The prognostic capability of inflammatory proteins in predicting peripheral artery disease related adverse events

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

Valproic Acid Induces Endothelial-to-Mesenchymal Transition-Like Phenotypic Switching

Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, is a widely used anticonvulsant drug that is currently undergoing clinical evaluation for anticancer therapy due to its anti-angiogenic potential. Endothelial cells (ECs) can transition into mesenchymal cells and this form of EC plasticity is called endothelial-to-mesenchymal transition (EndMT), which is widely implicated in several pathologies including cancer and organ fibrosis. However, the effect of VPA on EC plasticity and EndMT remains completely unknown. We report herein that VPA-treatment significantly inhibits tube formation, migration, nitric oxide production, proliferation and migration in ECs. A microscopic evaluation revealed, and qPCR, immunofluorescence and immunoblotting data confirmed EndMT-like phenotypic switching as well as an increased expression of pro-fibrotic genes in VPA-treated ECs. Furthermore, our data confirmed important and regulatory role played by TGFβ-signaling in VPA-induced EndMT. Our qPCR array data performed for 84 endothelial genes further supported our findings and demonstrated 28 significantly and differentially regulated genes mainly implicated in angiogenesis, endothelial function, EndMT and fibrosis. We, for the first time report that VPA-treatment associated EndMT contributes to the VPA-associated loss of endothelial function. Our data also suggest that VPA based therapeutics may exacerbate endothelial dysfunction and EndMT-related phenotype in patients undergoing anticonvulsant or anticancer therapy, warranting further investigation

Inflammatory Biomarkers to Predict Major Adverse Cardiovascular Events in Patients with Carotid Artery Stenosis

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

A machine learning algorithm for peripheral artery disease prognosis using biomarker data

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

The Clinical Utility of D-Dimer and Prothrombin Fragment (F1+2) for Peripheral Artery Disease: A Prospective Study

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

Urinary Cystatin C Has Prognostic Value in Peripheral Artery Disease

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

Serum Metabolic Signatures of Chronic Limb-Threatening Ischemia in Patients with Peripheral Artery Disease

Peripheral artery disease (PAD) is characterized by the atherosclerotic narrowing of lower limb vessels, leading to ischemic muscle pain in older persons. Some patients experience progression to advanced chronic limb-threatening ischemia (CLTI) with poor long-term survivorship. Herein, we performed serum metabolomics to reveal the mechanisms of PAD pathophysiology that may improve its diagnosis and prognosis to CLTI complementary to the ankle–brachial index (ABI) and clinical presentations. Non-targeted metabolite profiling of serum was performed by multisegment injection–capillary electrophoresis–mass spectrometry (MSI–CE–MS) from age and sex-matched, non-diabetic, PAD participants who were recruited and clinically stratified based on the Rutherford classification into CLTI (n = 18) and intermittent claudication (IC, n = 20). Compared to the non-PAD controls (n = 20), PAD patients had lower serum concentrations of creatine, histidine, lysine, oxoproline, monomethylarginine, as well as higher circulating phenylacetylglutamine (p < 0.05). Importantly, CLTI cases exhibited higher serum concentrations of carnitine, creatinine, cystine and trimethylamine-N-oxide along with lower circulating fatty acids relative to well matched IC patients. Most serum metabolites associated with PAD progression were also correlated with ABI (r = ±0.24−0.59, p < 0.05), whereas the ratio of stearic acid to carnitine, and arginine to propionylcarnitine differentiated CLTI from IC with good accuracy (AUC = 0.87, p = 4.0 × 10−5). This work provides new biochemical insights into PAD progression for the early detection and surveillance of high-risk patients who may require peripheral vascular intervention to prevent amputation and premature death

Perceived Challenges to Routine Uptake of the Ankle Brachial Index within Primary Care Practice

(1) Introduction: The ankle-brachial index (ABI) is the most widely used method of diagnosing peripheral arterial disease (PAD). However, the uptake of ABIs has been reported to be low in primary care settings across different various healthcare settings; however, this is yet to be investigated within the Canadian context. (2) Objective: Therefore, we sought to assess the rates of ABI usage as well as perceived barriers among primary care practitioners (PCPs) in Toronto, Canada. (3) Methods: A modified questionnaire was electronically sent to 257 PCPs in the Greater Toronto Area (GTA). Questions pertained to frequency, feasibility, utility, and barriers associated with ABI usage in clinical practice. Responses were collected and tallied. (4) Results: A total of 52 PCPs completed the questionnaire. 79% of PCPs did not routinely perform ABIs within their clinical practice, and 56% deemed ABI usage as unfeasible. Constraints in time and staff personnel, as well as complexity of ABI result interpretation, were cited as the major perceived barriers to ABI usage. The overwhelming majority of PCPs viewed alternative forms of diagnosis, such as a blood test for PAD, as being preferable to ABI, as such an approach would enhance diagnostic simplicity and efficiency. (5) Conclusion: ABI usage rates are poor within primary care practices in Toronto, Canada. Alternative approaches for diagnosing PAD may result in greater adoption rates among PCPs and therefore improve the identification of patients with PAD