Analysen von Proben der Etablierungsphase von BeLOVE

Aging is associated with an increased risk of vascular disease caused by the rupture of inflamed cholesterol plaques in arteries. When plaques become inflamed, they can rupture or erode, leading to blood clots that occlude the arteries and cause heart attacks and strokes. One possible driver is clonal haematopoiesis (CH) — a phenomenon in which mutations arise in blood-forming haematopoietic stem cells (HSCs) during aging, and promote the proliferation of blood-cell populations bearing these mutations at the expense of wild-type blood lineages. CH occurs in an age-related manner in up to 20% of the elderly population and typically affects leukemia-associated genes (1, 2). CH is nowadays accepted as a premalignant state for hematologic neoplasms. Surprisingly, large population based sequencing studies provided evidence that the mutations were also associated with greater overall mortality, largely reflecting an increase in myocardial infarctions and ischemic strokes (1-3). Causality was assessed in preclinical models of Tet2-deficient mice, which showed accelerated development of atherosclerosis driven by an altered function of the NLRP3/IL1β- inflammasome of clonally derived monocytes/macrophages (4). Collectively, these data suggest aberrant chronic inflammation as a critical commonality between cancer and cardiovascular disease development.

Preliminary evidence of the project partners investigating >350 patients included in the Proscis study  (5) indicate that a VAF (variant allele frequency) of CH >1% occurs as often as 44.5% in stroke patients and CHIP (ie, CH of “indeterminate potential” with a VAF>2%) occurs in over 30% of stroke patients. Of particular interest, there are major qualitative difference compared to CH in cancer patients with a comparable age profile: TET2 mutations are observed in 15% of stroke patients (vs. 6% in cancer patients) and stroke patients harbour frequently more than 1 gene mutation. Individuals with CHIP have a doubled risk of worsened heart failure outcomes independent of traditional cardiovascular risk factors (6), possibly related to inflammasome alterations. In fact, peripheral blood monocytes of patients with chronic postischemic heart failure who carry DNMT3A or TET2 CHIP-driver sequence variations displayed increased expression of proinflammatory cytokines, interleukin 6 receptor, and cellular receptor CD163, as well as the NLRP3 inflammasome complex and other genes involved in cytokine release syndrome (7).

Here, we propose to test the hypothesis that CH is associated with endothelial dysfunction, small vessel disease, and inflammation and correlates with poor long-term outcome and increased risk of recurrent cardiovascular events in patients at high vascular risk included in the BeLOVE trial.

We plan to analyse all 1,903 patients from all 5 strata included in BeLOVE 1.0. Whole-blood DNA will be subjected to targeted deep sequencing by the group of Prof. Frederik Damm at the Dept. Oncology CVK. Using a customized sequencing panel, covering all relevant genes described in CH in combination with unique molecular identifiers and dual indices during library preparation, that allow for bioinformatic correction of sequencing errors, rare somatic clones with allele frequencies as low as 0.25% will be detected reliably. Sequencing will be carried out at the BIH sequencing core facility on an Illumina NovaSeq platform. After bioinformatic analysis, the molecular data will be correlated with clinical data. Even with a conservative estimate of CH in only 20% over the full cohort, our analysis will have sufficient power

• to assess the prevalence of CH and analyse the affected genes in all patient strata of BeLOVE
• to investigate the impact of CH on the risk of recurrent cardiovascular events
• to investigate the association of CH with
  • biomarkers of ED and inflammation and correlate with long-term outcomes. For example, there seems to be a significant IL6 x CH interaction which can be analyzed  in our cohort (8)
  • cognitive and functional outcome
  • measures of atherosclerosis (IMT, ABI etc)
  • measures of atrial and ventricular structure and function
  • measures of microvascular and endothelial, and vascular function
  • metabolic and immune phenotypes in BeLOVE
  • silent ischemic events on cardiac and brain MRIs
  • cerebral small vessel disease assessed by WML on brain MRI

References

1.            Jaiswal S, Natarajan P, Silver AJ, et al. Clonal Hematopoiesis and Risk of Atherosclerotic Cardiovascular Disease. The New England journal of medicine. 2017;377(2):111-21. doi:10.1056/NEJMoa1701719

2.            Frick M, Chan W, Arends CM, et al. Role of Donor Clonal Hematopoiesis in Allogeneic Hematopoietic Stem-Cell Transplantation. J Clin Oncol. 2019;37(5):375-85. doi:10.1200/JCO.2018.79.2184

3.            Tall AR, Levine RL. Cardiovascular disease: Commonality with cancer. Nature. 2017;543(7643):45-7. doi:10.1038/nature21505

4.            Fuster JJ, MacLauchlan S, Zuriaga MA, et al. Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice. Science. 2017;355(6327):842-7. doi:10.1126/science.aag1381

5.            Liman TG, Zietemann V, Wiedmann S, et al. Prediction of vascular risk after stroke - protocol and pilot data of the Prospective Cohort with Incident Stroke (PROSCIS). International journal of stroke : official journal of the International Stroke Society. 2013;8(6):484-90. doi:10.1111/j.1747-4949.2012.00871.x

6.            Libby P, Jaiswal S, Lin AE, Ebert BL. CHIPping Away at the Pathogenesis of Heart Failure. JAMA Cardiol. 2019;4(1):5-6. doi:10.1001/jamacardio.2018.4039

7.            Abplanalp WT, Cremer S, John D, et al. Clonal Hematopoiesis-Driver DNMT3A Mutations Alter Immune Cells in Heart Failure. Circ Res. 2021;128(2):216-28. doi:10.1161/CIRCRESAHA.120.317104

8.            Bick AG, Pirruccello JP, Griffin GK, et al. Genetic Interleukin 6 Signaling Deficiency Attenuates Cardiovascular Risk in Clonal Hematopoiesis. Circulation. 2020;141(2):124-31. doi:10.1161/CIRCULATIONAHA.119.044362

Immune mechanisms play a pivotal role in cardiovascular disease genesis and progression. For example, atherosclerosis is increasingly understood as an inflammatory disease since innate as well as adaptive immunity are crucially involved in atherogenesis and atherosclerosis progression (1). Therefore, further characterization of biomarkers and mechanisms of immunopathology to identify new therapeutic targets is a promising research focus for better cardiovascular disease prevention and prognosis (2).

An immune mechanism particularly not well understood is the role of circulating autoantibodies in cardiovascular disease (3).

We have recently summarized how antibodies that activate the β₁-AR (β₁-adrenoreceptor) can induce heart failure in animal models. These antibodies are often found in patients with heart failure secondary to varying etiologies. Their binding to the β₁ receptor leads to prolonged receptor activation with subsequent induction of cellular dysfunction, apoptosis, and arrhythmias. β-blocker therapy while highly effective for heart failure, may not be sufficient treatment for patients who have β₁ receptor autoantibodies. Removal of these autoantibodies by immunoadsorption has been shown to improve heart failure in small studies. Neutralization of autoantibodies through the intravenous application of small soluble molecules, such as peptides or aptamers, which specifically target and neutralize β₁-AR autoantibodies are emerging as a therapeutic strategy (4).

Some antibodies produced by certain B-cell subsets might be directly involved in promoting atherogenesis by targeting endogenous antigens presented during plaque formation and vascular stress (5, 6). Autoantibodies may also promote the genesis and progression of cardiovascular disease by interaction with receptors for endogenous molecules involved in cardiovascular function and homeostasis. Antibodies activating the angiotensin 1 receptor promote arterial hypertension (7) and cause vascular allograft rejections (8) and circulating b-1-adrenergic receptor-antibodies increase the risk for atrial fibrillation (9). Autoantibodies specific for adrenergic or cholinergic receptors (10) as well as the angiotensin receptor 1 (11) and stabilin 1 (12) have been found to be present in patients with chronic heart failure. While the impact of these autoantibodies on pathogenesis and clinical course needs to be further investigated, analysis of circulating autoantibodies could also promote the understanding of the complex role of other receptors, like PAR1 and PAR2, which might be critically involved in vascular inflammation and atherogenesis but also thrombus formation (13, 14). Recent evidence from us describe descriminating signatures of autoantibodies targeting G protein-coupled receptors (GPCR) in healthy donors compared to patients with systemic sclerosis, Alzheimer's disease, and ovarian cancer (15). Furthermore, unpublished data from members of the BeLOVE consortium show that autoantibodies targeting GPCR (applied above and planed in the BeLOVE samples) can discriminate patients with severe Covid-19 from patients with unstable lung disease or healthy controls. Also, in the Proscis-B cohort [5] we analyzed endothelial antibodies in 489 patients with ischemic stroke and demonstrated that high levels of antiboides against VEGF2R, VEGF-B, and C3aR are associated with poor long-term outcome after ischemic stroke even after adjusting for age, sex, etiological subtypes, stroke severity, prestroke dependency, and vascular risk factors (unpublished results).

Here, we propose to test the hypotheses that circulating autoantibodies binding to receptors of endogenous molecules involved in cardiac and vascular function

• can serve as new biomarkers for disease progression or regression as determined by multiple measures for cardiovascular function and by measuring the clinical outcome (endpoints) and
• can clarify the role of different signaling pathways in patients with high cardiovascular risk

Circulating antibodies specific for receptors of the RAAS, adrenergic and cholinergic and several other endogenous molecules (see appendix) will be analysed using ELISA (CellTrend GmbH, Luckenwalde, Germany) in the blood samples of all 1,903 patients from all 5 strata included in BeLOVE 1.0 and will be correlated with clinical data.

References

1.            Wolf D, Ley K. Immunity and Inflammation in Atherosclerosis. Circ Res. 2019;124(2):315-27. doi:10.1161/CIRCRESAHA.118.313591

2.            Soehnlein O, Libby P. Targeting inflammation in atherosclerosis - from experimental insights to the clinic. Nat Rev Drug Discov. 2021. doi:10.1038/s41573-021-00198-1

3.            Meier LA, Binstadt BA. The Contribution of Autoantibodies to Inflammatory Cardiovascular Pathology. Front Immunol. 2018;9:911. doi:10.3389/fimmu.2018.00911

4.            Dungen HD, Dordevic A, Felix SB, et al. beta1-Adrenoreceptor Autoantibodies in Heart Failure: Physiology and Therapeutic Implications. Circ Heart Fail. 2020;13(1):e006155. doi:10.1161/CIRCHEARTFAILURE.119.006155

5.            Tsiantoulas D, Diehl CJ, Witztum JL, Binder CJ. B cells and humoral immunity in atherosclerosis. Circ Res. 2014;114(11):1743-56. doi:10.1161/CIRCRESAHA.113.301145

6.            Sage AP, Tsiantoulas D, Binder CJ, Mallat Z. The role of B cells in atherosclerosis. Nature reviews. Cardiology. 2019;16(3):180-96. doi:10.1038/s41569-018-0106-9

7.            Drummond GR, Vinh A, Guzik TJ, Sobey CG. Immune mechanisms of hypertension. Nat Rev Immunol. 2019;19(8):517-32. doi:10.1038/s41577-019-0160-5

8.            Dragun D, Muller DN, Brasen JH, et al. Angiotensin II type 1-receptor activating antibodies in renal-allograft rejection. The New England journal of medicine. 2005;352(6):558-69. doi:10.1056/NEJMoa035717

9.            Shang L, Zhang L, Shao M, et al. Elevated beta1-Adrenergic Receptor Autoantibody Levels Increase Atrial Fibrillation Susceptibility by Promoting Atrial Fibrosis. Front Physiol. 2020;11:76. doi:10.3389/fphys.2020.00076

10.         Kaya Z, Leib C, Katus HA. Autoantibodies in heart failure and cardiac dysfunction. Circ Res. 2012;110(1):145-58. doi:10.1161/CIRCRESAHA.111.243360

11.         Wang X, Zhang Y, Zhang J, et al. Multiple Autoantibodies against Cardiovascular Receptors as Biomarkers in Hypertensive Heart Disease. Cardiology. 2019;142(1):47-55. doi:10.1159/000497189

12.         Lund A, Giil LM, Slettom G, Nygaard O, Heidecke H, Nordrehaug JE. Antibodies to receptors are associated with biomarkers of inflammation and myocardial damage in heart failure. International journal of cardiology. 2018;250:253-9. doi:10.1016/j.ijcard.2017.10.013

13.         Ramachandran R, Noorbakhsh F, Defea K, Hollenberg MD. Targeting proteinase-activated receptors: therapeutic potential and challenges. Nat Rev Drug Discov. 2012;11(1):69-86. doi:10.1038/nrd3615

14.         Kremers BMM, Ten Cate H, Spronk HMH. Pleiotropic effects of the hemostatic system. Journal of thrombosis and haemostasis : JTH. 2018. doi:10.1111/jth.14161

15.         Cabral-Marques O, Marques A, Giil LM, et al. GPCR-specific autoantibody signatures are associated with physiological and pathological immune homeostasis. Nature communications. 2018;9(1):5224. doi:10.1038/s41467-018-07598-9