Aim: Insulin resistance and type 2 diabetes are independent risk factors for cardiovascular diseases. Levels of C-peptide

are increased in these patients and its role in the atherosclerosis progression was studied in vitro and in vivo over the

past years. To evaluate the possible use of C-peptide as cardiovascular biomarkers, we designed an observational study

in which we enrolled patients with mono- or poly-vascular atherosclerotic disease.

Methods: We recruited 431 patients with stable atherosclerosis and performed a yearly follow-up to estimate the

cardiovascular and total mortality and cardiovascular events.

Results: We performed a mean follow-up of 56months on 268 patients. A multivariate Cox analysis showed that

C-peptide significantly increased the risk of cardiovascular mortality [Hazard Ratio: 1.29 (95% confidence interval:

1.02-1.65, p<0.03513)] after adjustment for age, sex, diabetes treatment, estimated glomerular filtration rate and

known diabetes status. Furthermore, levels of C-peptide were significantly correlated with metabolic parameters and

atherogenic factors.

Conclusion: C-peptide was associated with cardiovascular mortality independently of known diabetes status in a cohort

of patients with chronic atherosclerotic disease. Future studies using C-peptide into a reclassification approach might be

undertaken to consider its potential as a cardiovascular disease biomarker

Ballanti, Marta

Cardellini, Marina

Cardolini, Iris

Davato, Francesca

Farcomeni, Alessio

Federici, Massimo

Grappasonni, Giulia

Guglielmi, Valeria

Ippoliti, Arnaldo

Menghini, Rossella

Morelli, Monica

Pecchioli, Chiara

Porzio, Ottavia

Rizza, Stefano

English

Insulin resistance and type 2 diabetes are independent risk factors for cardiovascular diseases. Levels of C-peptide are increased in these patients and its role in the atherosclerosis progression was studied in vitro and in vivo over the past years. To evaluate the possible use of C-peptide as cardiovascular biomarkers, we designed an observational study in which we enrolled patients with mono- or poly-vascular atherosclerotic disease

Cardellini, M

Farcomeni, A

Ballanti, M

Morelli, M

Davato, F

Cardolini, I

Grappasonni, G

Rizza, S

Guglielmi, V

Porzio, O

Pecchioli, C

Menghini, R

Ippoliti, A

Federici, M

Name not available

C-peptide: A predictor of cardiovascular mortality in subjects with established atherosclerotic disease

CARDELLINI, MARINA

FARCOMENI, ALESSIO

BALLANTI, MARTA

DAVATO, FRANCESCA

RIZZA, STEFANO

PORZIO, OTTAVIA

PECCHIOLI, CHIARA

MENGHINI, ROSSELLA

IPPOLITI, ARNALDO

FEDERICI, MASSIMO

Aim: Insulin resistance and type 2 diabetes are independent risk factors for cardiovascular diseases. Levels of C-peptide
are increased in these patients and its role in the atherosclerosis progression was studied in vitro and in vivo over the
past years. To evaluate the possible use of C-peptide as cardiovascular biomarkers, we designed an observational study
in which we enrolled patients with mono- or poly-vascular atherosclerotic disease.
Methods: We recruited 431 patients with stable atherosclerosis and performed a yearly follow-up to estimate the
cardiovascular and total mortality and cardiovascular events.
Results: We performed a mean follow-up of 56months on 268 patients. A multivariate Cox analysis showed that
C-peptide significantly increased the risk of cardiovascular mortality [Hazard Ratio: 1.29 (95% confidence interval:
1.02-1.65, p<0.03513)] after adjustment for age, sex, diabetes treatment, estimated glomerular filtration rate and
known diabetes status. Furthermore, levels of C-peptide were significantly correlated with metabolic parameters and
atherogenic factors.
Conclusion: C-peptide was associated with cardiovascular mortality independently of known diabetes status in a cohort
of patients with chronic atherosclerotic disease. Future studies using C-peptide into a reclassification approach might be
undertaken to consider its potential as a cardiovascular disease biomarker

FARCOMENI, Alessio

Archivio della ricerca- Università di Roma La Sapienza

https://doi.org/10.1177/1479164117710446Diabetes & Vascular Disease Research 1 –5© The Author(s) 2017Reprints and permissions: sagepub.co.uk/journalsPermissions.navDOI: 10.1177/14791641 7710446journals.sagepub.com/home/dvrIntroductionInsulin resistance is a hallmark and risk factor for cardio-vascular atherosclerotic disease and its complications such as myocardial infarction and stroke.1,2 Insulin regulates several anabolic and catabolic processes that influence lipid and glucose metabolism with an impact on cardiovas-cular homeostasis. The interaction among insulin, its met-abolic functions and cardiovascular events is an intensive area of research given the implications on the potential discovery of new biomarkers and therapeutic targets.3Among many conventional biomarkers for cardiovascu-lar disease, C-peptide is a potential unifier for insulin resist-ance and defective insulin secretion with cardiovascular diseases (CVD). Recent studies suggested that C-peptide exhibit biological activity, induces vascular smooth muscle cells migration and infiltration of inflammatory cells in in vitro studies while promoting atherosclerosis progression in a mouse model.4–7 Finally, the LUdwigshafen RIsk and Cardiovascular Health (LURIC) study identified an asso-ciation between C-peptide and cardiovascular mortality.8 C-peptide: A predictor of cardiovascular mortality in subjects with established atherosclerotic diseaseMarina Cardellini1,2, Alessio Farcomeni3, Marta Ballanti1,  Monica Morelli4, Francesca Davato1, Iris Cardolini1,  Giulia Grappasonni1, Stefano Rizza1,2, Valeria Guglielmi1,  Ottavia Porzio4,5, Chiara Pecchioli2, Rossella Menghini1,  Arnaldo Ippoliti4 and Massimo Federici1,2AbstractAim: Insulin resistance and type 2 diabetes are independent risk factors for cardiovascular diseases. Levels of C-peptide are increased in these patients and its role in the atherosclerosis progression was studied in vitro and in vivo over the past years. To evaluate the possible use of C-peptide as cardiovascular biomarkers, we designed an observational study in which we enrolled patients with mono- or poly-vascular atherosclerotic disease.Methods: We recruited 431 patients with stable atherosclerosis and performed a yearly follow-up to estimate the cardiovascular and total mortality and cardiovascular events.Results: We performed a mean follow-up of 56 months on 268 patients. A multivariate Cox analysis showed that C-peptide significantly increased the risk of cardiovascular mortality [Hazard Ratio: 1.29 (95% confidence interval: 1.02−1.65, p < 0.03513)] after adjustment for age, sex, diabetes treatment, estimated glomerular filtration rate and known diabetes status. Furthermore, levels of C-peptide were significantly correlated with metabolic parameters and atherogenic factors.Conclusion: C-peptide was associated with cardiovascular mortality independently of known diabetes status in a cohort of patients with chronic atherosclerotic disease. Future studies using C-peptide into a reclassification approach might be undertaken to consider its potential as a cardiovascular disease biomarker.KeywordsCardiovascular diseases, atherosclerosis, biomarkers, C-peptide1 Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy2Center for Atherosclerosis, Policlinico Tor Vergata, Rome, Italy3 Department of Public Health and Infectious Diseases, University of Rome La Sapienza, Rome, Italy4 Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy5 Medical Laboratory Unit, Bambino Gesù Children’s Hospital and Research Institute, IRCCS, Rome, ItalyCorresponding author:Massimo Federici, Department of Systems Medicine, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy. Email: federicm@uniroma2.it710446 DVR0010.1177/1479164117710446Diabetes & Vascular Disease ResearchCardellini et al.research-article2017Original Article2 Diabetes & Vascular Disease Research 00(0)In this study, we identified C-peptide as an important risk predictor for cardiovascular mortality in subjects with sta-ble atherosclerosis.MethodsDescription of patientsFour hundred and thirty-one patients with mono- or poly-vascular atherosclerosis disease were recruited at the Center for Atherosclerosis of the Policlinico Tor Vergata in Rome, for a clinical and therapeutical assessment. Patients were enrolled in the Tor Vergata Atherosclerosis Registry (TVAR) from 2007, listed on the International Standard Randomised Controlled Trial Number (ISRCTN) registry with study ID ISRCTN42405215. Patients were diagnosed with established and documented atheroscle-rotic vascular disease at the atherosclerosis ambulatory clinic that includes cardiology, diabetology and vascular surgery specialists. All the patients have had a major CardioVascular (CV) event or undergone a vascular pro-cedure for significant vascular stenosis. Exclusion criteria included liver disease, renal insufficiency, heart failure, coagulopathy or any other severe systemic disease. Subjects were also excluded if they had positive blood tests for HIV, hepatitis B or hepatitis C.Cerebrovascular disease was defined clinically by doc-umented history of a previous cerebrovascular disease event or by documented carotid plaques (50% luminal obstruction). Coronary artery disease (CAD) was defined by documented history of a previous acute myocardial infarction, presence of clinical signs and coronary angiog-raphy. Peripheral vascular disease was defined by a history of intermittent claudication, angiographic documentation of atherosclerotic luminal obstruction of the peripheral arteries or a history of a peripheral arterial intervention for atherosclerotic disease (angioplasty or surgery).The follow-up was performed every year by phone interview. The primary endpoint was major cardiovascular events (cardiovascular death, nonfatal stroke, nonfatal myocardial infarction and peripheral vascular surgical pro-cedures) and the secondary endpoint was death for any other diseases. Information on nonfatal events was sought yearly from study participants by phone interviews and confirmed by review of hospital records. In case of fatal events, information was obtained from their primary care physicians or from death certificates. In 2014, we per-formed an analysis on the status of the patients who answered to the follow-up. Prospective data were available for 268 subjects for an average follow-up of 56 months, 80 patients did not answer to phone interview, 32 patients had missing data on C-peptide and 51 patients were excluded because followed for less than 1 year. At recruitment, all patients underwent thorough medical history, clinical examination and anthropometric measurements.Diabetes treatmentForty-one of the 268 subjects with available follow-up were under diabetes treatments: 10 were under combined insulin and metformin treatment, 3 of them were on insulin therapy, 13 were on metformin and 15 on secretagogues.Informed consent and ethics committee approvalAn informed written consent was obtained from all partici-pants. The study was approved by the local ethics commit-tee and the reported investigations were carried out in accordance with the principles of the Declaration of Helsinki as revised in 2000.Laboratory measurementsFasting glucose, total cholesterol, high-density lipoprotein and low-density lipoprotein cholesterol, triglycerides, creatinine, urea, aminotransferases and gamma-glutamyl-transferase were assessed by standard immunoenzymatic methods. Insulin and C-peptide levels were measured by immunochemiluminescence assay (ADVIA Centaur XP, Siemens Healthineers Diagnostics) whereas haemoglobin A1c percentage by high-performance liquid chromatogra-phy method. High-sensitivity C-reactive protein was measured by nephelometric immunoassay (Dimension Vista 1500, Siemens Healthineers Diagnostics).Statistical analysisPatients’ characteristics were reported as mean and standard deviation (SD) for continuous variables and frequencies and percentages for categorical ones. Variables correlations were carried out using Spearman correlation. Time-to-event data were analysed by means of log-rank test or univariate Cox models for categorical or continuous variables, respec-tively. Significance of C-peptide was also assessed at multi-variate Cox modelling, after adjusting for possible relevant confounders. The final multivariable Cox model was chosen with forward stepwise regression based on Akaike’s infor-mation criterion (AIC), which guarantees a final parsimoni-ous model with best prediction performance. Of note, all potential predictors were considered for final inclusion.Final predictive scores were obtained as linear combina-tions of the most relevant predictors, where the weights were obtained by rounding the Cox regression coefficients. Predictive performance was assessed by means of time-dependent ROC curves and net reclassification index (NRI).ResultsClinical and biochemical characteristics of the 431 patients enrolled in the TVAR are shown in Table 1. We performed Cardellini et al. 3a follow-up for all-cause and cardiovascular event and mortality for a mean of 56.17 ± 28.52 months on 268 patients. During follow-up, 66 nonfatal cardiovascular events (24.6%) and 44 deaths (16.5%) occurred, 20 of which were due to cardiovascular causes (7.5%). Clinical and biochemical characteristics of the 268 patients are summarized in Table 1. Among the several parameters and biochemical variables available, only age was found inde-pendently associated with cardiovascular death with uni-variate Cox analysis. On the basis of this result, we performed a bivariate Cox analysis, after adjustment for age, and we found that C-peptide and gender were inde-pendently associated with cardiovascular death (Table 1).A multivariate Cox analysis showed that C-peptide, included as continuous variable, increased the risk in car-diovascular mortality per SD by [Hazard Ratio: 1.38 (95% confidence interval (CI): 1.057−1.801, p = 0.018)] after adjustment for age, sex, antidiabetic therapy, estimated glomerular filtration rate (eGFR) and known diabetes sta-tus, suggesting that C-peptide is a cardiovascular mortality predictor independently from a history of diabetes (Table 2). Effects of C-peptide were not significantly different between men and women (p = 0.208 for cardiovascular mortality and p = 0.71 for total mortality).In our cohort, we found that levels of C-peptide were significantly correlated with metabolic parameters such as fasting plasma glucose (R = 0.327, p < 0.0001; adjusted for sex and age R = 0.333, p < 0.0001), homeostatic model assessment for insulin resistance (HOMA IR; R = 0.693, p < 0.0001; adjusted for sex and age R = 0.690, p < 0.0001), body mass index (BMI; R = 0.402, p < 0.0001; adjusted for sex and age R = 0.394, p < 0.0001) and insulin-like growth factor 1 (IGF1; R = 0.206, p < 0.0001; adjusted for sex and age R = 0.195, p = 0.002). Levels of C-peptide were also significantly correlated with atherogenic factors such as high-density lipoprotein (HDL; R = −0.247, p < 0.0001; adjusted for sex and age R = −0.228, p < 0.001), triglycer-ides (r = 0.302, p < 0.0001; adjusted for sex and age R = 0.302, p < 0.0001) and high-sensitivity C-reactive pro-tein (hs-CRP) (R = 0.246, p < 0.0001; adjusted for sex and age R = 0.191, p < 0.0001).Finally, we build a predictive score for CVD mortality as follows: we gave −2 points to females, 0.25 points for each year of age, 0.33 points for each unit of C-peptide, −0.1 points for each unit of BMI and 0.002 points for each unit of hs-CRP. The resulting score predicted CVD at 60 months with an AUC of 0.920 (95% CI: 0.778–1.000), p < 0.0001. It shall be noted that a score which did not Table 1. Clinical characteristics of the patients included in TVAR registry and the patients included in the follow-up analysis.TVAR registry TVAR follow-up HR p HR adjusted for agepN (M/F) 431 (323/108) 268 (200/68) 0.15 0.061 0.12 0.036Age (years) 70.78 ± 7.72 70.93 ± 7.55 1.23 <0.001 – –BMI (kg/m2) 26.55 ± 4.01 26.49 ± 4.12 0.91 0.12 0.93 0.331Waist circumference (cm) 97.57 ± 11.10 97.75 ± 11.42 1.01 0.52 1.03 0.245Systolic blood pressure (mmHg) 137.70 ± 19.10 138.14 ± 19.21 1.01 0.57 0.99 0.899Diastolic blood pressure (mmHg) 80.67 ± 9.66 80.76 ± 10.21 1.02 0.36 1.04 0.143Total cholesterol (mg/dL) 185.59 ± 46.18 184.78 ± 44.26 0.99 0.19 0.99 0.320HDL cholesterol (mg/dL) 42.87 ± 13.16 43.41 ± 12.00 1.00 0.86 0.98 0.441LDL cholesterol (mg/dL) 113.47 ± 39.46 112.34 ± 36.23 0.99 0.15 0.99 0.250Triglycerides (mg/dL) 158.20 ± 130.30 150.74 ± 94.90 1.00 0.82 1.00 0.087Fasting plasma glucose (mg/dL) 106.48 ± 30.67 107.89 ± 28.52 1.00 0.94 1.01 0.232Fasting plasma insulin (uU/mL) 13.37 ± 13.46 13.01 ± 14.55 1.00 0.54 0.99 0.264HbA1c (mmol/mol) 41.32 ± 9.4 41.24 ± 8.27 1.02 0.43 1.04 0.265C-peptide (ng/mL) 3.17 ± 1.66 3.01 ± 1.19 1.17 0.15 1.36 0.009HOMA IR 3.82 ± 4.32 3.44 ± 3.2 1.01 0.86 1.10 0.110hs-CRP (mg/dL) 4.12 ± 10.60 4.49 ± 11.35 1.00 0.75 1.00 0.477Fibrinogen (mg/dL) 399.59 ± 399.37 357.12 ± 84.84 1.00 0.08 1.00 0.057ESR (mm/h) 25.34 ± 23.71 23.46 ± 22.95 1.01 0.81 1.00 0.744TNF-alpha (pg/mL) 11.34 ± 16.27 8.94 ± 9.93 1.01 0.73 0.99 0.883Glucose Tolerance (0/1/2/3) % 28.6/33.3/17.2/18.1 32.1/32.4/19.2/16.3 1.41 0.12 1.35 0.199Smoking (no/ex/yes) % 29.3/29.0/41.7 23.5/30.9/45.6 0.85 0.82 1.01 0.978Hypertension (no/yes) % 19.0/81.0 13.1/86.9 0.67 0.48 0.49 0.207TVAR: Tor Vergata Atherosclerosis Registry; BMI: body mass index; HDL: high-density lipoprotein; LDL: low-density lipoprotein; HbA1c: haemoglo-bin A1c; HOMA IR: homeostatic model assessment for insulin resistance; hs-CRP: high-sensitivity C-reactive protein; ESR: erythrocyte sedimenta-tion rate; TNF-alpha: tumour necrosis factor-alpha.Hazard Ratio for cardiovascular mortality is reported.4 Diabetes & Vascular Disease Research 00(0)include C-peptide would yield an Area Under Curve (AUC) of only 0.865 (95% CI: 0.738−0.979). The ½ NRI of C-peptide was 0.281, which was very large despite not being significant (p = 0.153).When we analysed all-cause mortality, we found that C-peptide did not have a significant effect, as HR of C-peptide was estimated as 1.091 (95% CI: 0.900–1.323, p = 0.376), after adjusting for age, sex, antidiabetic ther-apy, eGFR and known diabetes status.DiscussionThe major findings from this analysis of the TVAR con-cern the role of C-peptide as a biomarker for cardiovascu-lar mortality and its potential use to improve cardiovascular prediction. In our study, C-peptide resulted the most sig-nificant predictor of cardiovascular mortality in subjects with established atherosclerosis and high cardiovascular risk. Previous studies suggested a role for C-peptide as biomarker for cardiovascular and non-cardiovascular mor-tality.8 In our analysis, we did not find an association with non-cardiovascular mortality, a result potentially depend-ing on the different inclusion criteria between TVAR and LURIC study.The association between cardiovascular mortality and levels of C-peptide might be explained by the direct role of the C-peptide in promoting atherogenesis. Marx and col-laborators showed that C-peptide may facilitate the recruit-ment of inflammatory cells into early lesions7 and promote lesion progression by inducing smooth muscle cell prolif-eration5 in vitro. Furthermore, ApolipoproteinE (ApoE)-deficient mice on a high fat diet treated with C-peptide injections for 12 weeks showed more macrophages and smooth muscle cells in atherosclerotic lesions compared with controls.4Our study has some limitations; in fact, our database did not include information on the status of microvascu-lar complications; therefore, we cannot assess whether C-peptide levels increase as a consequence of reduced renal excretion or extrarenal causes. However, adjust-ment for eGFR did not change our results, although it is intriguing to hypothesize that both decline in renal excretion and increased production may lead to increased C-peptide signalling.In conclusion, C-peptide was associated with cardio-vascular mortality independently of known diabetes status in a cohort of patients with mono- or poly-vascular chronic atherosclerotic disease. Future studies using C-peptide into a reclassification approach might be undertaken to consider its potential as a CVD biomarker.AcknowledgementsM.C., M.B., M.M., F.D., I.C., G.G., S.R., V.G., O.P., C.P., R.M. and A.I. performed clinical and biochemical experiments. M.C. and A.F performed statistical analysis, contributed to data inter-pretation, generated tables and wrote the article. M.F. conceived the study and wrote the discussion. All authors discussed the data and commented on the article before submission.Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.FundingThis research was in part funded by MIUR PRIN 20123BJ89E, MIUR PRIN 2015MPESJS_004 and Fondazione Roma Non Communicable Diseases 2014 Call to M.F.References 1. Rask-Madsen C and Kahn CR. Tissue-specific insulin sign-aling, metabolic syndrome, and cardiovascular disease. Arterioscler Thromb Vasc Biol 2012; 32: 2052–2059. 2. Rask-Madsen C and King GL. Vascular complications of diabetes: mechanisms of injury and protective factors. Cell Metab 2013; 17: 20–33. 3. Laakso M and Kuusisto J. Insulin resistance and hyper-glycaemia in cardiovascular disease development. Nat Rev Endocrinol 2014; 10: 293–302. 4. Vasic D, Marx N, Sukhova G, et al. C-peptide promotes lesion development in a mouse model of arteriosclerosis. J Cell Mol Med 2012; 16: 927–935.Table 2. Multivariate analysis for cardiovascular mortality and all-cause mortality (also adjusted for known disease status, accompanying antidiabetic therapy and eGFR).Cardiovascular mortality HR 2.5% 97.5% pC-peptide 1.380 1.057 1.801 0.018Age 1.252 1.050 1.492 0.012Gender 0.128 0.034 0.475 0.021All-cause mortality HR 2.5% 97.5% pC-peptide 1.091 0.900 1.323 0.376Age 1.129 1.048 1.217 0.001Gender 0.202 0.072 0.564 0.002Cardellini et al. 5 5. Walcher D, Babiak C, Poletek P, et al. C-Peptide induces vascular smooth muscle cell proliferation: involvement of SRC-kinase, phosphatidylinositol 3-kinase, and extra-cellular signal-regulated kinase 1/2. Circ Res 2006; 99: 1181–1187. 6. Walcher D, Aleksic M, Jerg V, et al. C-peptide induces chemotaxis of human CD4-positive cells: involvement of pertussis toxin-sensitive G-proteins and phosphoinositide 3-kinase. Diabetes 2004; 53: 1664–1670. 7. Marx N, Walcher D, Raichle C, et al. C-peptide colocal-izes with macrophages in early arteriosclerotic lesions of diabetic subjects and induces monocyte chemot-axis in vitro. Arterioscler Thromb Vasc Biol 2004; 24: 540–545. 8. Marx N, Silbernagel G, Brandenburg V, et al. C-peptide levels are associated with mortality and cardiovascular mor-tality in patients undergoing angiography: the LURIC study. Diabetes Care 2013; 36: 708–714.

C-peptide: a predictor of cardiovascular mortality in subjects with established atherosclerotic disease

https://iris.uniroma1.it/retrieve/handle/11573/1007594/754379/Cardellini_C-peptide_2017.pdf

C-peptide: a predictor of cardiovascular mortality in subjects with established atherosclerotic disease

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