If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
CPP-II are formed in the blood under high mineral stress conditions.
•
CPP-II induce inflammation and promote vascular calcification.
•
The size of CPP-II represents the mineral buffering capacity of blood.
•
The size of CPP-II is associated with mortality in PAD patients.
Abstract
Background and aims
Secondary calciprotein particles (CPP-II) induce inflammation and contribute to vascular calcification. CPP-II size is associated with vascular calcification in patients with chronic kidney disease (CKD) and all-cause mortality in hemodialysis patients. Here, we investigate for the first time a possible role of CPP-II size in patients with peripheral artery disease (PAD) without severe CKD.
Methods
We measured the hydrodynamic radius (Rh) of CPP-II by using dynamic light scattering in a cohort of 281 PAD patients. Mortality was evaluated over a period of ten years by central death registry queries. 35% of patients died during the observation period (median of 8.8 (6.2–9.0) years). Cox-regression analyses were performed to estimate hazard ratios (HR) and 95% confidence intervals (CI) and to allow for multivariable adjustment.
Results
The mean CPP-II size was 188 (162-218) nm. Older patients, patients with reduced kidney function, and those with media sclerosis had larger CPP-II (p < 0.001, p = 0.008, and p = 0.043, retrospectively). There was no association between CPP-II size and overall atherosclerotic disease burden (p = 0.551). CPP-II size was independently significantly associated with all-cause (HR 1.33 (CI 1.01–1.74), p = 0.039) and cardiovascular mortality (HR 1.52 (CI 1.05–2.20), p = 0.026) in multivariable regression analyses.
Conclusions
Large CPP-II size is associated with mortality in PAD patients and might be a new feasible biomarker for the presence of media sclerosis in this patient population.
]. Subsequently, these mineral-loaded proteins (mainly fetuin-A) aggregate to form amorphous, so-called primary calciprotein particles (CPP-I). Their role is to prevent excess minerals from damaging endothelial cells (mineral stress), which ultimately leads to vascular calcification [
]. Under high mineral stress conditions, such as hyperphosphatemia in chronic kidney disease (CKD), these amorphous particles undergo topological transformation into crystalline secondary CPP (CPP-II). These larger CPP-II particles induce inflammatory pathways and contribute to vascular calcification. [
], Formation of larger amounts of CPP-II therefore can be viewed as a sign of overload of the endogenous CPP system. Taken together, the CPP system serves as a buffering system against high mineral concentrations, thus protecting endothelial cells from mineral stress and damage [
The buffering capacity of the CPP system can be tested in vitro by adding high concentrations of calcium and phosphate to patient serum, thus accelerating the physiologically occurring formation of CPP-II by approximately two orders of magnitude (from weeks to hours). The buffering capacity of the CPP system in vitro can then be described using several different parameters: The most widespread measure is the time needed for half-maximum transition of CPP-I to CPP-II, which is called the “T50” time [
]. Conceptually, the longer the T50 time, the higher the buffering capacity of serum to mineral loading. Recently, our working group found that low T50 associates with mortality in patients with ischemic heart failure with reduced ejection fraction (HFrEF) [
Another in vitro parameter of the CPP buffering capacity is the ultimate size (hydrodynamic radius) of CPP-II. Conceptually, the lower the buffering capacity of the CPP system, the larger the aggregates of CPP-II will get. Indeed, CPP-II size (indicating low buffering capacity) has been associated with vascular calcification in patients with CKD [
Associations of serum calciprotein particle size and transformation time with arterial calcification, arterial stiffness, and mortality in incident hemodialysis patients.
]. This cohort included patients with peripheral artery disease (PAD) without planned revascularization procedures or critical limb ischemia at baseline. Key exclusion criteria included vasculitis, oncologic disease, and end-stage kidney disease (chronic kidney disease stage 5D). The study was approved by the ethics committee of the Medical University of Vienna and complied with the Declaration of Helsinki, including current revisions. The procedures were following institutional guidelines, and all subjects gave written informed consent before inclusion in the study. Blood samples obtained at the one-year follow-up point in time were used in this study (three samples missing).
2.2 Peripheral vascular measurement
Ankle-brachial index (ABI) measurements were performed by trained technicians using non-invasive Doppler sonographic measurements (ELCAT VL5000, Wolfratshausen, Germany). Systolic blood pressure (SBP) was measured in both arms (brachial arteries) and both ankles (dorsal pedal arteries and posterior tibial arteries). ABI was calculated according to the TASC criteria [
] by dividing the higher ankle pressure by the most elevated brachial pressure. PAD was defined as an ABI below 0.9 or previous peripheral revascularization. Patients were classified as showing signs of media sclerosis in the case of non-compressible ankle arteries during doppler sonographic measurements (ABI >1.4). In those patients, toe pressures were measured to verify the possibility of media sclerosis. Axial imaging including computer tomography angiography and magnetic resonance angiography was not performed in this cohort. PAD was clinically staged by the self-reported pain-free walking distance (Fontaine classification).
2.3 Definition of cardiovascular co-morbidities
Arterial hypertension was defined as documentation of systolic blood pressure of ≥140 mmHg and/or diastolic blood pressure of ≥90 mmHg in at least two measurements [
Guidelines for the management of arterial hypertension: the task force for the management of arterial hypertension of the European society of hypertension (ESH) and of the European society of cardiology (ESC).
] or active use of any antihypertensive medication. A standardized oral glucose tolerance test (75g glucose) was performed in patients without known diabetes. Diabetes mellitus type 2 was defined as a fasting plasma glucose level over 7.0 mmol/L (126 mg/dL), glucose level over 11.1 mmol/L (200 mg/dL) after standardized oral glucose tolerance test [
], glycated hemoglobin (HbA1c) over 6.5% (48 mmol/mol) or intake of anti-diabetic medication. Prediabetes was defined by the results of the oral glucose tolerance test as either a fasting plasma glucose of 5.55–6.94 mmol/L (100–125 mg/dL), a 2-h glucose level of 7.77–11.05 mmol/L (140–199 mg/dL), or an HbA1c of 5.7–6.4% (39–46 mmol/mol). Smoking was defined as current smoking. Former smoking was defined as previous smoking of at least 100 cigarettes. Smoking one pack of cigarettes (20 pieces) per day was defined as one pack-year. Body mass index (BMI) was calculated as body weight in kg divided by squared body height in meters (kg/m2). Fasting blood samples for standard metabolic laboratory markers were analyzed by the institutions’ central laboratory at the study visit. The estimated glomerular filtration rate (eGFR) was calculated by the Chronic Kidney Disease Epidemiology (CKD-EPI) equation [
2.4 Sample collection and measurement of CPP-II size
Fasting blood samples were collected at the study visit and stored at −80 °C until measurement. A central freezer surveillance system monitored the storage temperature. CPP-II size was measured as described by Chen et al. [
] In brief, the serum samples were thawed at 4 °C over night and spiked with standardized solutions of calcium and phosphate, to achieve end concentrations of 10 and 6 mM, respectively, allowing CPP-II formation. Subsequently, the hydrodynamic radius (Rh) of CPP-II was measured using 3D-dynamic light scattering (3D-DLS). Because CPP-II size was measured after prolonged incubation (600 min) of serum, when turbidity of samples (i.e. CPP-II generation) has reached a plateau (typically after 300–500 min in the majority of samples), the CPP-II size reported here is seen as end-product of the transformation and aggregation process and CPP-II size is therefore termed ultimate CPP-II size here. Five extreme outliers were excluded from further analysis.
2.5 Mortality assessment
Mortality was assessed by central death registry queries over ten years (Statistics Austria) in the VMC Vienna cohort. International Statistical Classification of Disease and Related Health Problems 10th revision (ICD-10) codes were retrieved from the central death registry and verified by hospital or autopsy reports as available to quantify cardiovascular mortality. Cardiovascular mortality was defined by the ICD-10 diseases of the circulatory system (I00-I99 code).
2.6 Statistical analysis
Continuous data were subjected to Kolmogorov-Smirnov test to evaluate their distribution. Normally distributed data are presented as mean ± standard deviation (SD), while non-normal data are shown as median (25th-75th percentile). Variables were log-transformed for parametric statistics if needed. Student's unpaired t-test and χ2-test were used as applicable. Analysis of variance (ANOVA) or Kruskal-Wallis test were used to evaluate differences between multiple groups. Univariable associations were estimated by the Pearson correlation coefficient (R). 95% confidence intervals (CI) were generated by conversion into z-scores and subsequently, calculation of CI by linear Cox-regression analyses. The Kaplan-Meier method was used for survival curves and statistically compared by the log-rank test. Multivariable adjustment and effect size calculation was performed by Cox proportional hazards regression analysis. The effect size for CPP-II is given as hazard ratio (HR) per one SD and 95% CI. An alpha level of p < 0.05 (two-tailed) was considered statistically significant. SPSS 28 (IBM, Chicago, IL, USA) was used for all statistical analyses and GraphPad Prism 8 (GraphPad Software Inc., La Jolla, CA, USA) for the graphical presentation of survival.
3. Results
3.1 Study population
This study consists of 281 patients with PAD (32% women) with underlying cardiovascular risk factors such as arterial hypertension (95%), type 2 diabetes mellitus (47%), and active or previous smoking (85%). Those risk factors were equally distributed among CPP-II tertiles as shown in Table 1. The mean age was 70 ± 10 years and the mean eGFR was 66.5 ml/min/1.73 m2. Larger CPP-II were present in older patients (p < 0.001) and those with reduced kidney function (p = 0.008), while smaller CPP-II are seen in PAD patients with elevated BMI (p = 0.002) and increased low density lipoprotein cholesterol (LDL-C) (p < 0.001). CPP-II size was 188 (162-218) nm in this cohort.
Table 1Baseline characteristics according to CPP-II size tertiles.
overall cohort
1st tertile
2nd tertile
3rd tertile
p-value
n
281
93
94
94
CPP-II (nm)
195.2 ± 43.3
152.8 ± 12.7
188.1 ± 9.3
244.3 ± 33.5
Age (years)
70 ± 10
65 ± 9
71 ± 9
72 ± 10
<0.001
Female, n (%)
91 (32.4)
30 (32.3)
31 (33.0)
30 (31.9)
0.987
Body mass index (kg/m2)
27.7 ± 4.1
28.8 ± 4.0
27.8 ± 4.1
26.7 ± 4.1
0.002
HbA1c (mmol/mol)
43 (39, 49)
44 (39, 51)
43 (39, 46)
42 (39, 48)
0.270
LDL-C (mmol/L)
2.30 (1.96, 2.91)
2.61 (2.08, 3.28)
2.28 (2.00, 2.85)
2.11 (1.85, 2.61)
<0.001
HDL-C (mmol/L)
1.27 (1.09, 1.55)
1.22 (1.09, 1.42)
1.32 (1.06, 1.55)
1.35 (1.11, 1.58)
0.030
Triglycerides (mmol/L)
1.51 (1.06, 2.19)
2.00 (1.48, 2.70)
1.51 (1.08, 2.01)
1.15 (0.94, 1.51)
<0.001
Statin usage (%)
249 (88.6)
80 (86)
82 (87.2)
87 (92.6)
0.326
C-reactive protein (mg/L)
3.0 (1.5, 5.3)
3.4 (1.9, 6.1)
3.0 (1.4, 4.9)
2.4 (1.2, 5.2)
0.091
eGFR (ml/min/1.73 m2)
66.5 ± 18.6
71.2 ± 17.7
65.3 ± 17.4
63.0 ± 20.0
0.008
Ankle brachial index
0.77 ± 0.21
0.78 ± 0.20
0.76 ± 0.21
0.76 ± 0.21
0.744
Hypertension (%)
266 (94.7)
88 (94.6)
90 (95.7)
88 (93.6)
0.810
Diabetes mellitus (%)
131 (46.6)
49 (52.7)
40 (42.6)
42 (44.7)
0.343
RAAS blockage (%)
214 (76.2)
69 (74.2)
76 (80.9)
69 (73.4)
0.421
Smoking – active (%)
103 (36.7)
40 (43.5)
29 (31.5)
34 (36.2)
0.239
Data are mean ± SD or median (25,75 percentile) or n (%). CPP-II, secondary calciprotein particles; eGFR, estimated glomerular filtration rate according to CKD-EPI equation; HbA1c, glycated hemoglobin A1c, HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; RAAS, renin angiotensin aldosterone system. Differences were analyzed by ANOVA, Kruskal-Wallis and Chi-Square Test between CPP-II size tertiles as appropriate. An alpha-level of p < 0.05 (two-tailed) was considered statistically significant.
In univariable correlation analyses (Table 2A), CPP-II size was associated with patient age and lower BMI. Furthermore, we observed an inverse association between CPP-II size and LDL-cholesterol as well as fasting triglycerides. CPP-II size showed a weak association with the patients' C-reactive protein (CRP) level. CPP-II size was inversely linked to eGFR. Patients’ HbA1c showed no correlation with CPP-II size. In multivariable stepwise regression analysis, only BMI, LDL-C, triglycerides, CRP, and eGFR remained statistically linked to CPP-II size as shown in Table 2B.
Table 2(A) Pearson correlation analyses for CPP-II size. (B) Cox regression multivariable analyses final model for univariable associations for CPP-II size.
Table 2(A) Pearson correlation analyses for CPP-II size. (B) Cox regression multivariable analyses final model for univariable associations for CPP-II size.
3.3 CPP-II size and atherosclerosis
CPP-II particles were equally distributed between asymptomatic patients and those with claudication symptoms (p = 0.374). The size of CPP-II was furthermore not associated with the patients’ ankle-brachial index in those without media sclerosis (p = 0.217, N = 196) according to ankle-brachial index categories. The presence of additional known coronary and/or carotid artery disease did not influence CPP-II size (p = 0.551) as shown in Fig. 1A. However, larger CPP-II were measured in PAD patients with media sclerosis (p = 0.043) as shown in Fig. 1B.
Fig. 1Association of atherosclerotic burden (A) and presence of mediasclerosis (B) with CPP-II size.
During the study, 35.2% of patients died after a median of 8.8 (6.2–9.0) years, 55% of which due to a cardiovascular cause. CPP-II size was significantly associated with long-term all-cause mortality (HR 1.56 (95% CI 1.25–1.97, p < 0.001). The association of CPP-II size and mortality remained significant after multivariable adjustment for cardiovascular risk factors including patient age, sex, BMI, HbA1c, LDL-cholesterol, SBP, smoking status, eGFR, and CRP (HR 1.34 (1.02–1.76), p = 0.035) as shown in Table 3. This association sustained further adjustment for media sclerosis (HR 1.33 (1.01–1.74), p = 0.039; Table 3). Significant associations between CPP-II size and cardiovascular mortality were seen in univariable analysis (HR 1.66 (1.23–2.26), p < 0.001) and after multivariable adjustment for cardiovascular risk factors (HR 1.51 (1.05–2.18), p = 0.026) as well as media sclerosis (HR 1.52 (1.05–2.20), p = 0.026) as shown in Table 3. Fig. 2 depicts the Kaplan-Meier curves for all-cause (2A) and cardiovascular survival (2B) according to CPP-II size tertiles.
Table 3Multivariable adjusted model of mortality by CPP-II size.
All-cause mortality
Cardiovascular mortality
Hazard ratio (CI)
p-value
Hazard ratio (CI)
p-value
Unadjusted model
1.56 (1.25–1.97)
<0.001
1.66 (1.23–2.26)
<0.001
Multivariable model
1 + CV risk factors
1.34 (1.02–1.76)
0.035
1.51 (1.05–2.18)
0.026
2 + media sclerosis
1.33 (1.01–1.74)
0.039
1.52 (1.05–2.20)
0.026
Cox regression analyses for all-cause and cardiovascular mortality by CPP-II size. HR, hazard ratio; CI, 95% confidence interval; cardiovascular (cv) risk factors included age, sex, BMI, SBP, smoking, LDL-C, eGFR, HbA1c, and CRP. CPP-II, secondary calciprotein particles; CRP – c reactive protein, eGFR – estimated glomerular filtration rate according to CKD-EPI; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure; smoking – smoking status. Multivariable model were adjusted stepwise adding the shown co-variates.
The main finding of this study is that CPP-II size associates with all-cause mortality as well as cardiovascular mortality in PAD patients. CPP-II size was associated with commonly recognized cardiovascular risk factors such as LDL-cholesterol, triglycerides, and eGFR in univariable analysis in PAD patients, all of which are associated with a pro-inflammatory state [
Use of measures of inflammation and kidney function for prediction of atherosclerotic vascular disease events and death in patients with CKD: findings from the CRIC study.
]. However, CPP-II size remained an independent risk factor for mortality in multivariable analysis. This might suggest that large CPP-II contribute to systemic inflammation and thus vascular damage, but are independent of other pro-inflammatory factors studied here. Consistent with this notion, it has been observed that CPP induce inflammation, and inflammation contributes to vascular calcification [
Assessment of the mineral buffering capacity of serum is an emerging prognostic marker for cardiovascular and overall mortality. Several different but probably complementary parameters of this mineral buffering capacity can be measured in vitro. Until recently, the majority of studies reported associations between the time for half-maximum transition from primary (CPP-I) to secondary (CPP-II) calciprotein particles. [
], Associations between the size of CPP-II and patient characteristics and outcomes have been reported in two studies so far, in a cohort of advanced CKD [
Associations of serum calciprotein particle size and transformation time with arterial calcification, arterial stiffness, and mortality in incident hemodialysis patients.
]. In both reports as well as in our current study, 3D-DLS was used to study CPP-II size, rendering these studies likely comparable from a methodological point of view. We found a lower CPP-II size in PAD patients (∼190 nm) compared to results of patients with advanced CKD without (∼210 nm) and with (∼370 nm) prevalent vascular calcification [
], PAD patients studied here showed larger CPP-II sizes (∼170 nm vs. 190 nm). Although these comparisons are only indirect and have to be interpreted with great caution, this observation still fits well to the clinically perceived degree of overall morbidity and also differing survival of these patient groups.
We found larger CPP-II sizes in PAD patients with prevalent mediasclerosis, whereas the group of Chen and colleagues reported divergent results on the association between CPP-II size and vascular calcification and/or stiffness in CKD patients [
Associations of serum calciprotein particle size and transformation time with arterial calcification, arterial stiffness, and mortality in incident hemodialysis patients.
]. So currently, the relationship between CPP-II size and already existing vascular calcification is unclear. It is important to note that vascular calcification and high arterial stiffness can be viewed as manifestations which occur late in the process of vascular damage. Some patients who experience high mineral stress and have low mineral buffering capacity leading to vascular damage may experience cardiovascular events and die prematurely, so the rather weak and inconsistent association between CPP-II size and vascular calcification might be the result of a survivorship bias. Nevertheless, large CPP-II size was found to associate with mortality in the PAD patients studied here as well as hemodialysis patients reported earlier [
Associations of serum calciprotein particle size and transformation time with arterial calcification, arterial stiffness, and mortality in incident hemodialysis patients.
]. Therefore, CPP-II size is not only a prognostic marker for mortality in PAD patients, but CPP-II probably mediate vascular damage directly. It is worthwhile to notice that the PAD patients in this cohort exhibited a narrow eGFR range, excluding patients with advanced kidney function impairment. Therefore, mineral buffering capacity appears to be a relevant prognostic marker also in patients with preserved kidney function, which is in line with previous cohorts [
This study has several limitations: First, further longitudinal studies in PAD patients are desirable to corroborate the findings reported here. Second, in an observational study, treatment effect over time might influence the individual patient outcome. Third, the diagnosis of media sclerosis was based on functional patient assessment and computer tomography angiography scans were not performed for the study.
In conclusion, mineral buffering capacity as determined by CPP-II size was associated with cardiovascular and all-cause mortality in PAD patients.
CRediT authorship contribution statement
Marija Bojic: Conceptualization, Methodology, Formal analysis, Writing - Original Draft, Writing - Review & Editing, Visualization. Daniel Cejka: Supervision, Writing - Review & Editing. Bernhard Bielesz: Resources, Writing - Review & Editing. Gerit-Holger Schernthaner: Conceptualization,Validation, Investigation, Resources, Writing - Original Draft, Writing - Review & Editing, Project administration. Clemens Höbaus: Conceptualization, Methodology,Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References
Jahnen-Dechent W.
Smith E.R.
Nature's remedy to phosphate woes: calciprotein particles regulate systemic mineral metabolism.
Associations of serum calciprotein particle size and transformation time with arterial calcification, arterial stiffness, and mortality in incident hemodialysis patients.
Guidelines for the management of arterial hypertension: the task force for the management of arterial hypertension of the European society of hypertension (ESH) and of the European society of cardiology (ESC).
Use of measures of inflammation and kidney function for prediction of atherosclerotic vascular disease events and death in patients with CKD: findings from the CRIC study.
00083-7&id=ga1.jpg){kind=link}
00083-7&id=gr1.jpg){kind=link}
00083-7&id=gr2.jpg){kind=link}