PCSK9, A Promising Novel Target for Age-Related Cardiovascular Dysfunction

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SUMMARY
Cardiovascular diseases (CVDs) are the leading cause of death among elderly people.Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important regulator of cholesterol metabolism.Herein, we investigated the role of PCSK9 in age-related CVD.Both in humans and rats, blood PCSK9 level correlated positively with increasing age and the development of cardiovascular dysfunction.Age-related fatty degeneration of liver tissue positively correlated with serum PCSK9 levels in the rat model, while development of age-related nonalcoholic fatty liver disease correlated with cardiovascular functional impairment.Network analysis identified PCSK9 as an important factor in age-associated lipid alterations and it correlated positively with intimamedia thickness, a clinical parameter of CVD risk.PCSK9 inhibition with alirocumab effectively reduced the CVD progression in aging rats, suggesting that PCSK9 plays an important role in cardiovascular aging.T he average life expectancy is continuously increasing worldwide, and the population over 65 years of age is expected to double in the coming decades.The prevalence of cardiovascular and liver diseases, including vascular dysfunction, fatty liver, and heart failure, increases with age 1,2 and is expected to further increase over the next decades.
Cardiovascular aging is characterized by atherosclerosis and the remodeling of the myocardium such as hypertrophy and fibrosis, and will eventually culminate in the development of heart failure, 3 while hallmarks of liver aging are fatty degeneration, fibrosis, and inflammation. 2The prevalence of nonalcoholic fatty liver disease (NAFLD) increases with age, 4 and NAFLD is associated with increased cardiovascular risk, including heart failure. 4,5Major pathophysiological events in age-related cardiovascular disorders include, but are not limited to, oxidative/nitrative stress 6 and mitochondrial dysfunction. 7,8Ultimately, these pathways culminate in cardiovascular dysfunction and remodeling of the cardiac tissue with characteristic changes in the extracellular matrix and finally with the development of myocardial hypertrophy and fibrosis. 9Despite the need for effective drug therapies to prevent or treat aging-associated heart failure, therapeutic options are largely limited for this condition, especially taking the prevalence of heart failure with preserved ejection fraction into account.
Aging is associated with dysregulation of cholesterol metabolism. 10Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of a class of proteinase K-like serine proteases first described in cerebellar cells as a regulator of apoptosis. 11Later, PCSK9 was identified as one of the most important regulators of cholesterol metabolism via the regulation of the expression of low-density lipoprotein receptor (LDLR) in the liver, where it binds to liver LDLRs, thereby promoting their degradation. 12As a result, lowdensity lipoprotein (LDL) cholesterol uptake is reduced by the lower number of available LDLRs, which will eventually lead to hypercholesterolemia. 13 PCSK9 is expressed in different organs, and while it is most abundant in the liver, it is expressed at lower levels in the heart, too. 12Levels of PCSK9 depend on many factors including, for example, age, sex, diurnal rhythm, nutrition, and comorbidities. 14PCSK9 expression is regulated by many factors, with one of the most important among them being the sterol regulatory element-binding proteins (SREBPs). 15SREBPs are activated by oxidative stress, 16 during inflammation, 17 or by aging, [17][18][19] contributing to altered lipid metabolism and steatosis. 17   first in targeted patient populations of familial hypercholesterolemia and followed by cardiovascular diseases, in which recommended cholesterol levels were not met or cardiovascular risk remained high despite optimal drug therapy. 14,212][23][24][25] Recently, a number of studies investigated the pleiotropic effects of PCSK9 inhibitors beyond their lipid-lowering properties.PCSK9 stimulates oxidized LDL formation 26 and participates in a bidirectional crosstalk with reactive oxygen species formation, 27 leads to endothelial activation/damage, 28 and is a critical immune response regulator in sepsis. 29On the other hand, PCSK9 inhibition proved to have antioxidant effects in alcohol-induced liver disease 30 or in H 2 O 2 -induced oxidative damage, 31 was able to reduce oxidized LDL-induced myocardial injury, 32 and was recently shown to reduce mortality or serious complications and inflammatory response in COVID-19 patients. 33though representing a major group of Western societies, elderly people are often underrepresented in clinical trials of lipid-lowering therapies. 34spite available data about the beneficial effects   was considered statistically significant.

PCSK9 in Aging Heart
To perform differential expression analysis for the transcriptomic data, the raw count files from HISAT2 were used as an input to the DESeq2 package in R

RESULTS
HUMAN STUDY.B a s i c c h a r a c t e r i s t i c s o f t h e s t u d y g r o u p s .According to our predefined inclusion and exclusion criteria (Figure 1A), 100 patients were included in the young group and 109 in the elderly group of the final study population.Both groups were characterized by a modest female predominance (Table 1).The subjects in the young group had higher height, lower weight, and correspondingly lower BMI.
The prevalence of hypertension, hyperlipidemia, and diabetes mellitus was significantly higher in the elderly group.There were more current smokers among the young.Elderly individuals presented with higher serum levels of total cholesterol, triglyceride, and LDL cholesterol.High-density lipoprotein levels were similar between the 2 groups (Table 1).The medication list for the study groups is listed in Supplemental Table 1.
Echocardiography data of the study groups.Two-dimensional and speckle-tracking echocardiographic data are summarized in Table 2. Higher values of interventricular septal, left ventricular (LV) posterior wall thicknesses, and LV mass index (Figure 1B) were present in the elderly group, while LV end-diastolic volume did not differ.However, LV systolic function was significantly lower based on the reduced values of ejection fraction and global longitudinal strain (GLS) as well (Figure 1B).The ratio of early mitral inflow velocity to late mitral inflow velocity (E/A) was lower, E-wave deceleration time was longer, average early diastolic mitral annular velocity (e 0 ) was lower, and E/e 0 values were higher, while left  2).

I n c r e a s e d P C S K 9 l e v e l s w e r e a s s o c i a t e d w i t h
a g e -r e l a t e d c a r d i o v a s c u l a r d y s f u n c t i o n .Plasma PCSK9 levels were significantly higher in the elderly group (Figure 1B).The network from iNetModels database showed that PCSK9 and age both positively correlated with serum triglyceride and LDL cholesterol, and age positively correlated with total cholesterol level (Figure 1C).In the pooled population of the Budakalász Study, higher PCSK9 levels were associated with higher LV mass index and reduced LV GLS (Figure 1D).In a multivariable linear regression model, age, sex, BMI, and hyperlipidemia were independent predictors of plasma PCSK9 levels (Table 3).In another model, PCSK9 level and hypertension were found to be independent predictors of E/e 0 (ie, diastolic dysfunction) (Table 4).Representative echocardiographic apical 4-chamber view images and corresponding speckle-tracking analysis to determine GLS in the young and the elderly are shown in Figure 1E.In a separate analysis, PCSK9 gene expression levels were very low in heart compared with liver in the human RNA sequencing data obtained from the Genotype-Tissue Expression Project Portal (Supplemental Table 2).Cardiac gene expression levels of PCSK9 in human and rat hearts showed very low levels of PCSK9 expression in hearts (Supplemental Table 3).Protein levels of PCSK9 were higher in aging hearts (Supplemental Table 3).On the other hand, single-nuclei transcriptomics showed that PCSK9 was only expressed in <0.07%, while troponin T, as a reference gene, was expressed in almost all of the ventricular cardiomyocyte cells.Spatial transcriptomics data also showed that PCSK9 level in heart was very low compared with troponin T (Supplemental Figure 1).2).
Aging led to characteristic changes in the liver.PCSK9 levels were significantly increased in the aging liver (Figure 2A).Liver LDLR expression was decreased in the aging animals, which was in line with the observed increase in serum total cholesterol and oxidized LDL levels (Figures 2B and 2C).Alirocumab significantly increased the LDLR expression and led to a decrease in total cholesterol and oxidized LDL levels (Figures 2B and 2C).   1 and 2.  5B).

Matyas et al
On the other hand, fibrotic remodeling of the myocardium was observed in aging animals (Figure 6A).These changes were supported with a shift in messenger RNA expression profile (Figures 6B   and 6C).PCSK9 inhibitor treatment prevented further progression of fibrotic remodeling in the heart (Figures 6A and 6C).
A l i r o c u m a b a t t e n u a t e d l i p i d p e r o x i d a t i o n , o x i d a t i v e s t r e s s , a n d c e l l d e a t h .Increasing age was associated with higher levels of oxidative (lipid peroxidation) and nitrative stress (Figure 7A) and an altered gene expression profile for genes related to oxidative stress and cell death (Figure 7B).Cell death markers PARP1 and caspase 3 showed increased activity in aging hearts (Figure 7C).Gene expression studies further supported increased oxidative stress, proinflammatory pathways, and vascular injury in the aging myocardium (Figures 7D and 7E).Alirocumab treatment effectively reduced oxidative/nitrative stress and cell death in aging hearts (Figure 7).Abbreviations as in Tables 1, 2, and 3.  Others reported the liver as the major site of PCSK9 production with several orders of magnitude more production than in any other organ. 37Comparable to these data, our animal studies showed abundant levels of PCSK9 in the liver, while it was further increased during aging, leading to lower expression of LDLR and dysregulation of cholesterol metabolism in the animals.Our data suggested that expression levels of PCSK9 in the cardiac tissue were very low in human bulk, single-nuclei, and spatial transcriptomic data as well as in mouse and rat tissues.Consistently with the important role of PCSK9 production in the liver, Food and Drug Administration-approved inclisiran, which targets PCSK9 synthesis in the liver, reduces LDL cholesterol levels to similar extent as the monoclonal antibodies targeting PCSK9 in humans (by approximately 50%). 38e relationship between elevated PCSK9 levels and hepatic fat accumulation and cardiovascular health is of significant research interest.[41][42] Increased PCSK9 levels were observed to be associated with hepatic fat accumulation in humans. 20reover, disarrangement of LDLR expression is associated with hepatic steatosis 37 and LDLR is thought to be a major regulator of liver steatosis. 43On the other hand, gain-of-function mutations of PCSK9 result in familial hypercholesterolemia and increased cardiovascular risk, while loss-of-function mutations are associated with better lipid profile and confer to lower cardiovascular risk. 44Importantly, PCSK9 inhibition was shown to protect against alcoholic 30 and nonalcoholic liver steatosis. 43 Recently, PCSK9 inhibitors were shown to have added benefit in the treatment of cardiovascular diseases.Monoclonal antibodies targeted against the circulating plasma PCSK9 lowered cardiovascular risk on top of conventional lipid-lowering therapies. 22,24though modulating LDL cholesterol levels is the major site of effect of PCSK9, 21 additional direct or indirect effects may include a proinflammatory effect on white blood cells 14 or macrophages, 45 regulation of vascular endothelial and smooth muscle cell proliferation, 27 increasing the expression of oxidized LDLRs in endothelium 27 or macrophages, 46 mitochondrial DNA damage, 47 and inflammation. 46Activation of the oxidized LDLR resulted in the induction of PCSK9 expression and decreased function in cardiomyocytes. 48Our animal study confirmed the dysregulation of cholesterol metabolism in aging, while it was paralleled with increased oxidized LDL levels, a major contributor to cardiomyocyte and vascular stress. 49Alongside these, myocardial lipid      The very low (close to zero) local PCSK9 expression in cardiac tissues (both in young and aging) and high hepatic expression, which is further increased with age, suggest that the liver is the primary source for age-related increased serum PCSK9 levels and is possibly the primary site of action of PCSK9 inhibitors.However, direct cardiovascular effects of the drug cannot be ruled out either.

PCSK9 in Aging Heart
STUDY LIMITATIONS.The human study was a community-based screening sample not designed    Although multiple analyses showed negligible levels of PCSK9 in young and aging hearts, enzyme-linked immunosorbent assay showed very low but detectable levels of PCSK9 protein in aging rat hearts compared with the young hearts.A possible explanation is that, despite our best efforts to perfuse the hearts, remaining blood in heart tissue samples could

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J Am Coll Cardiol Basic Trans Science 2023;8:1334-1353) Published by Elsevier on behalf of the American College of Cardiology Foundation.This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).
Moreover, circulating PCSK9 levels are associated with hepatic fat content and NAFLD severity. 20PCSK9 was recently identified as a potential therapeutic target to treat hypercholesterolemia, A B B R E V I A T I O N S A N D A C R O N Y M S BMI = body mass index DT = deceleration time ÀdP/dt = minimal slope of maximal rate of left ventricular Therapeutics, University of Kansas Medical Center, Kansas City, Kansas, USA; and the i Department of Internal Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA.*Drs Matyas and Trojnar contributed equally to this work.The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors' institutions and Food and Drug Administration guidelines, including patient consent where appropriate.For more information, visit the Author Center.Manuscript received March 1, 2023; revised manuscript received June 26, 2023, accepted June 27, 2023.

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FIGURE 1 3 PCSK9
FIGURE 1 Increased PCSK9 Levels Are Associated With Age-Related Cardiovascular Dysfunction

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A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L .8 , N O . 1 0 , 2 0 2 Alcoholism and conformed to the National Institutes of Health guidelines on animal experiments (Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health [publication 86-23, revised 1985]).The housing of rats and treatment protocol with PCSK9 inhibitor alirocumab and sample harvesting are described in the Supplemental Appendix.

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0.3 (R Foundation for Statistical Computing), and genes with P value <0.05 were considered as significantly differentially expressed genes.The gene-level statistics (log2 fold changes and P value) from DESeq2 were then used to perform functional analysis, together with the gene-set collection from the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology downloaded from the Enrichr library.The functional analyses were performed using the PIANO package in R. The Benjamin-Hochberg false discovery rate was used for the adjustment.Kyoto Encyclopedia of Genes and Genomes or Gene Ontology terms with false discovery rate <0.05 were considered significant.Visualization of the results was done using Seaborn package in Python 3.7 (Python Software Foundation).Statistical analysis was performed in R version 4.0.3 or in GraphPad Prism 7.0 for Windows (Graph-Pad Software).Results with P < 0.05 were accepted as statistically significant.
atrial peak left atrial longitudinal strain (LA PALS) was reduced in the elderly, pointing at LV diastolic dysfunction.Correspondingly, left atrial volume was significantly higher.Concerning the right heart, the right ventricle was larger in the elderly group, but the right atrial volume and right ventricular systolic function (quantified by tricuspid annular plane systolic excursion [TAPSE]) were similar.The peak velocity of the tricuspid regurgitation jet was higher among the elderly individuals, suggesting elevated pulmonary artery systolic pressure (Table ANIMAL STUDY.S e r u m P C S K 9 l e v e l s c o r r e l a t e d w i t h c a r d i a c d y s f u n c t i o n .Serum PCSK9 levels were significantly increased in the aging animals and its level correlated positively with LV mass values (Figure 1F).Serum PCSK9 levels correlated with the development of systolic dysfunction (ejection fraction, slope of end-systolic pressurevolume relationship [ESPVR], preload recruitable stroke work [PRSW], GLS, longitudinal systolic strain rate [LSr]) and diastolic (left ventricular diastolic time constant [Tau Weiss ], minimal slope of maximal rate of left ventricular pressure rise [ÀdP/ dt], longitudinal early systolic strain rate [LSrE], mitral isovolumic relaxation time [IVRT], mitral deceleration time [DT]) (Figure 1G).Of note, PCSK9 protein and gene expression values were very low in the rat heart in comparison with the liver, which was further supported by independent mouse data (Supplemental Table The aging liver was characterized by fatty, inflammatory, and fibrotic changes (NAFLD activity score [NAS]) (Figures 2D to 2F), along with increased oxidative stress (Figure 2G).Gene expression studies showed overexpression of inflammatory, oxidative stress, and fibrosis-related genes (Figure 2H).Alirocumab effectively reduced the fat accumulation and inflammatory and fibrotic changes in the aging liver (Figures 2D to 2H).Of note, serum PCSK9 levels positively correlated with liver fat content (Figure 2F).The drug treatment had no adverse effects on the liver, represented by unchanged aspartate and alanine transaminase levels in the rats (Supplemental Figure 2).NAFLD development correlated with cardiovascular functional impairment.Severity of NAFLD status (NAS) positively correlated with increasing LV mass values (Figure 3A) and correlated with the impairment of different systolic (ejection fraction, slope of ESPVR, PRSW, GLS, LSr) and diastolic (Tau Weiss , ÀdP/dt, LSrE, IVRT, DT) parameters (Figure 3B).Moreover, liver triglyceride levels correlated positively with increasing LV mass values (Figure 3C) and correlated with cardiovascular systolic and diastolic dysfunction (Figure 3D).Alirocumab improved age-related cardiovascular dysfunction.Conventional echocardiography showed impaired systolic (ejection fraction, cardiac output, fractional area change) and diastolic (mitral IVRT and DT) function in aging animals (Figure 4A).Speckletracking strain analysis supported serious impairment of contractility (global circumferential strain, circumferential strain rate, GLS, LSr) and diastolic dysfunction (circumferential early diastolic strain rate, LSrE) of the aging animals (Figure 4B).Hemodynamic investigation showed significant decrease of conventional systolic functional parameters such as cardiac output, stroke work, and maximal rate of left ventricular pressure rise (dP/dt) in the aging group (Figure 4C), while detailed analysis of pressurevolume loops revealed severe impairment of contractility (slope of ESPVR, PRSW, maximal rate of left ventricular pressure rise and end-diastolic volume relationship [dP/dt-EDV]) in the aging rats (Figure 4C).Diastolic dysfunction in aging (Tau Weiss , LV end-diastolic pressure, ÀdP/dt) was associated

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A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L .8 , N O . 1 arterial pressure values and increased peripheral resistance (Figure 4C), leading to a mismatch in ventriculoarterial coupling (Figure 4C).Hemodynamics, including systolic-diastolic function, contractility, and vascular function, were all improved by the drug treatment (Figure 4C).Alirocumab improved myocardial remodeling.Aging was associated with cardiac hypertrophy, represented by increased cardiomyocyte diameter (Figures 5A and 5B), alteration of messenger RNA expression profile (Figure 5C), and the reactivation of the fetal gene program (Figure 5D).Higher serum B-type natriuretic peptide levels indicated the development of chronic heart failure in this group (Figure 5E).Alirocumab treatment decreased cardiac hypertrophy in aging (Figures 5A and Alirocumab attenuated aging-associated mitochondrial dysfunction.The functional analysis of messenger RNA transcriptomic data revealed significant alteration and decreased expression of mitochondrion-related pathways according to the Gene Ontology Cellular Component terms (Figure8A) and by Kyoto Encyclopedia of Genes and Genomes pathway analysis (Figures8B and 8D).Subsequently, measurement of the activity of mitochondrial complexes showed severe mitochondrial dysfunction in the aging heart (Figure8C).Alirocumab treatment significantly improved mitochondrial function in the aged hearts (Figure8C).DISCUSSIONHerein, we investigated the role of PCSK9 in cardiovascular dysfunction in advanced aging.We found that: 1) advanced age was associated with an increase of serum and liver PCSK9 levels; 2) blood level of PCSK9 correlated positively with the degree of cardiovascular dysfunction both in humans and experimental animals; 3) age-related fatty degeneration of liver positively correlated with serum PCSK9 levels in the rat model; 4) development of age-related NAFLD correlated with cardiovascular dysfunction; and 5) treatment with the PCSK9 inhibitor alirocumab attenuated serum lipid abnormalities, cardiac dysfunction, and cardiac and liver remodeling in aging animals, at least in part, by improving mitochondrial function and lowering oxidative/nitrative stress and inflammation.Our study is the first to investigate plasma PCSK9 levels in a community-based screening sample, comparing young and elderly subgroups of patients not taking statins or other lipid-lowering agents.We performed a detailed characterization of systolic and diastolic cardiac function, including advanced echocardiographic parameters (ie, LV GLS and PALS).In this relatively low-risk population, we showed that elderly individuals present with a mildly decreased LV ejection fraction; however, with a pronounced LV hypertrophy and deterioration of longitudinal myocardial deformation and diastolic dysfunction.The association of these echocardiographic presentations with long-term all-cause mortality was recently established in the same community-based sample.

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FIGURE 2 3 PCSK9
FIGURE 2 Increased PCSK9 Levels Are Associated With Age-Related Liver Changes However, little is known about the role of PCSK9 in age-related steatohepatitis.In our current animal study, we observed increased levels of PCSK9 and the development of NAFLD features of the liver including fat accumulation, inflammation, and oxidative stress.Importantly, fat accumulation in the liver showed positive correlation with serum PCSK9 levels.In concert with the previous observations, degeneration of the liver (represented by NAS and fat content) correlated with the impairment of cardiovascular function in rats.

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A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L .8 , N O . 1 peroxidation, oxidative stress, and cell death markers were up in our aging group, probably leading to altered mitochondrial metabolism and subsequently impaired mitochondrial function and cellular senescence with characteristic changes of the myocardial transcriptome.PCSK9 inhibitor alirocumab exerted antiinflammatory effects in the aging liver, which is in line with a recent report in which silencing PCSK9 repressed oxidized LDLR expression and inflammatory cell activation. 50Moreover, it prevented hepatic LDLR degradation and effectively improved cholesterol levels in aging animals, thereby possibly removing the most important precursor molecule for oxidized LDL production, without causing liver injury or having adverse effect on aging-associated pathophysiological processes.Most importantly, age-

FIGURE 3 1 . 3 PCSK9
FIGURE 3 NAFLD Development Correlates With the Impairment of Cardiovascular Dysfunction

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A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L .8 , N O . 1 attenuated by the drug, most likely by attenuation of the myocardial lipid peroxidation, oxidative stress, and mitochondrial dysfunction and consequent remodeling (hypertrophy and fibrosis) of the heart.A possible explanation for the beneficial cellular and cardiac effects may also involve a better hepatic status including less inflammation, halted fatty degeneration, and overall better cholesterol and oxidative

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FIGURE 9 3 PCSK9
FIGURE 9 Proposed Mechanism of Beneficial Effects of PCSK9 Inhibition on Aging-Related Cardiovascular Dysfunction

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Values are median (IQR) or mean AE SD.The characteristics of the 2 groups were compared using unpaired Student's t test or Mann-Whitney U test, as appropriate.A ¼ late mitral inflow velocity; AV ¼ aortic valve; DT ¼ E-wave deceleration time; E

TABLE 3
Univariable and Multivariable Linear Regression Analysis to Determine the Predictors of Plasma PCSK9 Levels (n ¼ 209) VIF ¼ variance inflation factor; other abbreviations as in Tables

TABLE 4
Univariable and Multivariable Linear Regression Analysis to Determine the Predictors of Average E/e 0 (n ¼ 198)