Role of LCN2 in a murine model of hindlimb ischemia and in peripheral artery disease patients, and its potential regulation by miR-138-5P.

BACKGROUND AND AIMS: Peripheral arterial disease (PAD) is a leading cause of morbimortality worldwide. Lipocalin-2 (LCN2) has been associated with higher risk of amputation or mortality in PAD and might be involved in muscle regeneration. Our aim is to unravel the role of LCN2 in skeletal muscle repair and PAD. METHODS AND RESULTS: WT and Lcn2-/- mice underwent hindlimb ischemia. Blood and crural muscles were analyzed at the inflammatory and regenerative phases. At day 2, Lcn2-/- male mice, but not females, showed increased blood and soleus muscle neutrophils, and elevated circulating pro-inflammatory monocytes (p < 0.05), while locally, total infiltrating macrophages were reduced (p < 0.05). Moreover, Lcn2-/- soleus displayed an elevation of Cxcl1 (p < 0.001), and Cxcr2 (p < 0.01 in males), and a decrease in Ccl5 (p < 0.05). At day 15, Lcn2 deficiency delayed muscle recovery, with higher density of regenerating myocytes (p < 0.04) and arterioles (αSMA+, p < 0.025). Reverse target prediction analysis identified miR-138-5p as a potential regulator of LCN2, showing an inverse correlation with Lcn2 mRNA in skeletal muscles (rho = -0.58, p < 0.01). In vitro, miR-138-5p mimic reduced Lcn2 expression and luciferase activity in murine macrophages (p < 0.05). Finally, in human serum miR-138-5p was inversely correlated with LCN2 (p ≤ 0.001 adjusted, n = 318), and associated with PAD (Odds ratio 0.634, p = 0.02, adjusted, PAD n = 264, control n = 54). CONCLUSIONS: This study suggests a possible dual role of LCN2 in acute and chronic conditions, with a probable role in restraining inflammation early after skeletal muscle ischemia, while being associated with vascular damage in PAD, and identifies miR-138-5p as one potential post-transcriptional regulator of LCN2.

Targeting and delivery of microRNA-targeting antisense oligonucleotides in cardiovascular diseases.

Discovered three decades ago, microRNAs (miRNAs) are now recognized as key players in the pathophysiology of multiple human diseases, including those affecting the cardiovascular system. As such, miRNAs have emerged as promising therapeutic targets for preventing the onset and/or progression of several cardiovascular diseases. Anti-miRNA antisense oligonucleotides or "antagomirs" precisely block the activity of specific miRNAs and are therefore a promising therapeutic strategy to repress pathological miRNAs. In this review, we describe advancements in antisense oligonucleotide chemistry that have significantly improved efficacy and safety. Moreover, we summarize recent approaches for the targeted delivery of antagomirs to cardiovascular tissues, highlighting major advantages as well as limitations of viral (i.e., adenovirus, adeno-associated virus, and lentivirus) and non-viral (i.e., liposomes, extracellular vesicles, and polymer nanoparticles) delivery systems. We discuss recent preclinical studies that use targeted antagomir delivery systems to treat three major cardiovascular diseases (atherosclerosis, myocardial infarction, and cardiac hypertrophy, including hypertrophy caused by hypertension), highlighting therapeutic results and discussing challenges that limit clinical applicability.

Lipocalin-2 and Calprotectin Potential Prognosis Biomarkers in Peripheral Arterial Disease.

OBJECTIVE: Peripheral arterial disease (PAD) is the most prevalent cardiovascular (CV) condition globally. Despite the high CV risk of PAD patients, no reliable predictors of adverse clinical evolution are yet available. In this regard, previous transcriptomic analyses revealed increased expression of calprotectin (S100A8/A9) and lipocalin-2 (LCN2) in circulating extracellular vesicles (EVs) of patients with PAD. The aim of this study was to determine the prognostic value of LCN2 and calprotectin for CV risk assessment in PAD. METHODS: LCN2 and the S100A9 subunit of calprotectin were examined in human femoral plaques by immunohistochemistry and qPCR. LCN2 and calprotectin were determined by ELISA in PAD (CHN cohort, n = 331, Fontaine II-IV, serum), and PAD diagnosed by population based screening (VIVA trial, n = 413, the majority Fontaine 0-I, plasma). Patients were followed up for a mean of four years, recording the primary outcomes; CV death or amputation in the CHN cohort and CV death or major lower limb events (MALE) in the VIVA population. Secondary outcomes were all cause death or amputation, and all cause death or MALE, respectively. RESULTS: LCN2 and S100A9 were detected in human plaques in regions rich in inflammatory cells. LCN2 and calprotectin levels were 70% and 64% lower in plasma than in serum. In the CHN cohort, high serum levels of LCN2 and calprotectin increased the risk of primary and secondary outcomes 5.6 fold (p < .001) and 1.8 fold (p = .034), respectively, after covariable adjustment. Similarly, elevated plasma levels of LCN2 and calprotectin increased by three fold the risk of primary and secondary outcomes (p < .001) in the VIVA cohort. Moreover, addition of the combined variable to basal models, considering clinically relevant risk factors, improved reclassification for the primary outcome in both cohorts (p ≤ .024). CONCLUSION: Combined assessment of the inflammatory biomarkers LCN2 and calprotectin might be useful for risk stratification in advanced and early PAD.

The Role of Circulating Biomarkers in Peripheral Arterial Disease.

Peripheral arterial disease (PAD) of the lower extremities is a chronic illness predominantly of atherosclerotic aetiology, associated to traditional cardiovascular (CV) risk factors. It is one of the most prevalent CV conditions worldwide in subjects >65 years, estimated to increase greatly with the aging of the population, becoming a severe socioeconomic problem in the future. The narrowing and thrombotic occlusion of the lower limb arteries impairs the walking function as the disease progresses, increasing the risk of CV events (myocardial infarction and stroke), amputation and death. Despite its poor prognosis, PAD patients are scarcely identified until the disease is advanced, highlighting the need for reliable biomarkers for PAD patient stratification, that might also contribute to define more personalized medical treatments. In this review, we will discuss the usefulness of inflammatory molecules, matrix metalloproteinases (MMPs), and cardiac damage markers, as well as novel components of the liquid biopsy, extracellular vesicles (EVs), and non-coding RNAs for lower limb PAD identification, stratification, and outcome assessment. We will also explore the potential of machine learning methods to build prediction models to refine PAD assessment. In this line, the usefulness of multimarker approaches to evaluate this complex multifactorial disease will be also discussed.

Functional and transcriptomic analysis of extracellular vesicles identifies calprotectin as a new prognostic marker in peripheral arterial disease (PAD).

Peripheral arterial disease (PAD) is associated with a high risk of cardiovascular events and death and is postulated to be a critical socioeconomic cost in the future. Extracellular vesicles (EVs) have emerged as potential candidates for new biomarker discovery related to their protein and nucleic acid cargo. In search of new prognostic and therapeutic targets in PAD, we determined the prothrombotic activity, the cellular origin and the transcriptomic profile of circulating EVs. This prospective study included control and PAD patients. Coagulation time (Procoag-PPL kit), EVs cellular origin and phosphatidylserine exposure were determined by flow cytometry in platelet-free plasma (n = 45 PAD). Transcriptomic profiles of medium/large EVs were generated using the MARS-Seq RNA-Seq protocol (n = 12/group). The serum concentration of the differentially expressed gene S100A9, in serum calprotectin (S100A8/A9), was validated by ELISA in control (n = 100) and PAD patients (n = 317). S100A9 was also determined in EVs and tissues of human atherosclerotic plaques (n = 3). Circulating EVs of PAD patients were mainly of platelet origin, predominantly Annexin V positive and were associated with the procoagulant activity of platelet-free plasma. Transcriptomic analysis of EVs identified 15 differentially expressed genes. Among them, serum calprotectin was elevated in PAD patients (p < 0.05) and associated with increased amputation risk before and after covariate adjustment (mean follow-up 3.6 years, p < 0.01). The combination of calprotectin with hs-CRP in the multivariate analysis further improved risk stratification (p < 0.01). Furthermore, S100A9 was also expressed in femoral plaque derived EVs and tissues. In summary, we found that PAD patients release EVs, mainly of platelet origin, highly positive for AnnexinV and rich in transcripts related to platelet biology and immune responses. Amputation risk prediction improved with calprotectin and was significantly higher when combined with hs-CRP. Our results suggest that EVs can be a promising component of liquid biopsy to identify the molecular signature of PAD patients.