Galina K. Sukhova

BACKGROUND: Clonal hematopoiesis of indeterminate potential (CHIP), which is defined as the presence of an expanded somatic blood-cell clone in persons without other hematologic abnormalities, is common among older persons and is associated with an increased risk of hematologic cancer. We previously found preliminary evidence for an association between CHIP and atherosclerotic cardiovascular disease, but the nature of this association was unclear. METHODS: We used whole-exome sequencing to detect the presence of CHIP in peripheral-blood cells and associated such presence with coronary heart disease using samples from four case-control studies that together enrolled 4726 participants with coronary heart disease and 3529 controls. To assess causality, we perturbed the function of Tet2, the second most commonly mutated gene linked to clonal hematopoiesis, in the hematopoietic cells of atherosclerosis-prone mice. RESULTS: In nested case-control analyses from two prospective cohorts, carriers of CHIP had a risk of coronary heart disease that was 1.9 times as great as in noncarriers (95% confidence interval [CI], 1.4 to 2.7). In two retrospective case-control cohorts for the evaluation of early-onset myocardial infarction, participants with CHIP had a risk of myocardial infarction that was 4.0 times as great as in noncarriers (95% CI, 2.4 to 6.7). Mutations in DNMT3A, TET2, ASXL1, and JAK2 were each individually associated with coronary heart disease. CHIP carriers with these mutations also had increased coronary-artery calcification, a marker of coronary atherosclerosis burden. Hypercholesterolemia-prone mice that were engrafted with bone marrow obtained from homozygous or heterozygous Tet2 knockout mice had larger atherosclerotic lesions in the aortic root and aorta than did mice that had received control bone marrow. Analyses of macrophages from Tet2 knockout mice showed elevated expression of several chemokine and cytokine genes that contribute to atherosclerosis. CONCLUSIONS: The presence of CHIP in peripheral-blood cells was associated with nearly a doubling in the risk of coronary heart disease in humans and with accelerated atherosclerosis in mice. (Funded by the National Institutes of Health and others.).

Dysregulated extracellular matrix (ECM) metabolism may contribute to vascular remodeling during the development and complication of human atherosclerotic lesions. We in- vestigated the expression of matrix metalloproteinases (MMPs), a family of enzymes that degrade ECM compo- nents in human atherosclerotic plaques (n =30) and in uninvolved arterial specimens (n=11). We studied members of all three MMP classes (interstitial coilagenase, MMP-1; gel- atinases, MMP-2 and MMP-9; and stromelysin, MMP-3) and their endogenous inhibitors (TIMPs 1 and 2) by immu- nocytochemistry, zymography, and immunoprecipitation. Normal arteries stained uniformly for 72-kD gelatinase and TIMPs. In contrast, plaques' shoulders and regions of foam cell accumulation displayed locally increased expression of 92-kD gelatinase, stromelysin, and interstitial collagenase. However, the mere presence of MMP does not establish their catalytic capacity, as the zymogens lack activity, and TIMPs may block activated MMPs. All plaque extracts con- tained activated forms of gelatinases determined zymo- graphically and by degradation of 3H-collagen type IV. To test directly whether atheromata actually contain active ma- trix-degrading enzymes in situ, we devised a method which allows the detection and microscopic localization of MMP enzymatic activity directly in tissue sections. In situ zymog- raphy revealed gelatinolytic and caseinolytic activity in fro- zen sections of atherosclerotic but not of uninvolved arterial tissues. The MMP inhibitors, EDTA and 1,10-phenanthroline, as well as recombinant TIMP-1, reduced these activities which colocalized with regions of increased immunoreactive MMP expression, i.e., the shoulders, core, and microvascula- ture of the plaques. Focal overexpression of activated MMP may promote destabilization and complication of atherosclerotic plaques and provide novel targets for therapeutic in- tervention. (