Association of lower vitamin D levels with inflammation and leucocytes parameters in patients with and without diabetes mellitus undergoing coronary angiography.

BACKGROUND: Diabetes mellitus has been associated with a chronic low-grade inflammation and a higher risk of cardiovascular and infectious disease, that could be prevented by the effects of vitamin D. We aimed at evaluating the impact of vitamin D levels on the biomarkers of acute-phase response, inflammation and glucose metabolism in a large cohort of diabetic patients with cardiovascular disease. MATERIALS AND METHODS: Consecutive patients undergoing coronary angiography were included. Diabetes mellitus was defined as previous diagnosis, specific treatment administration (oral drug or insulin), fasting glycaemia >6.99 mmol/L or HbA1c >48 mmol/L. Glucose parameters, white blood cells, Neutrophil-to-Lymphocyte Ratio (NLR), Monocyte-to-Lymphocyte Ratio (MLR), C-reactive protein (CRP) and vitamin D were measured at admission. Vitamin D levels were measured by chemiluminescence immunoassay kit LIAISON® Vitamin D assay (Diasorin Inc). RESULTS: We included 1472 diabetic patients and 2499 non-diabetic patients that were divided according to vitamin D tertiles. Among diabetic patients, lower levels of vitamin D were associated with female gender (P = .02), obesity (P = .004), active smoking and acute presentation (P < .001) and with a more atherogenic metabolic profile. The levels of white blood cells, leucocytes subfamilies, and inflammatory parameters significantly correlated with vitamin D levels in both patients with and without diabetes (diabetic: P = .012 for WBC, P = .004 for NLR and P < .001 for MLR and C-reactive protein, non-diabetic: P < .001 for WBC; NLR, MLR and C-reactive protein, respectively). Among diabetic patients, results were confirmed at multivariate analysis with no significant interaction according to glycaemic control. CONCLUSION: The present study demonstrates that, among patients with cardiovascular disease, vitamin D deficiency is associated with metabolic dysregulation and with an elevation of cellular and humoural inflammatory parameters, especially among diabetics, although not being dependent from glycaemic control.

Low hemoglobin predicts high-platelet reactivity and major cardiovascular ischemic events at long-term follow-up among ACS patients receiving dual antiplatelet therapy with ticagrelor.

BACKGROUND: Reduced levels of hemoglobin (Hb) represent an established marker of impaired outcomes and increased cardiovascular risk in patients with coronary artery disease, challenging the management of dual antiplatelet therapy (DAPT). However, while anemia has emerged as an independent predictor of suboptimal platelet inhibition in patients receiving clopidogrel, no study has so far evaluated the impact of Hb levels on high-on treatment platelet reactivity (HRPR) with ticagrelor and their prognostic consequences, that were the aim of the present study. METHODS: Patients on DAPT with ASA + Ticagrelor (90 mg/twice a day) after percutaneous coronary revascularization for ACS were scheduled for platelet function assessment 30-90 days post-discharge. Aggregation tests were performed by multiple electrode aggregometry. Suboptimal platelet inhibition (HRPR-high residual platelet reactivity was defined if above the lower limit of normality (417 AU*min). The primary study endpoint was defined as the occurrence of major cardiovascular events (a composite of cardiovascular death, recurrent acute coronary syndrome [MI], target vessel revascularization) at longest available follow-up. RESULTS: We included 397 patients that were divided according to tertiles values of Hb (< 12.7, 12-7-14.09, ≥14.1 g/dl). Patients with lower Hb were older and displayed a more severe cardiovascular risk profile. Mean levels of platelet reactivity were enhanced in patients with lower Hb after stimulation with TRAP peptide (TRAP test, p = .03) and ADP (p = .02). Elevated platelet reactivity (HRPR) on Ticagrelor was more frequent among patients with reduced Hb (16.4% vs. 12% vs. 5.4%, p = .005, adjusted OR [95%CI] = 1.71[0.996;3.01], p = .056). At a mean follow-up of 820.9 ± 553.4 days, 21.4% of the patients experienced the primary composite endpoint, with a higher rate of events in patients with lower Hb (27.6% vs. 22.6% vs. 13.5%, p = .006, adjusted HR [95%CI] = 1.51[1.12; 2.03], p = .006), mainly driven by a higher rate of recurrent ACS. After correction for baseline differences lower Hb tertiles but not HRPR emerged as independent predictor of MACE (adjusted HR [95%CI] = 0.98[0.50; 1.92], p = .95). CONCLUSIONS: In the present study, we demonstrated that among patients on DAPT with ASA and ticagrelor after PCI for ACS, lower Hb levels are independently associated with a higher rate of HRPR and an increased rate of major ischemic events, and especially for recurrent ACS, although with no impact on survival. Neutral prognostic effect of HRPR was observed across Hb tertiles.

Impact of renin angiotensin system inhibitors on homocysteine levels and platelets reactivity in patients on dual antiplatelet therapy.

BACKGROUND AND AIMS: Dual antiplatelet therapy (DAPT) and Renin-angiotensin system inhibitors (RASi) represent the cornerstone in the treatment of patients undergoing percutaneous coronary interventions (PCI), mainly after an acute ischemic event. However, high-on treatment residual platelet reactivity (HRPR), is not infrequent despite optimal medical treatment. Homocysteine (Hcy) is a metabolite of methionine catabolism linked to atherothrombosis. Recently, a potential crosstalk between RAS and Hcy has been suggested, potentially favouring platelet aggregation and cardiovascular disease.Therefore, we aimed to investigate the impact of RASi on Hcy levels and platelet aggregation in patients on DAPT after PCI. METHODS AND RESULTS: Patients undergoing PCI on DAPT with ASA plus an ADP-antagonist (clopidogrel, ticagrelor or prasugrel), were included. RASi comprised angiotensin converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARB). Aggregation tests were performed by Multiple Electrode Aggregometry. We included 1210 patients, of whom 862 (71.2%) were on treatment with RASi. Overall, DAPT composition was ASA+clopidogrel in 566 (46.8%) patients, ASA+ticagrelor in 428 (35.4%) and ASA+prasugrel in 216 (17.9%). Median values of Hcy were higher in RASi patients (p = 0.006), who displayed a higher percentage of Hcy above the median value (52.4% vs. 44.8%, p = 0.019, adjustedOR [95%CI] = 1.40 [1.04-1.88], p = 0.027). No differences in HRPR rate were found according to RASi use for ASPI test (3.6% vs. 3.3%, p = 0.88) and ADP test (25.6% vs. 24.3%,p = 0.62; adjustedOR [95%CI] = 1.23 [0.89-1.70], p = 0.220) and according to ADP-antagonist type. A direct linear relationship was observed between platelet reactivity and Hcy in both patients receiving RASi and untreated ones, with higher values of platelet aggregation being observed in patients with Hcy above the median, independently from RASi administration and DAPT strategy. CONCLUSION: In patients on DAPT after PCI, RASi treatment did not emerge as an independent predictor of HRPR. However, the levels of Hcy were significantly elevated in patients on RASi and related to higher values of platelet reactivity, independently from the DAPT strategy.

Determinants of vitamin D activation in patients with acute coronary syndromes and its correlation with inflammatory markers.

BACKGROUND AND AIM: Vitamin D deficiency is a pandemic disorder affecting over 1 billion of subjects worldwide. Calcitriol (1,25(OH)2D) represents the perpetrator of the several systemic effects of vitamin D, including the anti-inflammatory, antithrombotic and anti-atherosclerotic actions, potentially preventing acute cardiovascular ischemic events. Variability in the transformation of vitamin D into 1,25(OH)2D has been suggested to modulate its cardioprotective benefits, however, the determinants of the levels of calcitriol and their impact on the cardiovascular risk have been seldom addressed and were, therefore, the aim of the present study. METHODS AND RESULTS: A consecutive cohort of patients undergoing coronary angiography for acute coronary syndrome (ACS) were included. The levels of 25 and 1,25(OH)2 D were assessed at admission by chemiluminescence immunoassay kit LIAISON® Vitamin D assay (Diasorin Inc) and LIAISON® XL. Hypovitaminosis D was defined as 25(OH)D < 10 ng/ml, whereas calcitriol deficiency as 1,25(OH)2D < 19.9 pg/ml. We included in our study 228 patients, divided according to median values of 1,25(OH)2D (

Association between vitamin D deficiency and serum Homocysteine levels and its relationship with coronary artery disease.

Homocysteine (Hcy) elevation and vitamin D deficiency have emerged as potential markers of coronary artery disease (CAD). However, even tough hypovitaminosis D has been suggested to interfere with Hcy catabolism, no study has so far addressed the interaction of vitamin D and Hcy and their impact on CAD, that was the aim of present study. A cohort of consecutive patients undergoing coronary angiography in a single center were included and analyzed within the year 2019. Significant CAD was defined as at least 1 vessel stenosis > 50%, while severe CAD as left main and/or three-vessel disease. Hcy and vitamin D levels were assesssed at admission. We included 3150 patients undergoing coronary angiography at our centre, who were divided according to the quartiles values of vitamin D. Patients with lower levels of Vitamin D displayed a higher cardiovascular risk profile and a higher prevalence of CAD. We observed an inverse linear relationship between lower levels of vitamin D and higher Hcy (r = - 0.092, p < 0.001) and a higher prevalence of hyperhomocysteinemia in patients with lower quartiles values of vitamin D (p < 0.001). By forward conditional regression model, low vitamin D appeared as independent predictors of Homocysteine levels above the median (OR[95%CI] = 1.79[1.37-2.33], p < 0.001). In addition, patients with low vitamin D (below the median) and increased Hcy displayed a non-significantly higher rate of CAD (81% vs 77.7%, p = 0.13, adjusted OR[95%CI] = 1.16[0.88-1.54], p = 0.29) but a significant increase in the rate of severe left main/3-vessel CAD (37.4% vs 30.5%, p = 0.005, adjusted OR[95%CI] = 1.29[1.02-1.67], p = 0.04). Among patients with vitamin D levels above the median, Hcy levels did not impact on the prevalence and extent of CAD (77.7 vs 77.2%, p = 0.81, adjusted OR[95%CI] = 0.94[0.73-1.20], p = 0.60 for CAD and 31.8% vs 27.7%, p = 0.08, adjusted OR[95%CI] = 0.97[0.75-1.25], p = 0.81 for severe left main/3-vessel CAD). No significant interaction between Hcy and vitamin D with CAD or severe CAD was observed. The present study shows an independent inverse linear relationship between vitamin D and Hcy values. Moreover, the association of Hcy with the extent of CAD was significant only among patients with hypovitaminosis D, and not in the cohort of subjects with vitamin D levels above the median, suggesting that a normal vitamin D status can prevent the deleterious effects of hyperhomocysteinemia on coronary atherosclerosis, a hypothesis that certainly needs further confirmation in larger randomized trials.

Differential Therapeutic Effect of Extracellular Vesicles Derived by Bone Marrow and Adipose Mesenchymal Stem Cells on Wound Healing of Diabetic Ulcers and Correlation to Their Cargoes.

Extracellular vesicles (EVs) derived from mesenchymal stem cells isolated from both bone marrow (BMSCs) and adipose tissue (ADSCs) show potential therapeutic effects. These vesicles often show a similar beneficial effect on tissue regeneration, but in some contexts, they exert different biological properties. To date, a comparison of their molecular cargo that could explain the different biological effect is not available. Here, we demonstrated that ADSC-EVs, and not BMSC-EVs, promote wound healing on a murine model of diabetic wounds. Besides a general similarity, the bioinformatic analysis of their protein and miRNA cargo highlighted important differences between these two types of EVs. Molecules present exclusively in ADSC-EVs were highly correlated to angiogenesis, whereas those expressed in BMSC-EVs were preferentially involved in cellular proliferation. Finally, in vitro analysis confirmed that both ADSC and BMSC-EVs exploited beneficial effect on cells involved in skin wound healing such as fibroblasts, keratinocytes and endothelial cells, but through different cellular processes. Consistent with the bioinformatic analyses, BMSC-EVs were shown to mainly promote proliferation, whereas ADSC-EVs demonstrated a major effect on angiogenesis. Taken together, these results provide deeper comparative information on the cargo of ADSC-EVs and BMSC-EVs and the impact on regenerative processes essential for diabetic wound healing.

Impact of active smoking on the immature platelet fraction and its relationship with the extent of coronary artery disease.

INTRODUCTION: Smoking represents a major cardiovascular risk factor, due to the induction of oxidative stress and low-grade, continuous, inflammation that contribute to promote atherothrombosis. However, the mechanisms leading to increased platelet aggregability associated with smoking are only partially defined. A potential role has been hypothesized for immature platelets, a younger and potentially more reactive fraction, previously associated with the main determinants of coronary artery disease (CAD). Therefore, the aim of our study was to define the impact of smoking on the immature platelet fraction (IPF) and its relationship with prevalence and extent of coronary artery disease. METHODS: We enrolled a cohort of consecutive patients undergoing coronary angiography in a single centre. Significant CAD was defined as at least 1 vessel stenosis >50%, while severe CAD was defined as left main and/or three-vessel disease. IPF was measured at admission by routine blood cell count (Sysmex XE-2100). RESULTS: We included in our study 2553 patients who were divided according to smoking status (active smokers: 512; nonactive smokers: 2041). Smokers were younger, more frequent males, with lower rate of diabetes mellitus, previous PCI and previous CABG (P < .001, respectively) and were in treatment less often with ARB, BB, nitrates, statins, ASA, clopidogrel, CCB and diuretics (P < .001, respectively) as compared to nonactive smokers. Higher percentage of smokers was observed in patients with higher IPF values, and at multivariate analysis, active smoking resulted as an independent predictor of higher IPF (adjusted OR [95% CI] = 1.59[1.03-2.45], P = .035). Among smokers, higher IPF was associated with lower ejection fraction (P = .034), percentage of acute coronary syndrome (P = .002) and platelet count (P < .001) compared to ones with lower IPF. However, the IPF (according to quartiles values) was not associated with the prevalence and extent of CAD (82.5%, 80.4%, 86.1% and 80.9%, from 1st to 4th quartile, respectively, adjusted OR[95% CI] = 0.98[0.79-1.23], P = .89) and severe CAD (31%, 31.1%, 39.1% and 35.2%, from 1st to 4th quartile, respectively, adjusted OR[95% CI] = 1.03[0.86-1.23], P = .76). CONCLUSION: The present study shows an independent association between active smoking and the levels of immature platelet fraction in patients undergoing coronary angiography. However, among active smokers, IPF did not result as an independent predictor of CAD or severe CAD.

Vitamin D levels condition the outcome benefits of renin-angiotensin system inhibitors (RASI) among patients undergoing percutaneous coronary intervention.

BACKGROUND: Vitamin D deficiency is estimated as the most common medical condition worldwide, with severe implications on survival and on several inflammatory, immune-mediated and thrombotic disorders, and especially for cardiovascular disease. Recent studies have suggested that vitamin D could directly regulate the Renin-Angiotensin System (RAS) activity, therefore potentially interfering with the pharmacological effects of RAS Inhibitors (RASI), an issue that has seldom been explored. Therefore, the aim of the present study was to evaluate the prognostic impact of the use of RASI according to vitamin D levels among patients with coronary artery disease (CAD) undergoing percutaneous coronary intervention (PCI). METHODS: Consecutive patients undergoing PCI were included. Main clinical features and chemistry parameters were assessed at admission. Vitamin D levels were measured by chemiluminescence immunoassay kit LIAISON® Vitamin D assay (Diasorin Inc). Severe deficiency was defined for 25(OH)D < 10 ng/mL. The primary study endpoint was defined as the occurrence of major cardiovascular events (MACE, a composite of death, recurrent Myocardial Infarction (MI) and target vessel revascularization) at the longest available follow-up. RESULTS: We included a total of 705 patients, that were divided according to vitamin D tertiles (< 12.7 ng/mL; 12.7-21.59 ng/mL; ≥21.6 ng/mL) and use of RASI. RASI therapy was significantly associated to arterial hypertension, creatinine, lower 25(OH)D, use of statins, diuretics, ASA and ticagrelor across vitamin D tertiles. At a median follow-up of 996 [377-1552] days, MACE occurred in 174 (24.7 %) patients. Severe hypovitaminosis D was significantly associated with a higher rate of MACE (HR[95 %CI] = 0.75[0.62-0.91], p = 0.004). The use of RASI significantly lowered the rate of MACE in patients with lower vitamin D (I tertile: 41.3 % vs 25.9 %, adjusted HR[95 %CI] = 0.43[0.26-0.73], p = 0.002); whilst a non-significant effect was observed for II and III tertiles values (18.6 %vs 29.5 %, adjusted HR[95 %CI] = 1.16[0.57-2.34], p = 0.69, and 21.2 % vs 12.6 %, adjusted HR[95 %CI] = 1.1[0.46-2.62], p = 0.83) (p int = 0.04). A similar prognostic interaction for RASI and vitamin D was observed for cardiovascular mortality and MI (p int = 0.03). CONCLUSION: Among patients undergoing PCI, the use of RASI was associated with lower risk of MACE only among patients with lower levels of vitamin D. Future larger studies are certainly warranted in order to define the prognostic implications of vitamin D supplementation on the RAS system modulation, especially among patients treated with RASI.

Homocysteine levels and platelet reactivity in coronary artery disease patients treated with ticagrelor.

BACKGROUND AND AIM: Recurrent atherothrombotic events have been reported in certain higher risk subsets of patients even with ticagrelor, a potent first-line antiplatelet agent for the management of patients with acute coronary syndrome (ACS). Hyperhomocysteinemia is a known determinant of platelet function abnormalities. Therefore, the aim of our study was to evaluate the impact of homocysteine (Hcy) levels on platelet reactivity in patients receiving Ticagrelor. METHODS AND RESULTS: Patients with ACS undergoing percutaneous coronary revascularization and on dual antiplatelet therapy with ASA + Ticagrelor (90mg/twice a day) were scheduled for platelet function assessment 30-90 days post-discharge. Aggregation tests were performed by Multiple Electrode Aggregometry (MEA). Suboptimal platelet inhibition HRPR-high residual platelet reactivity was defined if above the lower limit of normality (417 AU*min). We included 432 patients, divided according to Hcy tertiles. Higher Hcy levels were associated with age, renal failure, creatinine levels and use diuretics (p < 0.001). Patients with higher Hcy levels displayed a higher platelet reactivity at COL test (p = 0.002), and ADP test (p = 0.04), with a linear relationship between Hcy and platelet aggregation after stimulation with collagen (r = 0.202, p < 0.001), thrombin receptor peptide (r = 0.104, p = 0.05) and ADP (r = 0.145, p = 0.006). However, Hcy levels did not significantly affect the rate of HRPR with Ticagrelor (9.9% vs 13.7% vs 10.7%, p = 0.89; adjusted OR [95% CI] = [0.616-1.51], p = 0.99). CONCLUSIONS: Among patients with ACS, despite the elevated platelet reactivity associated to hyperhomocysteinemia, DAPT with ticagrelor could overcome such phenomenon, achieving an adequate platelet inhibition in the majority of the patients.

Impact of gender on immature platelet count and its relationship with coronary artery disease.

The impact of platelet parameters on the cardiovascular risk is still debated. Gender differences in platelet volume indexes and turnover have been previously reported, potentially conditioning their role in the development of coronary artery disease (CAD). However, few studies have addressed, so far, the impact of gender on the immature platelet fraction (IPF) and count (IPC) and their relationship with CAD. We enrolled consecutive patients undergoing coronary angiography in a single centre. IPF and platelet indexes were measured at admission. Significant CAD was defined as the presence of at least one coronary stenosis more than 50%. A total of 2550 patients were included, 1835 (72%) were males, and 715 (28%) were females. Female patients were older (p < 0.001), with lower BMI (p = 0.002), lower prevalence of active smoking (p < 0.001), previous MI, previous PCI and CABG (p = 0.001, p = 0.001, p < 0.001), whilst a higher prevalence of renal failure (p = 0.02), acute presentation (p < 0.001) and CAD (p < 0.001). Platelet count was higher in females (p < 0.001), as well as the IPC levels (838.38 ± 562.05 vs 792.24 ± 535.66, p = 0.05) with no difference in the levels of immature platelet fraction (3.67 ± 2.68% vs 3.74 ± 2.6%, p = 0.55) or the prevalence of patients with IPF ≥ 3rd tertile (33.7% vs 35.2%, p = 0.26). At multivariate analysis, after correction for baseline confounders, gender did not emerge as an independent predictor of higher IPF (adjusted OR [95% CI] = 0.82 [0.64-1.06], p = 0.13). When dividing our patients according to the levels of IPF, in women we observed an inverse association between IPF ≥ 3rd tertile and coronary calcifications (p = 0.025) and a higher prevalence of restenosis (p = 0.003), but no difference in CAD (65.6% vs 66.9%, p = 0.71) or severe CAD (28.1% vs 24.7%, p = 0.31). In males, the IPF ≥ 3rd tertile related with a lower TIMI flow (p = 0.001). Males with lower IPF had a significantly higher percentage of CAD (87.7% vs 83.3%, p = 0.007; adjusted OR: 0.699 [95% CI] = [0.54-0.91], p = 0.008) but not for severe CAD (36.5% vs 39.9%, p = 0.134). The present study shows that among patients undergoing coronary angiography, gender is not associated to the levels of immature platelet fraction. Moreover, we found no association between IPF and the prevalence and extent of CAD in female gender, whereas in male gender the IPF was inversely related with the prevalence of CAD.

Impact of aging on immature platelet count and its relationship with coronary artery disease.

Despite the fact that elderly patients represent a prevalent and challenging population in the current practice, few data exist on the impact of platelet parameters on cardiovascular risk in these patients. Therefore, the aim of the present study was to evaluate the impact of age on the immature platelet count (IPC) and their relationship with CAD. We included a total of 2236 consecutive patients undergoing coronary angiography in a single center. Elderly patients (age ≥ 75 years) were 756 (33.7%). IPC was measured at admission. Elderly patients were more often females (p < .001), with lower BMI and prevalence of smokers (p < .001), and a more complex cardiovascular risk profile and coronary disease (p = .02). Platelet count decreased with aging (p = .05), whereas no difference in the mean IPC was found between patients < or ≥75 years. In fact, advanced age did not emerge as an independent predictor of IPC above III tertile (≥8.6*10^6/ml), (adjusted OR[95%CI] = 0.97[0.78-1.21], p = .79). When considering elderly patients according to tertiles values of IPC (<5.1,5.1-8.59; ≥8.6*10^6/ml), we found no impact of IPC on the prevalence of CAD (81.1% vs 84.5% vs 81.5%, p = .92; adjusted OR[95%CI] = 1.08[0.67-1.72], p = .75) and its extent (37.7% vs 34.5% vs 40.2%, p = .57; adjusted OR[95%CI] = 1.22[0.85-1.73], p = .28). However, we observed a higher rate of calcified and type C lesions in elderly patients with higher IPC (p = .03 and p < .001, respectively). Therefore, advanced age is not associated with higher immature platelet count and the prevalence and severity of CAD. Moreover, IPC does not contribute to explain the higher prevalence and extent of coronary artery disease observed in elderly patients.

Relationship between adenosine A2a receptor polymorphism rs5751876 and fractional flow reserve during percutaneous coronary intervention.

Fractional flow reserve (FFR) assessed during adenosine-induced maximal hyperemia has emerged as a useful tool for the guidance of percutaneous coronary interventions (PCI). However, interindividual variability in the response to adenosine has been claimed as a major limitation to the use of adenosine for the measurement of FFR, carrying the risk of underestimating the severity of coronary stenoses, with potential negative prognostic consequences. Genetic variants of the adenosine receptor A2a (ADORA2A gene), located in the coronary circulation, have been involved in the modulation of the hyperemic response to adenosine. However, no study has so far evaluated the impact of the single nucleotide polymorphism rs5751876 of ADORA2A on the measurement of FFR in patients undergoing percutaneous coronary intervention that was, therefore, the aim of our study. We included patients undergoing coronary angiography and FFR assessment for intermediate (40-70%) coronary lesions. FFR measurement was performed by pressure-recording guidewire (Prime Wire, Volcano), after induction of hyperemia with intracoronary boli of adenosine (from 60 to 1440 µg, with dose doubling at each step). Restriction fragment length polymorphism (RFLP) analysis was performed to assess the presence of rs5751876 C>T polymorphism of ADORA2a receptor. We included 204 patients undergoing FFR measurement of 231 coronary lesions. A total of 134 patients carried the polymorphism (T allele), of whom 41 (30.6%) in homozygosis (T/T).Main clinical and angiographic features did not differ according to ADORA2A genotype. The rs5751876 C>T polymorphism did not affect mean FFR values (p = 0.91), the percentage of positive FFR (p = 0.54) and the duration of maximal hyperemia. However, the time to recovery to baseline FFR values was more prolonged among the T-allele carriers as compared to wild-type patients (p = 0.04). Based on these results, in patients with intermediate coronary stenoses undergoing FFR assessment with adenosine, the polymorphism rs5751876 of ADORA2A does not affect the peak hyperemic response to adenosine and the results of FFR. However, a more prolonged effect of adenosine was observed in T-carriers.

Oral Delivery of mRNA Vaccine by Plant-Derived Extracellular Vesicle Carriers.

mRNA-based vaccines were effective in contrasting SARS-CoV-2 infection. However, they presented several limitations of storage and supply chain, and their parenteral administration elicited a limited mucosal IgA immune response. Extracellular vesicles (EVs) have been recognized as a mechanism of cell-to-cell communication well-preserved in all life kingdoms, including plants. Their membrane confers protection from enzyme degradation to encapsulated nucleic acids favoring their transfer between cells. In the present study, EVs derived from the juice of an edible plant (Citrus sinensis) (oEVs) were investigated as carriers of an orally administered mRNA vaccine coding for the S1 protein subunit of SARS-CoV-2 with gastro-resistant oral capsule formulation. The mRNA loaded into oEVs was protected and was stable at room temperature for one year after lyophilization and encapsulation. Rats immunized via gavage administration developed a humoral immune response with the production of specific IgM, IgG, and IgA, which represent the first mucosal barrier in the adaptive immune response. The vaccination also triggered the generation of blocking antibodies and specific lymphocyte activation. In conclusion, the formulation of lyophilized mRNA-containing oEVs represents an efficient delivery strategy for oral vaccines due to their stability at room temperature, optimal mucosal absorption, and the ability to trigger an immune response.

Plant-Derived Extracellular Vesicles as a Delivery Platform for RNA-Based Vaccine: Feasibility Study of an Oral and Intranasal SARS-CoV-2 Vaccine.

Plant-derived extracellular vesicles (EVs) may represent a platform for the delivery of RNA-based vaccines, exploiting their natural membrane envelope to protect and deliver nucleic acids. Here, EVs extracted from orange (Citrus sinensis) juice (oEVs) were investigated as carriers for oral and intranasal SARS-CoV-2 mRNA vaccine. oEVs were efficiently loaded with different mRNA molecules (coding N, subunit 1 and full S proteins) and the mRNA was protected from degrading stress (including RNase and simulated gastric fluid), delivered to target cells and translated into protein. APC cells stimulated with oEVs loaded with mRNAs induced T lymphocyte activation in vitro. The immunization of mice with oEVs loaded with S1 mRNA via different routes of administration including intramuscular, oral and intranasal stimulated a humoral immune response with production of specific IgM and IgG blocking antibodies and a T cell immune response, as suggested by IFN-γ production by spleen lymphocytes stimulated with S peptide. Oral and intranasal administration also triggered the production of specific IgA, the mucosal barrier in the adaptive immune response. In conclusion, plant-derived EVs represent a useful platform for mRNA-based vaccines administered not only parentally but also orally and intranasally.

Optimized Protocol for Plasma-Derived Extracellular Vesicles Loading with Synthetic miRNA Mimic Using Electroporation.

Extracellular vesicles (EVs) are a population of particles naturally released by cells to transport biological messages, including nucleic acids. Thus, EVs represent an ideal vehicle to deliver therapeutic miRNAs. The current challenge is the development of efficient protocols to load EVs with exogenous miRNAs. Human plasma is an abundant source of EVs which can be manipulated for therapeutic applications. Despite numerous techniques are currently available to load EVs, all of them present issues which limit their clinical application. Among all, electroporation was shown to be superior to other protocols and to efficiently load plasma-derived EVs with miRNAs. However, also the electroporation procedure presents issues that can reduce the miRNA delivery. In this chapter, we describe a protocol to isolate EVs from human plasma, to load synthetic miRNA mimics using electroporation, to evaluate EV integrity and miRNA loading into EVs. Finally, the analysis of EV functionality allows to investigate the ability of engineered EVs to transfer the miRNAs to target cells and to exploit their biological effects.

Impact of uric acid on immature platelet fraction in patients undergoing percutaneous coronary intervention.

INTRODUCTION: Reticulated platelets have been involved in the pathophysiology of coronary artery disease (CAD). Immature platelet fraction (IPF) allows their measurement in daily clinical practice, although the factors conditioning their elevation are still largely unexplored. Serum uric acid (SUA) is the end product of purine metabolism, displaying a pro-oxidant and pro-inflammatory action and increasing the cardiovascular risk. The aim of our study was to investigate the impact of SUA on IPF levels in patients undergoing percutaneous coronary intervention (PCI), and their relationship with CAD. METHODS: We enrolled a cohort of consecutive patients undergoing coronary angiography in a single center. Hyperuricemia was defined by SUA ≥ 6.5 mg/dl. Significant CAD was defined as at least 1 vessel stenosis >50%, while severe CAD was defined as left main and/or three-vessel disease. IPF was measured at admission by routine blood cells count (A Sysmex XE-2100). RESULTS: We included in our study 2217 patients, of whom 544 had high levels of SUA, while 1673 had normal levels. Hyperuricemics were older, with higher percentage of renal failure, hypertension, dilated cardiomyopathy or valvular disease as indication to angiography, higher levels of creatinine and C-reactive protein (p < 0.001, respectively) when compared to normouricemics. Immature platelet fraction (IPF) was significant higher in hyperuricemic patients (3.96% vs 3.59%, p = 0.004). A progressive direct increase in the IPF values was observed in relation to SUA levels (r = 0.101, p < 0.001), although at multivariate analysis, hyperuricemia did not result as an independent predictor of IPC in 3rd tertile (adjusted OR [95%CI] = 1.21 [0.85-1.71] P = 0.288). When stratifying hyperuricemics and normouricemics according to IPF tertiles (<2.3%; 2.3-3.9%; ≥ 4%), reticulated platelets were not associated to the prevalence of CAD (SUA ≥6.5:80.9 vs 79.3% vs 78.6%, p = 0.60; SUA < 6.5: 79.3% vs 81.3% vs 78.9%, p = 0.878) or severe CAD (SUA ≥6.5: 34.9% vs 38.9% vs 35.2%, p = 0.99; SUA < 6.5: 30.4% vs 33.5% vs 34%, p = 0.192), and the results were confirmed at multivariate analysis for CAD (SUA ≥6.5: adjusted OR [95%CI] = 1.11 [0.81-1.51] P = 0.524, SUA < 6.5: adjusted OR [95%CI] = 0.89 [0.75-1.05] P = 0.170) or severe CAD (SUA ≥6.5: adjusted OR [95%CI] = 1.03 [0.81-1.31] P = 0.795; SUA < 6.5: adjusted OR [95%CI] = 1.10 [0.96-1.26] P = 0.192). CONCLUSIONS: In the present study we found a direct relationship between SUA levels and IPF values; however, hyperuricemia did not result as an independent predictor of higher IPF tertile values. Neither in hyperuricemics nor in normouricemics higher IPF were independently associated to the prevalence of CAD or severe CAD.

Impact of the Polymorphism rs5751876 of the Purinergic Receptor ADORA2A on Periprocedural Myocardial Infarction in Patients Undergoing Percutaneous Coronary Intervention.

AIM: Periprocedural myocardial infarction (PMI), a severe complication of Percutaneous Coronary Intervention (PCI) procedures, has a negative prognostic effect, both at short and long-term follow-up. So far, adenosine's role in preventing PMI has shown contrasting results. A genetic variant of ADORA2A receptor, 1976 C＞T, has been suggested as a potential determinant of the interindividual response to adenosine, thus conditioning its potential benefits on PMI. In our study, we investigated whether the ADORA2A 1976 C＞T polymorphism is associated with PMI occurrence in patients undergoing coronary stenting. METHODS: The study included consecutive patients undergoing PCI at the Azienda Ospedaliera-Universitaria "Maggiore della Carità," Novara, Italy, between January 2010 and January 2016. Their genetic status was assessed using polymerase chain reaction (PCR) and restriction-fragment-length-polymorphism technique. Myonecrosis biomarkers were measured at intervals from 6 to 48 hours. PMI was defined as CKMB increased 3 times over the Upper Limit of Normal (ULN), or 50% of pre-PCI value; periprocedural myonecrosis was defined as troponin I increased 3 times over the ULN or by 50% of the baseline value. RESULTS: We included 1,104 patients undergoing PCI, 863 (78.2%) of whom carried the ADORA2A T-allele. No difference was found for the main demographic, clinical features, or biochemistry parameters. However, C-carriers had lower statin therapy use (p=0.008) and lower HDL-cholesterol levels (p=0.01). Homozygous C/C patients had more frequent multivessel disease (p=0.03), longer lesions (p=0.01) and Type C lesions (p=0.01), thus requiring more complex procedures. After correction for baseline confounding factors at multivariate analysis, there was no difference in myocardial necrosis according to the ADORA2A genotype (p=0.40). In contrast, PMI tended to increase in the homozygous C/C population (p=0.06), but this trend was attenuated at multivariate analysis after correction for baseline confounding factors (C/C: OR[95%CI]=1.52 [0.88-2.6], p=0.14). CONCLUSIONS: Our study showed that the polymorphism rs5751876 of the ADORA2A receptor is associated with a higher prevalence of complex coronary lesions and multivessel disease. However, it does not significantly influence the occurrence of periprocedural MI or myonecrosis.

Benefits with drug-coated balloon as compared to a conventional revascularization strategy for the treatment of coronary and non-coronary arterial disease: a comprehensive meta-analysis of 45 randomized trials.

BACKGROUND: Drug-coated balloons (DCB) have shown promising results for the percutaneous treatment of de novo and restenotic lesions, involving both the coronary and femoropopliteal district. However, clinical outcomes data associated with the use of this devices are still unclear, with potential warnings on increased mortality being raised from initial studies. We aimed at performing an updated and comprehensive meta-analysis comparing DCB with conventional percutaneous revascularization strategies for the treatment of coronary (CAD) or peripheral artery disease (PAD). METHODS: Literature and main scientific session abstracts were searched for studies comparing DCB vs a standard percutaneous revascularization strategy, with or without stenting, for the treatment of CAD and PAD. The primary efficacy endpoint was mortality. Secondary endpoints were recurrent acute ischemic events (myocardial infarction or amputation) or target lesion revascularization (TLR). RESULTS: We included 45 randomized trials, (CAD: 27 studies, PAD: 18 studies) with an overall population of 7718 patients, (56.4%) randomized to a DCB strategy. At a mean follow-up of19.3 ± 15.2 months, death occurred in 5.8% of the patients, with no significant difference between DCB or conventionally treated patients (5.9% vs 5.7%, OR[95%CI] = 0.89[0.71,1.11], p = 0.31; phet = 0.43). We observed a non-significant reduction in recurrent acute ischemic events, whereas the use of DCB significantly reduced the rate of TLR, with larger benefits observed in patients with PAD and respect to balloon-only angioplasty, while being lower in comparison with stent implantation. No significant interaction was observed with de novo lesions or in-stent restenosis. CONCLUSIONS: Based on the current meta-analysis, the use of drug-coated balloons for the percutaneous treatment of CAD and PAD is associated to a comparable risk of mortality and recurrent acute ischemic events as compared to a conventional revascularization strategy, although offering larger benefits in terms of TLR, especially when compared with balloon-only angioplasty and in femoropopliteal disease.

Impact of Age on the Functional Evaluation of Intermediate Coronary Stenoses With Instantaneous Wave-Free Ratio and Fractional Flow Reserve.

The optimal strategy for assessing the ischemic significance of intermediate coronary stenoses with adenosine-induced fractional flow reserve (FFR) and instantaneous wave-free ratio (iFR) is still debated. Few studies have previously assessed the impact of age on FFR and iFR, which was the aim of our study. Patients undergoing FFR and iFR evaluation for intermediate (40%-70%) coronary lesions were included and divided according to age. Fractional flow reserve was performed by intracoronary boluses of adenosine (60-1440 µg). Instantaneous wave-free ratio was automatically calculated. Among 148 patients undergoing FFR measurement of 166 lesions, 45.3% were ≥70 years. Elderly patients had higher minimal lumen diameter (P = .03). We also observed a linear relationship between iFR and FFR independently of age. Fractional flow reserve values were higher in the elderly patients, whereas iFR was not related to age. A total of 33 lesions had a positive iFR with no difference for age (17.3% vs 22%, P = .56), while FFR <0.80 was more infrequent in the elderly patients (17.1% vs 34.8%, P = .02). In intermediate coronary stenoses, iFR and FFR correlation is unaffected by age. Fractional flow reserve is higher in the elderly patients, whereas iFR is less affected by age. Future large-scale studies are needed to define whether iFR should be the preferred choice in elderly patients.

Prognostic impact of Vitamin D deficiency in patients with coronary artery disease undergoing percutaneous coronary intervention.

BACKGROUND: Whether Vitamin D deficiency represents an independent predictor of mortality and major cardiovascular events or rather the mirror of a more advanced clinical condition with increased comorbidities is still debated. We aimed at assessing the impact of vitamin D levels on the long-term outcomes among patients with coronary artery disease (CAD) undergoing percutaneous coronary intervention. METHODS: Consecutive patients from a single centre were included. Vitamin D levels were measured at admission by chemiluminescence immunoassay kit LIAISON® Vitamin D assay (Diasorin Inc). Severe deficiency was defined for 25(OH)D < 10 ng/ml. The primary study endpoint was overall mortality. Secondary endpoints were cardiovascular mortality, recurrent acute coronary syndrome or major cardiovascular events (a composite of death, recurrent MI and target vessel revascularization) at the longest available follow-up. RESULTS: We included a total of 705 patients, that were divided according to vitamin D tertiles (<12.7; 12.7-21.59; ≥21.6 ng/ml). Lower levels of Vitamin D were associated with renal failure (p=0.03), more severe coronary disease (p=0.001), diabetes mellitus and previous CABG (p<0.001), lower ejection fraction (p=0.02), acute presentation (p=0.04), use of statins (p=0.02), diuretics, nitrates and clopidogrel (p<0.001) and RASI (p=0.008). An inverse association was documented with BMI, glycemia, total cholesterol (p<0.001), creatinine and WBC (p=0.001). At a median follow-up of 996.5 [377-1552] days, 3.8% of the patients died. Vitamin D deficiency was significantly associated with overall mortality (7.6% vs 2.9% vs 0.4%, adjusted HR[95%CI]=3.6[1.43-8.9], p=0.006), MACE (adjusted HR[95%CI]=1.32[1.07-1.63], p=0.01) and the composite of death and MI (adjusted HR[95%CI]=1.3[1.03-1.65], p=0.03). A similarly increased risk was confirmed for all major higher-risk subsets of patients, with no significant interaction according to age, gender, diabetes mellitus or chronic kidney disease. CONCLUSION: Among patients undergoing percutaneous coronary interventions, lower levels of vitamin D are associated with an over 3-fold increased risk of mortality and major cardiovascular events. Future larger studies are certainly warranted in order to define the prognostic implications of cholecalciferol supplementation among high-risk patients with established coronary artery disease.