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Soluble interleukin 1 receptor-like 1 (ST2) is a circulating protein demonstrated to be associated with cardiovascular diseases; however, it has not been studied as a biomarker for peripheral artery disease (PAD). Using a prospectively recruited cohort of 476 patients (312 with PAD and 164 without PAD), we conducted a prognostic study of PAD using clinical/biomarker data. Plasma concentrations of three circulating proteins [ST2, cytokine-responsive gene-2 (CRG-2), vascular endothelial growth factor (VEGF)] were measured at baseline and the cohort was followed for 2 years. The outcome of interest was a 2-year major adverse limb event (MALE; composite of major amputation, vascular intervention, or acute limb ischemia). Using 10-fold cross-validation, a random forest model was trained using clinical characteristics and plasma ST2 levels. The primary model evaluation metric was the F1 score. Out of the three circulating proteins analyzed, ST2 was the only one that was statistically significantly higher in individuals with PAD compared to patients without PAD (mean concentration in plasma of 9.57 [SD 5.86] vs. 11.39 [SD 6.43] pg/mL, p < 0.001). Over a 2-year period, 28 (9%) patients with PAD experienced MALE. Our predictive model, incorporating clinical features and plasma ST2 levels, achieved an F1 score of 0.713 for forecasting 2-year MALE outcomes. Patients identified as high-risk by this model showed a significantly increased likelihood of developing MALE (HR 1.06, 95% CI 1.02–1.13, p = 0.003). By combining clinical characteristics and plasma ST2 levels, our proposed predictive model offers accurate risk assessment for 2-year MALE in PAD patients. This algorithm supports risk stratification in PAD, guiding clinical decisions regarding further vascular evaluation, specialist referrals, and appropriate medical or surgical interventions, thereby potentially enhancing patient outcomes.

Cytokine-induced neutrophil chemoattractant 1 (CINC-1), a cluster of differentiation 95 (CD95), fractalkine, and T-cell immunoglobulin and mucin domain 1 (TIM-1) are circulating proteins known to be involved in inflammation. While their roles have been studied in neurological conditions and cardiovascular diseases, their potential as peripheral artery disease (PAD) biomarkers remain unexplored. We conducted a cross-sectional diagnostic study using data from 476 recruited patients (164 without PAD and 312 with PAD). Plasma levels of CINC-1, CD95, fractalkine, and TIM-1 were measured at baseline. A PAD diagnosis was established at recruitment based on clinical exams and investigations, defined as an ankle-brachial index < 0.9 or toe-brachial index < 0.67 with absent/diminished pedal pulses. Using 10-fold cross-validation, we trained a random forest algorithm, incorporating clinical characteristics and biomarkers that showed differential expression in PAD versus non-PAD patients to predict a PAD diagnosis. Among the proteins tested, CINC-1, CD95, and fractalkine were elevated in PAD vs. non-PAD patients, forming a 3-biomarker panel. Our predictive model achieved an AUROC of 0.85 for a PAD diagnosis using clinical features and this 3-biomarker panel. By combining the clinical characteristics with these biomarkers, we developed an accurate predictive model for a PAD diagnosis. This algorithm can assist in PAD screening, risk stratification, and guiding clinical decisions regarding further vascular assessment, referrals, and medical/surgical management to potentially improve patient outcomes.

Summary: Peripheral artery disease (PAD) biomarkers have been studied in isolation; however, an algorithm that considers a protein panel to inform PAD prognosis may improve predictive accuracy. Biomarker-based prediction models were developed and evaluated using a model development (n = 270) and prospective validation cohort (n = 277). Plasma concentrations of 37 proteins were measured at baseline and the patients were followed for 2 years. The primary outcome was 2-year major adverse limb event (MALE; composite of vascular intervention or major amputation). Of the 37 proteins tested, 6 were differentially expressed in patients with vs. without PAD (ADAMTS13, ICAM-1, ANGPTL3, Alpha 1-microglobulin, GDF15, and endostatin). Using 10-fold cross-validation, we developed a random forest machine learning model that accurately predicts 2-year MALE in a prospective validation cohort of PAD patients using a 6-protein panel (AUROC 0.84). This algorithm can support PAD risk stratification, informing clinical decisions on further vascular evaluation and management.

Background and Objectives: Inflammatory proteins and their prognostic value in patients with carotid artery stenosis (CAS) have not been adequately studied. Herein, we identified CAS-specific biomarkers from a large pool of inflammatory proteins and assessed the ability of these biomarkers to predict adverse events in individuals with CAS. Materials and Methods: Samples of blood were prospectively obtained from 336 individuals (290 with CAS and 46 without CAS). Plasma concentrations of 29 inflammatory proteins were determined at recruitment, and the patients were followed for 24 months. The outcome of interest was a major adverse cardiovascular event (MACE; composite of stroke, myocardial infarction, or death). The differences in plasma protein concentrations between patients with vs. without a 2-year MACE were determined using the independent t-test or Mann–Whitney U test to identify CAS-specific prognostic biomarkers. Kaplan–Meier and Cox proportional hazards analyses with adjustment for baseline demographic and clinical characteristics were performed to assess the prognostic value of differentially expressed inflammatory proteins in predicting a 2-year MACE in patients with CAS. Results: The mean age of the cohort was 68.8 (SD 10.2) years and 39% were female. The plasma concentrations of two inflammatory proteins were significantly higher in individuals with a 2-year MACE relative to those without a 2-year MACE: IL-6 (5.07 (SD 4.66) vs. 3.36 (SD 4.04) pg/mL, p = 0.03) and CD163 (233.825 (SD 230.306) vs. 159.673 (SD 175.669) pg/mL, p = 0.033). Over a follow-up period of 2 years, individuals with elevated levels of IL-6 were more likely to develop MACE (HR 1.269 (95% CI 1.122–1.639), p = 0.042). Similarly, over a 2-year period, patients with high levels of CD163 were more likely to develop MACE (HR 1.413 (95% CI 1.022–1.954), p = 0.036). Conclusions: The plasma levels of inflammatory proteins IL-6 and CD163 are independently associated with adverse outcomes in individuals with CAS. These CAS-specific prognostic biomarkers may assist in the risk stratification of patients at an elevated risk of a MACE and subsequently guide further vascular evaluation, specialist referrals, and aggressive medical/surgical management, thereby improving outcomes for patients with CAS.

Abstract Blood-based adjunctive measures that can reliably predict abdominal aortic aneurysm (AAA)-related complications hold promise for mitigating the AAA disease burden. In this pilot study, we sought to evaluate the prognostic performance of complement factors in predicting AAA-related clinical outcomes. We recruited consecutive AAA patients (n = 75) and non-AAA patients (n = 75) presenting to St. Michael’s Hospital. Plasma levels of complement proteins were assessed at baseline, as well as prospectively measured regularly over a period of 2 years. The primary outcome was the incidence of rapidly progressing AAA (i.e. aortic expansion), defined as change in AAA diameter by either 0.5 cm in 6 months, or 1 cm in 12 months. Secondary outcomes included incidence of major adverse aortic events (MAAE) and major adverse cardiovascular events (MACE). All study outcomes (AAA diameter, MACE and MAAE) were obtained during follow-up. Multivariable adjusted Cox regression analyses were performed to assess the prognostic value of plasma C2 levels in patients with AAA regarding rapid aortic expansion and MAAE and MACE. Event-free survival rates of both groups were also compared. Compared to non-AAA patients, patients with AAA demonstrated significantly higher plasma concentrations of C1q, C4, Factor B, Factor H and Factor D, and significantly lower plasma concentrations of C2, C3, and C4b (p = 0.001). After a median of 24 months from initial baseline measurements, C2 was determined as the strongest predictor of rapid aortic expansion (HR 0.10, p = 0.040), MAAE (HR 0.09, p = 0.001) and MACE (HR 0.14, p = 0.011). Based on the data from the survival analysis, higher levels of C2 at admission in patients with AAA predicted greater risk for rapid aortic expansion and MAAE (not MACE). Plasma C2 has the potential to be a biomarker for predicting rapid aortic expansion, MAAE, and the eventual need for an aortic intervention in AAA patients.

Background: Angiogenesis plays an important role in peripheral artery disease (PAD) and angiogenesis-related proteins may act as prognostic biomarkers. This study assesses the potential for angiogenesis-related proteins to predict adverse events associated with PAD. Methods: This was a case-control study. Patients with PAD (n = 250) and without PAD (n = 125) provided blood samples and were followed prospectively for three years. Concentrations of 17 angiogenesis-related proteins were measured in plasma. The incidence of major adverse limb event (MALE), defined as a composite of major amputation or vascular intervention, was the primary outcome. Worsening PAD status, defined as a drop in ankle brachial index ≥ 0.15, was the secondary outcome. Multivariable regression adjusted for baseline characteristics was conducted to determine the prognostication value of angiogenesis-related proteins in predicting MALE. Findings: Relative to patients without PAD, 8 proteins related to angiogenesis were expressed differentially in PAD patients. Worsening PAD status and MALE were observed in 52 (14%) and 83 (22%) patients, respectively. Hepatocyte growth factor (HGF) was the most reliable predictor of MALE (adjusted HR 0.79, 95% CI 0.15–0.86). Compared to individuals with high HGF, patients with low HGF had a decreased three-year freedom from MALE [66% vs 88%, p = 0.001], major amputation [93% vs 98%, p = 0.023], vascular intervention [68% vs 88%, p = 0.001], and worsening PAD status [81% vs 91%, p = 0.006]. Interpretation: Measuring plasma levels of HGF in individuals with PAD can assist in identifying patients at elevated risk of adverse events related to PAD who may benefit from additional evaluation or treatment.

Abstract Neutrophil gelatinase-associated lipocalin (NGAL) is expressed in atherosclerotic plaques and implicated in the development of cardiovascular diseases. Peripheral arterial disease (PAD) is an atherosclerotic disease that often results in major cardiovascular events. This study aimed to prospectively examine the potential of urine NGAL (uNGAL) in predicting worsening PAD status and major adverse limb events (MALE). Baseline urine NGAL (uNGAL) and urine creatinine (uCr) concentrations were measured in PAD (n = 121) and non-PAD (n = 77) patients. Levels of uNGAL were normalized for urine creatinine (uNGAL/uCr). Outcomes included worsening PAD status, which was defined as a drop in ankle brachial index (ABI) > 0.15, and major adverse limb events (MALE), which was defined as a need for surgical revascularization or amputations. PAD patients had 2.30-fold higher levels of uNGAL/uCr [median (IQR) 31.8 (17.0–62.5) μg/g] in comparison to non-PAD patients [median (IQR) 73.3 (37.5–154.7) μg/g] (P = 0.011). Multivariate cox analysis showed that uNGAL/uCr levels were independently associated with predicting worsening PAD status and MALE outcomes. Cumulative survival analysis, over follow up period, demonstrated a direct correlation between elevated uNGAL/uCr levels and PAD disease progression and MALE outcomes. These data demonstrate an association between elevated uNGAL/uCr levels and worsening PAD disease status and MALE outcomes, indicating its potential for risk-stratification of PAD patients.

Summary: Background: Patients with peripheral arterial disease (PAD) often remain undiagnosed and therefore suboptimally managed. Here, we investigated the diagnostic and prognostic potential of fatty acid binding protein 3 (FABP3) in patients with PAD. Methods: In the discovery phase, 374 PAD and 184 non-PAD patients were recruited from vascular surgery ambulatory clinics at St. Michael's Hospital (Toronto, Ontario, Canada) between October 4, 2017 to October 29, 2018. The diagnostic ability of baseline FABP3 level was investigated through receiver operator characteristic (ROC) curves to determine two cutoff points: 1) an exclusionary “rule out” cutoff point, and 2) a confirmatory “rule in” cutoff point. Next, these cutoff points were confirmed in the external validation phase using a separate cohort of 312 patients (180 PAD and 132 non-PAD) recruited from ambulatory vascular surgery clinics at St. Michael's Hospital (Canada) between November 6, 2018–July 30, 2019. Cox regression analyses were used to explore the independent association between FABP3 and major adverse limb events (MALE – defined as need for arterial revascularization or major amputation) and decrease in ankle-brachial index (ABI -defined as drop ≥0.15) during 3 years of follow-up. Findings: In the discovery phase, FABP3 levels were significantly elevated in patients with PAD compared to non-PAD patients. ROC analysis demonstrated that FABP3 had an AUC of 0.83 (95% CI: 0.81–0.86, p-value < 0.001). FABP3 exclusionary cutoff was 3.55 ng/ml (sensitivity = 43%; specificity = 95%) – values that were confirmed in the external validation phase. Cox regression analysis demonstrated FABP3 to be an independent predictor of increase in MALE [HR = 1.14 (1.03–1.29); p-value = 0.010] and worsening PAD status (drop in ABI >0.15 [HR = 1.11 (1.02–1.19); p-value = 0.009]). Interpretation: Our findings suggested that FABP3 levels can be used as both a diagnostic and prognostic biomarker for PAD, and may facilitate risk stratification in select individuals for purposes of vascular evaluation or intensive medical management. Funding: Funding for this study was provided by the Bill and Vicky Blair Foundation.

BackgroundLevels of inflammatory proteins and their prognostic potential have been inadequately studied in patients with peripheral artery disease (PAD). In this study, we quantified and assessed the ability of inflammatory proteins in predicting PAD-related adverse events.MethodsIn this prospective case-control study, blood samples were collected from patients without PAD (n = 202) and patients with PAD (n = 275). The PAD cohort was stratified by disease severity based on ankle brachial index (ABI): mild (n = 49), moderate (n = 164), and severe (n = 62). Patients were followed for 2 years. Plasma concentrations of 5 inflammatory proteins were measured: Alpha-2-Macroglobulin (A2M), Fetuin A, Alpha-1-Acid Glycoprotein (AGP), Serum Amyloid P component (SAP), and Adipsin. The primary outcome of our study was major adverse limb event (MALE), defined as the need for vascular intervention (open or endovascular revascularization) or major amputation. The secondary outcome was worsening PAD status, defined as a drop in ABI greater than or equal to 0.15 over the study period. Multivariable logistic regression was performed to assess the prognostic value of inflammatory proteins in predicting MALE, adjusting for confounding variables.ResultsCompared to patients without PAD, three inflammatory proteins were differentially expressed in patients with PAD (AGP, Fetuin A, and SAP). The primary outcome (MALE) and secondary outcome (worsening PAD) status were noted in 69 (25%) and 60 (22%) patients, respectively. PAD-related adverse events occurred more frequently in severe PAD patients. Based on our data, the inflammatory protein AGP was the most reliable predictor of primary and secondary outcomes. On multivariable analysis, there was a significant association between AGP and MALE in all PAD disease states [mild: adjusted HR 1.13 (95% CI 1.05–1.47), moderate: adjusted HR 1.23 (95% CI 1.16–1.73), severe: adjusted HR 1.37 (95% CI 1.25–1.85)]. High levels of AGP were associated with lower 2-year MALE-free survival in all PAD disease states [mild (64% vs. 100%, p = 0.02), moderate (64% vs. 85%, p = 0.02), severe (55% vs. 88%, p = 0.02), all PAD (62% vs. 88%, p = 0.01)].ConclusionLevels of inflammatory protein AGP may help in risk stratifying PAD patients at high risk of MALE and worsening PAD status and subsequently facilitate further vascular evaluation and initiation of aggressive medical/surgical management.

Abstract Plasma levels of fatty acid binding protein 3 (pFABP3) are elevated in patients with peripheral artery disease (PAD). Since the kidney filters FABP3 from circulation, we investigated whether urinary fatty acid binding protein 3 (uFABP3) is associated with PAD, and also explored its potential as a diagnostic biomarker for this disease state. A total of 130 patients were recruited from outpatient clinics at St. Michael’s Hospital, comprising of 65 patients with PAD and 65 patients without PAD (non-PAD). Levels of uFABP3 normalized for urine creatinine (uFABP3/uCr) were 1.7-folds higher in patients with PAD [median (IQR) 4.41 (2.79–8.08)] compared with non-PAD controls [median (IQR) 2.49 (1.78–3.12), p-value = 0.001]. Subgroup analysis demonstrated no significant effect of cardiovascular risk factors (age, sex, hypertension, hypercholesteremia, diabetes and smoking) on uFABP3/uCr in both PAD and non-PAD patients. Spearmen correlation studies demonstrated a significant negative correlation between uFABP3/uCr and ABI (ρ = − 0.436; p-value = 0.001). Regression analysis demonstrated that uFABP3/Cr levels were associated with PAD independently of age, sex, hypercholesterolemia, smoking, prior history of coronary arterial disease and Estimated Glomerular Filtration rate (eGFR) [odds ratio: 2.34 (95% confidence interval: 1.47–3.75) p-value

BackgroundDespite its significant association with limb loss and death, peripheral artery disease (PAD) remains underdiagnosed and undertreated. The current accepted gold-standard for PAD screening, the ankle brachial index (ABI), is limited by operator dependence, erroneous interpretation, and unreliability in patients with diabetes. Fatty acid binding protein 3 (FABP3) is an intracellular protein that becomes released into circulation and excreted into urine following skeletal muscle injury. We examined the prognostic ability of urinary FABP3 (uFABP3) in predicting adverse PAD-related events.MethodsIn this prospective case-control study, urine samples were collected from patients with PAD (n = 142) and without PAD (n = 72). The cohort was followed for 2 years. uFABP3 was normalized to urinary creatinine (uCr) (uFABP3/uCr). The primary outcome was major adverse limb event (MALE; composite of vascular intervention [open or endovascular] or major limb amputation). The secondary outcome was worsening PAD status (drop in ABI≥0.15). Cox regression analyses with multivariable adjustment for baseline demographic and clinical variables were performed to assess the prognostic value of uFABP3/uCr with regards to predicting MALE and worsening PAD status.ResultsPatients with PAD had significantly higher median [IQR] uFABP3/uCr levels (3.46 [2.45–6.90] vs. 2.61 [1.98–4.62], p = 0.001). MALE and worsening PAD status were observed in 21 (10%) and 28 (14%) patients, respectively. uFABP3/uCr predicted MALE and worsening PAD status with adjusted hazard ratios (HR) of 1.28 (1.16–1.41, p = 0.001) and 1.16 (1.02–1.27, p = 0.021), respectively. Patients with high uFABP3/uCr had a lower 2-year freedom from MALE (86 vs. 96%, p = 0.047) and worsening PAD status (78 vs. 99%, p = 0.001). There was good discriminatory ability for uFABP3/uCr in predicting the primary outcome of MALE, with an area under the receiver operating characteristics curve (AUROC) of 0.78.ConclusionsMeasuring uFABP3/uCr levels in patients with PAD can help identify those at high risk of adverse PAD-related events. This study highlights the prognostic value of uFABP3 in risk-stratifying individuals for further diagnostic vascular evaluation or aggressive medical management.

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) 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. : Clinical Relevance: There is a pressing need for a simple, readily accessible, blood-based biomarker for PAD. In this study, we found elevated levels of FABP3 in patients with PAD. This increase in FABP3 was irrespective of history of coronary artery disease or diabetes. Furthermore, our data suggest that an increasing severity of PAD is associated with higher FABP3 levels. Subsequently, FABP3 may be a potential diagnostic biomarker for PAD. However, further studies are needed to confirm the capability of FABP3 to serve as a valid and reliable biomarker for PAD.

Abstract 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

N-terminal pro B-type natriuretic peptide (NT-proBNP), a cardiac disease biomarker, has been demonstrated to be a strong independent predictor of cardiovascular events in patients without heart failure. Patients with peripheral arterial disease (PAD) are at high risk of cardiovascular events and death. In this study, we investigated levels of NT-proBNP in patients with PAD compared to non-PAD controls. A total of 355 patients were recruited from outpatient clinics at a tertiary care hospital network. Plasma NT-proBNP levels were quantified using protein multiplex. There were 279 patients with both clinical and diagnostic features of PAD and 76 control patients without PAD (non-PAD cohort). Compared with non-PAD patients, median (IQR) NT-proBNP levels in PAD patients were significantly higher (225 ng/L (120-363) vs 285 ng/L (188-425), p- value = 0.001, respectively). Regression analysis demonstrated that NT-proBNP remained significantly higher in patients with PAD relative to non-PAD despite adjusting for age, sex, hypercholesterolemia, smoking and hypertension [odds ratio = 1.28 (1.07-1.54), p-value

Despite its association with adverse outcomes, peripheral artery disease (PAD) remains undertreated. Cystatin C is elevated in patients with renal disease and may be a marker of cardiovascular disease. We examined the prognostic ability of urinary Cystatin C (uCystatinC) in predicting adverse PAD-related events. In this prospective case-control study, urine samples were collected from patients with PAD (n = 121) and without PAD (n = 77). The cohort was followed for 2 years. uCystatinC was normalized to urinary creatinine (uCr) (uCystatinC/uCr; μg/g). The primary outcome was major adverse limb event (MALE; composite of vascular intervention (open or endovascular) or major limb amputation). The secondary outcome was worsening PAD status (drop in ABI ≥ 0.15). Multivariable Cox regression and Kaplan–Meier analyses were performed to assess the prognostic value of uCystatinC/uCr with regards to predicting MALE and worsening PAD status. Our analysis demonstrated that patients with PAD had significantly higher median [IQR] uCystatinC/uCr levels (24.9 μg/g [14.2–32.9] vs. 20.9 μg/g [11.1–27.8], p = 0.018). Worsening PAD status and MALE were observed in 39 (20%) and 34 (17%) patients, respectively. uCystatinC/uCr predicted worsening PAD status with a hazard ratio (HR) of 1.78 (95% CI 1.12–2.83, p = 0.015), which persisted after controlling for baseline demographic and clinical characteristics (adjusted HR 1.79 [95% CI 1.11–2.87], p = 0.017). Patients with high uCystatinC/uCr had a lower 2-year freedom from MALE (77% vs. 89%, p = 0.025) and worsening PAD status (63% vs. 87%, p = 0.001). Based on these data, higher uCystatinC/uCr levels are associated with adverse PAD-related events and have prognostic value in risk-stratifying individuals for further diagnostic vascular evaluation or aggressive medical management.

D-dimer and prothrombin fragment (F1+2) levels are elevated in patients with peripheral artery disease (PAD). We examined their prognostic potential in predicting decreasing ABI and major adverse limb events (MALE). A total of 206 patients were recruited from St. Michael’s Hospital and followed for two years. Baseline plasma concentrations of D-dimer and F1+2 were recorded. Pearson’s correlation was used to assess the correlation between the biomarkers and ABI at year 2. During follow-up, multivariable Cox proportional hazard analysis was performed to investigate their role in predicting decreasing ABI (defined as change in ABI > −0.15) and MALE (defined as the need for arterial intervention or major limb amputation). Cumulative survival was assessed using Kaplan–Meier analysis. Baseline D-dimer and F1+2 levels were elevated in PAD patients (median (IQR) 1.34 (0.80–2.20) for D-dimer and 3.60 (2.30–4.74) for F1+2; p = 0.001) compared to non-PAD controls (median (IQR) 0.69 (0.29–1.20) for D-dimer and 1.84 (1.17–3.09) for F1+2; p = 0.001). Both markers were negatively correlated with ABI at year 2 (r = −0.231 for D-dimer, r = −0.49 for F1+2; p = 0.001). Cox analysis demonstrated F1+2 and D-dimer to be independent predictors of PAD status (HR = 1.27, 95% CI = 1.15–1.54; p = 0.013 for D-dimer and HR = 1.28, 95% CI = 1.14–1.58; p = 0.019 for F1+2). Elevated baseline concentrations of D-dimer and F1+2 were associated with high incidence of decreasing ABI and 1- and 2-year event-free survival (62% and 86%, respectively). Combined analysis of D-dimer and F1+2 provides important prognostic information that facilitates risk stratification for future disease progression and MALE outcomes in patients with PAD.

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 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.

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.