Low PCSK-9 levels Are Associated with Favorable Neurologic Function after Resuscitation from out of Hospital Cardiac Arrest

Endotoxemia after cardiopulmonary resuscitation (CPR) is associated with unfavorable outcome. Proprotein convertase subtilisin/kexin type-9 (PCSK–9) regulates low-density lipoprotein receptors, which mediate the hepatic uptake of endotoxins. We hypothesized that PCSK–9 concentrations are associated with neurological outcome in patients after CPR. Successfully resuscitated out-of-hospital cardiac arrest patients were included prospectively (n = 79). PCSK–9 levels were measured on admission, 12 h and 24 h thereafter, and after rewarming. The primary outcome was favorable neurologic function at day 30, defined by cerebral performance categories (CPC 1–2 = favorable vs. CPC 3–5 = unfavorable). Receiver operating characteristic curve analysis was used to identify the PCSK–9 level cut-off for optimal discrimination between favorable and unfavorable 30-day neurologic function. Logistic regression models were calculated to estimate the effect of PCSK–9 levels on the primary outcome, given as odds ratio (OR) and 95% confidence interval (95%CI). PCSK–9 levels on admission were significantly lower in patients with favorable 30-day neurologic function (median 158 ng/mL, (quartiles: 124–225) vs. 207 ng/mL (174–259); p = 0.019). The optimally discriminating PCSK–9 level cut-off was 165 ng/mL. In patients with PCSK–9 levels ≥ 165 ng/mL, the odds of unfavorable neurological outcome were 4.7-fold higher compared to those with PCSK–9 levels < 165 ng/mL. In conclusion, low PCSK–9 levels were associated with favorable neurologic function.


Introduction
Proprotein convertase subtilisin/kexin type 9 (PCSK-9) regulates the expression of low-density lipoprotein (LDL) receptors (-R) on hepatocytes [1]. PCSK-9 binds to the LDL-R and facilitates the intracellular degradation of the receptor. Thereby, it reduces hepatic LDL-R expression, LDL uptake and consequently increases circulating LDL [2]. The discovery that people with gain-of-function mutations have hypercholesterolemia [3] and those with loss of function mutations have low levels of LDL and a lower risk to develop coronary events in spite of the presence other non-lipid-related

Materials and Methods
The independent review board of the Medical University of Vienna approved the study, which was performed in accordance with the Declaration of Helsinki. A waiver was obtained for informed consent at admission, and patients were informed of their study participation on regaining consciousness.
Adults (>18 years) with out-of-hospital cardiac arrest of presumed cardiac cause who had achieved the return of spontaneous circulation (ROSC) at admission to the intensive care unit (ICU) of the Department of Emergency Medicine at the Medical University of Vienna, were prospectively included in this study. All patients suffered from acute coronary syndrome and underwent percutaneous coronary intervention after admission. No-flow intervals were defined as the time from collapse to start of CPR, low-flow time as the time from CPR start to sustained ROSC and sustained ROSC as recovery of spontaneous circulation for more than 20 min. These intervals were established by immediate, structured interviews with the dispatch center, involved emergency physicians, paramedics, or bystander who performed the emergency call. All patients have undergone targeted temperature management with target temperatures between 32 and 34 • C for 24 h. Rewarming was performed at a rate of 0.25-0.5 • C. Resuscitation related parameters were analyzed and reported according to the Utstein recommendations [23,24]. Patient demographics, co-medication, and concomitant diseases were obtained by chart review. Laboratory data were obtained from the ISO-credited central laboratory at the General Hospital of Vienna. Blood samples were obtained at admission, 12 h and 24 h after admission and at rewarming. Ethylendiaminetetraacetic acid (EDTA) anti-coagulated blood was centrifuged at 2000 g for 10 min. Plasma was obtained and stored at -80 • C until batch analysis. PCSK-9 concentrations were measured by enzyme-linked immunoassays in EDTA plasma (Circulex human PCSK-9 Elisa, MBL International, Woburn, MA, USA).

Definition of Multi-Organ Failure (MOF):
Multi-organ failure was defined as a failure of two or more organs within the first 48 h after admission. Acute kidney failure was defined as an elevation of serum-creatinine ≥ 0.5 mg/dL within the first 48 h. Acute shock liver was defined as an elevation of glutamate oxalacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) > 10-100 fold within the first 48 h.

Endpoints
The primary outcome was favorable neurologic function at day 30 after resuscitation, assessed using the cerebral performance category (CPC) 5-points scale (CPC 1-2 = favorable neurologic function vs. CPC 3-5 = a composite of unfavorable neurologic function and death). CPC was assessed by study fellows through structured face-to-face or telephone interviews with the patient, the relatives, treating physicians, or nursing home members.
30-day mortality was the secondary outcome. We investigated the influence of PCSK-9 levels on these outcomes, but also associations with inflammatory and other resuscitation specific parameters.

Statistics
We present continuous data as median ± quartiles, and categorised data as absolute count and relative frequency. The Mann-Whitney U test was used to test for differences between two independent group medians. For categorised data we used a chi-squared test. Receiver operating characteristic curve (ROC) analysis was performed (i) to analyze specificity and sensitivity of PCSK-9 concentrations to predict neurological outcome and (ii) to identify the PCSK 9 level cut-off for optimal discrimination between favorable and unfavorable 30-day neurologic function (primary outcome). Additionally, we performed ROC-analysis for the secondary endpoint 30-day mortality. To estimate the association between PCSK-9 levels, potential confounders and primary outcome we used logistic regression analysis. We included covariables into the models based on clinical reasoning. Continuous variables were categorised and used as index variables. If applicable, we added a separate category for missing observations, otherwise no data-imputation for missing data was applied. We tested for linearity and interactions of the main effects using the likelihood ratio test. The log rank test was used to compare survival distributions between patients with PCSK-9 levels below and above the identified optimal cut-off at admission. Cox-regression analysis was used to estimate the association of PCSK-9 levels and the included co-variables with 30-day mortality and to assess potential confounding. The limited sample size precluded the calculation of multivariable regression models. Therefore, we calculated models only including sex and age to present adjusted odds (or hazard) ratios for these two factors. Moreover, we calculated multiple linear regression models including all covariables, because this procedure is more apt at dealing with small sample sizes (results are presented in the supplement, Table S1). For data management and analyses we used Stata 14 (Stata Corp, College Station, Tx, USA) and IBM SPSS Statistics (Version 26, IBM Corporation, NY, USA). Generally, a two-sided p-value less 0.05 was considered statistically significant.

Receiver Operating Characteristic Curve Analysis
The optimal PCSK-9 level cut-off for the discrimination between favorable and unfavorable neurologic function was 165 ng/mL based on ROC analysis (area under the curve (AUC) = 0.67; 95% confidence interval (CI) 0.55-0.80, Figure 2). Sensitivity for this cut-off was 79% and specificity was 56%.
The optimal PCSK-9 level cut-off for the discrimination between 30-day survival and 30-day mortality was 180 ng/mL based on ROC analysis (AUC = 0.67, 95% CI 0.54-0.79, Figure 2) with a sensitivity of 81% and a specificity of 51%.

Receiver Operating Characteristic Curve Analysis
The optimal PCSK-9 level cut-off for the discrimination between favorable and unfavorable neurologic function was 165 ng/mL based on ROC analysis (area under the curve (AUC) = 0.67; 95% confidence interval (CI) 0.55-0.80, Figure 2). Sensitivity for this cut-off was 79% and specificity was 56%.
In multivariable analysis we included PCSK-9 levels, initial heart rhythm, no-flow interval, CRP levels, age and gender. The size of the sample did not allow for a multivariable analysis containing all co-variables simultaneously, but multivariable analyses including each relevant co-variable separately could indicate possible confounding by age (p = 0.041) or initial rhythm (p = 0.031) (Table S1). We could not identify significant interactions. Statin intake may increase PCSK-9 concentrations [25], but in our study we did not observe any influence on PCSK-9 levels on the outcome.

Mortality
Thirty-day mortality was significantly higher in patients with PCSK-9 levels ≥180 ng/mL (18% vs. 46%; p = 0.006) (Figure 3). all co-variables simultaneously, but multivariable analyses including each relevant co-variable separately could indicate possible confounding by age (p = 0.041) or initial rhythm (p = 0.031) (Table  S1). We could not identify significant interactions. Statin intake may increase PCSK-9 concentrations [25], but in our study we did not observe any influence on PCSK-9 levels on the outcome.

Additional Analyses
Since both, CRP levels and PCSK-9 levels were associated with neurologic function, we categorized patients into four groups: low CRP and low PCSK-9, high CRP and low PCSK-9, low CRP and high PCSK-9, high CRP and high PCSK-9. For PCSK-9 the respective cutoffs for neurologic function (165 ng/mL) and for 30-day mortality (180 ng/mL) were used. For CRP levels no clear cutoff was identifiable in ROC analysis and, therefore, the upper limit of normal (reference range < 0.5 mg/dL) was used. There was a significant difference in the distribution of favorable and unfavorable neurologic outcome (p = 0.007) and 30-day mortality (p = 0.005) over these four groups (Table 2). Table 2. Outcome analysis for groups according to PCSK-9 and C-reactive protein levels.

Low CRP Low PCSK-9
High CRP Low PCSK-9 Low CRP High PCSK-9 High CRP High PCSK-9 In a post-hoc analysis we analyzed whether neuron-specific enolase or the neuron-specific protein S100, which were available for 52 and 51 patients (measured between 24 and 48 h after cardiac arrest), respectively, correlate with PCSK-9 levels. However, no such correlation could be identified.
Loss-of-function mutations but also low PCSK-9 concentrations were associated with lower mortality in septic patients, while high PCSK-9 levels were associated with organ dysfunction [11,14,26]. The assumed mechanism is a more rapid detoxification of bacterial lipids via higher LDL-R expression and consequently a reduced inflammatory response [11,14]. Given the similarities between sepsis and PCAS [27][28][29], we hypothesized that low PCSK-9 levels may be associated with favorable outcome after out-of-hospital cardiac arrest and indeed our findings confirm this hypothesis. Moreover, recently Lee et al. reported that in patients after cardiopulmonary resuscitation, total cholesterol, low-density lipoprotein, as well as high-density lipoprotein differed between those with favorable or unfavorable outcome, whereas especially high-density lipoprotein and total cholesterol were associated with clinical outcomes after regression analysis [30].
Based on the underlying mechanism, we expected lower levels of inflammatory biomarkers in patients with low PCSK-9 levels. However, we only observed lower CRP levels in the first 12 h after admission. Of note, the causes for inflammation after CPR are multifactorial including increased intestinal permeability, but also aspiration pneumonia, ischemic events, comorbidities, etc. Also, therapeutic interventions including temperature management or pharmacological treatment may affect the inflammatory response, which in sum may explain the missing association at later time points. Similarly, to our findings Vaahersalo et al. reported that interleukin-6 levels only at admission, but not at later time points predicted neurologic outcome [27].
Furthermore, recent data suggest that PCSK-9 could be involved in the release of pro-inflammatory cytokines after hypoxia and reperfusion [22]. Moreover, PCSK-9 also interacts with LOX-1, the receptor for oxidized LDL, which is upregulated in ischemic hearts [21,22]. Low levels of PCSK-9 (or inhibition of PCSK-9) could therefore exert beneficial effects-Of note, all patients included in this analysis had an acute coronary syndrome as cause of cardiac arrest. It is therefore possible, that in this selected cohort the associations of PCSK-9 levels with outcomes are especially pronounced. Thus, our findings should be confirmed in other cardiac arrest populations, including patients with non-cardiac cause of cardiac arrest.
PCSK-9 levels changed only marginally during the first 24 h, but increased significantly until rewarming in both groups, while CRP levels increased constantly. The increase in PCSK-9 levels is most likely caused by the inflammatory response [2]. Of note, during experimental endotoxemia in healthy volunteers PCSK-9 levels initially decreased by approximately 40% before they returned to baseline [15]. This observation ended after 24 h, but there were no increases in PCSK-9 in that period. Any possible increases in that model would take at least 24 h, which corresponds well with our results.
Astonishingly, the marked difference in neurologic outcome in patients having PCSK-9 concentrations < 165 ng/mL (or < 180 ng/mL for 30-day mortality) cannot be explained by differences in the "classical" CPR variables: bystander status, basic life-support, no-flow, low-flow intervals, time to sustained ROSC, and initial heart rhythm differed only marginally. It is noteworthy that only patients with sustained ROSC at admission were included in the trial.
In multivariate analysis none of the chosen factors remained significant. However, this may also be caused by the small sample size in the study and should be re-evaluated in a larger sample.
Based on our results, low PCSK-9 levels may have beneficial effects on the neurologic outcome after CPR. Thus, early pharmacologic inhibition of PCSK-9 may be an interesting treatment option to improve neurologic survival. Further research may be warranted to elucidate its therapeutic potential.
Limitations: this is a cohort study with its inherent limitations. The population was selected based on the inclusion and exclusion criteria, and we cannot exclude a potentially associated selection bias. The effects of targeted temperature management on PCSK-9 levels and metabolism are unknown. The sample size is limited and analysis in larger populations to confirm our results is warranted. We did not measure endotoxins or other lipoprotein levels. All patients were treated with target temperature management, which may influence PCSK-9 levels.

Conclusions
In conclusion, lower PCSK9 levels at admission were associated with favorable neurologic outcome after CPR.