Angiogenesis-related proteins as biomarkers for peripheral artery disease.

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.

Fatty acid binding protein 4 has prognostic value in peripheral artery disease.

OBJECTIVE: Peripheral artery disease (PAD) remains undertreated, despite its association with major amputation and mortality. This is partly due to a lack of available disease biomarkers. The intracellular protein fatty acid binding protein 4 (FABP4) is implicated in diabetes, obesity, and metabolic syndrome. Given that these risk factors are strong contributors to vascular disease, we assessed the prognostic ability of FABP4 in predicting PAD-related adverse limb events. METHODS: This was a prospective case-control study with 3 years of follow-up. Baseline serum FABP4 concentrations were measured in patients with PAD (n = 569) and without PAD (n = 279). The primary outcome was major adverse limb event (MALE; defined as a composite of vascular intervention or major amputation). The secondary outcome was worsening PAD status (drop in ankle-brachial index ≥0.15). Kaplan-Meier and Cox proportional hazards analyses adjusted for baseline characteristics were conducted to assess the ability of FABP4 to predict MALE and worsening PAD status. RESULTS: Patients with PAD were older and more likely to have cardiovascular risk factors compared with those without PAD. Over the study period, MALE and worsening PAD status occurred in 162 (19%) and 92 (11%) patients, respectively. Higher FABP4 levels were significantly associated with 3-year MALE (unadjusted hazard ratio [HR], 1.19; 95% confidence interval [CI], 1.04-1.27; adjusted HR, 1.18; 95% CI, 1.03-1.27; P = .022) and worsening PAD status (unadjusted HR, 1.18; 95% CI, 1.13-1.31; adjusted HR, 1.17; 95% CI, 1.12-1.28; P < .001). Three-year Kaplan-Meier survival analysis demonstrated that patients with high FABP4 levels had a decreased freedom from MALE (75% vs 88%; log rank = 22.6; P < .001), vascular intervention (77% vs 89%; log rank = 20.8; P < .001), and worsening PAD status (87% vs 91%; log rank = 6.16; P = .013). CONCLUSIONS: Individuals with higher serum concentrations of FABP4 are more likely to develop PAD-related adverse limb events. FABP4 has prognostic value in risk-stratifying patients for further vascular evaluation and management.

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

Background: Levels 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. Methods: In 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. Results: Compared 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)]. Conclusion: Levels 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.

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.

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.

Urinary Fatty Acid Binding Protein 3 Has Prognostic Value in Peripheral Artery Disease.

Background: Despite 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. Methods: In 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. Results: Patients 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. Conclusions: Measuring 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.

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.

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%.

Microfluidic Protein Imaging Platform: Study of Tau Protein Aggregation and Alzheimer's Drug Response.

Tau protein aggregation is identified as one of the key phenomena associated with the onset and progression of Alzheimer's disease. In the present study, we performed on-chip confocal imaging of tau protein aggregation and tau-drug interactions using a spiral-shaped passive micromixing platform. Numerical simulations and experiments were performed in order to validate the performance of the micromixer design. We performed molecular modeling of adenosine triphosphate (ATP)-induced tau aggregation in order to successfully validate the concept of helical tau filament formation. Tau aggregation and native tau restoration were realized using an immunofluorescence antibody assay. The dose-response behavior of an Alzheimer's drug, methylthioninium chloride (MTC), was monitored on-chip for defining the optimum concentration of the drug. The proposed device was tested for reliability and repeatability of on-chip tau imaging. The amount of the tau protein sample used in our experiments was significantly less than the usage for conventional techniques, and the whole protein-drug assay was realized in less than two hours. We identified that intensity-based tau imaging could be used to study Alzheimer's drug response. In addition, it was demonstrated that cell-free, microfluidic tau protein assays could be used as potential on-chip drug evaluation tools for Alzheimer's disease.

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.

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 TGFß-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.

The celecoxib derivatives AR-12 and AR-14 induce autophagy and clear prion-infected cells from prions.

Prion diseases are fatal infectious neurodegenerative disorders that affect both humans and animals. The autocatalytic conversion of the cellular prion protein (PrPC) into the pathologic isoform PrPSc is a key feature in prion pathogenesis. AR-12 is an IND-approved derivative of celecoxib that demonstrated preclinical activity against several microbial diseases. Recently, AR-12 has been shown to facilitate clearance of misfolded proteins. The latter proposes AR-12 to be a potential therapeutic agent for neurodegenerative disorders. In this study, we investigated the role of AR-12 and its derivatives in controlling prion infection. We tested AR-12 in prion infected neuronal and non-neuronal cell lines. Immunoblotting and confocal microscopy results showed that AR-12 and its analogue AR-14 reduced PrPSc levels after only 72 hours of treatment. Furthermore, infected cells were cured of PrPSc after exposure of AR-12 or AR-14 for only two weeks. We partially attribute the influence of the AR compounds on prion propagation to autophagy stimulation, in line with our previous findings that drug-induced stimulation of autophagy has anti-prion effects in vitro and in vivo. Taken together, this study demonstrates that AR-12 and the AR-14 analogue are potential new therapeutic agents for prion diseases and possibly protein misfolding disorders involving prion-like mechanisms.

Solvent-Free Green and Efficient One-Pot Synthesis of Dihydropyrano[3,2-c]chromene Derivatives.

A rapid, clean, and highly efficient method for synthesis of dihydropyrano[3,2-c]chromene derivatives by one-pot, three-component condensation of aromatic aldehydes, malononitrile, and 4-hydroxycoumarin using DABCO as catalyst in solvent-free neat conditions is described. The present method has the advantages of mild reaction conditions, short reaction times, easy isolation of products, and excellent yields.

DABCO Catalyzed Synthesis of Xanthene Derivatives in Aqueous Media.

The reaction of 5,5-dimethylcyclohexane-1,3-dione with various heteroarylaldehydes afforded the corresponding heteroaryl substituted xanthene derivatives 1(a-f). The reaction proceeds via the initial Knoevenagel, subsequent Michael, and final heterocyclization reactions using 1,4-diazabicyclo[2.2.2]octane (DABCO) as a catalyst in aqueous media. The synthesized heteroaryl substituted xanthenes 1(a-f) reacted with malononitrile to obtain different alkylidenes 2(a-f). Short reaction time, environmentally friendly procedure, avoiding of cumbersome apparatus, and excellent yields are the main advantages of this procedure which makes it more economic than the other conventional methods.

Microwave-assisted synthesis and biological evaluation of dihydropyrimidinone derivatives as anti-inflammatory, antibacterial, and antifungal agents.

A simple protocol for the efficient preparation of aryl and heteroaryl substituted dihydropyrimidinone has been achieved via initial Knoevenagel, subsequent addition, and final cyclization of aldehyde, ethylcyanoacetate, and guanidine nitrate in the presence of piperidine as a catalyst in solvent-free under microwave irradiation. The synthesized compounds showed a good anti-inflammatory, antibacterial, and antifungal activity.

An Efficient One-Pot Green Protocol for the Synthesis of 5-Unsubstituted 3,4-Dihydropyrimidin-2(1H)-Ones Using Recyclable Amberlyst 15 DRY as a Heterogeneous Catalyst via Three-Component Biginelli-Like Reaction.

An environmentally benign green protocol for the synthesis of 5-unsubstituted 3,4-dihydropyrimidin-2(1H)-ones using Amberlyst 15 DRY as a recyclable catalyst has been developed. The use of resinous, nontoxic, thermally stable, and inexpensive Amberlyst 15 DRY, as a recyclable heterogeneous catalyst, makes the process simple with negligible chemical waste. Among the various solid acid catalysts Amberlyst 15 DRY was found to be the most efficient catalyst with regard to reaction time, yield, and ease of work-up procedure.