Elevated expression of clonal hematopoiesis of indeterminate potential in patients with coronary endothelial dysfunction is associated with future cardiovascular events

Aims We aimed to test the hypothesis that the presence of clonal hematopoiesis of indeterminate potential (CHIP) in peripheral blood cells is associated with coronary endothelial dysfunction, enhanced inammatory markers, and major adverse cardiovascular events (MACE). Methods and results We compared targeted next generation sequencing (35 CHIP related genes) between patients with coronary endothelial dysfunction (n = 123) and controls (n=65). Coronary endothelial dysfunction was dened by ≥ 20% decrease in coronary artery diameter (CAD) or ≤ 50% increase in coronary blood ow (CBF) in response to acetylcholine injection compared to baseline. Plasma cytokine levels of Interleukin (IL)-6 and IL-8 were also assessed. Patients were subsequently followed for 12.2 ± 4.3 years. Clonal hematopoiesis relevant gene mutations were found in 1 individual in normal endothelial function group (1.5%) and 11 cases in endothelial dysfunction group (9.3%) (p = 0.04). Additionally, CHIP mutations were associated with an increased risk of MACE (OR = 4.08, P = 0.04). Mutations in ASXL1, DNMT3A and TET2 in the endothelial dysfunction group were also associated with increased levels of IL6 and IL-8 (P = 0.001, P = 0.003; respectively). Conclusion The current study demonstrates a high frequency of CHIP in patients with coronary endothelial dysfunction as well as an association between mutations in three most common epigenetic regulator genes and increased levels of IL-6 and IL-8. Therefore it infers a probable relationship between CHIP, endothelial dysfunction and cardiovascular adverse events.


Introduction
Endothelial dysfunction is the earliest feature of atherosclerosis, which is characterized by decreased bioavailability of nitric oxide (NO) (1). The endothelium is a prime site that is exposed to the atherogenic effects of traditional cardiovascular risk factors (diabetes, hypertension, dyslipidemia, and smoking), biochemical markers and other local anatomic/hemodynamic factors, leading to the development of atherosclerosis (2). Coronary endothelial dysfunction is associated with an increased risk of cardiovascular mortality and myocardial infarction (3). Although traditional cardiometabolic risk factors account for major factors resulting in impairment of endothelial function, many individuals with early coronary atherosclerosis and coronary endothelial dysfunction, especially at younger ages, do not have identi able comorbidities or risk factors, leading us to propose that unknown elements may play a role in endothelial dysfunction and subsequent cardiovascular diseases (4).
Clonal hematopoiesis of indeterminate potential (CHIP) is de ned by the presence of expanded somatic mutations in cancer related genes that arise in hematopoietic stem cells and expand with time, in apparently healthy individuals with no abnormalities in their blood counts (5). It has been demonstrated that approximately 1% of the population younger than 40 years of age has CHIP, compared to 30% of the population > 80 years (5). CHIP commonly involved epigenetic regulator genes including DNMT3A (DNA methyltransferase 3A), TET2 (Ten-eleven-translocation-2), and ASXL1 (Additional sex combs-like 1), all of which impact methylation and chromatin dynamics and are associated with in ammation (6). In patients with CHIP, a 40% increased risk of all-cause mortality surpasses the risk of developing hematological malignancies (< 1%/year), and is usually secondary to increased risk of atherosclerotic cardiovascular disease (7). Prior studies on CHIP (7-9) have focused on late stages of atherosclerosis and there is a lack of data to show the impact of CHIP on endothelial dysfunction, which can also be impaired by prevalent in ammation. Yet, patients with mutations in DNMT3A or TET2 are at higher risk of cardiovascular diseases, which is linked to IL-6 receptor activation (10,11). TET2 de cient-cells have also showed increased transcription of IL-6 via alternation in histone deacetylation (12). Based on this, we hypothesize that CHIP is associated with coronary endothelial dysfunction, leading to the development of overt cardiovascular disease. We carried out this study to explore the association between CHIP, in ammation, endothelial dysfunction and adverse cardiovascular events in patients and appropriately matched controls.

Methods
Mayo Clinic Institutional Review Board approved the current study. Written consent was obtained from all participants. The study was conducted in accordance with the guidelines of the Declaration of Helsinki.

Study population
In this study, 188 patients who underwent invasive coronary endothelial function testing at Mayo Clinic, Rochester, Minnesota, from 1993 to 2015 were enrolled. Patients presenting with chest pain without history of cardiovascular intervention, myocardial infarction, heart failure, known structural cardiac diseases or evidence of obstructive coronary artery disease on cardiac angiography were selected for physiological assessment of microcirculatory endothelial function by assessment of the change in diameter and blood ow of the coronary artery in response to administration of intracoronary graded infusion of acetylcholine (13,14). Based on the results, patients were categorized into two groups; patients with (n=123) and without (n=65) endothelial dysfunction.

Invasive coronary functional testing
The study protocol has been described in detail elsewhere (15,16). In brief, patients presenting to the catheterization laboratory with non-obstructive coronary artery (< 40% stenosis) underwent invasive coronary functional testing using an intracoronary Doppler guidewire. A Doppler guidewire (0.014-inch FloWire, Philips/Volcano Inc) was advanced 2-3 mm distal to the tip of 2.2 F coronary-infusion catheter (Ultrafuse, SciMed Life System) positioned into the mid-portion of the left anterior descending artery (LAD). In all patients, acetylcholine was selectively infused into the LAD at concentrations of 10 −6 , 10 −5 , and 10 −4 mol/L over 3 minutes at each concentration. Doppler measurements and coronary angiography were obtained after each infusion. CAD was measured in segment 5 mm distal to the tip of the Doppler wire. CBF was calculated from the Doppler-derived time velocity integral and vessel diameter, as previously described (17,18). Epicardial endothelial dysfunction was de ned as a decrease in CAD of > 20% in response to acetylcholine as compared to baseline; microvascular endothelial dysfunction was de ned as a maximal percentage increase in CBF in response to acetylcholine < 50% as compared to baseline (13,16). We de ned coronary endothelial dysfunction as either epicardial or microvascular endothelial dysfunction or both.

Clinical and biochemical data
For all participants prior to undergoing diagnostic tests, demographic and clinical characteristics were obtained through history and physical examination, as previously described (19,20). Data regarding age, sex, body mass index (BMI), smoking status (never/previous/current), hypertension, diabetes and hyperlipidemia were collected. Diabetes was de ned as a positive history of diabetes and/or consumption of antidiabetic medications; hypertension was de ned as positive history of hypertension and/or consumption of antihypertensive medications; hyperlipidemia was de ned as positive history of serum lipid pro le out of the normal range and/or usage of lipid lowering medications. Venous blood samples for routine biochemical tests (complete blood count, glucose, creatinine, lipid pro le) were obtained after overnight fasting before the procedure. Buffy coat and plasma samples extracted from whole blood were used for collection of DNA and measurement of cytokine expression levels, respectively. Using echocardiography, the percentage of left ventricular ejection fraction was determined and then compared between two study groups. A standardized questionnaire was administered to the patients to record occurrence of MACE and hematologic malignancies in average follow-up of 12.2 ± 4.3 years, as was previously described (19,21). Review of medical records was performed blindly by an independent investigator. Detection of clonal hematopoiesis by targeted capture assays DNA was extracted from buffy coat samples following the procedures in Qiagen's Puregene kit. We sequenced the entire coding regions of 35 genes using a customized 150Kb Agilent SureSelect panel.
Samples were paired-end sequenced (150 bp reads), using Illumina HiSeq 4000 sequencer with 96 samples per lane of ow cell. The median coverage depth per sample across the 35 genes was >1000X per nucleotide, allowing the detection of mutations with variant allelic fraction (VAF) as low as 1%. Raw variants were annotated using GATK Variant Annotator for variant quality, and Biological Reference Repository (BioR) was used for variant annotation (22). Variants with a Mapping Quality <20, read depth <10X, or found in <1% of reads were removed. Additionally, sequencing artifacts found in homopolymers were excluded. Finally, variants of signi cant interest were visually inspected using Integrative Genomics Viewer (IGV) (23).

De nitions related to Clonal Hematopoiesis Mutations
Variants were classi ed as CHIP related if they had a variant allele frequency of >2%, and exhibited a minor allele frequency of ≤0.1% in the Exome Aggregation Consortium Project (6), a database of known non-somatic variants (ExAC, http://exac.broadinstitute.org/), and were present in the Catalogue of Somatic Mutations in Cancer (COSMIC, https://cancer.sanger.ac.uk/cosmic) database. Additionally, if CHIP mutations were cited in COSMIC as somatic and identi ed in a hematologic malignancy (known to be pathogenic), we labeled them CH-PD (clonal hematopoiesis with a putative driver). Variants below minor allele threshold, and not present in COSMIC, were de ned as a variant of uncertain signi cance (VUS).

Cytokine level analysis
Plasma samples from 178 patients were tested for the levels of IL-6 and IL-8 cytokines, by the Cytokine Human Magnetic Kit (cat: LHC0001M, ThermoFisher Scienti c) (24). Ten patients were excluded due to missing plasma samples. The bead mix was prepared according to the manufacturer's protocol and samples analyzed in a multiplex array using ProcartaPlex magnetic beads via a Luminex ® 200™ instrument (Austin, TX). All plasma samples were run in duplicate. The data were analyzed using the instrument speci c software, xPONENT®. Statistical comparisons were tabulated using the nonparametric Welch's t test via Prism Software.

Statistical analysis
In current study, continuous variable are expressed as mean ± standard deviation (SD). For comparison of quantitative variables between two groups, student t-test was performed for normally distributed variables and Mann-Whitney U test (or Kruskal-Wallis test) for abnormal distributed data. Categorical variable are expressed as frequencies (%) and analyzed between different groups by the Chi-square test. Univariate and multivariate logistic regression analyses were performed to evaluate the independent association between CHIP and increased risk of MACE as well as association of existence of ASXL1, DNMT3A and TET2 mutations with MACE. For evaluation of association between levels of IL-6 and IL-8 and mutations in ASXL1, DNMT3A and TET2, all study participants were categorized into three groups: individuals with normal endothelial function, individuals with endothelial dysfunction, but without mutations in ASXL1, DNMT3A and TET2 genes and individuals with endothelial dysfunction and mutations in ASXL1, DNMT3A and TET2 genes. These genes were selected from CHIP and VUS groups. Results of regression model were reported as odd ratios (ORs) and 95% con dence interval (95% CIs). A P -value < 0.05 was assumed for statistical signi cance. All analyses were perform using SPSS software version 25.0 (SPSS Inc., Chicago, IL, USA), GraphPad Prism 6.07 (GraphPad Software, La Jolla, California, USA), and JMP 9 software (SAS Institute, Inc., Cary, NC).

Baseline characteristics of study participants
Data from 188 individuals were analyzed. Table 1 summarizes baseline clinical, demographic and biochemical characteristics of the study participants. There was no signi cant difference in age and sex, cardiovascular risk factors (diabetes, hyperlipidemia, and hypertension), medications use, lipid pro les and other laboratory test results between subjects with impaired versus normal endothelial function. In addition, after an average 12.2 ± 4.3 years follow-up, no hematologic malignancies were observed among study participants in either study group.

Association between CHIP and endothelial dysfunction
In the endothelial dysfunction group, ve individuals and in the control group one individual, were excluded because of the lack of su cient extracted DNA for analysis. In the endothelial dysfunction  (Figure 1 and Supplementary table 1); with the prevalence of VUS in CHIP genes being 10.6% vs 9.2%, respectively (P = 0.77). Among these, the most commonly described CHIP mutations including DNMT3A and ASXL1 (n=5) were found exclusively in patients with endothelial dysfunction.

Associations of CHIP and ASXL1+ DNMT3A+ TET2 mutations with MACE
Out of 188 individuals, 163 followed-up and completed the questionnaire. In univariate and multivariable logistic regression models, CHIP mutations were associated with an increased risk of subsequent MACE after adjusting for age, sex, hypertension, hyperlipidemia and diabetes ( OR = 3.69, P = 0.04 and OR = 4.08, P = 0.04; respectively) ( Table 2). Furthermore, in the endothelial dysfunction group, individual mutations in ASXL1, DNMT3A and TET2 were signi cantly associated with increased risk of MACE by univariate and multivariate regression analyses (OR = 4.46, P = 0.02 and OR = 6.17, P = 0.01; respectively) ( Table 2).

Association of IL6 and IL-8 level with ASXL1+DNMT3A+TET2 mutations
Assessment of cytokine levels showed that plasma levels of IL-6 and IL-8 were both signi cantly increased in patients with endothelial dysfunction and mutations in ASXL1, DNMT3A and TET2, vs. individuals with normal function of endothelium and individuals in endothelial dysfunction group without mutations in those three genes (IL-6 (median [ [7.42, 7.42], p=0.003, respectively) ( Figure 2).

Discussion
The current study demonstrates for the rst time a link between the presence of CHIP and early coronary arthrosclerosis, characterized by coronary endothelial dysfunction. Mutations in epigenetic regulator genes (ASXL1, DNMT3A and TET2) were speci cally associated with in ammatory biomarkers (increased plasma levels of IL-6 and IL-8). Moreover, the existence of this triad mutant CHIP was independently associated with MACE in follow-up observations. The current study therefore supports a potential role for CHIP in the mechanism of coronary artery disease starting at an early stage of atherosclerosis formation.
Vascular endothelial cells, which regulate vascular tone to maintain blood supply to the tissues and protect vessels from mechanical and chemical stress, constantly undergo injury and repair. Impaired repair damages hemostasis and initiates a variety of changes such as increased vascular permeability and cytokine release that promote atherogenesis. Recent evidence suggests that circulating endothelial progenitor cells arising from the bone marrow play an important role in the repair process of the injured endothelial layer, and impaired function of these cells contributes to endothelial dysfunction (25,26). In addition, circulating monocytes adhere to endothelium and in ltrate into the vessel wall, where they differentiate into various phenotypes of macrophages which play a crucial role in all stages of plaque formation and development of cardiovascular events (27,28).
With age, hematopoietic stem cells acquire mutations which can form clonal populations of mutant peripheral blood cells. Most carriers of these clones have normal blood counts, no evidence for an underlying hematological neoplasm, and in fact will never develop a hematologic neoplasm (CHIP); however they do have a signi cant increase in mortality (27) and cardiovascular diseases (7). Further, CHIP is associated with degenerative calci ed aortic valve stenosis (29) and with worse clinical outcomes in heart failure patients with ischemic cardiomyopathy (30).
The underlying mechanisms of the link between CHIP and cardiovascular diseases are being sought in cardiovascular research. It has been suggested that CHIP associated mutations can alter transcription of genes related to in ammatory pathways in peripheral blood cells such as monocytes, potentially augmenting in ammatory responses during the atherogenesis (10). Murine models of TET2 and DNMT3A loss-of-function mutations showed advanced cardiovascular diseases, potentially through accelerating in ammation (31). A recent observational study supported this hypothesis by showing that in DNMT3A and TET2 associated CHIP, the increased risk of cardiovascular events was mitigated in patients with IL-6 receptor mutations (11). It has been demonstrated that loss of TET2 function in macrophages mediates upregulation of several in ammatory markers such as IL-6 and IL-8 via alterations in DNA methylation, hydroxymethylation and histone deacetylation (7,32,33). Increased levels of both IL-6 and IL-8 in the microenvironment of vessels can initiate atherosclerosis, as phenotype transformation of cells results in proliferation of vascular smooth muscle cells, endothelial dysfunction and activation of pro-in ammatory macrophages (34). Speci cally, IL-8 overexpression accompanies increased endothelial permeability and early stages of plaque formation (35). Importantly, our analysis revealed a relationship between mutations in ASXL1, DNMT3A and TET2 genes in patients with endothelial dysfunction and increased plasma levels of IL-6 and IL-8. This suggests possible mechanistic role of these mutations in in ammatory pathways and initiation or progression of endothelial dysfunction.
In the current study, we observed an increased frequency of CHIP mutations in patients with early coronary atherosclerosis and endothelial dysfunction, especially involving ASXL1. Previous studies have demonstrated a potential role for CHIP in the late stage of coronary artery disease (7,9,30). The current study extends these previous observations by evaluating CHIP associated mutations in patients with the early stage of coronary artery disease prior to the development of any obstructive plaque characterized by coronary endothelial dysfunction. We observed an association of CHIP mutations in ASXL1, DNMT3A, CBL, FLT3-TDK and SETBP1 with coronary endothelial dysfunction. Moreover, the relationship of these mutations associated CHIP and existence of ASXL1, DNMT3A and TET2 mutations, individually in endothelial dysfunction group with occurrence of myocardial infarction, stroke and death appears to be causal linkage. Consistent with our ndings, association of cardiovascular disease with CHIP mutations in DNMT3A and ASXL1 has been reported in different studies (7,36).
DNMT3A and ASXL1 mutations associated CHIP observed in patients with endothelial dysfunction DNMT3A encodes DNA methyltransferase, which adds methyl groups to DNA. This is necessary for maturation of hematopoietic stem cells and their differentiation into different peripheral blood cells. Bone marrow-derived macrophages with loss of function in DNMT3A revealed increased expression of CXC chemokines and synthesis of pro-in ammatory cytokines, IL-6 and IL-1b (11). In addition, Mast cell activation and increased synthesis of interferon -γ via T cells were linked to DNMT3A de ciency. Interestingly, mutations in DNMT3A gene may also cause T cell polarization into pro-in ammatory and pro-atherogenic type (8,37).
Another common CHIP mutation in epigenetic regulator genes is ASXL1 gene, encoding additional sex combs like 1 protein which in uences histone modi cations and gene expression (38). Mutation in ASXL1 gene can result in inhibition of polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 (H3K27) tri-methylation. This effect causes dysregulation of hematopoietic cells. Loss of function of ASXL1 leading to CHIP has been investigated for its role in the development of malignant myeloid diseases such as myeloproliferative neoplasms, myelodysplastic syndromes, and acute myeloid leukemia. However, to date, the role of CHIP mutation in ASXL1 on the development of cardiovascular diseases is still unknown (39).
Based on our results, we postulate that the CHIP mutations may occur especially in epigenetic modulator genes such as ASXL1 of progenitor cells of monocyte or endothelial cells in bone marrow and clonally expand to the circulation (9). After migration of these cells to the arterial wall, expression of genes involved in pro-in ammatory mechanisms are elevated due to alternations in methylation of DNA and other epigenetic factors. This process may drive endothelial dysfunction and atherosclerosis formation.
The current study had several limitations. The plasma and buffy coat samples available to us for this current study were obtained at baseline at the time of presentation. Thus, we are unable to evaluate alternations in frequency of mutations overtime. Future studies with large sample size are required to explore underlying mechanisms linking the impact of CHIP on endothelial dysfunction to IL-6 and IL-8. Despite these limitations, the present study is strengthened by investigation of somatic mutations in hematopoietic cells in carefully phenotyped patients with endothelial dysfunction before developing obstructive coronary diseases for the rst time in cardiovascular research. Importantly, we assessed the independent association between these mutations and adverse cardiovascular events.
In conclusion, the current study revealed, for the rst time, increased somatic mutations related to CHIP in individuals with coronary endothelial dysfunction but without obstructive coronary disease, especially in ASXL1 gene, postulating a novel mechanism of developing coronary endothelial dysfunction. These mutations may contribute to the progression of cardiovascular diseases, leading to death, myocardial infarction and stroke. Furthermore, enhanced expression levels of IL-6 and IL-8 seems to be related to mutations in DNMT3A, ASXL1 and TET2, more than other gene mutations relevant CHIP. The current study supports a role for CHIP as a mechanism and potential therapeutic target for patients with early coronary atherosclerosis, and advances our understanding of the pathogenesis of the disease. Odds ratios (ORs) and 95% con dence intervals (CIs) were reported for each model. Figure 1