).
59
Watanabe R, Hanawa H, Yoshida T et al. Gene expression profiles of cardiomyocytes in rat autoimmune myocarditis by DNA microarray and increase of regenerating gene family. Transl. Res. 152, 119–127 (2008).
60
Fitzgibbons TP, Paolino J, Dagorn JC, Meyer TE. Usefulness of pancreatitis-associated protein, a novel biomarker, to predict severity of disease in ambulatory patients with heart failure. Am. J. Cardiol. 113, 123–126 (2014).
61
Ai J, Druhan LJ, Hunter MG, Loveland MJ, Avalos BR. LRG-accelerated differentiation defines unique G-CSFR
Biomarkers Med. (2014) 8(6)
future science group
Emerging biomarkers for heart failure: an update
A growing array of biological pathways underpins the syndrome we recognize as heart failure. These include both deleterious pathways promoting its development and progression, as well as compensatory cardioprotective pathways. Components of these pathways can be utilized as biomarkers of this condition to aid diagnosis, prognostication and potentially guide management. As our understanding of the pathophysiology of heart failure deepens further candidate biomarkers are being identified. We provide an overview of the more recently emerging biomarkers displaying potential promise for future clinical use. Keywords: biomarkers • emerging • heart failure • novel
Heart failure is associated with significant mortality, morbidity and economic cost [1] . Over the past 30 years, our understanding of the pathophysiology of heart failure has advanced greatly. It is now recognized as a systemic syndrome characterized by maladaptive neurohormonal, metabolic and inflammatory processes. Recognition of these pathways has prompted the evaluation of some of their components as biomarkers in both chronic heart failure and acutely decompensated heart failure. We have previously summarized the more novel additions to this growing portfolio of candidate biomarkers [2] . Many of the biomarkers previously discussed have progressed to the forefront of clinical heart failure research with much expectation surrounding them, particularly the midregional peptides, ST2 and galectin-3. In this paper, we review only the more recently identified plasma biomarkers that have emerged as potential additions to the heart failure biomarker portfolio since our last review in 2009 [2] . Tenascin-C Tenascin-C (TN-C) is an extracellular glycoprotein that is released in response to inflammatory tissue remodeling under various path-
10.2217/BMM.14.51 © 2014 Future Medicine Ltd
ological conditions such as acute myocardial infarction [3,4] , chronic kidney disease [5] , myocarditis [6,7] as well as idiopathic dilated [8] and hypertrophic cardiomyopathy [9] . TN-C loosens the attachment of cardiomyocytes to connective tissue and upregulates expression and activity of matrix metalloproteinases, which may promote degradation of connective tissue and contribute to left ventricular (LV) dilatation. Serum levels of TN-C independently predict LV remodeling and all-cause mortality post-MI [4,10] . Circulating serum TN-C was measured in 107 patients with heart failure secondary to dilated cardiomyopathy (DCM) [11] . Levels in DCM patients were higher than those in normal controls (p < 0.001), showed a significantly positive correlation with New York Heart Association (NYHA) functional class (p < 0.001), B-type natriuretic peptide (p < 0.001), cardiothoracic ratio on chest x-ray (p < 0.01) and a significantly negative correlation with left ventricular ejection fraction (p < 0.01) (Table 1) [11] . Similar results were noted in a study of 110 consecutive DCM patients hospitalized with acute decompensated heart failure [12] . Serum TN-C and BNP levels were checked at discharge and at a mean of 22.4 months follow-
Biomarkers Med. (2014) 8(6), 833–840
Jonathan R Dalzell*,1, Jane A Cannon2, Colette E Jackson1, Ninian N Lang1 & Roy S Gardner1 Scottish Advanced Heart Failure Unit, Golden Jubilee National Hospital, Glasgow, G81 4DY, UK 2 British Heart Foundation Cardiovascular Research Centre, University of Glasgow, Glasgow, G12 8TA, UK *Author for correspondence: Tel.: +44 141 330 2237 Fax: +44 141 330 6955 j.dalzell@ nhs.net 1
part of
ISSN 1752-0363
833
Review Dalzell, Cannon, Jackson, Lang & Gardner
Table 1. Summary of novel biomarkers in acute and chronic heart failure. Biomarker
Plasma levels in Independent decompensated HF prognostic information?
Levels Plasma levels altered with in CHF therapy?
Independent prognostic information?
Levels altered with therapy?
Tenascin-C
↑
U
U
FL1
U
U
U
↑
Yes
Yes
↑
No
U
Syndecan-4
U
U
U
↑
No
U
Midkine
U
CCL21
U
U
U
↑
Yes
U
U
U
↑
Yes
Yes
S100A8/A9
U
ACE2
↑
U
U
↑
Yes
U
U
Yes
↑
α-defensins
U
Yes
U
U
U
↑
Yes
U
PAP
U
U
U
↑
U
U
U
U
U
Yes
LRG
U
U
U
↑
sEng
U
U
U
↑
↑: Raised; CHF: Chronic heart failure; HF: Heart failure; U: Unknown.
up. The optimal cutoff value for serum TN-C levels predicting the combined end-point of rehospitalization and cardiac death (due to heart failure) was ≥78.4 ng/ml and ≥219 pg/ml for BNP. Patients with serum TN-C levels ≥78.4 ng/Ml at discharge had higher NYHA functional class, larger LV end-diastolic and end-systolic diameters, and higher plasma BNP levels [12] . Furthermore, combined serum TN-C levels with plasma BNP levels at discharge were a stronger predictor of cardiac events for heart failure than either single biomarker alone. Circulating levels were measured in 83 chronic heart failure patients and 30 healthy controls. TN-C concentrations were significantly raised in heart failure patients in whom they correlated significantly with BNP worsening EF. TN-C was an independent predictor of a composite of all-cause mortality or hospitalization over a 12-month follow-up period [13] . When considering markers indicative of reverse cardiac remodeling, 64 patients scheduled for cardiac resynchronization therapy (CRT) had serum biomarkers (including TN-C) checked at baseline and 6 months postimplant [14] . In total, 72% of patients were classified as ‘responders’ to device therapy (i.e., showed a >10% reduction in LV end-systolic volume at followup) and among these patients a significant decrease in circulating levels of TN-C (from 60 ± 40 ng/ml to 40 ± 30 ng/ml; p < 0.01) was observed. This suggests an important role of extracellular matrix modulation in the process of reverse ventricular remodeling in patients responding to CRT [14] . Follistatin Like-1 Follistatin like-1 (FL1) is an extracellular glycoprotein belonging to the BM-40/Osteonectin family. It
834
Biomarkers Med. (2014) 8(6)
was first identified as a TGF-β-induced protein from a mouse osteoblast cell line and is thought to work by neutralizing activins (members of the TGF-β family) that are implicated in diverse biological processes such as wound healing, inflammation and fibrosis [15,16] . It has since been found to be upregulated in cardio vascular injury models (where it protects cardiac myocytes from hypoxia and reperfusion injury) [17] and in cardiac myocytes in the failing heart, returning to normal after myocardial recovery. Myocardial FL1 levels are significantly raised in samples obtained from patients with advanced heart failure undergoing LVAD implantation [18] . Plasma concentrations were assessed in 83 patients with stable chronic heart failure and were found to be significantly raised compared with healthy controls. FL1 levels correlated with LV dimensions and mass, left atrial size and BNP concentrations [18] . FL1 was a predictor of mortality on univariate but not multivariate analysis [18] . Syndecan-4 Components of the extra cellular matrix, of which collagen is the best characterized to date, are now recognized to play significant role in myocardial remodeling. Proteoglycans represent a major component of the extracellular matrix. Syndecan-4 is a transmembrane proteoglycan and preclinical studies have suggested it plays a crucial role in the development of compensatory myocardial hypertrophy following experimental myocardial infarction or pressure overload [19–23] and circulating concentrations have previously been shown to be elevated in patients after acute myocardial infarction [24] . Plasma syndecan-4 was measured in 45 patients, predominantly (76%) NYHA II or better. Syndecan-4
future science group
Emerging biomarkers for heart failure: an update
concentrations were significantly raised in HF patients compared with healthy controls. Although this study is too small to derive meaningful conclusions, a negative correlation was seen between syndecan-4 levels and LVEF. Syndecan-4 levels predicted death or HF-related hospital admission over a 3-year follow-up period on univariate analysis only [25] . A prospective study of 68 patients with LV systolic dysfunction secondary to dilated cardiomyopathy similarly demonstrated an increase in circulating syndecan-4 concentrations with an identical correlation with LVEF [26] . A positive correlation was noted with LV end systolic and diastolic diameters. Midkine Midkine is a multifunctional growth factor with roles in angiogenesis, cell growth and inflammation. It is predominantly expressed in the kidney, GI tract and lung in humans but expression can also be induced by ischemia in the cerebral cortex, vascular endothelium and myocardium. Experimental data suggest that midkine plays a crucial role in the development of LV hypertrophy and adverse remodeling [27] . Plasma concentrations were assessed in 216 patients with chronic heart failure and 60 healthy controls [28] . Circulating midkine levels were significantly raised in chronic heart failure patients in whom they correlated with NYHA class but not with LV ejection fraction or plasma BNP. In a multivariable analysis that included age, NYHA class BNP and LVEF, midkine was an independent predictor of cardiovascular death, heart failure hospitalization over a mean follow-up of 653 ± 375 days. Plasma midkine is also significantly raised in stable patients (n = 134) post cardiac transplantation. Similar correlations were noted with NY-HA class, NT-proBNP, creatinine and cystatin C [29] . CCL21 CCL21 is one of a family of so called homeostatic chemokines playing a crucial role in inflammatory responses, particularly T cell migration in nonlymphoid tissue. Expression of CCL21 has been shown to be upregulated in the myocardium of patients (n = 29) with advanced heart failure undergoing cardiac transplantation [30] . Myocardial samples were also taken from nine patients with advanced heart failure at the time of left ventricular assist device implantation and again at subsequent transplantation. In these patients, the raised CCL21 expression seen at LVAD implantation was significantly attenuated following a period of LV assistance (mean 8 ± 1.7 months) at the time of transplantation [30] . In 150 patients with stable chronic heart failure, circulating CCL21 was significantly elevated compared
future science group
Review
with healthy controls and independently predicted allcause mortality, in a multivariable model that included NT-proBNP, over a mean follow-up period of 24 (±12) months [30] . Plasma concentrations were significantly positively correlated with NT-proBNP and age while a negative correlation with cardiac index was noted [30] . Interestingly, CCL21 levels were higher in the subgroup of patients with an etiology of coronary artery disease as compared with those with idiopathic dilated cardiomyopathy [30] . CCL21 concentrations were also studied in the CORONA (n = 1456) and GISSI-HF (n = 1145) trials [31] . Plasma CCL21 correlated positively with NTproBNP, age and CRP while a negative correlation was noted with estimated glomerular filtration rate. CCL21 was noted to be an independent predictor of all-cause and cardiovascular mortality in both trial cohorts in multivariable analyses that included NT-proBNP. S100A8/A9 S100A8/A9 is a proinflammatory extracellular heterodimic protein secreted by neutrophils, monocytes and macrophages, and exerts its effects via receptor of advanced glycation end products and toll-like receptors. Myocardial levels are raised in a murine model of post-MI heart failure and S100A8/A9 supplementation exacerbates myocardial ischemia-reperfusion injury [32] . Plasma concentrations were measured in 54 patients with chronic heart failure, 31 hypertensive patients without heart failure and 23 healthy controls [33] . Levels were significantly higher in the heart failure patients compared with the other two groups. Plasma levels correlated significantly with IL-2, IL-8 and creatinine. In a multivariable model that included BNP, S100A8/A9 was an independent predictor of all-cause mortality at 1 year, but not at 2 years, of follow-up. ACE2 ACE2 is an 805-amino acid transmembrane peptidase that negatively regulates the RAS by counterbalancing the actions of ACE. In humans, ACE2 is most abundant in the heart, kidney and testes [34] where it is predominantly membrane bound. It displays 42% sequence homology with ACE in its extracellular (catalytic) domain but is resistant to contemporary ACE inhibitors. ACE2 hydrolyses angiotensin II, for which it has the greatest affinity [35] , to angiotensin-(1–7) by cleaving the C-terminal amino acid [36] and has also been shown to hydrolyze numerous peptides in vitro via the same mechanism. ACE2 can undergo cleavage of its extracellular catalytic domain by the enzyme TACE with subsequent systemic release [37] . ACE2 knockout mice demonstrate a progressive, severe reduction in LV systolic function by 6 months of
www.futuremedicine.com
835
Review Dalzell, Cannon, Jackson, Lang & Gardner age along with an increase in myocardial angiotensin-II levels. These effects are abolished by additional deletion of the ACE gene [38] . A significantly quicker development of CHF following aortic constriction [38] and myocardial infarction compared with wild-type controls is also observed [39–42] . Overexpression of ACE2 in the rat heart significantly attenuates the deleterious myocardial effects of angiotensin II infusion [43] and also significantly reduces infarct size and preserves LV function in a rat model of myocardial infarction [44] . Infusion of soluble ACE2 improves right ventricular function in a murine model of right ventricular pressure overload [45] . ACE2 is upregulated in the myocardium of CHF patients on standard therapy [46,47] and ACE inhibitor or angiotensin receptor blocker therapy upregulates myocardial ACE2 levels in normal [48] and CHF [49,50] rats. Circulating ACE2 activity has been shown to be significantly raised in human chronic heart failure [51] . Positive correlations were seen with worsening NYHA class, worsening LVEF and plasma BNP concentrations. Plasma ACE2 activity independently predicted outcome in 113 patients with chronic heart failure over a median follow-up of 34 ± 17 months in a multivariable analysis which included NT-proBNP and LVEF [52] . Similar correlations and impressive prognostic power was noted in 111 patients with chronic heart failure secondary to Chagas’ disease [53] . Plasma ACE2 activity was measured within 12 h of admission and again 48–72 h after intensive contemporary treatment in 70 patients (of which 57 were followed up) with acute decompensated heart failure [54] . In this cohort ACE2 activity correlated with BNP; however, no correlation was noted with LV systolic or diastolic function. Somewhat paradoxically, when compared with the above data in chronic heart failure, a 50% increase from baseline ACE2 levels following treatment was associated with a significant reduction in the subsequent risk of death or requirement for transplantation or subsequent rehospitalization [54] . Clearly, larger studies are required to ascertain the significance of ACE2 activity levels and any potential confounders. Perhaps more importantly, the potential of therapeutic manipulation of this putative cardioprotective arm of the renin angiotensin system merits investigation. α-defensins α-defensins are peptides secreted by neutrophils as part of the innate inflammatory response and have been implicated in the progression of atherosclerosis [55] . Circulating levels were assessed in 194 patients with stable chronic heart failure and compared with 98 age-matched healthy controls [56] . Concentrations were significantly raised in patients with heart fail-
836
Biomarkers Med. (2014) 8(6)
ure in whom significant correlations were noted with NYHA status and less strongly with NT-proBNP. No correlation was noted with LVEF. In a multivariable model which included age, sex, NYHA class, LVEF and NT-proBNP, α-defensins concentrations were an independent predictor of all-cause mortality over a median follow-up of 2.6 years. The combination of high NT-proBNP and α-defensins levels provided incremental prognostic information compared with either biomarker alone. Pancreatitis-associated protein Pancreatitis-associated peptide (PAP) is a novel cytokine with putative anti-inflammatory properties [57] . It was initially discovered in pancreatic secretions from rats with experimental acute pancreatitis [58] . Myocardial PAP expression is markedly increased in a rat model of autoimmune myocarditis [59] . Plasma PAP was measured in 70 stable chronic heart failure patients and compared with 17 healthy controls. PAP concentrations were significantly raised in patients with heart failure in whom significant correlations were noted with BNP, NYHA class and pulmonary artery pressure [60] . Leucine-rich alpha-2-glycoprotein The trace protein leucine-rich alpha-2-glycoprotein (LRG) has a putative role in the inflammatory response and neutrophilic differentiation [61–64] . It has been postulated that LRG may have a role in the exaggerated inflammatory responses leading to myocardial fibrosis. Leading on from this, Watson et al. sampled coronary sinus serum from 11 stable patients undergoing cardiac catheterization and analyzed for LRG, TNF-α and IL-6 [65] . They also sampled peripheral venous blood from 40 asymptomatic hypertensive patients and 105 patients with a range of ventricular dysfunction. They found LRG levels were significantly correlated with BNP in hypertensive, asymptomatic left ventricular diastolic dysfunction, diastolic HF and systolic HF patient groups (p ≤ 0.05). LRG levels correlated with coronary sinus serum levels of TNF-α (p = 0.009) and IL-6 (p = 0.021). They concluded that LRG levels can accurately identify patients with HF and multivariable analyses confirmed LRG was a stronger identifier of heart failure than BNP, independent of age, sex, creatinine and BNP. There are no prognostic data available for LRG in heart failure as yet. Soluble endoglin The glycoprotein endoglin is a TGF-β1 coreceptor that is released into the circulation as soluble endoglin (sEng). TGF-β1 is a profibrogenic cytokine that
future science group
Emerging biomarkers for heart failure: an update
contributes to multiple fibroproliferative disorders including myocardial fibrosis associated with heart failure. In cardiac tissue, endoglin is expressed by endothelial cells, fibroblasts and fibroblast-like stromal cells of valve leaflets. sEng is also used as a marker of endothelial function and is known to regulate the expression and activity of endothelial NO synthase, reducing NO production and making it an important modulator of endothelial vascular tone. Recently the difference in levels of sEng between smokers and nonsmokers with HF has been explored [66] . Brachial artery flow-mediated dilatation (FMD) was recorded, as a measure of conduit vessel endothelial function, as well as sEng in 33 smokers and nonsmokers with HF versus controls. They found that smokers with HF had higher brachial FMD (p < 0.05) and lower sEng than nonsmokers with HF (p < 0.05), and values were comparable to nonsmokers without HF. It was concluded that HF appears to modulate the relationship between smoking exposure and endothelial biology with HF patients who smoke having preserved endothelial function and lower sEng compared with non-smokers with HF. A study of 82 consecutive patients referred for evaluation of suspected left ventricular dysfunction including right and left heart catheterization measured serum sEng levels and compared the results with calculated left ventricular end diastolic pressure (LVEDP) [67] . Patients with an LVEDP of ≥16mmHg had significantly greater serum sEng concentrations, compared with those with an LVEDP of ≤16 mmHg (p < 0.001) even after adjusting for age, sex, renal function and diuretic use. A reduction in pulmonary capillary wedge pressure correlated with a reduction in sEng following diuretic therapy (p = 0.008) suggesting a potential application as a marker of response to therapy [66] .
the targeting of advanced device therapy to appropriate patients. Mortality and morbidity remain high and further improvement is therefore necessary. Many of the biomarkers discussed here have shown promise in one or more of these categories. However, further work is required if we are to ascertain their relevance to contemporary heart failure management. The incremental impact on current clinical acumen arising from the prognostic information provided by this myriad of emerging biomarkers is not yet clear. Even the role of the more established heart failure biomarkers in guiding therapy is unclear with conflicting results in large randomized trials [68,69] . It would seem that our ability to simply correlate components of neurohormonal, inflammatory and other pathways with clinical outcomes has far exceeded progress in using the accrued information to improve care and outcomes in heart failure. The critical question remains: what clinically meaningful information is provided by these novel biomarkers that is not already provided by other established clinical parameters (e.g., NT-proBNP/BNP, peak VO2, serum sodium, 6-min walk test, invasive hemodynamic data, LVEF, QRS duration, among others)? It remains to be seen whether the apparent increase in prognostic power reported by many of these studies translates into a clinically meaningful benefit to patients. It is therefore crucial to comprehensively explore whether or not the incremental information provided by this expanding array of potential biomarkers has any translatable clinical impact on contemporary chronic heart failure management incremental to the aforementioned variables. It is also important that research focuses on the potential therapeutic manipulation of these pathways, which may allow the development of new disease-modifying therapies, and perhaps help to improve prognosis as well as effectively predict it.
Conclusion The use of biomarkers in heart failure has improved diagnosis and prognostication, as well as improving
Future perspective The ability to tailor specific therapy to the individual patient based upon a multibiomarker approach is an
Review
Executive summary Background • Heart failure is a complex systemic syndrome characterized by abnormalities of a spectrum of biological systems and pathways, components of which are potential biomarkers of the syndrome. • Established biomarkers, such as BNP, have improved the diagnosis and prognostication of heart failure. • The introduction of further biomarkers may further improve the management of these patients.
Conclusion • Any novel biomarkers should add to contemporary diagnostic and prognostic acumen whilst being cost effective. • Much work is required to ascertain which, if any, of these candidate biomarkers have a place in contemporary clinical practice and, if so, how they will be best utilized. • The improvement in the understanding of the pathophysiology of CHF should also prompt investigation into the development of novel disease modifying therapies.
future science group
www.futuremedicine.com
837
Review Dalzell, Cannon, Jackson, Lang & Gardner exciting prospect. Whether a multimarker approach will allow us to identify if particular pathways, be it neurohormonal or inflammatory, require more intensive inhibition from patient to patient remains to be seen. The rapidly evolving fields of genomics and proteomics will likely revolutionize the appliance of biomarkers not only in heart failure but medicine as a whole and may ultimately lead to a personalized approach to heart failure management.
Financial & competing interests disclosure
References
13
Yao HC, Han QF, Zhao AP, Yao DK, Wang LX. Prognostic values of serum tenascin-C in patients with ischaemic heart disease and heart failure. Heart Lung Circ. 22, 184–187 (2013).
14
Hessel MH, Bleeker GB, Bax JJ et al. Reverse ventricular remodelling after cardiac resynchronization therapy is associated with a reduction in serum tenascin-C and plasma matrix metalloproteinase-9 levels. Eur. J. Heart Fail. 9, 1058–1063 (2007).
15
Shibanuma M, Mashimo J, Mita A, Kuroki T, Nose K. Cloning from a mouse osteoblastic cell line of a set of transforming-growth-factor-beta 1-regulated genes, one of which seems to encode a follistatin-related polypeptide. Eur. J. Biochem. 217, 13–19 (1993).
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Papers of special note have been highlighted as: • of interest; •• of considerable interest 1
Mosterd A, Hoes AW. Clinical epidemiology of heart failure. Heart 93, 1137–1146 (2007).
2
Dalzell JR, Jackson CE, McDonagh TA, Gardner RS. Novel biomarkers in heart failure: an overview. Biomark. Med. 3, 453–463 (2009).
3
Nishioka T, Onishi K, Shimojo N et al. Tenascin-C may aggravate left ventricular remodeling and function after myocardial infarction in mice. Am. J. Physiol. Heart Circ. Physiol. 298, H1072–H1078 (2010).
4
Sato A, Aonuma K, Imanaka-Yoshida K et al. Serum tenascin-C might be a novel predictor of left ventricular remodeling and prognosis after acute myocardial infarction. J. Am. Coll. Cardiol. 47, 2319–2325 (2006).
16
Liabeuf S, Barreto DV, Kretschmer A et al. High circulating levels of large splice variants of tenascin-C is associated with mortality and cardiovascular disease in chronic kidney disease patients. Atherosclerosis 215, 116–124 (2011).
Kawabata D, Tanaka M, Fujii T et al. Ameliorative effects of follistatin-related protein/TSC-36/FSTL1 on joint inflammation in a mouse model of arthritis. Arthritis Rheum. 50, 660–668 (2004).
17
Oshima Y, Ouchi N, Sato K, Izumiya Y, Pimentel DR, Walsh K. Follistatin-like 1 is an Akt-regulated cardioprotective factor that is secreted by the heart. Circulation 117, 3099–3108 (2008).
18
El-Armouche A, Ouchi N, Tanaka K et al. Follistatin-like 1 in chronic systolic heart failure: a marker of left ventricular remodeling. Circ. Heart Fail. 4, 621–627 (2011).
5
6
7
838
Imanaka-Yoshida K, Hiroe M, Yasutomi Y et al. Tenascin-C is a useful marker for disease activity in myocarditis. J. Pathol. 197, 388–394 (2002). Morimoto S, Imanaka-Yoshida K, Hiramitsu S et al. Diagnostic utility of tenascin-C for evaluation of the activity of human acute myocarditis. J. Pathol. 205, 460–467 (2005).
8
Aso N, Tamura A, Nasu M. Circulating tenascin-C levels in patients with idiopathic dilated cardiomyopathy. Am. J. Cardiol. 94, 1468–1470 (2004).
9
Kitaoka H, Kubo T, Baba Y et al. Serum tenascin-C levels as a prognostic biomarker of heart failure events in patients with hypertrophic cardiomyopathy. J. Cardiol. 59, 209–214 (2012).
10
Sato A, Hiroe M, Akiyama D et al. Prognostic value of serum tenascin-C levels on long-term outcome after acute myocardial infarction. J. Card. Fail. 18, 480–486 (2012).
11
Terasaki F, Okamoto H, Onishi K et al. Higher serum tenascin-C levels reflect the severity of heart failure, left ventricular dysfunction and remodeling in patients with dilated cardiomyopathy. Circ. J. 71, 327–330 (2007).
12
Fujimoto N, Onishi K, Sato A et al. Incremental prognostic values of serum tenascin-C levels with blood B-type natriuretic peptide testing at discharge in patients with dilated cardiomyopathy and decompensated heart failure. J. Card. Fail. 15, 898–905 (2009).
Biomarkers Med. (2014) 8(6)
•
First description of chronic heart failure.
19
Finsen AV, Woldbaek PR, Li J et al. Increased syndecan expression following myocardial infarction indicates a role in cardiac remodeling. Physiol. Genomics 16, 301–308 (2004).
20
Finsen AV, Lunde IG, Sjaastad I et al. Syndecan-4 is essential for development of concentric myocardial hypertrophy via stretch-induced activation of the calcineurin-NFAT pathway. PLoS ONE 6, e28302 (2011).
21
Matsui Y, Ikesue M, Danzaki K et al. Syndecan-4 prevents cardiac rupture and dysfunction after myocardial infarction. Circ. Res. 108, 1328–1339 (2011).
22
Echtermeyer F, Harendza T, Hubrich S et al. Syndecan-4 signalling inhibits apoptosis and controls NFAT activity during myocardial damage and remodelling. Cardiovasc. Res. 92, 123–131 (2011).
23
Strand ME, Herum KM, Rana ZA et al. Innate immune signaling induces expression and shedding of the heparan sulfate proteoglycan syndecan-4 in cardiac fibroblasts and myocytes, affecting inflammation in the pressure-overloaded heart. FEBS J. 280, 2228–2247 (2013).
future science group
Emerging biomarkers for heart failure: an update
24
Kojima T, Takagi A, Maeda M et al. Plasma levels of syndecan-4 (ryudocan) are elevated in patients with acute myocardial infarction. Thromb. Haemost. 85, 793–799 (2001).
25
Takahashi R, Negishi K, Watanabe A et al. Serum syndecan-4 is a novel biomarker for patients with chronic heart failure. J. Cardiol. 57, 325–332 (2011).
26
Bielecka-Dabrowa A, von Haehling S, Aronow WS, Ahmed MI, Rysz J, Banach M. Heart failure biomarkers in patients with dilated cardiomyopathy. Int. J. Cardiol. 168, 2404–2410 (2013).
27
Netsu S, Shishido T, Kitahara T et al. Midkine exacerbates pressure overload-induced cardiac remodeling. Biochem. Biophys. Res. Commun. 443, 205–210 (2014).
28
Kitahara T, Shishido T, Suzuki S et al. Serum midkine as a predictor of cardiac events in patients with chronic heart failure. J. Card. Fail. 16, 308–313 (2010).
•
First description in chronic heart failure.
29
Przybylowski P, Malyszko J, Malyszko JS. Serum midkine is related to NYHA class and cystatin C in heart transplant recipients. Transplant Proc. 42, 3704–3707 (2010).
30
Yndestad A, Finsen AV, Ueland T et al. The homeostatic chemokine CCL21 predicts mortality and may play a pathogenic role in heart failure. PLoS ONE 7, e33038 (2012).
31
Ueland T, Nymo SH, Latini R et al. Investigators of the Controlled Rosuvastatin Multinational Study in Heart Failure (CORONA) trial; Investigators of the GISSI-Heart Failure (GISSI-HF) trial. CCL21 is associated with fatal outcomes in chronic heart failure: data from CORONA and GISSI-HF trials. Eur. J. Heart Fail. 15, 747–755 (2013).
••
Demonstrates the prognostic power of CCL21 in two large heart failure clinical trial cohorts.
32
Volz HC, Laohachewin D, Seidel C et al. S100A8/A9 aggravates post-ischemic heart failure through activation of RAGE-dependent NF-κB signaling. Basic Res. Cardiol. 107, 250 (2012).
33
Ma LP, Haugen E, Ikemoto M, Fujita M, Terasaki F, Fu M. S100A8/A9 complex as a new biomarker in prediction of mortality in elderly patients with severe heart failure. Int. J. Cardiol. 155, 26–32 (2012).
•
First description of chronic heart failure.
34
Donoghue M, Hsieh F, Baronas E et al. A novel angiotensinconverting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1–9. Circ. Res. 87, e1–e9 (2000).
35
Vickers C, Hales P, Kaushik V et al. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J. Biol. Chem. 277, 14838–14843 (2002).
36
Danilczyk U, Penninger JM. Angiotensin-converting enzyme II in the heart and the kidney. Circ. Res. 98, 463–471 (2006).
37
Lambert DW, Yarski M, Warner FJ et al. Tumor necrosis factor-alpha convertase (ADAM17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (SARS-CoV) receptor, angiotensinconverting enzyme-2 (ACE2). J. Biol. Chem. 280, 30113–30119 (2005).
future science group
38
Crackower MA, Sarao R, Oudit GY et al. Angiotensinconverting enzyme 2 is an essential regulator of heart function. Nature 417, 822–828 (2002).
39
Nakamura K, Koibuchi N, Nishimatsu H et al. Candesartan ameliorates cardiac dysfunction observed in angiotensinconverting enzyme 2-deficient mice. Hypertens. Res. 31, 1953–1961 (2008).
40
Gurley SB, Allred A, Le TH et al. Altered blood pressure responses and normal cardiac phenotype in ACE2-null mice. J. Clin. Invest. 116, 2218–2225 (2006).
41
Kassiri Z, Zhong J, Guo D et al. Loss of angiotensinconverting enzyme 2 accelerates maladaptive left ventricular remodeling in response to myocardial infarction. Circ. Heart Fail. 2, 446–455 (2009).
42
Yamamoto K, Ohishi M, Katsuya T et al. Deletion of angiotensin-converting enzyme 2 accelerates pressure overload-induced cardiac dysfunction by increasing local angiotensin II. Hypertension 47, 718–726 (2006).
43
Huentelman MJ, Grobe JL, Vazquez J et al. Protection from angiotensin II-induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats. Exp. Physiol. 90, 783–790 (2005).
44
Der Sarkissian S, Grobe JL, Yuan L et al. Cardiac overexpression of angiotensin converting enzyme 2 protects the heart from ischemia-induced pathophysiology. Hypertension 51, 712–718 (2008).
45
Johnson JA, West J, Maynard KB, Hemnes AR. ACE2 improves right ventricular function in a pressure overload model. PLoS ONE 6, e20828 (2011).
46
Burrell LM, Risvanis J, Kubota E et al. Myocardial infarction increases ACE2 expression in rat and humans. Eur. Heart J. 26, 369–375 (2005).
47
Goulter AB, Goddard MJ, Allen JC, Clark KL. ACE2 gene expression is up-regulated in the human failing heart. BMC Med. 2, 19 (2004).
48
Ferrario CM, Jessup J, Chappell MC et al. Effect of angiotensin-converting enzyme inhibition and angiotensin II receptor blockers on cardiac angiotensin-converting enzyme 2. Circulation 111, 2605–2610 (2005).
49
Ishiyama Y, Gallagher PE, Averill DB, Tallant EA, Brosnihan KB, Ferrario CM. Upregulation of angiotensinconverting enzyme 2 after myocardial infarction by blockade of angiotensin II receptors. Hypertension 43, 970–976 (2004).
50
Kaiqiang Ji, Minakawa M, Fukui K, Suzuki Y, Fukuda I. Olmesartan improves left ventricular function in pressureoverload hypertrophied rat heart by blocking angiotensin II receptor with synergic effects of upregulation of angiotensin converting enzyme 2. Ther. Adv. Cardiovasc. Dis. 3, 103–111 (2009).
51
Epelman S, Tang WH, Chen SY, Van Lente F, Francis GS, Sen S. Detection of soluble angiotensin-converting enzyme 2 in heart failure: insights into the endogenous counterregulatory pathway of the renin–angiotensin–aldosterone system. J. Am. Coll. Cardiol. 52, 750–754 (2008).
52
Epelman S, Shrestha K, Troughton RW et al. Soluble angiotensin-converting enzyme 2 in human heart failure:
www.futuremedicine.com
Review
839
Review Dalzell, Cannon, Jackson, Lang & Gardner relation with myocardial function and clinical outcomes. J. Card. Fail. 15, 565–571 (2009). •
840
signaling pathways downstream of PU.1 and C/EBPepsilon that modulate neutrophil activation. J. Leukoc. Biol. 83, 1277–1285 (2008).
First description of prognostic power in chronic heart failure.
62
53
Wang Y, Moreira Mda C, Heringer-Walther S et al. Plasma ACE2 activity is an independent prognostic marker in Chagas’ disease and equally potent as BNP. J. Card. Fail. 16, 157–163 (2010).
Codina R, Vanasse A, Kelekar A, Vezys V, Jemmerson R. Cytochrome c-induced lymphocyte death from the outside in: inhibition by serum leucine-rich alpha-2-glycoprotein-1. Apoptosis 15, 139–152 (2010).
63
54
Shao Z, Shrestha K, Borowski AG et al. Increasing serum soluble angiotensin-converting enzyme 2 activity after intensive medical therapy is associated with better prognosis in acute decompensated heart failure. J. Card. Fail. 19, 605–610 (2013).
O’Donnell LC, Druhan LJ, Avalos BR. Molecular characterization and expression analysis of leucine-rich alpha2-glycoprotein, a novel marker of granulocytic differentiation. J. Leukoc. Biol. 72, 478–485 (2002).
64
Shirai R, Hirano F, Ohkura N, Ikeda K, Inoue S. Upregulation of the expression of leucine-rich alpha(2)glycoprotein in hepatocytes by the mediators of acute-phase response. Biochem. Biophys. Res. Commun. 382, 776–779 (2009).
65
Watson CJ, Ledwidge MT, Phelan D et al. Proteomic analysis of coronary sinus serum reveals leucine-rich alpha2glycoprotein as a novel biomarker of ventricular dysfunction and heart failure. Circ. Heart Fail. 4, 188–197 (2011).
66
Heffernan KS, Kuvin JT, Patel AR, Karas RH, Kapur NK. Endothelial function and soluble endoglin in smokers with heart failure. Clin. Cardiol. 34, 729–733 (2011).
67
Kapur NK, Heffernan KS, Yunis AA et al. Usefulness of soluble endoglin as a noninvasive measure of left ventricular filling pressure in heart failure. Am. J. Cardiol. 106, 1770–1776 (2010).
68
Pfisterer M, Buser P, Rickli H et al. TIME-CHF Investigators. BNP-guided vs symptom-guided heart failure therapy: the Trial of Intensified vs Standard Medical Therapy in Elderly Patients With Congestive Heart Failure (TIMECHF) randomized trial. JAMA 301, 383–392 (2009).
69
Lainchbury JG, Troughton RW, Strangman KM et al. N-terminal pro-B-type natriuretic peptide-guided treatment for chronic heart failure: results from the BATTLESCARRED (NT-proBNP-Assisted Treatment To Lessen Serial Cardiac Readmissions and Death) trial. J. Am. Coll. Cardiol. 55, 53–60 (2009).
55
Bdeir K, Cane W, Canziani G et al. Defensin promotes the binding of lipoprotein(a) to vascular matrix. Blood 94, 2007–2019 (1999).
56
Christensen HM, Frystyk J, Faber J et al. α-defensins and outcome in patients with chronic heart failure. Eur. J. Heart Fail. 14, 387–394 (2012).
•
First description of chronic heart failure.
57
Closa D, Motoo Y, Iovanna JL. Pancreatitis-associated protein: from a lectin to an anti-inflammatory cytokine. World J. Gastroenterol. 13, 170–174 (2007).
58
Rouquier S, Verdier JM, Iovanna J, Dagorn JC, Giorgi D. Rat pancreatic stone protein messenger RNA. Abundant expression in mature exocrine cells, regulation by food content, and sequence identity with the endocrine reg transcript. J. Biol. Chem. 266, 786–791 (1991).
59
Watanabe R, Hanawa H, Yoshida T et al. Gene expression profiles of cardiomyocytes in rat autoimmune myocarditis by DNA microarray and increase of regenerating gene family. Transl. Res. 152, 119–127 (2008).
60
Fitzgibbons TP, Paolino J, Dagorn JC, Meyer TE. Usefulness of pancreatitis-associated protein, a novel biomarker, to predict severity of disease in ambulatory patients with heart failure. Am. J. Cardiol. 113, 123–126 (2014).
61
Ai J, Druhan LJ, Hunter MG, Loveland MJ, Avalos BR. LRG-accelerated differentiation defines unique G-CSFR
Biomarkers Med. (2014) 8(6)
future science group
