The pleiotropic benefits of statins include the ability to reduce CD47 and amplify the effect of pro-efferocytic therapies in atherosclerosis.

The pleiotropic benefits of statins may result from their impact on vascular inflammation. The molecular process underlying this phenomenon is not fully elucidated. Here, RNA sequencing designed to investigate gene expression patterns following CD47-SIRPα inhibition identifies a link between statins, efferocytosis, and vascular inflammation. In vivo and in vitro studies provide evidence that statins augment programmed cell removal by inhibiting the nuclear translocation of NFκB1 p50 and suppressing the expression of the critical 'don't eat me' molecule, CD47. Statins amplify the phagocytic capacity of macrophages, and thus the anti-atherosclerotic effects of CD47-SIRPα blockade, in an additive manner. Analyses of clinical biobank specimens suggest a similar link between statins and CD47 expression in humans, highlighting the potential translational implications. Taken together, our findings identify efferocytosis and CD47 as pivotal mediators of statin pleiotropy. In turn, statins amplify the anti-atherosclerotic effects of pro-phagocytic therapies independently of any lipid-lowering effect.

2021 Jeffrey M. Hoeg Award Lecture: Defining the Role of Efferocytosis in Cardiovascular Disease: A Focus on the CD47 (Cluster of Differentiation 47) Axis.

A key feature of atherogenesis is the accumulation of diseased and dying cells within the lesional necrotic core. While the burden of intraplaque apoptotic cells may be driven in part by an increase in programmed cell death, mounting evidence suggests that their presence may primarily be dictated by a defect in programmed cell removal, or efferocytosis. In this brief review, we will summarize the evidence suggesting that inflammation-dependent changes within the plaque render target cells inedible and reduce the appetite of lesional phagocytes. We will present the genetic causation studies, which indicate these phenomena promote lesion expansion and plaque vulnerability, and the interventional data which suggest that these processes can be reversed. Particular emphasis is provided related to the antiphagocytic CD47 (cluster of differentiation 47) do not eat me axis, which has emerged as a novel antiatherosclerotic translational target that is predicted to provide benefit independent of traditional cardiovascular risk factors.

Chitinase 3 like 1 is a regulator of smooth muscle cell physiology and atherosclerotic lesion stability.

AIMS: Atherosclerotic cerebrovascular disease underlies the majority of ischaemic strokes and is a major cause of death and disability. While plaque burden is a predictor of adverse outcomes, plaque vulnerability is increasingly recognized as a driver of lesion rupture and risk for clinical events. Defining the molecular regulators of carotid instability could inform the development of new biomarkers and/or translational targets for at-risk individuals. METHODS AND RESULTS: Using two independent human endarterectomy biobanks, we found that the understudied glycoprotein, chitinase 3 like 1 (CHI3L1), is up-regulated in patients with carotid disease compared to healthy controls. Further, CHI3L1 levels were found to stratify individuals based on symptomatology and histopathological evidence of an unstable fibrous cap. Gain- and loss-of-function studies in cultured human carotid artery smooth muscle cells (SMCs) showed that CHI3L1 prevents a number of maladaptive changes in that cell type, including phenotype switching towards a synthetic and hyperproliferative state. Using two murine models of carotid remodelling and lesion vulnerability, we found that knockdown of Chil1 resulted in larger neointimal lesions comprised by de-differentiated SMCs that failed to invest within and stabilize the fibrous cap. Exploratory mechanistic studies identified alterations in potential downstream regulatory genes, including large tumour suppressor kinase 2 (LATS2), which mediates macrophage marker and inflammatory cytokine expression on SMCs, and may explain how CHI3L1 modulates cellular plasticity. CONCLUSION: CHI3L1 is up-regulated in humans with carotid artery disease and appears to be a strong mediator of plaque vulnerability. Mechanistic studies suggest this change may be a context-dependent adaptive response meant to maintain vascular SMCs in a differentiated state and to prevent rupture of the fibrous cap. Part of this effect may be mediated through downstream suppression of LATS2. Future studies should determine how these changes occur at the molecular level, and whether this gene can be targeted as a novel translational therapy for subjects at risk of stroke.

18F-Fluorodeoxyglucose-Positron Emission Tomography Imaging Detects Response to Therapeutic Intervention and Plaque Vulnerability in a Murine Model of Advanced Atherosclerotic Disease-Brief Report.

OBJECTIVE: This study sought to determine whether 18F-fluorodeoxyglucose-positron emission tomography/computed tomography could be applied to a murine model of advanced atherosclerotic plaque vulnerability to detect response to therapeutic intervention and changes in lesion stability. Approach and Results: To analyze plaques susceptible to rupture, we fed ApoE-/- mice a high-fat diet and induced vulnerable lesions by cast placement over the carotid artery. After 9 weeks of treatment with orthogonal therapeutic agents (including lipid-lowering and proefferocytic therapies), we assessed vascular inflammation and several features of plaque vulnerability by 18F-fluorodeoxyglucose-positron emission tomography/computed tomography and histopathology, respectively. We observed that 18F-fluorodeoxyglucose-positron emission tomography/computed tomography had the capacity to resolve histopathologically proven changes in plaque stability after treatment. Moreover, mean target-to-background ratios correlated with multiple characteristics of lesion instability, including the corrected vulnerability index. CONCLUSIONS: These results suggest that the application of noninvasive 18F-fluorodeoxyglucose-positron emission tomography/computed tomography to a murine model can allow for the identification of vulnerable atherosclerotic plaques and their response to therapeutic intervention. This approach may prove useful as a drug discovery and prioritization method.

Clonally expanding smooth muscle cells promote atherosclerosis by escaping efferocytosis and activating the complement cascade.

Atherosclerosis is the process underlying heart attack and stroke. Despite decades of research, its pathogenesis remains unclear. Dogma suggests that atherosclerotic plaques expand primarily via the accumulation of cholesterol and inflammatory cells. However, recent evidence suggests that a substantial portion of the plaque may arise from a subset of "dedifferentiated" vascular smooth muscle cells (SMCs) which proliferate in a clonal fashion. Herein we use multicolor lineage-tracing models to confirm that the mature SMC can give rise to a hyperproliferative cell which appears to promote inflammation via elaboration of complement-dependent anaphylatoxins. Despite being extensively opsonized with prophagocytic complement fragments, we find that this cell also escapes immune surveillance by neighboring macrophages, thereby exacerbating its relative survival advantage. Mechanistic studies indicate this phenomenon results from a generalized opsonin-sensing defect acquired by macrophages during polarization. This defect coincides with the noncanonical up-regulation of so-called don't eat me molecules on inflamed phagocytes, which reduces their capacity for programmed cell removal (PrCR). Knockdown or knockout of the key antiphagocytic molecule CD47 restores the ability of macrophages to sense and clear opsonized targets in vitro, allowing for potent and targeted suppression of clonal SMC expansion in the plaque in vivo. Because integrated clinical and genomic analyses indicate that similar pathways are active in humans with cardiovascular disease, these studies suggest that the clonally expanding SMC may represent a translational target for treating atherosclerosis.

Pro-efferocytic nanoparticles are specifically taken up by lesional macrophages and prevent atherosclerosis.

Atherosclerosis is the process that underlies heart attack and stroke. A characteristic feature of the atherosclerotic plaque is the accumulation of apoptotic cells in the necrotic core. Prophagocytic antibody-based therapies are currently being explored to stimulate the phagocytic clearance of apoptotic cells; however, these therapies can cause off-target clearance of healthy tissues, which leads to toxicities such as anaemia. Here we developed a macrophage-specific nanotherapy based on single-walled carbon nanotubes loaded with a chemical inhibitor of the antiphagocytic CD47-SIRPα signalling axis. We demonstrate that these single-walled carbon nanotubes accumulate within the atherosclerotic plaque, reactivate lesional phagocytosis and reduce the plaque burden in atheroprone apolipoprotein-E-deficient mice without compromising safety, and thereby overcome a key translational barrier for this class of drugs. Single-cell RNA sequencing analysis reveals that prophagocytic single-walled carbon nanotubes decrease the expression of inflammatory genes linked to cytokine and chemokine pathways in lesional macrophages, which demonstrates the potential of 'Trojan horse' nanoparticles to prevent atherosclerotic cardiovascular disease.

Nanoparticle Therapy for Vascular Diseases.

Nanoparticles promise to advance strategies to treat vascular disease. Since being harnessed by the cancer field to deliver safer and more effective chemotherapeutics, nanoparticles have been translated into applications for cardiovascular disease. Systemic exposure and drug-drug interactions remain a concern for nearly all cardiovascular therapies, including statins, antithrombotic, and thrombolytic agents. Moreover, off-target effects and poor bioavailability have limited the development of completely new approaches to treat vascular disease. Through the rational design of nanoparticles, nano-based delivery systems enable more efficient delivery of a drug to its therapeutic target or even directly to the diseased site, overcoming biological barriers and enhancing a drug's therapeutic index. In addition, advances in molecular imaging have led to the development of theranostic nanoparticles that may simultaneously act as carriers of both therapeutic and imaging payloads. The following is a summary of nanoparticle therapy for atherosclerosis, thrombosis, and restenosis and an overview of recent major advances in the targeted treatment of vascular disease.

Initial Single-Center Experience With the Fully Repositionable Transfemoral Lotus Aortic Valve System.

BACKGROUND: Transcatheter aortic valve replacement (TAVR) has become the standard therapy for patients with severe symptomatic aortic stenosis and unacceptable high risk for surgical aortic valve replacement. Several different devices for TAVR have been introduced so far, each of them with unique features. AIMS: To analyze our first real-world experience with the second- generation, mechanically expanded, fully repositionable transfemoral Lotus TAVR device (Boston Scientific). RESULTS: Between August 2014 and January 2016, a total of 298 patients were scheduled for transfemoral TAVR at our institution. Among them, 145 patients received a CoreValve/CoreValve Evolut R device (Medtronic), 90 patients received a Sapien 3 device (Edwards Lifesciences), and 63 patients were scheduled for Lotus TAVR device implantation based on access vessel size and annulus diameter. Of the 63 subsequent patients scheduled, 62 were successfully implanted. Short-term Valve Academic Research Consortium (VARC)-2 results at 30 days showed a 6.4% all-cause mortality, a 3.2% major stroke rate, a 36.5% new permanent pacemaker rate and 1.6% major vascular complications. Of note was the near absence of significant residual aortic regurgitation in the presence of a favorable hemodynamic profile even in patients with difficult anatomy. CONCLUSION: Our first real-time experience with the fully repositionable Lotus TAVR system mimics the results of early clinical studies like REPRISE II. Further next-generation modifications in combination with gaining experience with this new device will likely reduce the need for new permanent pacemaker implantation and vascular complications in the future.

Heat-shock-protein 90 protects from downregulation of HIF-1α in calcineurin-induced myocardial hypertrophy.

AIM OF THE STUDY: Capillary/myocyte mismatch is a hallmark of maladaptive myocardial hypertrophy, but the exact mechanisms of this phenomenon remain unknown. We therefore aimed to evaluate the role of calcineurin A in the regulation of hypoxia-inducible factor-1 alpha (HIF-1 alpha) in a calcineurin overexpressing mouse model of myocardial hypertrophy. METHODS AND RESULTS: Mice overexpressing calcineurin A (CnATg) showed persistent upregulation of HIF-1 alpha protein without evidence of a reduction in capillary density despite progressive myocardial hypertrophy. Likewise, overexpression of calcineurin A in isolated cardiomyocytes induced upregulation of HIF-1 alpha protein. In contrast, NFAT-overexpression had no such effect, implying that NFAT-independent mechanisms were responsible for increased HIF-1 alpha levels. In addition, inhibition of HSP90 via the HSP90-inhibitor 17-AAG or siRNA abolished calcineurin A-induced upregulation of HIF-1 alpha. Consequently, upregulation of HIF-1 alpha target genes like VEGF-A, BNIP-3 or PGK-1 was also inhibited by either 17-AAG or siRNA directed against HSP90. Finally, when CnATg mice were treated with 17-AAG, they demonstrated reduced left ventricular function and capillary density. CONCLUSIONS: We describe here for the first time that overexpression of the phosphatase calcineurin A prevents the development of a capillary/myocyte mismatch despite progressive myocardial hypertrophy. This effect was mediated by HSP-90 induced stabilization of HIF-1 alpha. Further work is needed to understand this unexpected cardioprotective effect of calcineurin A.

Inter- not intraindividual differences in sTWEAK levels predict functional deterioration and mortality in patients with dilated cardiomyopathy.

BACKGROUND: TNF-like weak inducer of apoptosis (TWEAK) has been reported to predict mortality in patients with dilated cardiomyopathy. However, whether it can be used as a biomarker for disease monitoring or rather represents a risk factor for disease progression remains unclear. AIM OF THE STUDY: To evaluate the potential of sTWEAK as a biomarker in patients with dilated cardiomyopathy. RESULTS: We conducted a serial study of sTWEAK levels in 78 patients with dilated cardiomyopathy. Soluble TWEAK levels predicted not only a combined mortality/heart transplantation endpoint after 4 years (P = 0.0001), but also the risk for clinical deterioration (P = 0.0001). Compared to NT-proBNP, sTWEAK remained relatively stable in individual patients on follow-up indicating that inter- rather than intraindividual differences in sTWEAK levels predicted outcome. Finally, neither did the scavenger receptor sCD163 correlate with sTWEAK levels nor did its determination add additional information on outcome in patients with dilated cardiomyopathy. CONCLUSION: Soluble TWEAK levels in patients with dilated cardiomyopathy may not be of value for disease monitoring but may represent a risk factor for disease progression and death. Further research will be necessary to elucidate the exact role of sTWEAK as a potential modulator of immune response in the setting of dilated cardiomyopathy.

TNF-like weak inducer of apoptosis aggravates left ventricular dysfunction after myocardial infarction in mice.

BACKGROUND: TNF-like weak inducer of apoptosis (TWEAK) has recently been shown to be potentially involved in adverse cardiac remodeling. However, neither the exact role of TWEAK itself nor of its receptor Fn14 in this setting is known. AIM OF THE STUDY: To analyze the effects of sTWEAK on myocardial function and gene expression in response to experimental myocardial infarction in mice. RESULTS: TWEAK directly suppressed the expression of PGC-1α and genes of oxidative phosphorylation (OXPHOS) in cardiomyocytes. Systemic sTWEAK application after MI resulted in reduced left ventricular function and increased mortality without changes in interstitial fibrosis or infarct size. Molecular analysis revealed decreased phosphorylation of PI3K/Akt and ERK1/2 pathways associated with reduced expression of PGC-1α and PPARα. Likewise, expression of OXPHOS genes such as atp5O, cycs, cox5b, and ndufb5 was also reduced. Fn14-/- mice showed significantly improved left ventricular function and PGC-1α levels after MI compared to their respective WT littermates (Fn14+/+). Finally, inhibition of intrinsic TWEAK with anti-TWEAK antibodies resulted in improved left ventricular function and survival. CONCLUSIONS: TWEAK exerted maladaptive effects in mice after myocardial infarction most likely via direct effects on cardiomyocytes. Analysis of the potential mechanisms revealed that TWEAK reduced metabolic adaptations to increased cardiac workload by inhibition of PGC-1α.

MicroRNA-20a inhibits stress-induced cardiomyocyte apoptosis involving its novel target Egln3/PHD3.

Excessive stress, e.g. due to biomechanical overload or ischemia/reperfusion is a potent inductor of cardiomyocyte apoptosis, which contributes to maladaptive remodeling. Despite substantial progress in the understanding of the molecular pathophysiology, many components of the signaling pathways underlying remodeling in general and apoptosis in particular still remain unknown. Recent evidence suggests that microRNAs (miRs) play an important role in the heart's response to increased cardiac stress. To identify novel modulators of stress-dependent remodeling, we conducted a genome-wide miR-screen of mechanically stretched neonatal rat cardiomyocytes (NRCM). Out of 351 miRs, eight were significantly regulated by biomechanical stress, including microRNA-20a, which is part of the miR17-92 cluster. Interestingly, further expression analyses also revealed upregulation of microRNA-20a in an in vitro hypoxia/"reperfusion" model. Given the potential apoptosis-modulating properties of the miR17-92 cluster, we subjected NRCM to hypoxia and subsequent reoxygenation. AdmiR-20a significantly inhibited hypoxia-mediated apoptosis in a dose-dependent fashion, while targeted knockdown of miR-20a in NRCM induced cardiomyocyte apoptosis. Mechanistically, the antiapoptotic effect of miR-20a appears to be mediated through direct targeting and subsequent downregulation of the proapoptotic factor Egln3. Thus, miR-20a is upregulated in acute biomechanical stress as well as hypoxia and inhibits apoptosis in cardiomyocytes. These properties reveal miR-20a as a cardioprotective micro-RNA and a potential target for novel therapeutic strategies to prevent cardiac remodeling.