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AIMS: Traditional markers of cell senescence including p16, Lamin B1, and senescence-associated beta galactosidase (SAβG) suggest very high frequencies of senescent cells in atherosclerosis, while their removal via 'senolysis' has been reported to reduce atherogenesis. However, selective killing of a variety of different cell types can exacerbate atherosclerosis. We therefore examined the specificity of senescence markers in vascular smooth muscle cells (VSMCs) and the effects of genetic or pharmacological senolysis in atherosclerosis. METHODS AND RESULTS: We examined traditional senescence markers in human and mouse VSMCs in vitro, and in mouse atherosclerosis. p16 and SAβG increased and Lamin B1 decreased in replicative senescence and stress-induced premature senescence (SIPS) of cultured human VSMCs. In contrast, mouse VSMCs undergoing SIPS showed only modest p16 up-regulation, and proliferating mouse monocyte/macrophages also expressed p16 and SAβG. Single cell RNA-sequencing (scRNA-seq) of lineage-traced mice showed increased p16 expression in VSMC-derived cells in plaques vs. normal arteries, but p16 localized to Stem cell antigen-1 (Sca1)+ or macrophage-like populations. Activation of a p16-driven suicide gene to remove p16+ vessel wall- and/or bone marrow-derived cells increased apoptotic cells, but also induced inflammation and did not change plaque size or composition. In contrast, the senolytic ABT-263 selectively reduced senescent VSMCs in culture, and markedly reduced atherogenesis. However, ABT-263 did not reduce senescence markers in vivo, and significantly reduced monocyte and platelet counts and interleukin 6 as a marker of systemic inflammation. CONCLUSIONS: We show that genetic and pharmacological senolysis have variable effects on atherosclerosis, and may promote inflammation and non-specific effects respectively. In addition, traditional markers of cell senescence such as p16 have significant limitations to identify and remove senescent cells in atherosclerosis, suggesting that senescence studies in atherosclerosis and new senolytic drugs require more specific and lineage-restricted markers before ascribing their effects entirely to senolysis.

OBJECTIVE: Vascular inflammation underlies cardiovascular disease. Vascular smooth muscle cells (VSMCs) upregulate selective genes, including MMPs (matrix metalloproteinases) and proinflammatory cytokines upon local inflammation, which directly contribute to vascular disease and adverse clinical outcome. Identification of factors controlling VSMC responses to inflammation is therefore of considerable therapeutic importance. Here, we determine the role of Histone H3 lysine 9 di-methylation (H3K9me2), a repressive epigenetic mark that is reduced in atherosclerotic lesions, in regulating the VSMC inflammatory response. Approach and Results: We used VSMC-lineage tracing to reveal reduced H3K9me2 levels in VSMCs of arteries after injury and in atherosclerotic lesions compared with control vessels. Intriguingly, chromatin immunoprecipitation showed H3K9me2 enrichment at a subset of inflammation-responsive gene promoters, including MMP3, MMP9, MMP12, and IL6, in mouse and human VSMCs. Inhibition of G9A/GLP (G9A-like protein), the primary enzymes responsible for H3K9me2, significantly potentiated inflammation-induced gene induction in vitro and in vivo without altering NFκB (nuclear factor kappa-light-chain-enhancer of activated B cell) and MAPK (mitogen-activated protein kinase) signaling. Rather, reduced G9A/GLP activity enhanced inflammation-induced binding of transcription factors NFκB-p65 and cJUN to H3K9me2 target gene promoters MMP3 and IL6. Taken together, these results suggest that promoter-associated H3K9me2 directly attenuates the induction of target genes in response to inflammation in human VSMCs. CONCLUSIONS: This study implicates H3K9me2 in regulating the proinflammatory VSMC phenotype. Our findings suggest that reduced H3K9me2 in disease enhance binding of NFκB and AP-1 (activator protein-1) transcription factors at specific inflammation-responsive genes to augment proinflammatory stimuli in VSMC. Therefore, H3K9me2-regulation could be targeted clinically to limit expression of MMPs and IL6, which are induced in vascular disease.

OBJECTIVE: MARK4 (microtubule affinity-regulating kinase 4) regulates NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 3) inflammasome activation. The aim of the study is to examine the role of MARK4 in hematopoietic cells during atherosclerosis. METHODS AND RESULTS: We show increased MARK4 expression in human atherosclerotic lesions compared with adjacent areas. MARK4 is coexpressed with NLRP3, and they colocalize in areas enriched in CD68-positive but α-SMA (α-smooth muscle actin)-negative cells. Expression of MARK4 and NLRP3 in the atherosclerotic lesions is associated with the production of active IL (interleukin)-1β and IL-18. To directly assess the role of hematopoietic MARK4 in NLRP3 inflammasome activation and atherosclerotic plaque formation, Ldlr (low-density lipoprotein receptor)-deficient mice were lethally irradiated and reconstituted with either wild-type or Mark4-deficient bone marrow cells, and were subsequently fed a high-fat diet and cholesterol diet for 9 weeks. Mark4 deficiency in bone marrow cells led to a significant reduction of lesion size, together with decreased circulating levels of IL-18 and IFN-γ (interferon-γ). Furthermore, Mark4 deficiency in primary murine bone marrow-derived macrophages prevented cholesterol crystal-induced NLRP3 inflammasome activation, as revealed by reduced caspase-1 activity together with reduced production of IL-1β and IL-18. CONCLUSIONS: MARK4-dependent NLRP3 inflammasome activation in the hematopoietic cells regulates the development of atherosclerosis.

Supplemental Digital Content is available in the text., Background: The Asp358Ala variant (rs2228145; A>C) in the IL (interleukin)-6 receptor (IL6R) gene has been implicated in the development of abdominal aortic aneurysms (AAAs), but its effect on AAA growth over time is not known. We aimed to investigate the clinical association between the IL6R-Asp358Ala variant and AAA growth and to assess the effect of blocking the IL-6 signaling pathway in mouse models of aortic aneurysm rupture or dissection. Methods: Using data from 2863 participants with AAA from 9 prospective cohorts, age- and sex-adjusted mixed-effects linear regression models were used to estimate the association between the IL6R-Asp358Ala variant and annual change in AAA diameter (mm/y). In a series of complementary randomized trials in mice, the effect of blocking the IL-6 signaling pathways was assessed on plasma biomarkers, systolic blood pressure, aneurysm diameter, and time to aortic rupture and death. Results: After adjusting for age and sex, baseline aneurysm size was 0.55 mm (95% CI, 0.13–0.98 mm) smaller per copy of the minor allele [C] of the Asp358Ala variant. Change in AAA growth was −0.06 mm per year (−0.18 to 0.06) per copy of the minor allele; a result that was not statistically significant. Although all available worldwide data were used, the genetic analyses were not powered for an effect size as small as that observed. In 2 mouse models of AAA, selective blockage of the IL-6 trans-signaling pathway, but not combined blockage of both, the classical and trans-signaling pathways, was associated with improved survival (P

Aging is the largest risk factor for cardiovascular disease, yet the molecular mechanisms underlying vascular aging remain unclear. Mitochondrial DNA (mtDNA) damage is linked to aging, but whether mtDNA damage or mitochondrial dysfunction is present and directly promotes vascular aging is unknown. Furthermore, mechanistic studies in mice are severely hampered by long study times and lack of sensitive, repeatable and reproducible parameters of arterial aging at standardized early time points. We examined the time course of multiple invasive and noninvasive arterial physiological parameters and structural changes of arterial aging in mice, how aging affects vessel mitochondrial function, and the effects of gain or loss of mitochondrial function on vascular aging. Vascular aging was first detected by 44 weeks (wk) of age, with reduced carotid compliance and distensibility, increased β-stiffness index and increased aortic pulse wave velocity (PWV). Aortic collagen content and elastin breaks also increased at 44 wk. Arterial mtDNA copy number (mtCN) and the mtCN-regulatory proteins TFAM, PGC1α and Twinkle were reduced by 44 wk, associated with reduced mitochondrial respiration. Overexpression of the mitochondrial helicase Twinkle (Tw+ ) increased mtCN and improved mitochondrial respiration in arteries, and delayed physiological and structural aging in all parameters studied. Conversely, mice with defective mitochondrial polymerase-gamma (PolG) and reduced mtDNA integrity demonstrated accelerated vascular aging. Our study identifies multiple early and reproducible parameters for assessing vascular aging in mice. Arterial mitochondrial respiration reduces markedly with age, and reduced mtDNA integrity and mitochondrial function directly promote vascular aging.

OBJECTIVE: Mitochondrial DNA (mtDNA) damage is present in murine and human atherosclerotic plaques. However, whether endogenous levels of mtDNA damage are sufficient to cause mitochondrial dysfunction and whether decreasing mtDNA damage and improving mitochondrial respiration affects plaque burden or composition are unclear. We examined mitochondrial respiration in human atherosclerotic plaques and whether augmenting mitochondrial respiration affects atherogenesis. APPROACH AND RESULTS: Human atherosclerotic plaques showed marked mitochondrial dysfunction, manifested as reduced mtDNA copy number and oxygen consumption rate in fibrous cap and core regions. Vascular smooth muscle cells derived from plaques showed impaired mitochondrial respiration, reduced complex I expression, and increased mitophagy, which was induced by oxidized low-density lipoprotein. Apolipoprotein E-deficient (ApoE-/-) mice showed decreased mtDNA integrity and mitochondrial respiration, associated with increased mitochondrial reactive oxygen species. To determine whether alleviating mtDNA damage and increasing mitochondrial respiration affects atherogenesis, we studied ApoE-/- mice overexpressing the mitochondrial helicase Twinkle (Tw+/ApoE-/-). Tw+/ApoE-/- mice showed increased mtDNA integrity, copy number, respiratory complex abundance, and respiration. Tw+/ApoE-/- mice had decreased necrotic core and increased fibrous cap areas, and Tw+/ApoE-/- bone marrow transplantation also reduced core areas. Twinkle increased vascular smooth muscle cell mtDNA integrity and respiration. Twinkle also promoted vascular smooth muscle cell proliferation and protected both vascular smooth muscle cells and macrophages from oxidative stress-induced apoptosis. CONCLUSIONS: Endogenous mtDNA damage in mouse and human atherosclerosis is associated with significantly reduced mitochondrial respiration. Reducing mtDNA damage and increasing mitochondrial respiration decrease necrotic core and increase fibrous cap areas independently of changes in reactive oxygen species and may be a promising therapeutic strategy in atherosclerosis.

OBJECTIVE: To determine the role of regulatory B cell-derived interleukin (IL)-10 in atherosclerosis. APPROACH AND RESULTS: We created chimeric Ldlr(-/-) mice with a B cell-specific deficiency in IL-10, and confirmed that purified B cells stimulated with lipopolysaccharide failed to produce IL-10 compared with control Ldlr(-/-) chimeras. Mice lacking B-cell IL-10 demonstrated enhanced splenic B-cell numbers but no major differences in B-cell subsets, T cell or monocyte distribution, and unchanged body weights or serum cholesterol levels compared with control mice. After 8 weeks on high-fat diet, there were no differences in aortic root or aortic arch atherosclerosis. In addition to plaque size, plaque composition (macrophages, T cells, smooth muscle cells, and collagen) was similar between groups. CONCLUSIONS: In contrast to its prominent regulatory role in many immune-mediated diseases and its proposed modulatory role in atherosclerosis, B cell-derived IL-10 does not alter atherosclerosis in mice.

Objective- Recent studies suggested the occurrence of phenotypic switching of vascular smooth muscle cells (VSMCs) during the development of aortic aneurysm (AA). However, lineage-tracing studies are still lacking, and the behavior of VSMCs during the formation of dissecting AA is poorly understood. Approach and Results- We used multicolor lineage tracing of VSMCs to track their fate after injury in murine models of Ang II (angiotensin II)-induced dissecting AA. We also addressed the direct impact of autophagy on the response of VSMCs to AA dissection. Finally, we studied the relevance of these processes to human AAs. Here, we show that a subset of medial VSMCs undergoes clonal expansion and that VSMC outgrowths are observed in the adventitia and borders of the false channel during Ang II-induced development of dissecting AA. The clonally expanded VSMCs undergo phenotypic switching with downregulation of VSMC differentiation markers and upregulation of phagocytic markers, indicative of functional changes. In particular, autophagy and endoplasmic reticulum stress responses are activated in the injured VSMCs. Loss of autophagy in VSMCs through deletion of autophagy protein 5 gene ( Atg5) increases the susceptibility of VSMCs to death, enhances endoplasmic reticulum stress activation, and promotes IRE (inositol-requiring enzyme) 1α-dependent VSMC inflammation. These alterations culminate in increased severity of aortic disease and higher incidence of fatal AA dissection in mice with VSMC-restricted deletion of Atg5. We also report increased expression of autophagy and endoplasmic reticulum stress markers in VSMCs of human dissecting AAs. Conclusions- VSMCs undergo clonal expansion and phenotypic switching in Ang II-induced dissecting AAs in mice. We also identify a critical role for autophagy in regulating VSMC death and endoplasmic reticulum stress-dependent inflammation with important consequences for aortic wall homeostasis and repair.

Aims Quiescent, differentiated adult vascular smooth muscle cells (VSMCs) can be induced to proliferate and switch phenotype. Such plasticity underlies blood vessel homeostasis and contributes to vascular disease development. Oligoclonal VSMC contribution is a hallmark of end-stage vascular disease. Here we aim to understand cellular mechanisms underpinning generation of this VSMC oligoclonality. Methods and Results We investigate the dynamics of VSMC clone formation using confocal microscopy and single cell transcriptomics in VSMC-lineage-traced animal models. We find that activation of medial VSMC proliferation occurs at low frequency after vascular injury and that only a subset of expanding clones migrate, which together drives formation of oligoclonal neointimal lesions. VSMC contribution in small atherosclerotic lesions is typically from one or two clones, similar to observations in mature lesions. Low frequency (

OBJECTIVE: Vascular inflammation underlies cardiovascular disease. Vascular smooth muscle cells (VSMCs) upregulate selective genes, including MMPs (matrix metalloproteinases) and proinflammatory cytokines upon local inflammation, which directly contribute to vascular disease and adverse clinical outcome. Identification of factors controlling VSMC responses to inflammation is therefore of considerable therapeutic importance. Here, we determine the role of Histone H3 lysine 9 di-methylation (H3K9me2), a repressive epigenetic mark that is reduced in atherosclerotic lesions, in regulating the VSMC inflammatory response. Approach and Results: We used VSMC-lineage tracing to reveal reduced H3K9me2 levels in VSMCs of arteries after injury and in atherosclerotic lesions compared with control vessels. Intriguingly, chromatin immunoprecipitation showed H3K9me2 enrichment at a subset of inflammation-responsive gene promoters, including MMP3, MMP9, MMP12, and IL6, in mouse and human VSMCs. Inhibition of G9A/GLP (G9A-like protein), the primary enzymes responsible for H3K9me2, significantly potentiated inflammation-induced gene induction in vitro and in vivo without altering NFκB (nuclear factor kappa-light-chain-enhancer of activated B cell) and MAPK (mitogen-activated protein kinase) signaling. Rather, reduced G9A/GLP activity enhanced inflammation-induced binding of transcription factors NFκB-p65 and cJUN to H3K9me2 target gene promoters MMP3 and IL6. Taken together, these results suggest that promoter-associated H3K9me2 directly attenuates the induction of target genes in response to inflammation in human VSMCs. CONCLUSIONS: This study implicates H3K9me2 in regulating the proinflammatory VSMC phenotype. Our findings suggest that reduced H3K9me2 in disease enhance binding of NFκB and AP-1 (activator protein-1) transcription factors at specific inflammation-responsive genes to augment proinflammatory stimuli in VSMC. Therefore, H3K9me2-regulation could be targeted clinically to limit expression of MMPs and IL6, which are induced in vascular disease.

Aging is the largest risk factor for cardiovascular disease, yet the molecular mechanisms underlying vascular aging remain unclear. Mitochondrial DNA (mtDNA) damage is linked to aging, but whether mtDNA damage or mitochondrial dysfunction is present and directly promotes vascular aging is unknown. Furthermore, mechanistic studies in mice are severely hampered by long study times and lack of sensitive, repeatable and reproducible parameters of arterial aging at standardized early time points. We examined the time course of multiple invasive and noninvasive arterial physiological parameters and structural changes of arterial aging in mice, how aging affects vessel mitochondrial function, and the effects of gain or loss of mitochondrial function on vascular aging. Vascular aging was first detected by 44 weeks (wk) of age, with reduced carotid compliance and distensibility, increased β-stiffness index and increased aortic pulse wave velocity (PWV). Aortic collagen content and elastin breaks also increased at 44 wk. Arterial mtDNA copy number (mtCN) and the mtCN-regulatory proteins TFAM, PGC1α and Twinkle were reduced by 44 wk, associated with reduced mitochondrial respiration. Overexpression of the mitochondrial helicase Twinkle (Tw+ ) increased mtCN and improved mitochondrial respiration in arteries, and delayed physiological and structural aging in all parameters studied. Conversely, mice with defective mitochondrial polymerase-gamma (PolG) and reduced mtDNA integrity demonstrated accelerated vascular aging. Our study identifies multiple early and reproducible parameters for assessing vascular aging in mice. Arterial mitochondrial respiration reduces markedly with age, and reduced mtDNA integrity and mitochondrial function directly promote vascular aging.

OBJECTIVE: To determine the role of regulatory B cell-derived interleukin (IL)-10 in atherosclerosis. APPROACH AND RESULTS: We created chimeric Ldlr(-/-) mice with a B cell-specific deficiency in IL-10, and confirmed that purified B cells stimulated with lipopolysaccharide failed to produce IL-10 compared with control Ldlr(-/-) chimeras. Mice lacking B-cell IL-10 demonstrated enhanced splenic B-cell numbers but no major differences in B-cell subsets, T cell or monocyte distribution, and unchanged body weights or serum cholesterol levels compared with control mice. After 8 weeks on high-fat diet, there were no differences in aortic root or aortic arch atherosclerosis. In addition to plaque size, plaque composition (macrophages, T cells, smooth muscle cells, and collagen) was similar between groups. CONCLUSIONS: In contrast to its prominent regulatory role in many immune-mediated diseases and its proposed modulatory role in atherosclerosis, B cell-derived IL-10 does not alter atherosclerosis in mice.

Aims Quiescent, differentiated adult vascular smooth muscle cells (VSMCs) can be induced to proliferate and switch phenotype. Such plasticity underlies blood vessel homeostasis and contributes to vascular disease development. Oligoclonal VSMC contribution is a hallmark of end-stage vascular disease. Here, we aim to understand cellular mechanisms underpinning generation of this VSMC oligoclonality. Methods and results We investigate the dynamics of VSMC clone formation using confocal microscopy and single-cell transcriptomics in VSMC-lineage-traced animal models. We find that activation of medial VSMC proliferation occurs at low frequency after vascular injury and that only a subset of expanding clones migrate, which together drives formation of oligoclonal neointimal lesions. VSMC contribution in small atherosclerotic lesions is typically from one or two clones, similar to observations in mature lesions. Low frequency ( Conclusion Our data suggest that activation of proliferation at low frequency is a general, cell-intrinsic feature of VSMCs. We show that rare VSMCs in healthy arteries display VSMC phenotypic switching akin to that observed in pathological vessel remodelling and that this is a conserved feature of mouse and human healthy arteries. The increased proliferation of modulated VSMCs from healthy arteries suggests that these cells respond more readily to disease-inducing cues and could drive oligoclonal VSMC expansion.