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Extracellular vesicles (EVs) are critical in the initiation and progression of cardiovascular calcification, and immune cell infiltration and inflammation have a central role in this process. EVs egress from various cardiovascular cell types, which when acquiring specific properties, become calcifying. These calcifying EVs form nidi for microcalcification, which can progress to the macrocalcification lesions that are visualized clinically. We make the distinction between inflammatory-driven and mineral dysregulation-driven calcification, which both share EVs as a central initiator. In inflammation-mediated calcification, inflammation precedes microcalcification and results from EV release from macrophages. Local cellular crosstalk mediated by EVs as well as circulating EVs and other inflammatory nanoparticles, such as calciprotein particles and lipoproteins, are also critical in the progression of cardiovascular calcification. It is imperative that future work links the already established and to be discovered roles of inflammation and innate immunity in cardiovascular calcification to these key signaling and functional roles of these nanoparticles. It remains an understudied area with high potential to unravel mechanistic roles and has important implications in drug target research.

Introduction: Inflammation is causally related to atherothrombosis. Interleukin-1β (IL-1β) and interleukin-18 (IL-18) require NLRP3 inflammasome for activation and have downstream effects on interleukin-6 (IL-6), a marker previously associated with high risk of coronary artery and cerebrovascular disease (CCVD). However, data pertaining to peripheral artery disease (PAD) are sparse and could offer druggable targets in this disease. Methods: We conducted a prospective cohort study of 4248 patients with type 2 diabetes or metabolic syndrome and prior coronary artery disease who participated in the NIH-funded Cardiovascular Inflammation Reduction Trial (CIRT). Participants were followed for up to 5 years for incident CCVD and symptomatic PAD events. Randomized treatment with low-dose methotrexate (vs. placebo) had no effect on event rates or plasma levels of inflammatory biomarkers. Baseline levels of IL-1β, IL-18, and IL-6 were tested for association with incident vascular events. Kaplan-Meier curves and Cox proportional hazards models (adjusted for traditional risk factors) were estimated. Results: In multivariable adjusted analyses, hazard ratios for the lowest (referent) to highest baseline quartiles of IL-6 were 1.0, 1.5, 1.8, and 2.0 (p-trend Conclusion: In this contemporary cohort of secondary prevention patients, elevated IL-6 was associated with both incident CCVD and PAD. These data support exploration of direct IL-6 inhibition for PAD prevention, a strategy currently being pursued to reduce risk of coronary artery and cerebrovascular disease.

Introduction: Calcific aortic valve disease is a multifaceted disease in which pathogenic mineralisation of the valvular tissue occurs in tandem with fibrocalcific remodelling of the aortic valve, resulting in a wide variety of clinical presentations. Immunity, both innate and adaptive have been shown to play important roles in chronic cardiovascular diseases. Noteably, 15% of cells within healthy aortic valves are up to hematopoietic origin and these populations are further increased within calcified valves however, the role of the immune system within CAVD remains to be elucidated. Objectives: Investigate the prevalence and distribution of immune cells within CAVD. Methods & Results: Human calcified aortic heart valves were obtained from valve replacement surgeries. Immunohistological assessment of immune cells within the tissue was conducted, with the presence of T cells, B cells, macrophages and dendritic cells detected in the vicinity of calcific nodules. Flow cytometry analysis of cells isolated from the valve was conducted. 69% of cells isolated from the valve were CD45+, with 57% and 42% of CD45+ CD3+ presenting as CD8+ and CD4+ respectively. Within the CD8+ population, 56% of these were central memory CD8+ T cells, with 24% as activated CD8+ T cells. Likewise, within the CD4+ population 29.5% CD4+ cells were activated T cells and 55% central memory T cells. Small populations of naïve CD4+ and CD8+ T cells; 9% and 13% respectively, were present within the valve. CD45+, CD3+, CD4+/CD8+ cells were isolated via FACS and their global proteomic profile was analysed on the Thermo Scientific Lumos orbitrap mass spectrometer. IL-31RA was present within the T cell population, alongside MAPK pathway mediators were also present. Network analysis of T cell proteomics showed increased IL-4 and IL-13 pathways (p Conclusions: Prevalence of immune cells within the valve suggests an inflammatory role in the development of calcific aortic valve disease, with the prevalence of T cells within the valve suggesting an adaptive immune response may be central to the pathology of CAVD.

RATIONALE. Calcific Aortic Valve Disease (CAVD) affects more than 5.2 million people in the US. The only effective treatment is surgery and this comes with complications and no guarantee of long-term success. OBJECTIVE. Outcomes from pharmacological initiatives remain unsubstantiated and therefore the aim of this study is to determine if repurposing a selective XPO1 inhibitor drug (KPT-330) is beneficial in the treatment of CAVD. METHODS AND RESULTS. We show that KPT-330 prevents, attenuates and mitigates calcific nodule formation in heart valve interstitial cells (VICs) in vitro, and prevents CAVD in Klotho(−/−) mice. Using RNA-sequencing and Mass Spectrometry we show that KPT-330’s beneficial effect is mediated by inhibiting nuclear export of the transcription factor C/EBPβ in VICs, leading to repression of canonical Wnt signaling, in part through activation of the Wnt antagonist Axin1, and a subsequent decrease in pro-osteogenic markers and cell viability. CONCLUSIONS. Our findings have met a critical need to discover alterative, pharmacological-based therapies in the treatment of CAVD.

Aortic aneurysm/dissection (AAD) is a life-threatening disorder lacking effective pharmacotherapeutic remedies. Aldehyde dehydrogenase 2 (ALDH2) polymorphism is tied with various risk factors for AAD including hypertension, atherosclerosis, and hypercholesterolaemia although direct correlation between the two remains elusive.Two independent case-control studies were conducted involving 307 AAD patients and 399 healthy controls in two geographically distinct areas in China. Our data revealed that subjects carrying mutant ALDH2 gene possessed a ∼50% reduced risk of AAD compared with wild-type (WT) alleles. Using 3-aminopropionitrile fumarate (BAPN)- and angiotensin II (Ang II)-induced AAD animal models, inhibition of ALDH2 was found to retard development of AAD. Mechanistically, ALDH2 inhibition ablated pathological vascular smooth muscle cell (VSMC) phenotypical switch through interaction with myocardin, a determinant of VSMC contractile phenotype. Using microarray and bioinformatics analyses, ALDH2 deficiency was found to down-regulate miR-31-5p, which further altered myocardin mRNA level. Gain-of-function and loss-of-function studies verified that miR-31-5p significantly repressed myocardin level and aggravated pathological VSMC phenotypical switch and AAD, an effect that was blunted by ALDH2 inhibition. We next noted that ALDH2 deficiency increased Max expression and decreased miR-31-5p level. Moreover, ALDH2 mutation or inhibition down-regulated levels of miR-31-5p while promoting myocardin downstream contractile genes in the face of Ang II in primary human VSMCs.ALDH2 deficiency is associated with a lower risk of AAD in patients and mice, possibly via suppressing VSMC phenotypical switch in a miR-31-5p-myocardin-dependent manner. These findings favour a role for ALDH2 and miR-31-5p as novel targets for AAD therapy.

Calcific aortic valve disease (CAVD), and its clinical manifestation that is calcific aortic valve stenosis, is the leading cause for valve disease within the developed world, with no current pharmacological treatment available to delay or halt its progression. Characterized by progressive fibrotic remodelling and subsequent pathogenic mineralization of the valve leaflets, valve disease affects 2.5% of the western population, thus highlighting the need for urgent intervention. Whilst the pathobiology of valve disease is complex, involving genetic factors, lipid infiltration, and oxidative damage, the immune system is now being accepted to play a crucial role in pathogenesis and disease continuation. No longer considered a passive degenerative disease, CAVD is understood to be an active inflammatory process, involving a multitude of pro-inflammatory mechanisms, with both the adaptive and the innate immune system underpinning these complex mechanisms. Within the valve, 15% of cells evolve from haemopoietic origin, and this number greatly expands following inflammation, as macrophages, T lymphocytes, B lymphocytes, and innate immune cells infiltrate the valve, promoting further inflammation. Whether chronic immune infiltration or pathogenic clonal expansion of immune cells within the valve or a combination of the two is responsible for disease progression, it is clear that greater understanding of the immune systems role in valve disease is required to inform future treatment strategies for control of CAVD development.

Cellular heterogeneity of aortic valves complicates the mechanistic evaluation of the calcification processes in calcific aortic valve disease (CAVD), and animal disease models are lacking. In this study, we identify a disease-driver population (DDP) within valvular interstitial cells (VICs). Through stepwise single-cell analysis, phenotype-guided omic profiling, and network-based analysis, we characterize the DDP fingerprint as CD44

Aims Vascular calcification is a recognised predictor of cardiovascular risk in the diabetic patient, with DNA damage and accelerated senescence linked to oxidative stress associated pathological calcification. Having previously shown that systemic SIRT1 is reduced in diabetes, the aim was to establish whether SIRT1 is protective against a DNA damage-induced senescent and calcified phenotype in diabetic vascular smooth muscle cells (vSMCs). Methods and results Immunohistochemistry revealed decreased SIRT1 and increased DNA damage marker expression in diabetic calcified arteries compared to non-diabetic and non-calcified controls, strengthened by findings that vSMCs isolated from diabetic patients show elevated DNA damage and senescence, assessed by the Comet assay and telomere length. Hyperglycaemic conditions were used and induced DNA damage and enhanced senescence in vSMCs in vitro. Using H2O2 as a model of oxidative stress-induced DNA damage, pharmacological activation of SIRT1 reduced H2O2 DNA damage induced calcification, prevented not only DNA damage, as shown by reduced comet tail length, but also decreased yH2AX foci formation, and attenuated calcification. While ATM expression was reduced following DNA damage, in contrast, SIRT1 activation significantly increased ATM expression, phosphorylating both MRE11 and NBS1, thus allowing formation of the MRN complex and increasing activation of the DNA repair pathway. Conclusions DNA damage induced calcification is accelerated within a diabetic environment and can be attenuated in vitro by SIRT1 activation. This occurs through enhancement of the MRN repair complex within vSMCs and has therapeutic potential within the diabetic patient. Translational perspective Our study provides the first evidence that DNA damage is enhanced in the vasculature of the diabetic patient and that this process, in tandem with loss of SIRT1, exacerbates pathological smooth muscle cell calcification. We propose that current SIRT1 activators and their analogues may be useful as investigational tools, and further elucidation of downstream mechanisms of SIRT1 will aid the development of novel and more precise drug regimens.

Introduction Patients with type II diabetes have considerably higher risk of developing cardiovascular disease and often have an elevated prevalence of vascular calcification combined with a loss of SIRT1, which induces osteogenic differentiation within the smooth muscle. This study aims to determine whether the loss of SIRT1 contributes to the enhanced DNA damage associated with vascular calcification and diabetes. Methods Popliteal arteries, harvested from diabetic patients undergoing limb amputation, and non-atherosclerotic internal thoracic arteries, were stained for a range of DNA damage markers. Human coronary artery smooth muscle cells (vSMCs) were used for an in vitro calcification model (5mM βGP and 2.6mM CaCl2) in the presence or absence of a diabetic environment (high glucose (25mM), or control (5mM) DMEM respectively. DNA damage was induced using 200μM H2O2 and SIRT1 was activated or inhibited pharmacologically using SRT1720 or Sirtinol respectively. DNA damage was assessed via a comet assay, γH2A.X and p21 immunocytochemistry, whilst DNA damage-induced SIRT1 activation was assessed via nuclear fractionation and western blotting. Activation of the MRN DNA damage response pathway was confirmed by chIP qPCR and western blot analysis. Results Alizarin red, Alkaline phosphatase activity and RUNX2 and OCN qPCR confirmed development of calcification during H2O2 treatment, which was reduced with SIRT1 activation. Immunohistochemistry demonstrated enhanced DNA damage marker expression in diabetic vessels compared to non-diabetic control ITA tissue, while SMCs harvested from diabetic patients also showed a significant reduction in SIRT1 expression (p Conclusions This study demonstrates that SIRT1 protects against H2O2 induced DNA damage and subsequent calcification within a diabetic environment via activation of the MRN complex. The loss of SIRT1 within the calcified vessels of diabetic patients may contribute to a defective DNA repair mechanism, caused by the absence of SIRT1, resulting in a reduction of MRN deacetylation and thus activation of the DNA damage response, and could be an appropriate target for potential therapeutic intervention for vascular calcification. Conflict of interest N/A