An Ultrasound-Responsive Theranostic Cyclodextrin-Loaded Nanoparticle for Multimodal Imaging and Therapy for Atherosclerosis.

Atherosclerosis is a major cause of mortality and morbidity worldwide. Left undiagnosed and untreated, atherosclerotic plaques can rupture and cause cardiovascular complications such as myocardial infarction and stroke. Atherosclerotic plaques are composed of lipids, including oxidized low-density lipoproteins and cholesterol crystals, and immune cells, including macrophages. 2-Hydroxypropyl-beta-cyclodextrin (CD) is FDA-approved for capturing, solubilizing, and delivering lipophilic drugs in humans. It is also known to dissolve cholesterol crystals and decrease atherosclerotic plaque size. However, its low retention time necessitates high dosages for successful therapy. This study reports CD delivery via air-trapped polybutylcyanoacrylate nanoparticles (with diameters of 388 ± 34 nm) loaded with CD (CDNPs). The multimodal contrast ability of these nanoparticles after being loaded with IR780 dye in mice is demonstrated using ultrasound and near-infrared imaging. It is shown that CDNPs enhance the cellular uptake of CD in murine cells. In an ApoE-/- mouse model of atherosclerosis, treatment with CDNPs significantly improves the anti-atherosclerotic efficacy of CD. Ultrasound triggering further improves CD uptake, highlighting that CDNPs can be used for ultrasound imaging and ultrasound-responsive CD delivery. Thus, CDNPs represent a theranostic nanocarrier for potential application in patients with atherosclerosis.

Alarmin-activated B cells accelerate murine atherosclerosis after myocardial infarction via plasma cell-immunoglobulin-dependent mechanisms.

AIMS: Myocardial infarction (MI) accelerates atherosclerosis and greatly increases the risk of recurrent cardiovascular events for many years, in particular, strokes and MIs. Because B cell-derived autoantibodies produced in response to MI also persist for years, we investigated the role of B cells in adaptive immune responses to MI. METHODS AND RESULTS: We used an apolipoprotein-E-deficient (ApoE-/-) mouse model of MI-accelerated atherosclerosis to assess the importance of B cells. One week after inducing MI in atherosclerotic mice, we depleted B cells using an anti-CD20 antibody. This treatment prevented subsequent immunoglobulin G accumulation in plaques and MI-induced accelerated atherosclerosis. In gain of function experiments, we purified spleen B cells from mice 1 week after inducing MI and transferred these cells into atherosclerotic ApoE-/- mice, which greatly increased immunoglobulin G (IgG) accumulation in plaque and accelerated atherosclerosis. These B cells expressed many cytokines that promote humoural immunity and in addition, they formed germinal centres within the spleen where they differentiated into antibody-producing plasma cells. Specifically deleting Blimp-1 in B cells, the transcriptional regulator that drives their terminal differentiation into antibody-producing plasma cells prevented MI-accelerated atherosclerosis. Alarmins released from infarcted hearts were responsible for activating B cells via toll-like receptors and deleting MyD88, the canonical adaptor protein for inflammatory signalling downstream of toll-like receptors, prevented B-cell activation and MI-accelerated atherosclerosis. CONCLUSION: Our data implicate early B-cell activation and autoantibodies as a central cause for accelerated atherosclerosis post-MI and identifies novel therapeutic strategies towards preventing recurrent cardiovascular events such as MI and stroke.

Follicular B Cells Promote Atherosclerosis via T Cell-Mediated Differentiation Into Plasma Cells and Secreting Pathogenic Immunoglobulin G.

OBJECTIVE: B cells promote or protect development of atherosclerosis. In this study, we examined the role of MHCII (major histocompatibility II), CD40 (cluster of differentiation 40), and Blimp-1 (B-lymphocyte-induced maturation protein) expression by follicular B (FO B) cells in development of atherosclerosis together with the effects of IgG purified from atherosclerotic mice. APPROACH AND RESULTS: Using mixed chimeric Ldlr-/- mice whose B cells are deficient in MHCII or CD40, we demonstrate that these molecules are critical for the proatherogenic actions of FO B cells. During development of atherosclerosis, these deficiencies affected T-B cell interactions, germinal center B cells, plasma cells, and IgG. As FO B cells differentiating into plasma cells require Blimp-1, we also assessed its role in the development of atherosclerosis. Blimp-1-deficient B cells greatly attenuated atherosclerosis and immunoglobulin-including IgG production, preventing IgG accumulation in atherosclerotic lesions; Blimp-1 deletion also attenuated lesion proinflammatory cytokines, apoptotic cell numbers, and necrotic core. To determine the importance of IgG for atherosclerosis, we purified IgG from atherosclerotic mice. Their transfer but not IgG from nonatherosclerotic mice into Ldlr-/- mice whose B cells are Blimp-1-deficient increased atherosclerosis; transfer was associated with IgG accumulating in atherosclerotic lesions, increased lesion inflammatory cytokines, apoptotic cell numbers, and necrotic core size. CONCLUSIONS: The mechanism by which FO B cells promote atherosclerosis is highly dependent on their expression of MHCII, CD40, and Blimp-1. FO B cell differentiation into IgG-producing plasma cells also is critical for their proatherogenic actions. Targeting B-T cell interactions and pathogenic IgG may provide novel therapeutic strategies to prevent atherosclerosis and its adverse cardiovascular complications.

A novel mouse model of atherosclerotic plaque instability for drug testing and mechanistic/therapeutic discoveries using gene and microRNA expression profiling.

RATIONALE: The high morbidity/mortality of atherosclerosis is typically precipitated by plaque rupture and consequent thrombosis. However, research on underlying mechanisms and therapeutic approaches is limited by the lack of animal models that reproduce plaque instability observed in humans. OBJECTIVE: Development and use of a mouse model of plaque rupture that reflects the end stage of human atherosclerosis. METHODS AND RESULTS: On the basis of flow measurements and computational fluid dynamics, we applied a tandem stenosis to the carotid artery of apolipoprotein E-deficient mice on high-fat diet. At 7 weeks postoperatively, we observed intraplaque hemorrhage in ≈50% of mice, as well as disruption of fibrous caps, intraluminal thrombosis, neovascularization, and further characteristics typically seen in human unstable plaques. Administration of atorvastatin was associated with plaque stabilization and downregulation of monocyte chemoattractant protein-1 and ubiquitin. Microarray profiling of mRNA and microRNA (miR) and, in particular, its combined analysis demonstrated major differences in the hierarchical clustering of genes and miRs among nonatherosclerotic arteries, stable, and unstable plaques and allows the identification of distinct genes/miRs, potentially representing novel therapeutic targets for plaque stabilization. The feasibility of the described animal model as a discovery tool was established in a pilot approach, identifying a disintegrin and metalloprotease with thrombospondin motifs 4 (ADAMTS4) and miR-322 as potential pathogenic factors of plaque instability in mice and validated in human plaques. CONCLUSIONS: The newly described mouse model reflects human atherosclerotic plaque instability and represents a discovery tool toward the development and testing of therapeutic strategies aimed at preventing plaque rupture. Distinctly expressed genes and miRs can be linked to plaque instability.

Cytotoxic and proinflammatory CD8+ T lymphocytes promote development of vulnerable atherosclerotic plaques in apoE-deficient mice.

BACKGROUND: Heart attacks and strokes, leading causes of deaths globally, arise from thrombotic occlusion of ruptured vulnerable atherosclerotic plaques characterized by abundant apoptosis, large necrotic cores derived from inefficient apoptotic cell clearance, thin fibrous caps, and focal inflammation. The genesis of apoptosis and necrotic cores in these vulnerable atherosclerotic plaques remains unknown. Cytotoxic CD8(+) T lymphocytes represent up to 50% of leukocytes in advanced human plaques and dominate early immune responses in mouse lesions, yet their role in atherosclerosis also remains unresolved. METHODS AND RESULTS: CD8(+) T-lymphocyte depletion by CD8α or CD8ß monoclonal antibody in apolipoprotein E-deficient mice fed a high-fat diet ameliorated atherosclerosis by reducing lipid and macrophage accumulation, apoptosis, necrotic cores, and monocyte chemoattractant protein 1, interleukin 1ß, interferon γ, and vascular cell adhesion molecule 1. Transfer of CD8(+) T cells into lymphocyte-deficient, apolipoprotein E-deficient mice partially reconstituted CD8(+) T cells in lymphoid compartments and was associated with CD8(+) T-cell infiltration in lesions, increased lipid and macrophage accumulation, apoptotic cells, necrotic cores, and interleukin 1ß in atherosclerotic lesions. Transfer of CD8(+) T cells deficient in perforin, granzyme B, or tumor necrosis factor α but not interferon γ failed to increase atherosclerotic lesions despite partial reconstitution in the lymphoid system and the presence in atherosclerotic lesions. Macrophages, smooth muscle cells, and endothelial cells were identified as apoptotic targets. CONCLUSIONS: We conclude that CD8(+) T lymphocytes promote the development of vulnerable atherosclerotic plaques by perforin- and granzyme B-mediated apoptosis of macrophages, smooth muscle cells, and endothelial cells that, in turn, leads to necrotic core formation and further augments inflammation by tumor necrosis factor α secretion.

Inert 50-nm polystyrene nanoparticles that modify pulmonary dendritic cell function and inhibit allergic airway inflammation.

Nanoparticles are being developed for diverse biomedical applications, but there is concern about their potential to promote inflammation, particularly in the lung. Although a variety of ambient, anthropogenic and man-made nanoparticles can promote lung inflammation, little is known about the long-term immunomodulatory effects of inert noninflammatory nanoparticles. We previously showed polystyrene 50-nm nanoparticles coated with the neutral amino acid glycine (PS50G nanoparticles) are not inflammatory and are taken up preferentially by dendritic cells (DCs) in the periphery. We tested the effects of such nanoparticles on pulmonary DC function and the development of acute allergic airway inflammation. Surprisingly, exposure to PS50G nanoparticles did not exacerbate but instead inhibited key features of allergic airway inflammation including lung airway and parenchymal inflammation, airway epithelial mucus production, and serum allergen-specific IgE and allergen-specific Th2 cytokines in the lung-draining lymph node (LN) after allergen challenge 1 mo later. PS50G nanoparticles themselves did not induce lung oxidative stress or cardiac or lung inflammation. Mechanistically, PS50G nanoparticles did not impair peripheral allergen sensitization but exerted their effect at the lung allergen challenge phase by inhibiting expansion of CD11c(+)MHCII(hi) DCs in the lung and draining LN and allergen-laden CD11b(hi)MHCII(hi) DCs in the lung after allergen challenge. PS50G nanoparticles further suppressed the ability of CD11b(hi) DCs in the draining LN of allergen-challenged mice to induce proliferation of OVA-specific CD4(+) T cells. The discovery that a defined type of nanoparticle can inhibit, rather than promote, lung inflammation via modulation of DC function opens the door to the discovery of other nanoparticle types with exciting beneficial properties.

B1a B lymphocytes are atheroprotective by secreting natural IgM that increases IgM deposits and reduces necrotic cores in atherosclerotic lesions.

RATIONALE: Aggravated atherosclerosis in B lymphocyte-deficient chimeric mice and reduced atherosclerosis after transfer of unfractionated spleen B lymphocytes into splenectomized mice have led to the widely held notion that B lymphocytes are atheroprotective. However, B lymphocytes can be pathogenic, because their depletion by anti-CD20 antibody ameliorated atherosclerosis, and transfer of B2 lymphocytes aggravated atherosclerosis. These observations raise the question of the identity of the atheroprotective B-lymphocyte population. OBJECTIVE: The purpose of the study was to identify an atheroprotective B-lymphocyte subset and mechanisms by which they confer atheroprotection. METHODS AND RESULTS: Splenectomy of apolipoprotein E-deficient mice selectively reduced peritoneal B1a lymphocytes, plasma IgM, and oxidized low-density lipoprotein IgM levels and lesion IgM deposits. These reductions were accompanied by increased oil red O-stained atherosclerotic lesions and increased necrotic cores, oxidized low-density lipoproteins, and apoptotic cells in lesions. Plasma lipids, body weight, collagen, and smooth muscle content were unaffected. Transfer of B1a lymphocytes into splenectomized mice increased peritoneal B1a lymphocytes; restored plasma IgM, oxidized low-density lipoprotein IgM levels, and lesion IgM deposits; and potently attenuated atherosclerotic lesions, with reduced lesion necrotic cores, oxidized low-density lipoprotein, and apoptotic cells. In contrast, transfer of B1a lymphocytes that cannot secrete IgM failed to protect against atherosclerosis development in splenectomized mice despite reconstitution in the peritoneum. CONCLUSIONS: B1a lymphocytes are an atheroprotective B-lymphocyte population. Our data suggest that natural IgM secreted by these lymphocytes offers protection by depositing IgM in atherosclerotic lesions, which reduces the necrotic cores of lesions.

Fibulin-5 binds urokinase-type plasminogen activator and mediates urokinase-stimulated ß1-integrin-dependent cell migration.

uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human ß1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of ß1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from ß1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.

Myocarditis: causes, mechanisms, and evolving therapies.

INTRODUCTION: Myocarditis is a severe lymphocyte-mediated inflammatory disorder of the heart, mostly caused by viruses and immune checkpoint inhibitors (ICIs). Recently, myocarditis as a rare adverse event of mRNA vaccines for SARS-CoV-2 has caused global attention. The clinical consequences of myocarditis can be very severe, but specific treatment options are lacking or not yet clinically proven. AREAS COVERED: This paper offers a brief overview of the biology of viruses that frequently cause myocarditis, focusing on mechanisms important for viral entry and replication following host infection. Current and new potential therapeutic targets/strategies especially for viral myocarditis are reviewed systematically. In particular, the immune system in myocarditis is dissected with respect to infective viral and non-infective, ICI-induced myocarditis. EXPERT OPINION: Vaccination is an excellent emerging preventative strategy for viral myocarditis, but most vaccines still require further development. Anti-viral treatments that inhibit viral replication need to be considered following viral infection in host myocardium, as lower viral load reduces inflammation severity. Understanding how the immune system continues to damage the heart even after viral clearance will define novel therapeutic targets/strategies. We propose that viral myocarditis can be best treated using a combination of antiviral agents and immunotherapies that control cytotoxic T cell activity.

Association of constipation with increased risk of hypertension and cardiovascular events in elderly Australian patients.

The association between constipation and cardiovascular risk is unclear. This population-level matched cohort study compared the association of constipation with hypertension and incident cardiovascular events in 541,172 hospitalized patients aged ≥ 60 years. For each constipation admission, one exact age-matched non-constipated admission was randomly selected from all hospitalizations within 2 weeks to form the comparison cohort. The association of constipation with hypertension and cardiovascular events (myocardial infarction, angina, stroke and transient ischemic attack) were analysed using a series of binary logistic regressions adjusting for age, sex, cardiovascular risk factors, gastrointestinal disorders and sociological factors. Patients with constipation had a higher multivariate-adjusted risk for hypertension (odds ratio [OR], 1.96; 95% confidence interval [CI] 1.94-1.99; P < 0.001). Compared to patients with neither constipation nor hypertension, there was a higher multivariate-adjusted risk for cardiovascular events in patients with constipation alone (OR, 1.58; 95% CI 1.55-1.61; P < 0.001) or hypertension alone (OR, 6.12; 95% CI 5.99-6.26; P < 0.001). In patients with both constipation and hypertension, the risk for all cardiovascular events appeared to be additive (OR, 6.53; 95% CI 6.40-6.66; P < 0.001). In conclusion, among hospital patients aged 60 years or older, constipation is linked to an increased risk of hypertension and cardiovascular events. These findings suggest that interventions to address constipation may reduce cardiovascular risk in elderly patients.

Proteasome inhibition reduces plasma cell and antibody secretion, but not angiotensin II-induced hypertension.

Introduction: Depletion of mature B cells affords protection against experimental hypertension. However, whether B cell-mediated hypertension is dependent on differentiation into antibody-secreting cells (ASCs) remains unclear. Using the proteasome inhibitor, bortezomib, the present study tested the effect of ASC reduction on angiotensin II-induced hypertension. Methods: Male C57BL6/J mice were infused with angiotensin II (0.7 mg/kg/day; s.c.) for 28 days via osmotic minipump to induce hypertension. Normotensive control mice received saline infusion. Bortezomib (750 µg/kg) or vehicle (0.1% DMSO) was administered (i.v.) 3 days prior to minipump implantation, and twice weekly thereafter. Systolic blood pressure was measured weekly using tail-cuff plethysmography. Spleen and bone marrow B1 (CD19+B220-), B2 (B220+CD19+) and ASCs (CD138hiSca-1+Blimp-1+) were enumerated by flow cytometry. Serum immunoglobulins were quantified using a bead-based immunoassay. Results: Bortezomib treatment reduced splenic ASCs by ∼68% and ∼64% compared to vehicle treatment in normotensive (2.00 ± 0.30 vs. 0.64 ± 0.15 × 105 cells; n = 10-11) and hypertensive mice (0.52 ± 0.11 vs. 0.14 ± 0.02 × 105 cells; n = 9-11), respectively. Bone marrow ASCs were also reduced by bortezomib in both normotensive (4.75 ± 1.53 vs. 1.71 ± 0.41 × 103 cells; n = 9-11) and hypertensive mice (4.12 ± 0.82 vs. 0.89 ± 0.18 × 103 cells; n = 9-11). Consistent with ASC reductions, bortezomib reduced serum IgM and IgG2a in all mice. Despite these reductions in ASCs and antibody levels, bortezomib did not affect angiotensin II-induced hypertension over 28 days (vehicle: 182 ± 4 mmHg vs. bortezomib: 177 ± 7 mmHg; n = 9-11). Conclusion: Reductions in ASCs and circulating IgG2a and IgM did not ameliorate experimental hypertension, suggesting other immunoglobulin isotypes or B cell effector functions may promote angiotensin II-induced hypertension.

Quantitative proteomic landscape of unstable atherosclerosis identifies molecular signatures and therapeutic targets for plaque stabilization.

Atherosclerotic plaque rupture leading to myocardial infarction is a major global health burden. Applying the tandem stenosis (TS) mouse model, which distinctively exhibits the characteristics of human plaque instability/rupture, we use quantitative proteomics to understand and directly compare unstable and stable atherosclerosis. Our data highlight the disparate natures and define unique protein signatures of unstable and stable atherosclerosis. Key proteins and pathway networks are identified such as the innate immune system, and neutrophil degranulation. The latter includes calprotectin S100A8/A9, which we validate in mouse and human unstable plaques, and we demonstrate the plaque-stabilizing effects of its inhibition. Overall, we provide critical insights into the unique proteomic landscape of unstable atherosclerosis (as distinct from stable atherosclerosis and vascular tissue). We further establish the TS model as a reliable preclinical tool for the discovery and testing of plaque-stabilizing drugs. Finally, we provide a knowledge resource defining unstable atherosclerosis that will facilitate the identification and validation of long-sought-after therapeutic targets and drugs for plaque stabilization.

High-mobility group box protein 1 neutralization reduces development of diet-induced atherosclerosis in apolipoprotein e-deficient mice.

OBJECTIVE: High-mobility group box protein 1 (HMGB1) is a DNA-binding protein and cytokine highly expressed in atherosclerotic lesions, but its pathophysiological role in atherosclerosis is unknown. We investigated its role in the development of atherosclerosis in ApoE-/- mice. METHODS AND RESULTS: Apolipoprotein E-deficient (ApoE-/-) mice fed a high-fat diet were administered a monoclonal anti-HMGB1 neutralizing antibody, and the effects on lesion size, immune cell accumulation, and proinflammatory mediators were assessed using Oil Red O, immunohistochemistry, and real-time polymerase chain reaction. As with human atherosclerotic lesions, lesions in ApoE-/- mice expressed HMGB1. Treatment with the neutralizing antibody attenuated atherosclerosis by 55%. Macrophage accumulation was reduced by 43%, and vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression was attenuated by 48% and 72%, respectively. CD11c+ dendritic cells were reduced by 65%, and the mature (CD83+) population was reduced by 60%. Treatment also reduced CD4+ cells by nearly 50%. mRNAs in lesions encoding tumor necrosis factor-α and interleukin-1ß tended to be reduced. Mechanistically, HMGB1 stimulated macrophage migration in vitro and in vivo; in vivo, it markedly augmented the accumulation of F4/80+Gr-1(Ly-6C)+ macrophages and also increased F4/80+CD11b+ macrophage numbers. CONCLUSIONS: HMGB1 exerts proatherogenic effects augmenting lesion development by stimulating macrophage migration, modulating proinflammatory mediators, and encouraging the accumulation of immune and smooth muscle cells.

Conventional B2 B cell depletion ameliorates whereas its adoptive transfer aggravates atherosclerosis.

Atherosclerosis is a chronic inflammatory arterial disease characterized by focal accumulation of lipid and inflammatory cells. It is the number one cause of deaths in the Western world because of its complications of heart attacks and strokes. Statins are effective in only approximately one third of patients, underscoring the urgent need for additional therapies. B cells that accumulate in atherosclerotic lesions and the aortic adventitia of humans and mice are considered to protect against atherosclerosis development. Unexpectedly, we found that selective B cell depletion in apolipoprotein E-deficient (ApoE(-/-)) mice using a well-characterized mAb to mouse CD20 reduced atherosclerosis development and progression without affecting the hyperlipidemia imposed by a high-fat diet. Adoptive transfer of 5 × 10(6) or 5 × 10(7) conventional B2 B cells but not 5 × 10(6) B1 B cells to a lymphocyte-deficient ApoE(-/-) Rag-2(-/-) common cytokine receptor γ-chain-deficient mouse that was fed a high-fat diet augmented atherosclerosis by 72%. Transfer of 5 × 10(6) B2 B cells to an ApoE(-/-) mouse deficient only in B cells aggravated atherosclerosis by >300%. Our findings provide compelling evidence for the hitherto unrecognized proatherogenic role of conventional B2 cells. The data indicate that B2 cells can potently promote atherosclerosis development entirely on their own in the total absence of all other lymphocyte populations. Additionally, these B2 cells can also significantly augment atherosclerosis development in the presence of T cells and all other lymphocyte populations. Our findings raise the prospect of B cell depletion as a therapeutic approach to inhibit atherosclerosis development and progression in humans.

Current understanding of the role of B cell subsets and intimal and adventitial B cells in atherosclerosis.

PURPOSE OF REVIEW: Inflammation, in addition to high cholesterol is a major factor contributing to atherosclerosis-associated adverse cardiovascular events. Thus, there is a pressing need for additional therapeutic strategies to reduce inflammation, by targeting immune cells and cytokines. Here we review B cell subsets and adventitial and intimal B cells in atherosclerosis development and discuss potential B cell-targeted anti-inflammatory therapies for atherosclerosis. RECENT FINDINGS: B cell subsets can have opposing proatherogenic and atheroprotective roles in atherosclerosis. CD-20-targeted B cell depletion has been shown to decrease murine atherosclerotic lesions. The accumulation of intimal and adventitial B cells associated with atherosclerotic lesions is consistent with their participation in local inflammatory responses. As B2 B cells are proatherogenic, blocking its survival factor B cell activating factor may selectively delete this proatherogenic subset. SUMMARY: Both intimal and adventitial B cells appear important in atherosclerosis. B2 B cells are proatherogenic and other subsets such as regulatory B cells are antiatherogenic. Future B cell-targeted therapy for atherosclerosis should be customized to selectively deplete damaging B2 B cells while sparing or expanding protective B cell subsets.

The tandem stenosis mouse model: Towards understanding, imaging, and preventing atherosclerotic plaque instability and rupture.

The rupture of unstable atherosclerotic plaques is the major cause of cardiovascular mortality and morbidity. Despite significant limitations in our understanding and ability to identify unstable plaque pathology and prevent plaque rupture, most atherosclerosis research utilises preclinical animal models exhibiting stable atherosclerosis. Here, we introduce the tandem stenosis (TS) mouse model that reflects plaque instability and rupture, as seen in patients. The TS model involves dual ligation of the right carotid artery, leading to locally predefined unstable atherosclerosis in hypercholesterolaemic mice. It exhibits key characteristics of human unstable plaques, including plaque rupture, luminal thrombosis, intraplaque haemorrhage, large necrotic cores, thin or ruptured fibrous caps and extensive immune cell accumulation. Altogether, the TS model represents an ideal preclinical tool for improving our understanding of human plaque instability and rupture, for the development of imaging technologies to identify unstable plaques, and for the development and testing of plaque-stabilising treatments for the prevention of atherosclerotic plaque rupture. LINKED ARTICLES: This article is part of a themed issue on Preclinical Models for Cardiovascular disease research (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.5/issuetoc.

Crosstalk between cytotoxic CD8+ T cells and stressed cardiomyocytes triggers development of interstitial cardiac fibrosis in hypertensive mouse hearts.

Aims: Cardiac fibrosis is central to heart failure (HF), especially HF with preserved ejection fraction (HFpEF), often caused by hypertension. Despite fibrosis causing diastolic dysfunction and impaired electrical conduction, responsible for arrhythmia-induced sudden cardiac death, the mechanisms are poorly defined and effective therapies are lacking. Here we show that crosstalk between cardiac cytotoxic memory CD8+ T cells and overly stressed cardiomyocytes is essential for development of non-ischemic hypertensive cardiac fibrosis. Methods and results: CD8 T cell depletion in hypertensive mice, strongly attenuated CF, reduced cardiac apoptosis and improved ventricular relaxation. Interaction between cytotoxic memory CD8+ T cells and overly stressed cardiomyocytes is highly dependent on the CD8+ T cells expressing the innate stress-sensing receptor NKG2D and stressed cardiomyocytes expressing the NKG2D activating ligand RAE-1. The interaction between NKG2D and RAE-1 results in CD8+ T cell activation, release of perforin, cardiomyocyte apoptosis, increased numbers of TGF-ß1 expressing macrophages and fibrosis. Deleting NKG2D or perforin from CD8+ T cells greatly attenuates these effects. Activation of the cytoplasmic DNA-STING-TBK1-IRF3 signaling pathway in overly stressed cardiomyocytes is responsible for elevating RAE-1 and MCP-1, a macrophage attracting chemokine. Inhibiting STING activation greatly attenuates cardiomyocyte RAE-1 expression, the cardiomyocyte apoptosis, TGF-ß1 and fibrosis. Conclusion: Our data highlight a novel pathway by which CD8 T cells contribute to an early triggering mechanism in CF development; preventing CD8+ T cell activation by inhibiting the cardiomyocyte RAE-1-CD8+ T cell-NKG2D axis holds promise for novel therapeutic strategies to limit hypertensive cardiac fibrosis.