Identification and Evaluation of Blood-Based Biomarkers for Abdominal Aortic Aneurysm.

INTRODUCTION: Blood-based biomarkers for abdominal aortic aneurysm (AAA) have been studied individually; however, we considered a panel of proteins to investigate AAA prognosis and its potential to improve predictive accuracy. MATERIALS AND METHODS: Using a prospectively recruited cohort of patients with/without AAA (n = 452), we conducted a prognostic study to develop a model that accurately predicts AAA outcomes using clinical features and circulating biomarker levels. Serum concentrations of 9 biomarkers were measured at baseline, and the cohort was followed for 2 years. The primary outcome was major adverse aortic event (MAAE; composite of rapid AAA expansion [>0.5 cm/6 months or >1 cm/12 months], AAA intervention, or AAA rupture). Using 10-fold cross-validation, we trained a random forest model to predict 2 year MAAE using (1) clinical characteristics, (2) biomarkers, and (3) clinical characteristics and biomarkers. RESULTS: Two-year MAAE occurred in 114 (25%) patients. Two proteins were significantly elevated in patients with AAA compared with those without AAA (angiopoietin-2 and aggrecan), composing the protein panel. For predicting 2 year MAAE, our random forest model achieved area under the receiver operating characteristic curve (AUROC) 0.74 using clinical features alone, and the addition of the 2-protein panel improved performance to AUROC 0.86. CONCLUSIONS: Using a combination of clinical/biomarker data, we developed a model that accurately predicts 2 year MAAE.

A Systematic Review of Interleukins as Diagnostic and Prognostic Biomarkers for Peripheral Artery Disease.

Background: Peripheral artery disease (PAD) involves atherosclerosis of the lower extremity arteries and is a major contributor to limb loss and death worldwide. Several studies have demonstrated that interleukins (ILs) play an important role in the development and progression of PAD; however, a comprehensive literature review has not been performed. Methods: A systematic review was conducted and reported according to PRISMA guidelines. MEDLINE was searched from inception to 5 December 2022, and all studies assessing the association between ILs and PAD were included. Results: We included 17 studies from a pool of 771 unique articles. Five pro-inflammatory ILs (IL-1ß, IL-2, IL-5, IL-6, and IL-8) and one pro-atherogenic IL (IL-12) were positively correlated with PAD diagnosis and progression. In contrast, two anti-inflammatory ILs (IL-4 and IL-10) were protective against PAD diagnosis and adverse limb events. Specifically, IL-6 and IL-8 were the most strongly associated with PAD and can act as potential disease biomarkers to support the identification and treatment of PAD. Conclusions: Ongoing work to identify and validate diagnostic/prognostic inflammatory biomarkers for PAD has the potential to assist clinicians in identifying high-risk patients for further evaluation and management which could reduce the risk of adverse cardiovascular and limb events.

Current Biomarkers for Carotid Artery Stenosis: A Comprehensive Review of the Literature.

Carotid artery stenosis (CAS), an atherosclerotic disease of the carotid artery, is one of the leading causes of transient ischemic attacks (TIA) and cerebrovascular attacks (CVA). The atherogenic process of CAS affects a wide range of physiological processes, such as inflammation, endothelial cell function, smooth muscle cell migration and many more. The current gold-standard test for CAS is Doppler ultrasound; however, there is yet to be determined a strong, clinically validated biomarker in the blood that can diagnose patients with CAS and/or predict adverse outcomes in such patients. In this comprehensive literature review, we evaluated all of the current research on plasma and serum proteins that are current contenders for biomarkers for CAS. In this literature review, 36 proteins found as potential biomarkers for CAS were categorized in to the following nine categories based on protein function: (1) Inflammation and Immunity, (2) Lipid Metabolism, (3) Haemostasis, (4) Cardiovascular Markers, (5) Markers of Kidney Function, (6) Bone Health, (7) Cellular Structure, (8) Growth Factors, and (9) Hormones. This literature review is the most up-to-date and current comprehensive review of research on biomarkers of CAS, and the only review that demonstrated the several pathways that contribute to the initiation and progression of the disease. With this review, future studies can determine if any new markers, or a panel of the proteins explored in this study, may be contenders as diagnostic or prognostic markers for CAS.

Aspirin Resistance in Vascular Disease: A Review Highlighting the Critical Need for Improved Point-of-Care Testing and Personalized Therapy.

Aspirin resistance describes a phenomenon where patients receiving aspirin therapy do not respond favorably to treatment, and is categorized by continued incidence of adverse cardiovascular events and/or the lack of reduced platelet reactivity. Studies demonstrate that one in four patients with vascular disease are resistant to aspirin therapy, placing them at an almost four-fold increased risk of major adverse limb and adverse cardiovascular events. Despite the increased cardiovascular risk incurred by aspirin resistant patients, strategies to diagnose or overcome this resistance are yet to be clinically validated and integrated. Currently, five unique laboratory assays have shown promise for aspirin resistance testing: Light transmission aggregometry, Platelet Function Analyzer-100, Thromboelastography, Verify Now, and Platelet Works. Newer antiplatelet therapies such as Plavix and Ticagrelor have been tested as an alternative to overcome aspirin resistance (used both in combination with aspirin and alone) but have not proven to be superior to aspirin alone. A recent breakthrough discovery has demonstrated that rivaroxaban, an anticoagulant which functions by inhibiting active Factor X when taken in combination with aspirin, improves outcomes in patients with vascular disease. Current studies are determining how this new regime may benefit those who are considered aspirin resistant.

The Role of Fatty Acid Binding Protein 3 in Cardiovascular Diseases.

Fatty acid binding proteins (FABPs) are proteins found in the cytosol that contribute to disorders related to the cardiovascular system, including atherosclerosis and metabolic syndrome. Functionally, FABPs serve as intracellular lipid chaperones, interacting with hydrophobic ligands and mediating their transportation to sites of lipid metabolism. To date, nine unique members of the FABP family (FABP 1-9) have been identified and classified according to the tissue in which they are most highly expressed. In the literature, FABP3 has been shown to be a promising clinical biomarker for coronary and peripheral artery disease. Given the rising incidence of cardiovascular disease and its associated morbidity/mortality, identifying biomarkers for early diagnosis and treatment is critical. In this review, we highlight key discoveries and recent studies on the role of FABP3 in cardiovascular disorders, with a particular focus on its clinical relevance as a biomarker for peripheral artery disease.

Low-dose aspirin and rivaroxaban combination therapy to overcome aspirin non-sensitivity in patients with vascular disease.

Approximately 20% of vascular patients treated with acetyl salicylic acid (i.e., aspirin) demonstrate less than expected platelet inhibition - putting them at a four-fold increased risk of adverse cardiovascular events. Low-dose rivaroxaban (2.5 mg twice daily) in combination with low-dose aspirin has been shown to reduce adverse cardiovascular and limb events when compared to aspirin alone. In this study, light transmission aggregometry was used to measure arachidonic acid-induced platelet aggregation to evaluate the potential of combining low-dose rivaroxaban and aspirin in attenuating or overcoming aspirin non-sensitivity. In the discovery phase, 83 patients with peripheral arterial disease (PAD) taking 81 mg aspirin daily were recruited from the outpatient vascular surgery clinic at St Michael's Hospital between January to September 2021. 19 (23%) were determined to be non-sensitive to aspirin. After ex-vivo addition of 2.5 mg dosage equivalent of rivaroxaban, aspirin non-sensitivity was overcome in 11 (58%) of these 19 patients. In the validation phase, 58 patients with cardiovascular risk factors who were not previously prescribed aspirin were recruited. In this group, ex-vivo addition of 2.5 mg dosage equivalent of rivaroxaban significantly reduced arachidonic acid-induced platelet aggregation in the presence of aspirin. These results demonstrate the potential for low-dose rivaroxaban to overcome aspirin non-sensitivity in patients with PAD. Further studies are needed to evaluate and confirm these findings.

Aspirin nonsensitivity in patients with vascular disease: Assessment by light transmission aggregometry (aspirin nonsensitivity in vascular patients).

BACKGROUND: Aspirin is a key antiplatelet therapy for the prevention of thrombotic events in patients with cardiovascular disease. Studies suggest that ≈20% of patients with cardiac disease suffer from aspirin nonsensitivity, a phenomenon characterized by the inability of 81 mg aspirin to inhibit platelet aggregation and/or prevent adverse cardiovascular events. OBJECTIVES: To investigate aspirin nonsensitivity in patients with vascular disease and assess the consequences of aspirin nonsensitivity. METHODS: One hundred fifty patients presenting to St. Michael's Hospital's outpatient clinics with evidence of vascular disease (peripheral arterial disease or carotid artery stenosis) and a previous prescription of 81 mg of aspirin were recruited in this study. Light transmission aggregometry with arachidonic acid induction was used to determine sensitivity to aspirin. Patients with a maximum aggregation ≥20% in response to arachidonic acid were considered aspirin nonsensitive, as per previous studies. RESULTS: Of the 150 patients recruited, 36 patients (24%) were nonsensitive to 81 mg of aspirin. Of these 36 nonsensitive patients, 30 patients provided a urine sample for urine salicyluric acid analysis (a major metabolite of aspirin). Urine analysis demonstrated that 14 patients were compliant and 16 were noncompliant with their aspirin therapy. Major adverse cardiovascular events and major adverse limb events were significantly higher in the nonsensitive patients compared to sensitive patients (hazard ratio, 3.68; P < 0.001). CONCLUSION: These data highlight the high prevalence of aspirin nonsensitivity and noncompliance in patients with vascular disease and emphasizes the urgent need for improved medical management options for this patient population.

Plateletworks® as a Point-of-Care Test for ASA Non-Sensitivity.

Aspirin (ASA) therapy is proven to be effective in preventing adverse cardiovascular events; however, up to 30% of patients are non-sensitive to their prescribed ASA dosage. In this pilot study, we demonstrated, for the first time, how ASA non-sensitivity can be diagnosed using Plateletworks®, a point-of-care platelet function test. Patients prescribed 81 mg of ASA were recruited in a series of two successive phases-a discovery phase and a validation phase. In the discovery phase, a total of 60 patients were recruited to establish a cut-off point (COP) for ASA non-sensitivity using Plateletworks®. Each sample was simultaneously cross-referenced with a light transmission aggregometer (LTA). Our findings demonstrated that >52% maximal platelet aggregation using Plateletworks® had a sensitivity, specificity, and likelihood ratio of 80%, 70%, and 2.67, respectively, in predicting ASA non-sensitivity. This COP was validated in a secondary cohort of 40 patients prescribed 81 mg of ASA using Plateletworks® and LTA. Our data demonstrated that our established COP had a 91% sensitivity and 69% specificity in identifying ASA non-sensitivity using Plateletworks®. In summary, Plateletworks® is a point-of-care platelet function test that can appropriately diagnose ASA non-sensitive patients with a sensitivity exceeding 80%.

Ticagrelor as an Alternative Antiplatelet Therapy in Cardiac Patients Non-Sensitive to Aspirin.

Background and Objectives: Aspirin (acetylsalicylic acid-ASA) is a first-line antiplatelet therapy provided to patients with coronary artery disease (CAD). However, it has been demonstrated that 20-30% of these patients are non-sensitive to their ASA therapy. ASA non-sensitivity is a phenomenon where low-dose ASA (81-325 mg) does not completely inhibit arachidonic-acid-induced platelet aggregation, putting patients at risk of adverse cardio-thrombotic events. Ticagrelor is a P2Y12 receptor inhibitor and alternative antiplatelet that has been approved to reduce the risk of stroke, myocardial infarction, and overall cardiovascular-related death. In this study, we aimed to identify ASA non-sensitive patients and evaluate if they would be sensitive to ticagrelor. Materials and Methods: For this pilot study, thirty-eight patients with CAD taking 81 mg ASA were recruited. Blood samples were collected from each patient and platelet rich plasma (PRP) from each sample was isolated. Light-transmission aggregometry (LTA) was used to determine baseline ASA sensitivity in each patient using 0.5 mg/mL arachidonic acid as a platelet agonist. Patients with ≥20% maximal platelet aggregation after activation were considered ASA non-sensitive. Fresh PRP samples from all patients were then spiked with a clinical dosage of ticagrelor (3 µM-approximately equivalent to a loading dose of 180 mg ticagrelor). Sensitivity was determined using LTA and 5 µM ADP as a platelet agonist. Patients with ≥46% maximal platelet aggregation were considered ticagrelor non-sensitive. Results: Of the 38 CAD patients taking 81 mg ASA, 32% (12/38) were non-sensitive to their 81 mg ASA therapy. All 38 of the recruited patients (100%) were sensitive to ticagrelor ex vivo. In conclusion, we were able to identify ASA non-sensitivity using LTA and determine that ASA non-sensitive patients were sensitive to ticagrelor. Conclusions: Our results suggest that ticagrelor is a promising alternative therapy for patients who are non-sensitive to ASA.

Personalization of Aspirin Therapy Ex Vivo in Patients with Atherosclerosis Using Light Transmission Aggregometry.

Acetylsalicylic acid (ASA), also known as aspirin, appears to be ineffective in inhibiting platelet aggregation in 20-30% of patients. Light transmission aggregometry (LTA) is a gold standard platelet function assay. In this pilot study, we used LTA to personalize ASA therapy ex vivo in atherosclerotic patients. Patients were recruited who were on 81 mg ASA, presenting to ambulatory clinics at St. Michael's Hospital (n = 64), with evidence of atherosclerotic disease defined as clinical symptoms and diagnostic findings indicative of symptomatic peripheral arterial disease (PAD), with an ankle brachial index (ABI) of <0.9 (n = 52) or had diagnostic features of asymptomatic carotid arterial stenosis (CAS), with >50% stenosis of internal carotid artery on duplex ultrasound (n = 12). ASA compliance was assessed via multisegmented injection-capillary electrophoresis-mass spectrometry based on measuring the predominant urinary ASA metabolite, salicyluric acid. LTA with arachidonic acid was used to test for ASA sensitivity. Escalating ASA dosages of 162 mg and 325 mg were investigated ex vivo for ASA dose personalization. Of the 64 atherosclerotic patients recruited, 8 patients (13%) were non-compliant with ASA. Of ASA compliant patients (n = 56), 9 patients (14%) were non-sensitive to their 81 mg ASA dosage. Personalizing ASA therapy in 81 mg ASA non-sensitive patients with escalating dosages of ASA demonstrated that 6 patients became sensitive to a dosage equivalent to 162 mg ASA and 3 patients became sensitive to a dosage equivalent to 325 mg ASA. We were able to personalize ASA dosage ex vivo in all ASA non-sensitive patients with escalating dosages of ASA within 1 h of testing.

Fatty Acid Binding Protein 4-A Circulating Protein Associated with Peripheral Arterial Disease in Diabetic Patients.

Diabetic patients with peripheral arterial disease (PAD) often suffer from poor clinical outcomes such as limb-loss. Fatty acid binding protein 4 (FABP4) is mainly expressed by adipocytes and is known to play a significant role in the development of atherosclerosis. In this study, we sought to investigate whether FABP4 is associated with PAD in patients with type 2 diabetes mellitus (DM). FABP4 plasma levels were studied in 119 diabetic patients with PAD (DM-PAD) and 49 diabetic patients without PAD (DM-noPAD) presenting to St. Michael's Hospital between October 2017 and September 2018. Levels of FABP4 in DM-PAD patients (23.34 ± 15.27 ng/mL) were found to be over two-fold higher than the levels in DM-noPAD patients (10.3 ± 7.59 ng/mL). Regression analysis demonstrated a significant association between FABP4 levels and DM-PAD after adjusting for age, sex, prior history of coronary arterial disease and white blood cells count (OR, 2.77; 95% CI, 1.81-4.31; p-value = 0.001). Relative to DM-noPAD controls, plasma FABP4 levels in DM-PAD patients were noted to be inversely correlated with the ankle brachial index (ABI; r= -0.374, p-value < 0.001). The diagnostic ability of FABP4 was investigated using receiver operator curves (ROC) and area under the curve (AUC) analysis. FABP4 had an AUC of 0.79, which improved to 0.86 after adjusting for age, sex and prior history of coronary arterial disease. This raises a possibility of utilizing FABP4 as a biomarker for diagnosing PAD in diabetic patients.

MicroRNA Profile of Patients with Chronic Limb-Threatening Ischemia.

Chronic limb-threatening ischemia (CLTI) results in devastating complications such as lower-limb amputations. In this study, a genome-wide plasma microRNAs (miRNA) sequencing was performed to identify miRNA(s) associated with CLTI. Blood samples were collected from early stage CLTI patients (ABI < 0.9) and non-PAD controls (ABI ≥ 0.9) for 3 experiments: discovery phase (n = 23), confirmatory phase (n = 52) and validation phase (n = 20). In the discovery phase, next generation sequencing (NGS) was used to identify miRNA circulating in the plasma CLTI (n = 13) patients, compared to non-PAD controls (n = 10). Two down-regulated miRNAs (miRNA-6843-3p and miRNA-6766-5p) and three upregulated miRNAs (miRNA-1827, miRNA-320 and miRNA-98-3p) were identified (≥2-fold change). In the confirmatory phase, these 5 deregulated miRNAs were further investigated in non-PAD (n = 21) and CTLI (n = 31) patients using qRT-PCR. Only miRNA-1827 was found to be significantly upregulated (≥3-fold, p-value < 0. 001) in the CLTI group. Lastly, to minimize the influence of confounding factors, miRNA-1827 plasma levels were validated in a third cohort of CLTI patients (n = 10) matched to non-PAD controls (n = 10). Our analysis demonstrated that miRNA-1827 expression was increased in the CLTI cohort (≥2-folds, p-value < 0.001). In summary, circulating miRNA-1827 is significantly elevated in patients with CLTI.

Altered coagulation profile in peripheral artery disease patients.

OBJECTIVE: Peripheral artery disease patients have been shown to be more susceptible to thrombotic events compared to non-peripheral artery disease patients. Therefore, the aim of this study was to investigate the coagulation profile in peripheral artery disease patients with chronic limb threatening ischemia, moderate peripheral artery disease patients with claudication, and non-peripheral artery disease controls. METHODS: Chronic limb threatening ischemia patients were matched to peripheral artery disease patients with claudication and non-peripheral artery disease controls in a 1:1:1 ratio. Each patient had their cytokines, markers of thrombin generation, coagulation factors, natural anti-coagulants, fibrinolysis, and endothelial injury markers assessed. RESULTS: Markers of thrombin activation, thrombin Fragments F1 + 2 (Frag 1 + 2), and thrombin-anti-thrombin complex were found to be significantly elevated in all peripheral artery disease and chronic limb threatening ischemia patients relative to non-peripheral artery disease controls. Similarly, relative to non-peripheral artery disease controls, inflammatory markers including C-reactive protein, soluble platelet factor 4, and neutrophil gelatinase-associated lipocalin were also found to be significantly upregulated in chronic limb threatening ischemia patients, but not in peripheral artery disease patients with claudication. Furthermore, our data demonstrated significant increases in markers of endothelial injury in chronic limb threatening ischemia patients relative to non-peripheral artery disease controls. Finally, decreases in natural anti-coagulants (protein C and protein S) and coagulation factors FIX, FXI, and FXII were also observed in chronic limb threatening ischemia patients when compared with non-peripheral artery disease controls. CONCLUSIONS: Our data suggest that in relation to non-peripheral artery disease controls, chronic limb threatening ischemia patients are more hypercoagulable. However, peripheral artery disease patients with claudication appear to have similar levels of circulating procoagulant markers as non-peripheral artery disease patients. This may explain the increased risk of thrombotic events observed in chronic limb threatening ischemia patients.

Fatty acid binding protein 3 is associated with peripheral arterial disease.

BACKGROUND: Peripheral arterial disease (PAD) affects more than 150 million people worldwide and is associated with high rates of lower extremity amputation, myocardial infarction, stroke and death. Fatty acid binding protein 3 (FABP3) is released into circulation in patients with skeletal muscle injury. In this pilot study, we investigated a possible association between PAD and blood levels of FABP3. METHODS: Blood samples were collected from patients with clinical symptoms and diagnostic findings indicative of PAD (PAD group; ankle-brachial index [ABI] <0.9; n = 75) and in those without clinical or diagnostic features of PAD (non-PAD group; ABI >0.9; n = 75) presenting to vascular surgery ambulatory clinics at St. Michael's Hospital. Plasma samples were analyzed by protein multiplex to quantify FABP3 levels. RESULTS: PAD patients were found to have higher blood levels of FABP3 compared to patients without PAD (mean 3.90 ± 1.69 vs 2.03 ± 0.78; P < .001). A subgroup analysis demonstrated that the FABP3 levels were increased by almost two-fold in patients with PAD, independent of coronary artery disease (P < .001) or diabetes mellitus status (P < .001). Moreover, a significant negative correlation between FABP3 and the ABI was observed in PAD and patients without PAD matched groups (r = -0.51; P = .001). Last, immunohistochemistry demonstrated elevated expressions of FABP3 within skeletal muscle obtained from patients with the most severe form of PAD, chronic limb-threatening ischemia, when compared with patients without PAD. CONCLUSIONS: Patients with PAD have elevated plasma levels of FABP3. An increasing severity of PAD is associated with higher FABP3 levels.