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Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab®: A prospective cohort study
Nutrition, Metabolism & Cardiovascular Diseases (xxxx) xxx, xxx
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Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study Matteo Nicola D. Di Minno a,*, Marco Gentile b, Alessandro Di Minno c,d, Gabriella Iannuzzo b, Ilenia Calcaterra b, Alessio Buonaiuto b, Maria D. Di Taranto e, Carola Giacobbe e, Giuliana Fortunato e, Paolo O.F. Rubba b a
Department of Translational Medical Sciences, Federico II University, Naples, Italy Department of Clinical Medicine and Surgery, Federico II University, Naples, Italy Department of Pharmacy, Federico II University, Naples, Italy d Unit of Metabolomics and Cellular Biochemistry of Atherothrombosis, Centro Cardiologico Monzino IRCCS, Milan, Italy e Department of Molecular Medicine e Medical Biotechnologies, Federico II University, Naples, Italy b c
Received 24 December 2019; received in revised form 26 February 2020; accepted 28 February 2020 Handling Editor: D. Noto Available online - - -
KEYWORDS Carotid stiffness; Familial hypercholesterolemia; PCSK9 inhibitors
Abstract Background and aim: Protein convertase subtilisin kexin type 9 (PCSK-9) inhibitors demonstrated efficacy in cholesterol reduction and in the prevention of cardiovascular events. We evaluated changes in lipid profile and carotid stiffness in patients with familial hypercholesterolemia during 12 weeks of treatment with a PCSK-9 inhibitor, Evolocumab. Methods and results: Patients with familial hypercholesterolemia starting a treatment with Evolocumab were included. Total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), small dense LDL (assessed by LDL score) and carotid stiffness were evaluated before starting treatment with Evolocumab and during 12 weeks of treatment. Twenty-five subjects were enrolled (52% males, mean age 51.5 years). TC and LDL-C were reduced of 38% and 52%, respectively during treatment, with LDL score reduced of 46.1%. In parallel, carotid stiffness changed from 8.8 (IQR: 7.0e10.4) m/sec to 6.6 (IQR: 5.4e7.5) m/sec, corresponding to a median change of 21.4% (p < 0.0 01), with a significant increase in carotid distensibility (from 12.1, IQR: 8.73e19.3 kPA1 103 at T0 to 21.8, IQR: 16.6e31.8 kPA1 103 at T12w) corresponding to a median change of 62.8% (p < 0.001). A multivariate analysis showed that changes in LDL score were independently associated with changes in carotid stiffness (b Z 0.429, p Z 0.041). Conclusion: Small dense LDL reduction, as assessed by LDL score, is associated with changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab. ª 2020 Published by Elsevier B.V. on behalf of The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University.
Introduction
* Corresponding author. Department of Translational Medical Sciences Federico II University, Via S. Pansini 5, 80131 Naples, Italy. Fax: þ390817464323. E-mail address: [email protected] (M.N.D. Di Minno).
Several studies consistently showed that increased levels of low-density lipoprotein cholesterol (LDL-C) represent the main causative factor for atherosclerosis development [1]. Patients with high levels of LDL-C exhibit an increased
https://doi.org/10.1016/j.numecd.2020.02.018 0939-4753/ª 2020 Published by Elsevier B.V. on behalf of The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University.
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
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prevalence of subclinical atherosclerosis and a more rapid atherosclerosis progression [2]. This leads to a significantly higher cardiovascular (CV) risk in patients with high LDL-C levels [3]. Increased arterial stiffness is one of the earliest stages of the atherosclerotic process [4] and some studies reported an association between arterial stiffness and incident CV disease and mortality [5e7], particularly in patients with familial hypercholesterolemia (FH) [8]. On the other hand, some recent reports showed that lipid lowering treatment with statins is associated with a significant reduction in arterial stiffness [9,10]. Although for years the gold standard for lipid lowering therapy was represented by statin treatment, the target LDL-C is not always achieved, mainly in patients with very high levels of LDL-C [11]. More recently, PCSK-9 inhibitors showed efficacy in LDL-C reduction and in the prevention from CV events [12,13]. Conflicting results are available about the association between PCSK-9 plasma levels and arterial stiffness [14,15] and only data on 2 patients are available about the effect of PCSK-9 inhibitors on arterial stiffness [16]. The aim of our study was to prospectively assess changes in lipid profile and carotid stiffness in patients with FH before and after the treatment with a PCSK9 inhibitor, Evolocumab.
Methods From December 2017 to December 2018, within the framework of LIPIGEN, a national project on familial dyslipidemia, consecutive patients attending the lipid clinic of the Department of Clinical Medicine and Surgery, Federico II University Hospital with very high levels of LDL-C (above the 95th percentile when compared with a sex- and agematched general population), normal triglyceride levels and probable autosomal dominant transmission of hypercholesterolemia (DUTCH Lipid Clinic Network score 6) in the family were screened for inclusion in the present study [17]. The major inclusion criterion was the eligibility of patients to start a treatment with PCSK-9 according to ESC/ EAS guidelines [18]. Exclusion criteria were: age < 18 years, inability to understand or sign the informed consent, high level of transaminases (>3x upper normal limit), hypertriglyceridemia (>150 mg/dl), end-stage renal disease (filtration rate < 30 ml/min/mq), current malignant disease or a diagnosis of malignancy in the 2 years prior to the first visit, previous exposure to PCSK-9 inhibitors, presence of hypercholesterolemia secondary to other causes (hypothyroidism, hormone therapies, corticosteroids etc.). Patients with a clinical picture or family history of familial combined hyperlipidemia were excluded [19,20]. Patients enrolled in the study continued the ongoing lipid lowering therapy and added PCSK-9 inhibitor (Evolocumab 140 mg subcutaneous injection every 14 days).
M.N.D. Di Minno et al.
Study protocol After informed consent, a detailed medical history was recorded for each patient. Data about age, gender, previous and/or current medical conditions, current and past lipid lowering therapy, vascular risk factors were collected. Height was measured to the nearest 0.1 cm. Body weight was assessed by using an electronic beam scale with digital readout to the nearest 0.1 kg after emptying the bladder and with the subjects standing barefoot and wearing light indoor clothing. Body mass index (BMI) was calculated as body weight/(height2). Clinical diagnosis of FH was achieved using Dutch Lipid Clinic Network Score and genetic testing was performed to assess major causative mutations, including LDLR, APOB and PCSK9 genes as previously described [21e23]. Blood laboratory parameters The following blood values were evaluated at baseline (before starting PCSK-9 inhibitor) and 12 weeks (T12w) after treatment with Evolocumab: total cholesterol (TC), triglycerides (TG), HDL cholesterol (HDL-C), LDL-C, smalldense LDL (sd-LDL), creatininemia, AST, ALT, CPK, glycaemia. LDL particles separation was performed by electrophoretic Lipoprint System (Quantimetrix Inc., Redondo Beach, CA). The proportion of sd-LDL particles (subfractions 3e7) to the whole LDL area (subfractions 1e7) was also calculated in our sample and expressed as LDL score, with higher score values representing a higher sd-LDL particles content [24]. In addition, mean LDL particle diameter was calculated on the basis of the different areas under the curve of the 7 LDL species with different electrophoretic mobility [25]. Carotid stiffness Patients were asked to abstain from alcohol, tobacco and caffeine for at least 12 h on the day of examination. All study procedures were performed in a temperaturecontrolled room (23 C), after 10 min of rest in supine position. Carotid stiffness was evaluated in each patient before starting PCSK-9 inhibitor (T0) and repeated after 12 weeks of treatment (T12w) by the same operator blinded for ongoing treatment and cholesterol levels. To validate results on the long-term, carotid stiffness was evaluated also after 104 weeks of treatment (T104w). Common carotid artery scans (25 frames/s) were obtained by high-resolution ultrasound with a 10 MHz linear array transducer (MyLab25; ESAOTE, Florence, Italy) by a trained operator. Longitudinal scans were acquired from each common carotid artery (1 cm proximal to the carotid bulb in a region 1-cm wide and free of plaques) and automatically analyzed with Carotid Studio
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
Genotype and PCSK-9 inhibitors
(Cardiovascular Suite, Carotid studio, QUIPU Srl, Pisa, Italy), a well validated system based on contour tracking algorithm [26]. Arterial interfaces were automatically detected, with estimation of instantaneous mean diameter as the distance between far and near mediaeadventitia interfaces. The following parameters were obtained: carotid distension (DD), that is the stroke change in diameter, calculated as the difference between the systolic and diastolic diameter values; carotid distensibility coefficient Z DA/(A*DP), where A represents the diastolic lumen area, evaluated from the diameter values (assuming the cross-section of the artery to be circular), DA represents the stroke change in lumen area, DP the local pulse pressure obtained by tonometry. Carotid stiffness was calculated according to the MoenseKorteweg equation [27]. In particular, carotid distensibility was converted into carotid stiffness by using the equation: PWV Z [(DP )A)/ (DA) r)](1/2) where r is the blood density. This formula allows carotid stiffness to be obtained as carotid stiffness Z (r ) carotid distensibility) (1/2), expressed in m/s. The reproducibility of this scanning protocol was evaluated on a representative sample of 5 subjects randomly selected from the study population within 1 week from the first examination and the r value resulted Z 0.91. Carotid distensibility was defined as low if < 15 kPa1 x 103 [28]. Statistical analysis Statistical analysis was performed with the IBM SPSS 22 system (SPSS Inc., Chicago, IL, USA). Continuous data were expressed as mean standard deviation (SD) or as median value with inter-quartile range (IQR). The t-test was performed to compare continuous variables for paired samples and for independent samples. In case of values with a skewed non-Gaussian distribution, ManneWhitney U test was used to compare means. The c2 test or Fisher’s exact test were used to compare categorical variables. Relationships between continuous variables were examined using simple regressions Spearman’s correlation (rho) for nonparametric variables. All results were expressed as 2tailed values, p values < 0.05 being statistically significant. To evaluate potential sources of heterogeneity, a sensitivity analysis was performed stratifying patients according to major clinical and demographic characteristics and according to genotype of LDLR, APOB and PCSK9 genes. To adjust for potential confounders and to make predictions, linear regression analyses (stepwise method) were implemented with changes in carotid stiffness as dependent variable and age, gender, BMI, cardiovascular events, hypertension, diabetes and changes in LDL score as independent variables. Sample size As to sample size, with a pre-defined 25% reduction in carotid stiffness from baseline values to T12w, 22 subjects were needed to obtain an 80% power and a 5% a error. Also
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considering a drop-out risk, we aimed at enrolling 25 subjects in the present study. Results Twenty-five subjects (13 males and 12 females, mean age 51.5 14.5 years) with FH (20 heterozygotes for the mutation of LDLR, 2 compound heterozygotes for 2 different mutations of LDLR, 1 double heterozygote for mutations of LDLR and of PCSK9 and 2 subjects were without major mutation in LDLR, APOB, PCSK9 genes) were enrolled. The mean Dutch Lipid Clinic Network Score was 9.3. Nineteen patients (76%) were under statin treatment, whereas 6 reported statin intolerance. Among the 19 statin-treated patients, 1 was under simvastatin 40 mg, 3 rosuvastatin 40 mg, 5 rosuvastatin 20 mg, 1 pravastatin 40 mg, 1 fluvastatin 40 mg, 5 atorvastatin 40 mg, 3 atorvastatin 80 mg. Thus, 16 patients were under high intensity-statins treatment (atorvastatin 40 mg or 80 mg; rosuvastatin 20 mg or 40 mg), whereas the other 3 patients were under low-intensity statins (pravastatin, fluvastatin and simvastatin) because of intolerance to high intensity-statin. Ezetimibe was present as co-treatment in 16 subjects (68%) and as a stand-alone treatment in the 6 subjects (20%) with statin intolerance, whereas 3 patients (12%) were under statin-alone therapy without ezetimibe because of ezetimibe intolerance. Antiplatelet drugs were used by 9 subjects (36%). None had received a treatment with PCSK-9 inhibitor before study entry. Previous vascular events were reported by 7 subjects (28%), with coronary artery disease being reported in 6 cases and ischemic stroke in 1 case. Major baseline clinical and demographic characteristics of the study population are reported in Table 1. Serum cholesterol profile and lipoproteins levels After 12 weeks of treatment with PCSK-9 inhibitor (T12w) patients reported a significant reduction in levels of TC (from 279.6 69.9 to 181.8 64.8, p < 0.001) and LDL (201.0 69.5 to 103.0 58.0, p < 0.001). No significant changes were found for HDL (from 52.5 12.9 to 55.0 12.6, p Z 0.065) and TG (from 107.5, IQR: 79.3e146.3 to 109.0, IQR: 73.5e146.0, p Z 0.607). Overall, the median reduction was 37.7% for TC and 52.4% for LDLC. According to ESC/EAS guidelines [18] LDL-C target (<100 mg/dl for FH patients without CV risk factors and <70 mg/dl for high cardiovascular risk subjects), was achieved by 15 out of 25 patients (60%). LDL-C target was more frequently reported in patients receiving statin ezetimibe treatment combined with PCSK-9 inhibitor (73.7%) than in patients receiving PCSK-9 inhibitor alone (16.7%). LDL diameter changed from 268.3 Å 2.99 to 270.5 Å 2.40 (p Z 0.004) with changes in LDL score from 6.60 (IQR: 4.65e11.7) to 3.7 (IQR: 0.37e4.9) (p Z 0.001). The median reduction for LDL score was 46.1%.
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
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M.N.D. Di Minno et al.
Table 1 Clinical and demographic features of subjects with familial hypercholesterolemia starting a treatment with PCSK9 inhibitor. Variable
Study subjects (n Z 25)
Age (years) Age > 65 years Male gender Hypertension Cardiovascular events Obesity Diabetes Smoking habit Body Mass Index (BMI) (Kg/m2) DUTCH score Without major mutations Heterozygotes Compound/double heterozygotes Alanine aminotransferase (ALT) (IU/L) Aspartate aminotransferase (AST) (IU/L) Creatine phosphokinase (IU/L) Total Cholesterol (mg/dl) Triglycerides (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) LDL score LDL diameter (Å) Use of statins Statin intolerance Use of ezetimibe þ statin Use of ezetimibe alone Use of antiplatelet drugs
51.5 14.5 4 (16%) 13 (52.0%) 12 (48%) 7 (28%) 13 (52%) 2 (8%) 2 (8%) 26 4,8 9,32 3,64 2 (8%) 20 (80%) 3 (12%) 24 (IQR:21e26) 27 (IQR: 21,3e38,8) 110 (IQR: 90e171) 279,6 69,9 107,5 (IQR: 79.3e146,3) 53 12,93 201 69,50 6,6 (IQR:4,65e11,7) 268.3 2.99 19 (78%) 6 (22%) 16 (66%) 6 (22%) 9 (36%)
Note. Data are presented as number (%) for dichotomous variables, mean standard deviation for continuous variables with a normal distribution and median (interquartile range [IQR]) for nonparametric continuous variables.
As showed in Fig. 1, median cholesterol sub-fraction reduction was more pronounced in patients with LDLR heterozygote mutation or in patients without major mutations as compared to compound heterozygote/double heterozygote ones.
Carotid stiffness and distensibility During the 12-week treatment both systolic and diastolic blood pressure values remained stable, changing from 132 22 to 126 16 mmHg (p Z 0.051) and from 76 8 to 77 9 mmHg (p Z 0.550). At baseline assessment, the 25 subjects showed a carotid stiffness of 8.8 (IQR: 7.0e10.4) m/sec. After 12 weeks of treatment, carotid stiffness changed from 8.8 (IQR: 7.0e10.4) m/sec to 6.6 (IQR: 5.4e7.5) m/sec at T12w corresponding to a median change of 21.4% (p < 0.001). In parallel, a significant increase in carotid distensibility was observed from 12.1 (IQR: 8.73e19.3) kPA1 103 at T0 to 21.8 (IQR: 16.6e31.8) kPA1 103 at T12w corresponding to a median change of 62.8% (p < 0.001). The prevalence of low carotid distensibility changed from 60% at baseline to 20% at T12w (p Z 0.011). Changes in carotid stiffness and carotid distensibility showed an inverse significant correlation (rho Z 0.998, p < 0.001).
Changes in LDL-c levels and in LDL score showed a significant correlation with carotid stiffness (Fig. 2). Given the potential influence of clinical and demographic characteristics and concomitant cardiovascular risk factor on changes in carotid stiffness and carotid distensibility, we performed sub-group analyses entirely confirming results obtained in the primary analysis (Fig. 3). A more pronounced reduction of carotid stiffness and increase in carotid distensibility was observed in patients with heterozygote mutation of LDLR as compared to compound heterozygotes/double heterozygotes subjects (Fig. 4). In a multivariate analysis including age, gender, BMI cardiovascular events, we found that on trial LDL-c, hypertension, diabetes were independently associated with carotid stiffness and carotid distensibility (Table 2). Moreover, changes in LDL score were independently associated with changes in carotid stiffness from baseline values to T12w (b Z 0.429, p Z 0.041). At T104w, carotid stiffness evaluation was performed in 16 patients, because 6 patients were lost at follow-up, 1 patient had a new-onset arterial hypertension, 1 patient changed the lipid lowering treatment adding evinacumab and 1 patient dead because of laryngeal cancer. The long-term assessment showed a consistent and progressive improvement of carotid stiffness and carotid distensibility as compared to baseline values (Fig. 5).
Discussion In the present prospective study, we documented, for the first time, a reduction of carotid stiffness in patients with hypercholesterolemia treated with Evolocumab. After 12 weeks of treatment with Evolocumab, all patients showed a reduction of LDL-C levels. LDL-C target was reached in 74% of subjects receiving Evolocumabþ statin ezetimibe and in 17% of those treated with Evolocumabezetimibe. This finding confirms the widely recognized LDL-C reduction effect associated with PCSK-9 inhibitors treatment [29]. Besides these expected findings, in the present study, we also documented a significant reduction in LDL score. This parameter represents a method to quantify sd-LDL, known to have a greater susceptibility to oxidative modification, a higher affinity for the arterial wall and widely recognized as predictor of carotid atherosclerosis, stroke and coronary heart disease [25]. Interestingly, we found an extremely limited modification in LDL score in compound heterozygote/double heterozygote patients as compared to those heterozygote for single mutation of LDLR. This is plausible by a pathophysiological point of view, considering the mechanism of action of PCSK-9 inhibitors, which induce an up-regulation in LDL receptor expression, following the inhibition of PCSK9-mediated degradation. In patients with heterozygote/double heterozygote LDLR mutation, receptor function could be seriously compromised, so up-regulation of LDL receptor expression could be ineffective [29,30].
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
Genotype and PCSK-9 inhibitors
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Figure 1 Percent changes in cholesterol profile and lipoprotein levels during treatment with PCSK9 inhibitor stratified according to genotype.
Figure 2 Scatter plot of correlation between percent change in low-density lipoprotein cholesterol (LDL-C) and LDL score with percent change in carotid stiffness during treatment with PCSK-9 inhibitor.
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
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M.N.D. Di Minno et al.
Figure 3 Median percent changes in carotid stiffness and carotid distensibility from baseline values to 12 weeks of therapy with PCSK9 inhibitors in the study population stratified according to predefined study sub-groups. LDL: low-density lipoprotein; CV: cardiovascular; ASA: aspirin; elderly: age 65 years. All data are median (IQR) values of percent change.
Figure 4 Changes in carotid stiffness (label A) and carotid distensibility (label B) during treatment with PCSK9 inhibitor stratifying patients according to genotype.
Based on this evidence, it could be useful to extend genetic testing also to APOB and PCSK9 genes, at least in patients with a limited clinical response to PCSK-9 inhibitors. Besides the lipid-lowering effect of Evolocumab, in the present study we documented a significant improvement in arterial stiffness, an established predictor of cardiovascular and cerebrovascular morbidity and mortality [4e7], particularly in dyslipidaemic patients [8]. In detail, we specifically evaluated local stiffness and distensibility of the carotid artery, a predominantly elastic artery [31]. Although carotid-femoral pulse wave velocity (cf-PWV) is currently considered the gold standard for arterial stiffness assessment [27], carotid stiffness is recognized as an adequate marker for non-invasive
assessment of arterial stiffness [27]. Some studies reported the importance of carotid stiffness evaluation as an independent predictor of incident cardiovascular disease and/or mortality [6,7,32]. In the present study, we observed a >20% reduction in carotid stiffness, with a direct correlation with LDL-C and LDL score reduction. Interestingly, changes in carotid stiffness was confirmed at a long-term assessment showing a consistent and progressive improvement of carotid stiffness and carotid distensibility as compared to baseline values after 2 years of treatment. The exact mechanism by which PCSK-9 inhibitor exert a beneficial effect in improving carotid stiffness is still not completely described. Lowering LDL-C levels represents one mechanism by which carotid stiffness reduction can
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
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Table 2 Linear regression analysis. Association of clinical and laboratory parameters with carotid stiffness and distensibility.
LDL-c Diabetes Hypertension
Carotid Stiffness
Carotid Distensibility
b Z 0.511 p < 0.001 b Z 0.346 p Z 0.007 b Z 0.391 p Z 0.004
b Z 0.506 p Z 0.001 b Z 0.294 p Z 0.040 b Z 0.366 p Z 0.018
be achieved [9,10]. Several literature data suggested that lipid-lowering therapies, by reducing oxidative stress and balancing nitric oxide synthase (eNOS)/nitric oxide synthase (iNOS) ratio, lead to an improvement of endothelial function [33e35]. Thus, it can be expected that lipidlowering therapies may induce functional and structural modifications in artery wall, positively impacting on arterial stiffness. In line with this hypothesis, we observed a correlation between LDL levels and carotid stiffness and, such an association was confirmed after adjusting for potential confounders. However, it remains unknown whether this effect on carotid stiffness is mostly mediated by reduction of cholesterol levels or by other presently unknown mechanisms. In the present study, we documented that, after adjusting for potential confounders, changes in LDL score (expression of sd-LDL concentration) was a predictor of modifications in carotid stiffness. This finding is in line with data showing that sd-LDL are associated with arterial stiffness and predicts cardiovascular events [36]. By a pathophysiological point of view, we have to account that sd-LDL particles bind with lower affinity LDL receptor [37]. sd-LDL synthesis is consequent to delipidation of large LDL particles. The up-regulation of the LDL receptor, induced by inhibition of PCSK-9, enhances the binding potential, leading to a variation in LDL
diameter and composition. Following to LDL receptor upregulation, the clearance of circulating large LDL particles hampers de-lipidation and leads to a reduction in sdLDL [38]. Although these modifications seem to determine an improvement in carotid stiffness, to support this hypothesis, some basic science experiments should be conducted, also including direct effects of PCSK9 inhibitors on human endothelial cell culture. Besides evident consequence due to LDL-C reduction, possible ancillary effects induced by PCSK-9 inhibitors can be presumed. A recent study showed that PCSK-9 induces a pro-inflammatory response in macrophages [39] and a treatment with a PCSK-9 inhibitor was able to induce a decrease in monocyte migratory capacity, lipid content, as well as inflammatory responsiveness [40]. Overall, the above described favorable effects might determine a reduced cardiovascular risk. This hypothesis is mostly supported by results of the randomized, doubleblinded, placebo-controlled FOURIER trial showing that in 27.564 patients with atherosclerotic cardiovascular disease the association of Evolocumab to standard statin treatment significantly reduced the risk of fatal and non-fatal cardiovascular events (HR: 0.85, 95%CI: 0.79e0.92, p < 0.001) [12]. A further demonstration of a potential link between lipid-lowering effect and carotid stiffness derives from the evidence that, as compared to double/compound heterozygotes, patients with heterozygote mutation of LDLR reported a more pronounced LDL reduction during treatment with Evolocumab accompanied by a more significant reduction in carotid stiffness and increase in carotid distensibility. However, the limited number of patients double/compound heterozygote partly hampers
Figure 5 Changes in carotid stiffness and carotid distensibility from baseline values to week 12 and week 104 during treatment with PCSK9 inhibitor. )The analysis was performed on 16 patients with data at baseline, at week 12 and at week 104.
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
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generalizability of these results and suggests the need of further studies to definitely address this issue. Some potential limitations of the present study need to be discussed. Most of patients included in the present study have concomitant cardiovascular risk factors, potentially impacting on carotid stiffness. To overcome this potential source of heterogeneity, sub-group analyses were performed and showed that patients not receiving statins represented the only clinical subset reporting a less significant reduction in carotid stiffness and increase in carotid distensibility, thus suggesting a cooperation between statins and PCSK-9 inhibitors. This hypothesis is also supported by previous studies showing that PCSK-9 inhibitors reduced LDL-C levels of about 50% when used alone and about 70% when used together with statins [29]. A hypothetical mechanism of a synergic effect of association between statins and PCSK-9 inhibitors is due to increased plasma levels of PCSK-9 in patients treated with statins alone. This effect is secondary to the activation of sterol responsive element binding protein (SREBP) pathway because of the inhibition of cholesterol biosynthesis. Thus, patients receiving chronic statin therapy have higher levels of PCSK-9 and a more marked response to PCSK-9 inhibitor [41]. On the other hand, the significant improvement in carotid stiffness consistently found in all the other sub-groups considered, suggests that PCSK-9 treatment leads to a reduction of the cardiovascular risk regardless of the presence of concomitant cardiovascular risk factors. In our sample, we observed a trend toward a reduction in SBP during treatment with PCSK9 inhibitor. We excluded any modification in antihypertensive treatment during study period to exclude a potential impact on results. On the other hand, we cannot explain these changes as an ancillary effect of PCSK9 inhibition, considering that previous data showed that PCSK9 levels are unrelated to blood pressure [42]. Thus, this issue needs to be specifically addressed in future studies. The lack of a control group might represent a limitation of the present study. However, PCSK-9 can be used only in patients not achieving target LDL-C levels under maximal statin treatment. Thus, a potential control group should be represented by patients not eligible to treatment with PCSK-9 or refusing this treatment. These hypothetic controls are not likely to be comparable to cases. Overall, the pre-post observational design allows for partly overcome the intrinsic inter-individual heterogeneity, with each patient being the control of himself. A further potential limitation is represented by the small study sample and, in turn, the limited number of patients available for subgroup analyses. However, FH has a prevalence of 1:300 according to epidemiological data [3] and PCSK-9 inhibitors can be used only in patients not achieving target LDL-C levels under maximal lipid lowering treatment. Thus, this study enrolled a highly selected population with defined eligibility criteria with a sample size calculated to achieve a target power of 80%. In conclusion, this study showed, for the first time a long-standing and consistent effect of treatment with
M.N.D. Di Minno et al.
PCSK-9 inhibitor Evolocumab on carotid stiffness. Moreover, we found that small dense LDL reduction is related to changes in carotid stiffness, thus suggesting a potential impact for changes in lipoprotein composition as a mechanism for cardiovascular risk reduction. Further studies are needed to fully investigate underlying mechanisms and the impact on cardiovascular events. Source of funding No funds were received for this study. Declaration of Competing Interest All the Authors have nothing to declare. References [1] Rubba P, Gentile M, Marotta G, Iannuzzi A, Sodano M, De Simone B, et al. Causative mutations and premature cardiovascular disease in patients with heterozygous familial hypercholesterolaemia. Eur J Prev Cardiol 2017;24:1051e9. [2] Stitziel NO, Peloso GM, Abifadel M, Cefalu AB, Fouchier S, Motazacker MM, et al. Exome sequencing in suspected monogenic dyslipidemias. Circ Cardiovasc Genet 2015;8:343e50. [3] 2019 ESC/EAS guidelines for the management of dyslipidaemias: lipid modification to reduce cardiovascular risk. Atherosclerosis 2019;290:140e205. [4] Jadhav UM, Kadam NN. Non-invasive assessment of arterial stiffness by pulse-wave velocity correlates with endothelial dysfunction. Indian Heart J 2005;57:226e32. [5] Blacher J, Pannier B, Guerin AP, Marchais SJ, Safar ME, London GM. Carotid arterial stiffness as a predictor of cardiovascular and allcause mortality in end-stage renal disease. Hypertension 1998;32: 570e4. [6] van Sloten TT, Schram MT, van den Hurk K, Dekker JM, Nijpels G, Henry RM, et al. Local stiffness of the carotid and femoral artery is associated with incident cardiovascular events and all-cause mortality: the Hoorn study. J Am Coll Cardiol 2014;63:1739e47. [7] Yang EY, Chambless L, Sharrett AR, Virani SS, Liu X, Tang Z, et al. Carotid arterial wall characteristics are associated with incident ischemic stroke but not coronary heart disease in the Atherosclerosis Risk in Communities (ARIC) study. Stroke 2012;43:103e8. [8] Tada H, Kawashiri MA, Nohara A, Inazu A, Mabuchi H, Yamagishi M. Assessment of arterial stiffness in patients with familial hypercholesterolemia. J Clin Lipidol 2018;12:397e402 e2. [9] Canepa M, Artom N, Ameri P, Carbone F, Montecucco F, Ghigliotti G, et al. Short-term effect of rosuvastatin treatment on arterial stiffness in individuals with newly-diagnosed heterozygous familial hypercholesterolemia. Int J Cardiol 2018;255:215e20. [10] Oh PC, Han SH, Koh KK, Lee K, Seo JG, Suh SY, et al. Rosuvastatin treatment improves arterial stiffness with lowering blood pressure in healthy hypercholesterolemic patients. Int J Cardiol 2014; 176:1284e7. [11] Nicholls SJ, Brandrup-Wognsen G, Palmer M, Barter PJ. Metaanalysis of comparative efficacy of increasing dose of Atorvastatin versus Rosuvastatin versus Simvastatin on lowering levels of atherogenic lipids (from VOYAGER). Am J Cardiol 2010;105:69e76. [12] Giugliano RP, Pedersen TR, Park JG, De Ferrari GM, Gaciong ZA, Ceska R, et al. Clinical efficacy and safety of achieving very low LDL-cholesterol concentrations with the PCSK9 inhibitor evolocumab: a prespecified secondary analysis of the FOURIER trial. Lancet 2017;390:1962e71. [13] Schwartz GG, Steg PG, Szarek M, Bhatt DL, Bittner VA, Diaz R, et al. Alirocumab and cardiovascular outcomes after acute coronary syndrome. N Engl J Med 2018;379:2097e107. [14] Han J, Wang X, Ye P, Cao R, Yang X, Xiao W, et al. Plasma PCSK9 levels are unrelated to arterial stiffness in a community-based, 4.8-year prospective study. J Hum Hypertens 2017;31:720e4.
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
Genotype and PCSK-9 inhibitors [15] Ruscica M, Ferri N, Fogacci F, Rosticci M, Botta M, Marchiano S, et al. Circulating levels of proprotein convertase subtilisin/kexin type 9 and arterial stiffness in a large population sample: data from the brisighella heart study. J Am Heart Assoc 2017;6. [16] Cicero AFG, Toth PP, Fogacci F, Virdis A, Borghi C. Improvement in arterial stiffness after short-term treatment with PCSK9 inhibitors. Nutr Metabol Cardiovasc Dis e NMCD 2019;29:527e9. [17] Pirillo A, Garlaschelli K, Arca M, Averna M, Bertolini S, Calandra S, et al. Spectrum of mutations in Italian patients with familial hypercholesterolemia: new results from the LIPIGEN study. Atherosclerosis Suppl 2017;29:17e24. [18] Catapano AL, Graham I, De Backer G, Wiklund O, Chapman MJ, Drexel H, et al. 2016 ESC/EAS guidelines for the management of dyslipidaemias. Eur Heart J 2016;37:2999e3058. [19] Di Taranto MD, Staiano A, D’Agostino MN, D’Angelo A, Bloise E, Morgante A, et al. Association of USF1 and APOA5 polymorphisms with familial combined hyperlipidemia in an Italian population. Mol Cell Probes 2015;29:19e24. [20] Staiano A, Di Taranto M, Bloise E, D’Agostino M, D’Angelo A, Marotta G, et al. Investigation of single nucleotide polymorphisms associated to familial combined hyperlipidemia with random forests. In: Neural Nets and Surroundings Smart Innovation, Systems and Technologies Book Series; 2013. p. 169e78. [21] Casula M, Olmastroni E, Pirillo A, Catapano AL. Evaluation of the performance of Dutch Lipid Clinic Network score in an Italian FH population: the LIPIGEN study. Atherosclerosis 2018;277:413e8. [22] Di Taranto MD, Benito-Vicente A, Giacobbe C, Uribe KB, Rubba P, Etxebarria A, et al. Identification and in vitro characterization of two new PCSK9 Gain of Function variants found in patients with Familial Hypercholesterolemia. Sci Rep 2017;7:15282. [23] Ruotolo A, Di Taranto MD, D’Agostino MN, Marotta G, Gentile M, Nunziata M, et al. The novel variant p.Ser465Leu in the PCSK9 gene does not account for the decreased LDLR activity in members of a FH family. Clin Chem Lab Med 2014;52:e175e8. [24] Hoefner DM, Hodel SD, O’Brien JF, Branum EL, Sun D, Meissner I, et al. Development of a rapid, quantitative method for LDL subfractionation with use of the Quantimetrix Lipoprint LDL System. Clin Chem 2001;47:266e74. [25] Gentile M, Panico S, Mattiello A, Ubaldi S, Iannuzzo G, De Michele M, et al. Association between small dense LDL and early atherosclerosis in a sample of menopausal women. Clin Chim Acta - Int J Clin Chem 2013;426:1e5. [26] Bianchini E, Bozec E, Gemignani V, Faita F, Giannarelli C, Ghiadoni L, et al. Assessment of carotid stiffness and intima-media thickness from ultrasound data: comparison between two methods. J Ultrasound Med e Off J Am Inst Ultrasound Med 2010; 29:1169e75. [27] Laurent S, Cockcroft J, Van Bortel L, Boutouyrie P, Giannattasio C, Hayoz D, et al. Expert consensus document on arterial stiffness: methodological issues and clinical applications. Eur Heart J 2006; 27:2588e605. [28] Laurent S, Caviezel B, Beck L, Girerd X, Billaud E, Boutouyrie P, et al. Carotid artery distensibility and distending pressure in hypertensive humans. Hypertension 1994;23:878e83.
9 [29] McKenney JM. Understanding PCSK9 and anti-PCSK9 therapies. J Clin Lipidol 2015;9:170e86. [30] Budha NR, Leabman M, Jin JY, Wada DR, Baruch A, Peng K, et al. Modeling and simulation to support phase 2 dose selection for RG7652, a fully human monoclonal antibody against proprotein convertase subtilisin/kexin type 9. AAPS J 2015;17:881e90. [31] Bruno RM, Cartoni G, Stea F, Armenia S, Bianchini E, Buralli S, et al. Carotid and aortic stiffness in essential hypertension and their relation with target organ damage: the CATOD study. J Hypertens 2017;35:310e8. [32] Mattace-Raso FU, van der Cammen TJ, Hofman A, van Popele NM, Bos ML, Schalekamp MA, et al. Arterial stiffness and risk of coronary heart disease and stroke: the Rotterdam Study. Circulation 2006;113:657e63. [33] Brunetti ND, Maulucci G, Casavecchia GP, Distaso C, De Gennaro L, Pellegrino PL, et al. Improvement in endothelium dysfunction in diabetics treated with statins: a randomized comparison of atorvastatin 20 mg versus rosuvastatin 10 mg. J Intervent Cardiol 2007;20:481e7. [34] Gong X, Ma Y, Ruan Y, Fu G, Wu S. Long-term atorvastatin improves age-related endothelial dysfunction by ameliorating oxidative stress and normalizing eNOS/iNOS imbalance in rat aorta. Exp Gerontol 2014;52:9e17. [35] Vlachopoulos C, Aznaouridis K, Dagre A, Vasiliadou C, Masoura C, Stefanadi E, et al. Protective effect of atorvastatin on acute systemic inflammation-induced endothelial dysfunction in hypercholesterolaemic subjects. Eur Heart J 2007;28:2102e9. [36] Li G, Wu HK, Wu XW, Cao Z, Tu YC, Ma Y, et al. Small dense low density lipoprotein-cholesterol and cholesterol ratios to predict arterial stiffness progression in normotensive subjects over a 5year period. Lipids Health Dis 2018;17:27. [37] Galeano NF, Al-Haideri M, Keyserman F, Rumsey SC, Deckelbaum RJ. Small dense low density lipoprotein has increased affinity for LDL receptor-independent cell surface binding sites: a potential mechanism for increased atherogenicity. J Lipid Res 1998;39:1263e73. [38] Diffenderfer MR, Schaefer EJ. The composition and metabolism of large and small LDL. Curr Opin Lipidol 2014;25:221e6. [39] Ricci C, Ruscica M, Camera M, Rossetti L, Macchi C, Colciago A, et al. PCSK9 induces a pro-inflammatory response in macrophages. Sci Rep 2018;8:2267. [40] Bernelot Moens SJ, Neele AE, Kroon J, van der Valk FM, Van den Bossche J, Hoeksema MA, et al. PCSK9 monoclonal antibodies reverse the pro-inflammatory profile of monocytes in familial hypercholesterolaemia. Eur Heart J 2017;38:1584e93. [41] Dubuc G, Chamberland A, Wassef H, Davignon J, Seidah NG, Bernier L, et al. Statins upregulate PCSK9, the gene encoding the proprotein convertase neural apoptosis-regulated convertase-1 implicated in familial hypercholesterolemia. Arterioscler Thromb Vasc Biol 2004;24:1454e9. [42] Yang SH, Du Y, Li S, Zhang Y, Xu RX, Zhu CG, et al. Plasma PCSK9 level is unrelated to blood pressure and not associated independently with carotid intima-media thickness in hypertensives. Hypertens Res e Off J Jpn Soc Hypertens 2016;39:598e605.
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
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Nutrition, Metabolism & Cardiovascular Diseases journal homepage: www.elsevier.com/locate/nmcd
Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study Matteo Nicola D. Di Minno a,*, Marco Gentile b, Alessandro Di Minno c,d, Gabriella Iannuzzo b, Ilenia Calcaterra b, Alessio Buonaiuto b, Maria D. Di Taranto e, Carola Giacobbe e, Giuliana Fortunato e, Paolo O.F. Rubba b a
Department of Translational Medical Sciences, Federico II University, Naples, Italy Department of Clinical Medicine and Surgery, Federico II University, Naples, Italy Department of Pharmacy, Federico II University, Naples, Italy d Unit of Metabolomics and Cellular Biochemistry of Atherothrombosis, Centro Cardiologico Monzino IRCCS, Milan, Italy e Department of Molecular Medicine e Medical Biotechnologies, Federico II University, Naples, Italy b c
Received 24 December 2019; received in revised form 26 February 2020; accepted 28 February 2020 Handling Editor: D. Noto Available online - - -
KEYWORDS Carotid stiffness; Familial hypercholesterolemia; PCSK9 inhibitors
Abstract Background and aim: Protein convertase subtilisin kexin type 9 (PCSK-9) inhibitors demonstrated efficacy in cholesterol reduction and in the prevention of cardiovascular events. We evaluated changes in lipid profile and carotid stiffness in patients with familial hypercholesterolemia during 12 weeks of treatment with a PCSK-9 inhibitor, Evolocumab. Methods and results: Patients with familial hypercholesterolemia starting a treatment with Evolocumab were included. Total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), small dense LDL (assessed by LDL score) and carotid stiffness were evaluated before starting treatment with Evolocumab and during 12 weeks of treatment. Twenty-five subjects were enrolled (52% males, mean age 51.5 years). TC and LDL-C were reduced of 38% and 52%, respectively during treatment, with LDL score reduced of 46.1%. In parallel, carotid stiffness changed from 8.8 (IQR: 7.0e10.4) m/sec to 6.6 (IQR: 5.4e7.5) m/sec, corresponding to a median change of 21.4% (p < 0.0 01), with a significant increase in carotid distensibility (from 12.1, IQR: 8.73e19.3 kPA1 103 at T0 to 21.8, IQR: 16.6e31.8 kPA1 103 at T12w) corresponding to a median change of 62.8% (p < 0.001). A multivariate analysis showed that changes in LDL score were independently associated with changes in carotid stiffness (b Z 0.429, p Z 0.041). Conclusion: Small dense LDL reduction, as assessed by LDL score, is associated with changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab. ª 2020 Published by Elsevier B.V. on behalf of The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University.
Introduction
* Corresponding author. Department of Translational Medical Sciences Federico II University, Via S. Pansini 5, 80131 Naples, Italy. Fax: þ390817464323. E-mail address: [email protected] (M.N.D. Di Minno).
Several studies consistently showed that increased levels of low-density lipoprotein cholesterol (LDL-C) represent the main causative factor for atherosclerosis development [1]. Patients with high levels of LDL-C exhibit an increased
https://doi.org/10.1016/j.numecd.2020.02.018 0939-4753/ª 2020 Published by Elsevier B.V. on behalf of The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University.
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
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prevalence of subclinical atherosclerosis and a more rapid atherosclerosis progression [2]. This leads to a significantly higher cardiovascular (CV) risk in patients with high LDL-C levels [3]. Increased arterial stiffness is one of the earliest stages of the atherosclerotic process [4] and some studies reported an association between arterial stiffness and incident CV disease and mortality [5e7], particularly in patients with familial hypercholesterolemia (FH) [8]. On the other hand, some recent reports showed that lipid lowering treatment with statins is associated with a significant reduction in arterial stiffness [9,10]. Although for years the gold standard for lipid lowering therapy was represented by statin treatment, the target LDL-C is not always achieved, mainly in patients with very high levels of LDL-C [11]. More recently, PCSK-9 inhibitors showed efficacy in LDL-C reduction and in the prevention from CV events [12,13]. Conflicting results are available about the association between PCSK-9 plasma levels and arterial stiffness [14,15] and only data on 2 patients are available about the effect of PCSK-9 inhibitors on arterial stiffness [16]. The aim of our study was to prospectively assess changes in lipid profile and carotid stiffness in patients with FH before and after the treatment with a PCSK9 inhibitor, Evolocumab.
Methods From December 2017 to December 2018, within the framework of LIPIGEN, a national project on familial dyslipidemia, consecutive patients attending the lipid clinic of the Department of Clinical Medicine and Surgery, Federico II University Hospital with very high levels of LDL-C (above the 95th percentile when compared with a sex- and agematched general population), normal triglyceride levels and probable autosomal dominant transmission of hypercholesterolemia (DUTCH Lipid Clinic Network score 6) in the family were screened for inclusion in the present study [17]. The major inclusion criterion was the eligibility of patients to start a treatment with PCSK-9 according to ESC/ EAS guidelines [18]. Exclusion criteria were: age < 18 years, inability to understand or sign the informed consent, high level of transaminases (>3x upper normal limit), hypertriglyceridemia (>150 mg/dl), end-stage renal disease (filtration rate < 30 ml/min/mq), current malignant disease or a diagnosis of malignancy in the 2 years prior to the first visit, previous exposure to PCSK-9 inhibitors, presence of hypercholesterolemia secondary to other causes (hypothyroidism, hormone therapies, corticosteroids etc.). Patients with a clinical picture or family history of familial combined hyperlipidemia were excluded [19,20]. Patients enrolled in the study continued the ongoing lipid lowering therapy and added PCSK-9 inhibitor (Evolocumab 140 mg subcutaneous injection every 14 days).
M.N.D. Di Minno et al.
Study protocol After informed consent, a detailed medical history was recorded for each patient. Data about age, gender, previous and/or current medical conditions, current and past lipid lowering therapy, vascular risk factors were collected. Height was measured to the nearest 0.1 cm. Body weight was assessed by using an electronic beam scale with digital readout to the nearest 0.1 kg after emptying the bladder and with the subjects standing barefoot and wearing light indoor clothing. Body mass index (BMI) was calculated as body weight/(height2). Clinical diagnosis of FH was achieved using Dutch Lipid Clinic Network Score and genetic testing was performed to assess major causative mutations, including LDLR, APOB and PCSK9 genes as previously described [21e23]. Blood laboratory parameters The following blood values were evaluated at baseline (before starting PCSK-9 inhibitor) and 12 weeks (T12w) after treatment with Evolocumab: total cholesterol (TC), triglycerides (TG), HDL cholesterol (HDL-C), LDL-C, smalldense LDL (sd-LDL), creatininemia, AST, ALT, CPK, glycaemia. LDL particles separation was performed by electrophoretic Lipoprint System (Quantimetrix Inc., Redondo Beach, CA). The proportion of sd-LDL particles (subfractions 3e7) to the whole LDL area (subfractions 1e7) was also calculated in our sample and expressed as LDL score, with higher score values representing a higher sd-LDL particles content [24]. In addition, mean LDL particle diameter was calculated on the basis of the different areas under the curve of the 7 LDL species with different electrophoretic mobility [25]. Carotid stiffness Patients were asked to abstain from alcohol, tobacco and caffeine for at least 12 h on the day of examination. All study procedures were performed in a temperaturecontrolled room (23 C), after 10 min of rest in supine position. Carotid stiffness was evaluated in each patient before starting PCSK-9 inhibitor (T0) and repeated after 12 weeks of treatment (T12w) by the same operator blinded for ongoing treatment and cholesterol levels. To validate results on the long-term, carotid stiffness was evaluated also after 104 weeks of treatment (T104w). Common carotid artery scans (25 frames/s) were obtained by high-resolution ultrasound with a 10 MHz linear array transducer (MyLab25; ESAOTE, Florence, Italy) by a trained operator. Longitudinal scans were acquired from each common carotid artery (1 cm proximal to the carotid bulb in a region 1-cm wide and free of plaques) and automatically analyzed with Carotid Studio
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
Genotype and PCSK-9 inhibitors
(Cardiovascular Suite, Carotid studio, QUIPU Srl, Pisa, Italy), a well validated system based on contour tracking algorithm [26]. Arterial interfaces were automatically detected, with estimation of instantaneous mean diameter as the distance between far and near mediaeadventitia interfaces. The following parameters were obtained: carotid distension (DD), that is the stroke change in diameter, calculated as the difference between the systolic and diastolic diameter values; carotid distensibility coefficient Z DA/(A*DP), where A represents the diastolic lumen area, evaluated from the diameter values (assuming the cross-section of the artery to be circular), DA represents the stroke change in lumen area, DP the local pulse pressure obtained by tonometry. Carotid stiffness was calculated according to the MoenseKorteweg equation [27]. In particular, carotid distensibility was converted into carotid stiffness by using the equation: PWV Z [(DP )A)/ (DA) r)](1/2) where r is the blood density. This formula allows carotid stiffness to be obtained as carotid stiffness Z (r ) carotid distensibility) (1/2), expressed in m/s. The reproducibility of this scanning protocol was evaluated on a representative sample of 5 subjects randomly selected from the study population within 1 week from the first examination and the r value resulted Z 0.91. Carotid distensibility was defined as low if < 15 kPa1 x 103 [28]. Statistical analysis Statistical analysis was performed with the IBM SPSS 22 system (SPSS Inc., Chicago, IL, USA). Continuous data were expressed as mean standard deviation (SD) or as median value with inter-quartile range (IQR). The t-test was performed to compare continuous variables for paired samples and for independent samples. In case of values with a skewed non-Gaussian distribution, ManneWhitney U test was used to compare means. The c2 test or Fisher’s exact test were used to compare categorical variables. Relationships between continuous variables were examined using simple regressions Spearman’s correlation (rho) for nonparametric variables. All results were expressed as 2tailed values, p values < 0.05 being statistically significant. To evaluate potential sources of heterogeneity, a sensitivity analysis was performed stratifying patients according to major clinical and demographic characteristics and according to genotype of LDLR, APOB and PCSK9 genes. To adjust for potential confounders and to make predictions, linear regression analyses (stepwise method) were implemented with changes in carotid stiffness as dependent variable and age, gender, BMI, cardiovascular events, hypertension, diabetes and changes in LDL score as independent variables. Sample size As to sample size, with a pre-defined 25% reduction in carotid stiffness from baseline values to T12w, 22 subjects were needed to obtain an 80% power and a 5% a error. Also
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considering a drop-out risk, we aimed at enrolling 25 subjects in the present study. Results Twenty-five subjects (13 males and 12 females, mean age 51.5 14.5 years) with FH (20 heterozygotes for the mutation of LDLR, 2 compound heterozygotes for 2 different mutations of LDLR, 1 double heterozygote for mutations of LDLR and of PCSK9 and 2 subjects were without major mutation in LDLR, APOB, PCSK9 genes) were enrolled. The mean Dutch Lipid Clinic Network Score was 9.3. Nineteen patients (76%) were under statin treatment, whereas 6 reported statin intolerance. Among the 19 statin-treated patients, 1 was under simvastatin 40 mg, 3 rosuvastatin 40 mg, 5 rosuvastatin 20 mg, 1 pravastatin 40 mg, 1 fluvastatin 40 mg, 5 atorvastatin 40 mg, 3 atorvastatin 80 mg. Thus, 16 patients were under high intensity-statins treatment (atorvastatin 40 mg or 80 mg; rosuvastatin 20 mg or 40 mg), whereas the other 3 patients were under low-intensity statins (pravastatin, fluvastatin and simvastatin) because of intolerance to high intensity-statin. Ezetimibe was present as co-treatment in 16 subjects (68%) and as a stand-alone treatment in the 6 subjects (20%) with statin intolerance, whereas 3 patients (12%) were under statin-alone therapy without ezetimibe because of ezetimibe intolerance. Antiplatelet drugs were used by 9 subjects (36%). None had received a treatment with PCSK-9 inhibitor before study entry. Previous vascular events were reported by 7 subjects (28%), with coronary artery disease being reported in 6 cases and ischemic stroke in 1 case. Major baseline clinical and demographic characteristics of the study population are reported in Table 1. Serum cholesterol profile and lipoproteins levels After 12 weeks of treatment with PCSK-9 inhibitor (T12w) patients reported a significant reduction in levels of TC (from 279.6 69.9 to 181.8 64.8, p < 0.001) and LDL (201.0 69.5 to 103.0 58.0, p < 0.001). No significant changes were found for HDL (from 52.5 12.9 to 55.0 12.6, p Z 0.065) and TG (from 107.5, IQR: 79.3e146.3 to 109.0, IQR: 73.5e146.0, p Z 0.607). Overall, the median reduction was 37.7% for TC and 52.4% for LDLC. According to ESC/EAS guidelines [18] LDL-C target (<100 mg/dl for FH patients without CV risk factors and <70 mg/dl for high cardiovascular risk subjects), was achieved by 15 out of 25 patients (60%). LDL-C target was more frequently reported in patients receiving statin ezetimibe treatment combined with PCSK-9 inhibitor (73.7%) than in patients receiving PCSK-9 inhibitor alone (16.7%). LDL diameter changed from 268.3 Å 2.99 to 270.5 Å 2.40 (p Z 0.004) with changes in LDL score from 6.60 (IQR: 4.65e11.7) to 3.7 (IQR: 0.37e4.9) (p Z 0.001). The median reduction for LDL score was 46.1%.
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
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M.N.D. Di Minno et al.
Table 1 Clinical and demographic features of subjects with familial hypercholesterolemia starting a treatment with PCSK9 inhibitor. Variable
Study subjects (n Z 25)
Age (years) Age > 65 years Male gender Hypertension Cardiovascular events Obesity Diabetes Smoking habit Body Mass Index (BMI) (Kg/m2) DUTCH score Without major mutations Heterozygotes Compound/double heterozygotes Alanine aminotransferase (ALT) (IU/L) Aspartate aminotransferase (AST) (IU/L) Creatine phosphokinase (IU/L) Total Cholesterol (mg/dl) Triglycerides (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) LDL score LDL diameter (Å) Use of statins Statin intolerance Use of ezetimibe þ statin Use of ezetimibe alone Use of antiplatelet drugs
51.5 14.5 4 (16%) 13 (52.0%) 12 (48%) 7 (28%) 13 (52%) 2 (8%) 2 (8%) 26 4,8 9,32 3,64 2 (8%) 20 (80%) 3 (12%) 24 (IQR:21e26) 27 (IQR: 21,3e38,8) 110 (IQR: 90e171) 279,6 69,9 107,5 (IQR: 79.3e146,3) 53 12,93 201 69,50 6,6 (IQR:4,65e11,7) 268.3 2.99 19 (78%) 6 (22%) 16 (66%) 6 (22%) 9 (36%)
Note. Data are presented as number (%) for dichotomous variables, mean standard deviation for continuous variables with a normal distribution and median (interquartile range [IQR]) for nonparametric continuous variables.
As showed in Fig. 1, median cholesterol sub-fraction reduction was more pronounced in patients with LDLR heterozygote mutation or in patients without major mutations as compared to compound heterozygote/double heterozygote ones.
Carotid stiffness and distensibility During the 12-week treatment both systolic and diastolic blood pressure values remained stable, changing from 132 22 to 126 16 mmHg (p Z 0.051) and from 76 8 to 77 9 mmHg (p Z 0.550). At baseline assessment, the 25 subjects showed a carotid stiffness of 8.8 (IQR: 7.0e10.4) m/sec. After 12 weeks of treatment, carotid stiffness changed from 8.8 (IQR: 7.0e10.4) m/sec to 6.6 (IQR: 5.4e7.5) m/sec at T12w corresponding to a median change of 21.4% (p < 0.001). In parallel, a significant increase in carotid distensibility was observed from 12.1 (IQR: 8.73e19.3) kPA1 103 at T0 to 21.8 (IQR: 16.6e31.8) kPA1 103 at T12w corresponding to a median change of 62.8% (p < 0.001). The prevalence of low carotid distensibility changed from 60% at baseline to 20% at T12w (p Z 0.011). Changes in carotid stiffness and carotid distensibility showed an inverse significant correlation (rho Z 0.998, p < 0.001).
Changes in LDL-c levels and in LDL score showed a significant correlation with carotid stiffness (Fig. 2). Given the potential influence of clinical and demographic characteristics and concomitant cardiovascular risk factor on changes in carotid stiffness and carotid distensibility, we performed sub-group analyses entirely confirming results obtained in the primary analysis (Fig. 3). A more pronounced reduction of carotid stiffness and increase in carotid distensibility was observed in patients with heterozygote mutation of LDLR as compared to compound heterozygotes/double heterozygotes subjects (Fig. 4). In a multivariate analysis including age, gender, BMI cardiovascular events, we found that on trial LDL-c, hypertension, diabetes were independently associated with carotid stiffness and carotid distensibility (Table 2). Moreover, changes in LDL score were independently associated with changes in carotid stiffness from baseline values to T12w (b Z 0.429, p Z 0.041). At T104w, carotid stiffness evaluation was performed in 16 patients, because 6 patients were lost at follow-up, 1 patient had a new-onset arterial hypertension, 1 patient changed the lipid lowering treatment adding evinacumab and 1 patient dead because of laryngeal cancer. The long-term assessment showed a consistent and progressive improvement of carotid stiffness and carotid distensibility as compared to baseline values (Fig. 5).
Discussion In the present prospective study, we documented, for the first time, a reduction of carotid stiffness in patients with hypercholesterolemia treated with Evolocumab. After 12 weeks of treatment with Evolocumab, all patients showed a reduction of LDL-C levels. LDL-C target was reached in 74% of subjects receiving Evolocumabþ statin ezetimibe and in 17% of those treated with Evolocumabezetimibe. This finding confirms the widely recognized LDL-C reduction effect associated with PCSK-9 inhibitors treatment [29]. Besides these expected findings, in the present study, we also documented a significant reduction in LDL score. This parameter represents a method to quantify sd-LDL, known to have a greater susceptibility to oxidative modification, a higher affinity for the arterial wall and widely recognized as predictor of carotid atherosclerosis, stroke and coronary heart disease [25]. Interestingly, we found an extremely limited modification in LDL score in compound heterozygote/double heterozygote patients as compared to those heterozygote for single mutation of LDLR. This is plausible by a pathophysiological point of view, considering the mechanism of action of PCSK-9 inhibitors, which induce an up-regulation in LDL receptor expression, following the inhibition of PCSK9-mediated degradation. In patients with heterozygote/double heterozygote LDLR mutation, receptor function could be seriously compromised, so up-regulation of LDL receptor expression could be ineffective [29,30].
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
Genotype and PCSK-9 inhibitors
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Figure 1 Percent changes in cholesterol profile and lipoprotein levels during treatment with PCSK9 inhibitor stratified according to genotype.
Figure 2 Scatter plot of correlation between percent change in low-density lipoprotein cholesterol (LDL-C) and LDL score with percent change in carotid stiffness during treatment with PCSK-9 inhibitor.
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
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M.N.D. Di Minno et al.
Figure 3 Median percent changes in carotid stiffness and carotid distensibility from baseline values to 12 weeks of therapy with PCSK9 inhibitors in the study population stratified according to predefined study sub-groups. LDL: low-density lipoprotein; CV: cardiovascular; ASA: aspirin; elderly: age 65 years. All data are median (IQR) values of percent change.
Figure 4 Changes in carotid stiffness (label A) and carotid distensibility (label B) during treatment with PCSK9 inhibitor stratifying patients according to genotype.
Based on this evidence, it could be useful to extend genetic testing also to APOB and PCSK9 genes, at least in patients with a limited clinical response to PCSK-9 inhibitors. Besides the lipid-lowering effect of Evolocumab, in the present study we documented a significant improvement in arterial stiffness, an established predictor of cardiovascular and cerebrovascular morbidity and mortality [4e7], particularly in dyslipidaemic patients [8]. In detail, we specifically evaluated local stiffness and distensibility of the carotid artery, a predominantly elastic artery [31]. Although carotid-femoral pulse wave velocity (cf-PWV) is currently considered the gold standard for arterial stiffness assessment [27], carotid stiffness is recognized as an adequate marker for non-invasive
assessment of arterial stiffness [27]. Some studies reported the importance of carotid stiffness evaluation as an independent predictor of incident cardiovascular disease and/or mortality [6,7,32]. In the present study, we observed a >20% reduction in carotid stiffness, with a direct correlation with LDL-C and LDL score reduction. Interestingly, changes in carotid stiffness was confirmed at a long-term assessment showing a consistent and progressive improvement of carotid stiffness and carotid distensibility as compared to baseline values after 2 years of treatment. The exact mechanism by which PCSK-9 inhibitor exert a beneficial effect in improving carotid stiffness is still not completely described. Lowering LDL-C levels represents one mechanism by which carotid stiffness reduction can
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
Genotype and PCSK-9 inhibitors
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Table 2 Linear regression analysis. Association of clinical and laboratory parameters with carotid stiffness and distensibility.
LDL-c Diabetes Hypertension
Carotid Stiffness
Carotid Distensibility
b Z 0.511 p < 0.001 b Z 0.346 p Z 0.007 b Z 0.391 p Z 0.004
b Z 0.506 p Z 0.001 b Z 0.294 p Z 0.040 b Z 0.366 p Z 0.018
be achieved [9,10]. Several literature data suggested that lipid-lowering therapies, by reducing oxidative stress and balancing nitric oxide synthase (eNOS)/nitric oxide synthase (iNOS) ratio, lead to an improvement of endothelial function [33e35]. Thus, it can be expected that lipidlowering therapies may induce functional and structural modifications in artery wall, positively impacting on arterial stiffness. In line with this hypothesis, we observed a correlation between LDL levels and carotid stiffness and, such an association was confirmed after adjusting for potential confounders. However, it remains unknown whether this effect on carotid stiffness is mostly mediated by reduction of cholesterol levels or by other presently unknown mechanisms. In the present study, we documented that, after adjusting for potential confounders, changes in LDL score (expression of sd-LDL concentration) was a predictor of modifications in carotid stiffness. This finding is in line with data showing that sd-LDL are associated with arterial stiffness and predicts cardiovascular events [36]. By a pathophysiological point of view, we have to account that sd-LDL particles bind with lower affinity LDL receptor [37]. sd-LDL synthesis is consequent to delipidation of large LDL particles. The up-regulation of the LDL receptor, induced by inhibition of PCSK-9, enhances the binding potential, leading to a variation in LDL
diameter and composition. Following to LDL receptor upregulation, the clearance of circulating large LDL particles hampers de-lipidation and leads to a reduction in sdLDL [38]. Although these modifications seem to determine an improvement in carotid stiffness, to support this hypothesis, some basic science experiments should be conducted, also including direct effects of PCSK9 inhibitors on human endothelial cell culture. Besides evident consequence due to LDL-C reduction, possible ancillary effects induced by PCSK-9 inhibitors can be presumed. A recent study showed that PCSK-9 induces a pro-inflammatory response in macrophages [39] and a treatment with a PCSK-9 inhibitor was able to induce a decrease in monocyte migratory capacity, lipid content, as well as inflammatory responsiveness [40]. Overall, the above described favorable effects might determine a reduced cardiovascular risk. This hypothesis is mostly supported by results of the randomized, doubleblinded, placebo-controlled FOURIER trial showing that in 27.564 patients with atherosclerotic cardiovascular disease the association of Evolocumab to standard statin treatment significantly reduced the risk of fatal and non-fatal cardiovascular events (HR: 0.85, 95%CI: 0.79e0.92, p < 0.001) [12]. A further demonstration of a potential link between lipid-lowering effect and carotid stiffness derives from the evidence that, as compared to double/compound heterozygotes, patients with heterozygote mutation of LDLR reported a more pronounced LDL reduction during treatment with Evolocumab accompanied by a more significant reduction in carotid stiffness and increase in carotid distensibility. However, the limited number of patients double/compound heterozygote partly hampers
Figure 5 Changes in carotid stiffness and carotid distensibility from baseline values to week 12 and week 104 during treatment with PCSK9 inhibitor. )The analysis was performed on 16 patients with data at baseline, at week 12 and at week 104.
Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018
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generalizability of these results and suggests the need of further studies to definitely address this issue. Some potential limitations of the present study need to be discussed. Most of patients included in the present study have concomitant cardiovascular risk factors, potentially impacting on carotid stiffness. To overcome this potential source of heterogeneity, sub-group analyses were performed and showed that patients not receiving statins represented the only clinical subset reporting a less significant reduction in carotid stiffness and increase in carotid distensibility, thus suggesting a cooperation between statins and PCSK-9 inhibitors. This hypothesis is also supported by previous studies showing that PCSK-9 inhibitors reduced LDL-C levels of about 50% when used alone and about 70% when used together with statins [29]. A hypothetical mechanism of a synergic effect of association between statins and PCSK-9 inhibitors is due to increased plasma levels of PCSK-9 in patients treated with statins alone. This effect is secondary to the activation of sterol responsive element binding protein (SREBP) pathway because of the inhibition of cholesterol biosynthesis. Thus, patients receiving chronic statin therapy have higher levels of PCSK-9 and a more marked response to PCSK-9 inhibitor [41]. On the other hand, the significant improvement in carotid stiffness consistently found in all the other sub-groups considered, suggests that PCSK-9 treatment leads to a reduction of the cardiovascular risk regardless of the presence of concomitant cardiovascular risk factors. In our sample, we observed a trend toward a reduction in SBP during treatment with PCSK9 inhibitor. We excluded any modification in antihypertensive treatment during study period to exclude a potential impact on results. On the other hand, we cannot explain these changes as an ancillary effect of PCSK9 inhibition, considering that previous data showed that PCSK9 levels are unrelated to blood pressure [42]. Thus, this issue needs to be specifically addressed in future studies. The lack of a control group might represent a limitation of the present study. However, PCSK-9 can be used only in patients not achieving target LDL-C levels under maximal statin treatment. Thus, a potential control group should be represented by patients not eligible to treatment with PCSK-9 or refusing this treatment. These hypothetic controls are not likely to be comparable to cases. Overall, the pre-post observational design allows for partly overcome the intrinsic inter-individual heterogeneity, with each patient being the control of himself. A further potential limitation is represented by the small study sample and, in turn, the limited number of patients available for subgroup analyses. However, FH has a prevalence of 1:300 according to epidemiological data [3] and PCSK-9 inhibitors can be used only in patients not achieving target LDL-C levels under maximal lipid lowering treatment. Thus, this study enrolled a highly selected population with defined eligibility criteria with a sample size calculated to achieve a target power of 80%. In conclusion, this study showed, for the first time a long-standing and consistent effect of treatment with
M.N.D. Di Minno et al.
PCSK-9 inhibitor Evolocumab on carotid stiffness. Moreover, we found that small dense LDL reduction is related to changes in carotid stiffness, thus suggesting a potential impact for changes in lipoprotein composition as a mechanism for cardiovascular risk reduction. Further studies are needed to fully investigate underlying mechanisms and the impact on cardiovascular events. Source of funding No funds were received for this study. Declaration of Competing Interest All the Authors have nothing to declare. References [1] Rubba P, Gentile M, Marotta G, Iannuzzi A, Sodano M, De Simone B, et al. Causative mutations and premature cardiovascular disease in patients with heterozygous familial hypercholesterolaemia. Eur J Prev Cardiol 2017;24:1051e9. [2] Stitziel NO, Peloso GM, Abifadel M, Cefalu AB, Fouchier S, Motazacker MM, et al. Exome sequencing in suspected monogenic dyslipidemias. Circ Cardiovasc Genet 2015;8:343e50. [3] 2019 ESC/EAS guidelines for the management of dyslipidaemias: lipid modification to reduce cardiovascular risk. Atherosclerosis 2019;290:140e205. [4] Jadhav UM, Kadam NN. Non-invasive assessment of arterial stiffness by pulse-wave velocity correlates with endothelial dysfunction. Indian Heart J 2005;57:226e32. [5] Blacher J, Pannier B, Guerin AP, Marchais SJ, Safar ME, London GM. Carotid arterial stiffness as a predictor of cardiovascular and allcause mortality in end-stage renal disease. Hypertension 1998;32: 570e4. [6] van Sloten TT, Schram MT, van den Hurk K, Dekker JM, Nijpels G, Henry RM, et al. Local stiffness of the carotid and femoral artery is associated with incident cardiovascular events and all-cause mortality: the Hoorn study. J Am Coll Cardiol 2014;63:1739e47. [7] Yang EY, Chambless L, Sharrett AR, Virani SS, Liu X, Tang Z, et al. Carotid arterial wall characteristics are associated with incident ischemic stroke but not coronary heart disease in the Atherosclerosis Risk in Communities (ARIC) study. Stroke 2012;43:103e8. [8] Tada H, Kawashiri MA, Nohara A, Inazu A, Mabuchi H, Yamagishi M. Assessment of arterial stiffness in patients with familial hypercholesterolemia. J Clin Lipidol 2018;12:397e402 e2. [9] Canepa M, Artom N, Ameri P, Carbone F, Montecucco F, Ghigliotti G, et al. Short-term effect of rosuvastatin treatment on arterial stiffness in individuals with newly-diagnosed heterozygous familial hypercholesterolemia. Int J Cardiol 2018;255:215e20. [10] Oh PC, Han SH, Koh KK, Lee K, Seo JG, Suh SY, et al. Rosuvastatin treatment improves arterial stiffness with lowering blood pressure in healthy hypercholesterolemic patients. Int J Cardiol 2014; 176:1284e7. [11] Nicholls SJ, Brandrup-Wognsen G, Palmer M, Barter PJ. Metaanalysis of comparative efficacy of increasing dose of Atorvastatin versus Rosuvastatin versus Simvastatin on lowering levels of atherogenic lipids (from VOYAGER). Am J Cardiol 2010;105:69e76. [12] Giugliano RP, Pedersen TR, Park JG, De Ferrari GM, Gaciong ZA, Ceska R, et al. Clinical efficacy and safety of achieving very low LDL-cholesterol concentrations with the PCSK9 inhibitor evolocumab: a prespecified secondary analysis of the FOURIER trial. Lancet 2017;390:1962e71. [13] Schwartz GG, Steg PG, Szarek M, Bhatt DL, Bittner VA, Diaz R, et al. Alirocumab and cardiovascular outcomes after acute coronary syndrome. N Engl J Med 2018;379:2097e107. [14] Han J, Wang X, Ye P, Cao R, Yang X, Xiao W, et al. Plasma PCSK9 levels are unrelated to arterial stiffness in a community-based, 4.8-year prospective study. J Hum Hypertens 2017;31:720e4.
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Please cite this article as: Di Minno MND et al., Changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab: A prospective cohort study, Nutrition, Metabolism & Cardiovascular Diseases, https://doi.org/10.1016/ j.numecd.2020.02.018