Upon performing multiple imputation, a stepwise backward elimination model selection approach using Akaike’s information criteria was used to identify the optimal model. The resulting model, comprised of 14 predictors mostly at the county level, provides an additional 8% explanatory power to capture the variability in 30-day risk-standardized AMI mortality rates, which already account for patient-level clinical differences. SES factors may be an important feature for inclusion in future risk-adjustment models, which will have system and policy implications for distributing resources to hospitals, such as reimbursements. It also serves as a stepping stone to identify and address long-standing SES-related inequities.The U.S. Centers for Medicare and Medicaid Services’ (CMS’s) Hospital Compare (HC) data provides a collection of risk-adjusted hospital performance metrics intended to allow comparison of hospital-provided care. However, CMS does not adjust for socioeconomic status (SES) factors, which have been found to be associated with disparate health outcomes. Associations between county-level SES factors and CMS’s risk-adjusted 30-day acute myocardial infarction (AMI) mortality rates are explored for  Social determinants of health consist of the circumstances, settings, and environments in which people are born, develop, play, learn, live, and work . ExampleSES factors can inform a patient’s health status and risks , as highRisk adjustment methods, which have become common approaches to measurement of quality of care, aim to provide a fair metric of hospital performance by incorporating information about patient characteristics . MetricsThis manuscript focuses on an acute myocardial infarction (AMI) mortality measure to demonstrate the relevance of SES factors on patient health and hospital performance. Risk-adjusted AMI mortality is a metric used by CMS in the form of a 30-day, risk-standardized mortality measure adjusted by patient characteristics and comorbidities . MultiplIncorporation of such factors in patient risk adjustment modeling methods is imperative. CMS’s 30-day AMI mortality model does not consider the patients’ SES, even as it can be another determinant to their health outcomes. In this study, we aim to assess whether county-level SES factors are relevant to explain the variability within CMS’s 30-day risk-standardized AMI mortality hospital performance metric in the U.S., and quantify these associations. Incorporation of SES factors into such hospital performance metrics is a necessary step toward increased health equity and improved AMI-related mortality outcomes.The two primary data sources utilized in this study were the 2017–2018 Area Health Resources Files (AHRF) , which aIn addition to the aforementioned data, crime data were obtained from the Federal Bureau of Investigation’s (FBI) Uniform Crime Reporting (UCR) Program. The UCR Program provides annual counts of county-level arrests and reported crimes from over 18,000 law enforcement agencies throughout the US .n = 2462 hospitals with available 30-day risk-standardized AMI mortality rates. Safety net hospitals not included in the HC data primarily consisted of children’s hospitals, behavioral health/psychiatric hospitals, and rehabilitation hospitals.The Institute of Medicine defines safety net hospitals (SNH) as “providers that organize and deliver a significant level of both health care and other health-related services to the uninsured, Medicaid and other vulnerable populations” . UnfortuAll records in the CMS HC, including datasets concerning complications and deaths in hospitals and unplanned hospital visits with a 30-day risk-standardized AMI mortality rate, were included in the study. This resulted in children’s hospitals and Department of Defense hospitals being excluded.The outcome variable of interest is the 30-day risk-standardized AMI mortality rate. While the list of possible SES factors that could have been considered is fairly large, a set of relevant SES factors was identified and is listed in p-values from the resulting model were used to assess each SES factor’s significance at α = 0.05. Variance inflation factors (VIFs) were also explored for the final model to assess potential problems regarding multicollinearity in variable/model interpretation.Income and population density variables were log-transformed due to high skewness, and other count variables were transformed to population rates. Multiple imputation by chained equations (MICE) was used to impute missing data . In ordeFourteen SES predictors were included upon model selection for the SES-adjusted 30-day risk-standardized AMI mortality analysis. p < 0.001) with a ratio of sample size to covariates of 188. VIFs were consistently low, with only one value above 5 , a majority of VIFs smaller than 2, and a mean VIF of 2.5. Under a commonly used rule-of-thumb of VIF = 10, these values were considered sufficiently low to explore within-model variable significance.The AIC-based optimal model was able to explain 7.5% of the variability in the 30-day risk-standardized AMI-related mortality outcome  and where residents aged 60 and older grew by 15% of more over the 2000–2010 period  were associated with significantly higher 30-day risk-standardized AMI mortality rates. Manufacturing counties showed a non-significant positive association with 30-day risk-standardized AMI mortality rates (p = 0.122). Counties with higher per capita personal income (p = 0.009) and higher ratios of income to poverty level ≥ 2.00 (p = 0.011) were associated with statistically significantly lower 30-day risk-standardized AMI mortality rates. Counties with a larger percentage of its population who are Medicaid-eligible, which is more often associated with younger populations, were significantly negatively associated with 30-day risk-standardized AMI mortality rates (p = 0.016), whereas there was a non-significant positive association between dual Medicare/Medicaid eligibility and 30-day risk-standardized AMI mortality rates (p = 0.109).Counties coded as mining-focused (p = 0.002), as well as larger percentages of the population who are Asian (p = 0.021), black/African American (p = 0.024), and ‘Other’ race (p = 0.0709), compared to white populations, were all associated with higher 30-day risk-standardized AMI mortality rates, though the latter group was non-statistically significant. The magnitude was substantially worse for veterans, who appear to be more associated with worse 30-day risk-standardized AMI mortality rates for hospitals in the counties where they reside.Counties with larger populations of veterans (p = 0.033), whereas counties with higher violent crime rates were associated with higher 30-day risk-standardized AMI mortality rates (p = 0.012). Finally, designation of the hospital as a safety net facility was non-significantly associated with worse 30-day risk-standardized AMI mortality rates (p = 0.127).Counties with larger population densities were associated with lower 30-day risk-standardized AMI mortality rates , veteran population, race , and violent crime rate were significant contributing factors to explain higher 30-day risk-standardized AMI mortality rates. Conversely, the SES factors of population density, income, and Medicaid eligibility were associated with lower 30-day risk-standardized AMI mortality rates.Counties with larger retirement communities were associated with worse 30-day risk-standardized AMI mortality, even as this metric already accounts for age differentials at the patient level. Results from a study that examined data from the longitudinal Health and Retirement Study (HRS) in the U.S. concluded that retirement status was associated with a higher incidence of AMI as compared to those working full-time, controlling for age and sex . RetiremLarger proportions of individuals identified with certain races  were associated with higher 30-day risk-standardized AMI mortality rates compared to white individuals. This aligns with studies which have shown that African Americans experience worse clinical conditions than whites, including being more likely to be treated by less qualified physicians  and treaPopulation density was found to be associated with better 30-day risk-standardized AMI mortality rates. There could be confounders in this association due to populations with healthier habits living in more densely populated areas, as well as populations with higher income within metropolitan areas when compared to less densely populated rural areas. Violent crimes, which is a proxy for lack of perceived safety, were found to be statistically significantly associated with higher 30-day risk-standardized AMI mortality rates. This could be associated with fewer resources or less qualified physicians willing to work in unsafe areas. It could also relate to the responsiveness of emergency services in unsafe locations, or waiting times at potentially busy emergency rooms. This aligns with hospital safety net designation, which is associated with lower incomes of the populations they serve, and, therefore, such hospitals are also subject to fewer resources. Those hospitals, which may be subject to more stringent budgetary constraints, may also find it difficult to compete for highly qualified personnel when compared to those who serve other populations. However, we found safety net hospital designation to be relevant for inclusion in the final model on the basis of AIC, but non-significantly positively associated with higher 30-day risk-standardized AMI mortality rates. This indicates that safety net designation may be less relevant to 30-day risk-standardized AMI mortality upon accounting for other  neighborhood-level factors. However, safety net hospital designation had a low pseudo-R-squared (0.035) when regressed against the remaining covariates (but not against the outcome).Finally, while county-level SES factors were found to be associated with 30-day risk-standardized AMI mortality, patient-level SES factors are likely to provide additional, more granular information to capture the inherent heterogeneity in patient-level SES characteristics. Capturing and accounting for both county-level and patient-level SES factors may further enhance risk-adjustment models.Numerous studies identify socioeconomic factors that relate to AMI-related mortality, but oftentimes focus on a single SES, or focus on a very limited group of those factors ,15,16,18SES factors identified in this study account for about 8% of the total variability in 30-day AMI mortality rates , reflecting that there is additional, unaccounted SES-sourced rate heterogeneity that should be incorporated in AMI mortality risk-adjustment models. This is the case despite the study limitation stemming from the different geographical scope of 30-day risk-standardized AMI mortality rates for hospitals (which relate to the populations they serve) and county covariates . Some hospitals, especially those located along county boundaries, may serve large populations from neighboring counties. This may more greatly affect hospitals located in smaller, rural locations compared to those located in major metropolitan areas. Furthermore, some patients may prefer to travel to have better access to care in a different hospital than that of their county. Using county-level information may be best for hospitals located in counties with low intra-county variability in SES factors.Since this study relies on county-level information (and the limitations therein), some additional SES factors, including gender or sexual orientation, were not explored. Such information would not be readily available but should be explored by CMS. However, note that factors such as patient’s age or comorbidities are already included by CMS in the calculation of the 30-day risk-standardized AMI mortality metric. Although individual-level factors such as clinical conditions were already accounted for at the patient level in the current 30-day risk-adjusted AMI mortality rates, the use of patient-level (rather than county-level) SES factors is likely to provide additional granular information to capture the inherent heterogeneity in patient-level SES characteristics.All data used precede COVID-19, which constitutes a disruptive event to AMI mortality rates. However, all data were not synchronous, due to the heterogeneity of sources. This results in potential mismatches between covariate information across time. However, we do not anticipate this to be highly relevant, since most covariates used in this study are expected to be relatively stable over short periods of time.When comparing individual-level and county-level SES factors, there may be both overlapping and non-overlapping components, so both approaches accounting for SES are complementary. For example, personal income may be relevant at both the individual and county levels. Two individuals with equal income may experience different AMI-related health outcomes depending on whether they live in poorer or wealthier counties (county-level SES factor), while two individuals with different income may also experience different AMI-related outcomes when living in the same county (patient-level SES factor). Individual income may define personal affordability of resources, while county income characteristics may define their general availability.While individual clinical factors are a substantial component of 30-day risk-standardized AMI mortality, SES factors are also associated with heterogeneity of this outcome. County-level SES factors were found to be associated with 30-day risk-standardized AMI mortality, and could be used in conjunction with patient-level SES factors to capture inherent heterogeneity in patient-level health metrics.While income and wealth redistribution policies  and increased social, environmental, and community investments  can palliate some of the SES disparities identified, it may take decades or generations, unfortunately, before such disparities are no longer present or influential on health-related outcomes. Differences in 30-day risk-standardized AMI mortality are, in part, a result of these underlying SES disparities, highlighting that patient clinical characteristics are not the only relevant factors influencing health outcomes. Until relevant policies are implemented and the disparity gap is eliminated or greatly reduced, one immediate change that can be made is the incorporation of SES factors in CMS’s 30-day AMI mortality metric, for example. This remedy, in turn, may help towards bridging existing SES disparity gaps in health outcomes. Accounting for county-level SES factors is an essential improvement to hospital performance metrics, and capturing and accounting for SES factors can further enhance risk-adjustment models. This can result in fairer hospital evaluations, comparisons, and reimbursements via programs such as HVBP, and can act as a stepping stone towards addressing long-standing SES-related inequities.


Despite these promising results, validation via clinical trials is yet to be performed. The scope of statins in their pleiotropic range of actions is still not completely explored, and studies are still needed to enlighten different useful aspects of such drugs.Statins are a wide category of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor drugs extensively prescribed for hypercholesterolemia. In fact, many studies showed beneficial effects of these agents on a variety of related illnesses, which include increased atherosclerotic plaque stability, decreased proliferation of vascular smooth muscle, platelet aggregation, the dampening of vascular inflammation, and also anabolic effects on bone tissue. Therefore, these drugs are considered as pleiotropic agents having different clinical applications other than those for which they were initially developed. Controlled drug delivery is an efficient way of delivery in tissue engineering. Amongst different controlled release formulations, nanofibers are a novel, alternative, widely used agent because of their unique properties. These include their sustained release of drug, a high drug-loading capacity, flexible shapes with a high surface-to-volume ratio, and superior porosity. Electrospinning is an economic and a simple method employed to produce nanofibers. In this report, studies related to statin nanofiber applications have been reviewed and their results have been summarized. Four different applications of statin nanofibers have been reported, including bone generation, endothelial stenosis and thrombosis, peripheral nerve injury, and anti-inflammatory action. Studies carried out both in vitro and in vivo showed effectiveness of statins in bone healing, aneurysm, and the healing of sciatic nerve injury. In addition, statins showed apoptosis effects and anti-inflammatory effects, with dose-dependent reduction of IL-6 and dose-independent reduction of TNF- The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) have been in use for more than 30 years to decrease the rate of occurrence of coronary artery disease and stroke. Despite the introduction of newer agents –3, statiThere are several methodologies which can be employed to alleviate the effects of neointimal hyperplasia. Amongst them, the new drug-eluting stents (DESs) have a significant potential in reducing this condition. Therefore, use of stents and statins for coronary artery disease (CAD) is common with statin treatment, consistent with their lipid-lowering, antioxidative actions, and anti-inflammatory and antithrombotic effects useful for treating patients at different stages of the CAD disease .κB ligand (RANKL), a known osteoclast formation molecule. Statins simulate the production of OPG and as a result, they are recommended as bone generator and integrator agents [Another effect of statins is their potential for osseointegration. Studies have shown the effects of statins on the activation of osteoblasts and reduction of osteoclasts both in vivo and in vitro, along with increased expression of bone morphogenetic protein-2 (BMP-2) , followir agents , 22.Electrospinning is simple, economical, and versatile and provides a method in which polymer solution or melt undergo stretching and elongation through electrostatically driven jets, thereby forming polymer nanofibers (NFs). This method consists of three main parts including a source of high voltage, a syringe pump, and a conductive collector . The diaControlled drug delivery is an efficient and modern way of delivery in tissues and in tissue engineering processes , 28. DruInorganic materials including metals, surface-treated/ceramic-coated metals, and bioactive ceramics  have been widely used in bone tissue engineering and actively bind to the bone. Since the bone composition is mainly of type I collagen and inorganic crystals of carbonate-substituted HA , those implanted surfaces containing such materials (synthetic HA) will reveal osteoinductive properties. Adding these materials to electrospun NFs will improve the adhesion and differentiation of cells. This was shown in a study where after adding collagen/n-HA to PLGA NFs increased osseointegration and bone regeneration . In addiCurrently, several statin nanofibers have been synthesized for different purposes, especially bone regeneration and endothelial stenosis reduction. We have reviewed literature studies and identified a number of studies related to the clinical application of statin nanofibers. We review and summarize a number of such studies here.β-TCP) and simvastatin (SIM), showed better bone regeneration in comparison to the control group (pure NFs) with sustained drug release [ε-caprolactone) (PCL) and poly(glycerol sebacate) (PGS) loaded with hydroxyapatite nanoparticles (HANPs) and simvastatin (SIM) (PCL-PGS-HA-SIM). This study revealed that the presence of SIM increased MC3T3-E1 osteoblast cell proliferation rate more in comparison to that of PCL-PGS and PCL-PGS-HA, which was confirmed via kit-8 (CCK-8) assay. Also, PCL-PGS-HA-SIM showed improved cell adhesion and bone extracellular matrix (ECM) secretion after 6 days compared to PCL-PGS and PCL-PGS-HA [2/PANI) and SIM (PCL-PTS) using the electrospinning method. PCL-PTS showed the most MC3T3-E1 proliferation after 5 days, compared to the similar proliferation rates in all the different samples taken. PCL-PTS releases SIM gradually in a controlled manner within the first 5 days, beyond which SIM concentration and consequently cell proliferation increase simultaneously. All formulations in this study  showed a significant adhesion rate and an increase in ECM rates, demonstrating successful mineralization, after 5 days [Kalani et al. evaluated the osteogenesis differentiation of human adipose-derived stem cells (hADSCs). To this purpose, they prepared slow-release core-sheath poly/silk fibroin nanofibers (PVA-SF core-shell NFs) with rosuvastatin (RSV) by a coaxial electrospinning process. After culturing the hADSCs on RSV-loaded NFs, these NFs have significantly upregulated osteogenesis markers such as Col 1, RUNX2, alkaline phosphatase (ALP) (two early osteogenesis markers), and osteocalcin (OCN) (a late osteogenesis marker) compared to free-drug NFs. Also, ARS staining confirmed the osteogenesis by showing calcium formation. Therefore, the PVA-SF core-shell NFs, with sustained release of RSV, provide suitable applications in bone tissue engineering . In anot release . AnotherL-PGS-HA   embedded in poly(L-lactide-co-caprolactone) (PLCL) nanofiber-covered stents within vascular endothelial cells (HUVECs). The study revealed that these NFs showed controlled release and could induce HUVEC proliferation in a dose-dependent manner. The effect was dependent on concentration, as with 5 ± 1 nM of AtvCa or PLCL-AtvCa10 nanofiber, HUVEC proliferation was increased, whereas with 12 nM AtvCa, it decreased  Figure .A heparin-rosuvastatin (Rosu)-loaded P(LLA-CL) nanofiber-covered stent tested in smooth muscle cells (SMCs) showed that by increasing the Rosu concentration, the SMC proliferation decreased . AnotherIn a DSA follow-up study with a rabbit aneurysm model, aneurysms completely disappeared after one month of administration. All atorvastatin calcium (AtvCa) in the PLCL nanofiber-covered stents was fully embedded on the artery, with the exception of PLCL-AtvCa5. Among the various formulations, PLCL-AtvCa10 gave the best results, including reendothelization and intimal hyperplasia .μM rosuvastatin solution (400 : 100 μL) [In the study by Liu et al., an aneurysm was induced in the right common carotid artery (CCA) of the rabbit. A heparin-rosuvastatin-loaded P(LLA-CL) nanofiber-covered stent was then placed on the aneurysms. The DSA follow-up showed that after 30 days, aneurysms decreased and gradually disappeared. No stenosis in the CCAs was found in either the short- or long-term follow-ups. This was also seen among different formulations. The best effectiveness in reducing in-stent stenosis and thrombosis was shown by Rosu 100 with 15% heparin and 20  100 μL)   treatment has remained a serious medical problem till date. A sciatic nerve crush injury rat model was used by Haidar et al. to validate the efficacy of composite nanofibers incorporating alpha lipoic acid (ALA) and atorvastatin (ATR) . They evors well .Herein, studies related to various in vitro  and in vFor bone regeneration, simvastatin and rosuvastatin in in vitro studies applied to two different cell lines hADSCs and MC3T3-E1 showed induction of cell proliferation, adhesion, and osteogenesis differentiation. Furthermore, in vivo studies in which simvastatin nanofibers were applied to calvarial or femoral defects revealed bone healing ability, with one exception. This particular study showed no difference between the statin nanofiber and free polymer nanofiber, and further investigation needs to be done.In vitro studies in the field of endothelial stenosis and thrombosis demonstrated that atorvastatin and rosuvastatin NFs-covered stents caused HUVEC proliferation and adhesion in a dose-dependent manner. In one study, a low dose (5 ± 1 nM) of AtvCa increased proliferation, whereas at a higher dose (12 nM), HUVEC proliferation decreased. Addition of an anticoagulant agent like heparin to the formulation showed a synergistic effect. Moreover, addition of paclitaxel showed inhibition in SMC proliferation. Reduction of SMC proliferation, anticoagulation, RBC attachment inhibition, reendothelization, and intima coverage increased VEGF-A levels, with simultaneous decrease in platelet adhesion. This was confirmed from other in vitro findings, which overall confirm the antithrombosis and reendothelization actions. In all in vivo studies in rabbits, where CCA aneurysm was induced, disappearance of the clot was observed at particular time durations.α and IL-6 secretions. While the reduction of IL-6 occurred in a dose-dependent manner, TNF-α reduction was not dose related. This anti-inflammatory effect is also detected in some endothelial stenosis and bone regeneration studies.One study on atorvastatin NFs revealed that anti-inflammatory effect of these agents is related to the decrease in both TNF-Finally, experiments involving peripheral nerve injuries like sciatic nerve crush have also been treated by a NF formulation containing atorvastatin and ALA.Following these studies, it can be concluded that statin NFs, specifically atorvastatin, simvastatin, and rosuvastatin, are effective in healing bone fracture, aneurysm, and sciatic nerve crush as a result of their sustained drug release profile, although clinical trials still need to be performed to confirm these data. Therefore, these collective results reveal that statins are pleiotropic agents that are effective in various different fields apart from hypercholesterolemia. In addition, more studies are still needed to discover new applications of these promising agents.


During the ongoing coronavirus disease 2019 (COVID-19) outbreak, robust detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key element for clinical management and to interrupt transmission chains. We organized an external quality assessment (EQA) of molecular detection of SARS-CoV-2 for European expert laboratories. An EQA panel composed of 12 samples, containing either SARS-CoV-2 at different concentrations to evaluate sensitivity or other respiratory viruses to evaluate specificity of SARS-CoV-2 testing, was distributed to 68 laboratories in 35 countries. During the ongoing coronavirus disease 2019 (COVID-19) outbreak, robust detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key element for clinical management and to interrupt transmission chains. We organized an external quality assessment (EQA) of molecular detection of SARS-CoV-2 for European expert laboratories. An EQA panel composed of 12 samples, containing either SARS-CoV-2 at different concentrations to evaluate sensitivity or other respiratory viruses to evaluate specificity of SARS-CoV-2 testing, was distributed to 68 laboratories in 35 countries. Specificity samples included seasonal human coronaviruses hCoV-229E, hCoV-NL63, and hCoV-OC43, as well as Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, and human influenza viruses A and B. Sensitivity results differed among laboratories, particularly for low-concentration SARS-CoV-2 samples. Results indicated that performance was mostly independent of the selection of specific extraction or PCR methods. As both specific medications and approved vaccines are not available yet, public health strategies need to focus on containment and mitigation measures. Robust detection of acute SARS-CoV-2-infected individuals, typically done by real-time reverse transcription PCR (rRT-PCR), is crucial for clinical management, surveillance and to interrupt transmission chains  has infected over 66 million individuals and caused more than 1,500,000 deaths globally . All samples were heat-inactivated following Matheeussen et al. . Two independent laboratories validated the composition of the EQA panel and the result submission platform before distribution to all other EQA participants.TC) values, extraction methods and type of PCR(s). Laboratories were asked to treat and evaluate the EQA samples according to their routine diagnostic workflow, which could include the use of multiple assays. To ensure uniform interpretation, a sample was scored as positive if at least one SARS-CoV-2 test gave a positive result. No TC threshold was applied.Result reporting was done via an online submission form by which laboratories could score their samples  and provide information such as cycle threshold , different steps of the diagnostic workflow were analyzed individually. For this purpose, each variable was analyzed statistically, including the results of all EQA participants that provided reliable information for it. For some variables, such as applied PCR assays, we both tested for a general trend of correlation between the variable and the number of correct results and compared the amounts of correct results between specifically selected fractions of the variable, e.g., comparing the results between two specific real-time RT-PCR assays.Sixty-eight laboratories from 35 countries, i.e., 28 of 30 European Union/European Economic Area countries, five of seven European Union preaccession countries, and two other European countries, reported EQA results .P < 0.001; Spearman correlation test)  . CT valuntration . Sixty-tntration .As the performance in an EQA is the outcome of different steps in the diagnostic workflow combined, preferably different workflows should be compared. These steps comprise, e.g., resuspension of the panel samples, extraction method/kit used, change in nucleic acid (NA) concentration during extraction, and type of PCR assay, including the genomic target and the number of different genomic targets/tests performed. Among the participants of this EQA, there was an extensive variety of rRT-PCR methods that was used . The EQA performance was not correlated with the type of extraction kit used  and ranged between 87.9% and 93.8% correct results . In most extraction protocols NA concentration is increased. However, the extent of NA concentration was not correlated with diagnostic sensitivity .The performance among laboratories conducting manual NA extraction (92.4% correct tests) and laboratories conducting automated extraction (89.8% correct tests) did not differ significantly . However, results were significantly better with the best-performing test , Corman N assay (P = 0.008), and the Viasure ORF1 assay (P = 0.011). The performances among commercial tests (84.6% correct results) and in-house tests (85.7% correct results) did not differ significantly . As transcription in coronaviruses typically varies among different genomic and subgenomic regions, target sites of the applied assays may affect the diagnostic sensitivity   .The overall performance within this EQA was variable. Most false results were reported for low-concentration SARS-CoV-2 samples. While infectious SARS-CoV-2 patients commonly have high viral titers around the first day of illness , 10 an oTen laboratories showed problematic EQA performances, including five laboratories which had false-positive results. Notably, the laboratories reporting false-positive results applied both extraction kits and rRT-PCR tests also used by other laboratories. False-positive test results could thus be a consequence of contamination during sample handling and extraction or lot-specific contamination of rRT-PCR kits or oligonucleotides, as recently reported . Either Compared to the performance of first-line, routine clinical laboratories in a recently published SARS-CoV-2 EQA, the sensitivity of participants in our EQA was lower . HoweverNotably, the results indicate that performance can be increased by harmonized workflows, rather than by the selection of specific extraction or rRT-PCR kits. None of the individual components of the workflow that were analyzed for this study were able to significantly influence the overall performance of a laboratory.The conduction of follow-up EQAs will be essential to further support European laboratories to systematically improve and maintain diagnostic capabilities, while the need for a robust global detection capability requires global EQA programs.


GBA) are the most prevalent genetic risk factor for Lewy body disorders (LBD)—collectively Parkinson’s disease, Parkinson’s disease dementia and dementia with Lewy bodies. Despite this genetic association, it remains unclear how GBA mutations increase susceptibility to develop LBD. We investigated relationships between LBD-specific glucocerebrosidase deficits, GBA-related pathways, and α-synuclein levels in brain tissue from LBD and controls, with and without GBA mutations. We show that LBD is characterised by altered sphingolipid metabolism with prominent elevation of ceramide species, regardless of GBA mutations. Since extracellular vesicles (EV) could be involved in LBD pathogenesis by spreading disease-linked lipids and proteins, we investigated EV derived from post-mortem cerebrospinal fluid (CSF) and brain tissue from GBA mutation carriers and non-carriers. EV purified from LBD CSF and frontal cortex were heavily loaded with ceramides and neurodegeneration-linked proteins including alpha-synuclein and tau. Our in vitro studies demonstrate that LBD EV constitute a “pathological package” capable of inducing aggregation of wild-type alpha-synuclein, mediated through a combination of alpha-synuclein–ceramide interaction and the presence of pathological forms of alpha-synuclein. Together, our findings indicate that abnormalities in ceramide metabolism are a feature of LBD, constituting a promising source of biomarkers, and that GBA mutations likely accelerate the pathological process occurring in sporadic LBD through endolysosomal deficiency.Mutations in glucocerebrosidase (The online version contains supplementary material available at 10.1007/s00401-021-02367-3. GBA) gene mutations are the most prevalent genetic risk factors for LBD -labelled alpha-synuclein by growing the expression cells in M9 minimal media -SOFAST-HMQC [15N-dimension [resulting from 256 IPAP (in-phase/antiphase) increments]  .1H-90° pulse length, the number of scans, and the dilution factor  and 12 GBA mutation non-carriers [6 LBD  and 6 controls] [EV were purified from frontal cortex tissue from 13 ontrols]   (Online Resource Table 4). Gene Ontology (GO) enrichment analysis of the frontal cortex EV proteomes confirmed the high purity of the samples  using immuno-electron microscopy. We detected alpha-synuclein in both patient and control samples (9 LBD and 4 control samples screened), in association with vesicles typically 40–200 nm diameter . Nevertheless, exploiting the more sensitive -NMR spectra [GBA WT: F94, L113, Y125; PDD GBA MT: I112; DLB GBA WT: L113). The observation of effects within the carboxy-terminus of alpha-synuclein might indicate no direct vesicle interaction since this interaction occurs via the amino-terminus [2+ binding to this region enhancing its hydrophobicity has been reported [As LBD EV are filled with potentially neurotoxic lipid species and neurodegeneration-linked proteins, we thought to explore the interaction of wild-type alpha-synuclein with LBD EV. We used solution NMR spectroscopy, a technique used previously to interrogate alpha-synuclein–biological membrane binding at the atomic level . Additio spectra  indicateterminus ; howeverreported , 52.GBA MT-derived vesicles. Overall, in both samples, a significant loss of signal intensity could be observed, indicating the presence of an NMR-indiscernible large molecular alpha-synuclein species, presumably fibrils. These aggregation-inducing effects of vesicles were particularly strong for the DLB sample analysed, which showed only very few and weak resonances , and oxidative stress response/alpha-synuclein chaperone activity (PARK7/DJ1) [The observation of the distinctive cleavage patterns in the different PDD EV prompted us to identify proteases within EV . Notably, several different cellular proteases previously implicated in alpha-synuclein processing were identified that have the potential to modulate alpha-synuclein aggregation , 10. Foropensity . The DLBvity PARK/DJ1 [7, RK7/DJ1)   assay using GBA mutations and an increased risk of developing PD and DLB is well established [GBA mutations do not develop LBD, with the estimated risk of developing parkinsonism at 80 years being 9.1% [GBA mutations on the brain lipidome in LBD as we hypothesised that GBA mutations may be associated with the loss of homeostasis in lipid metabolism. We extended our hypothesis to brain secreted EV, as pathological changes in neural cells may lead to an altered composition of the EV lipidome and proteome and consequently impact disease signalling and spread to other cells.The association between ablished , 82, buting 9.1% . In the post-mortem tissues [GBA mutation status, indicating elevated ceramides are a marker of LBD. Although ceramide levels are similar in post-mortem GBA mutation carriers and non-carriers, the rate of ceramide accumulation may be accelerated in GBA mutation carriers, and this is suggested in plasma and serum studies in LBD. Notably, increased levels of ceramides have been found in plasma of PD cases without GBA mutation [GBA mutation carriers demonstrated elevated ceramides in serum compared to non-carriers [GBA mutation carriers and non-carriers; however, carriers of severe GBA mutations tended to have higher CSF ceramide, indicating the biochemical link between GBA mutations and elevated disease risk [Our study clearly indicates the specific accumulation of ceramide species in LBD signifying abnormal sphingolipid metabolism. This observation corroborates other reports of changes in sphingolipid composition of LBD  tissues , 21, tho tissues , 93. Var tissues . Our lipmutation , and PD carriers . No signase risk .Our targeted lipidomic approach specifically analysing the levels of hexosylceramides and in particular glucosylceramides that are a substrate for GBA, indicates no change in overall levels compared to controls. This latter finding indicates that, despite mutation of GBA leading to significantly reduced enzyme activity, there is sufficient enzyme activity remaining in heterozygous mutation carriers to effectively metabolise glucosylceramides. These findings correspond with previous animal and cell work demonstrating that heterozygous mutation leads to only mild changes and retained ability to handle cellular glucosylceramide species . Our resSince EV mediate transfer of alpha-synuclein species between cells, we investigated the lipid composition of EV. Global lipidomic analysis of CSF EV revealed that LBD EV are laden with ceramide species and show markedly elevated ceramide levels when compared to control EV, indicating that the presence of elevated ceramide species is an indicator of an LBD related process. While the global lipidomic approach to ceramide species in bulk tissue samples showed modest but significant changes, the elevations of ceramides in CSF EV suggest that EV may provide a more specific indicator of ceramide dysmetabolism with changes in ceramide levels associated with EV production pathways. If ceramide dysmetabolism is an indication of underlying pathological processes associated with LBD , then reductions of specific ceramide levels in CSF EV may provide an indicator of therapeutic effectiveness with agents targeting pathological processes in LBD.GBA mutation carriers [GBA mutation contributes to an earlier overload of ER compared to typical late onset cases [GBA mutation carriers shown here and elsewhere [We have previously shown that altered endoplasmic reticulum (ER) stress and unfolded protein response is present in LBD and in carriers , and suget cases , 74, 75.et cases , ceramidet cases . Alpha-set cases . As ceraet cases , upregullsewhere  may alsolsewhere . A patholsewhere , 59, 95.Sphingolipids are important molecules in cellular signal transduction. Ceramide is the backbone of complex glycosphingolipids, and ceramide signalling can culminate in apoptosis, autophagy and cell cycle arrest. Cellular ceramide levels need to be balanced to regulate cell death and proliferation, a feature of a key tumour suppressor . AccumulGBA mutation carriers and non-carriers. We also demonstrate that alpha-synuclein is present in EV in LBD and control samples, but with no significant changes in alpha-synuclein levels between patients and controls using ELISA and western blot analyses. It is therefore possible that an altered structure of alpha-synuclein in EV, such as fibrils, truncation, or strong alpha-synuclein–EV membrane binding detected by our NMR analysis, may be ways in which EV act as seeds for alpha-synuclein aggregation. The high abundance of heat shock 70 kDa protein (HSPA4) in DLB EV determined by mass spectrometry analysis (see Online Resource Table 4) might be an indicator of misfolded alpha-synuclein since HSPA4 can directly bind to alpha-synuclein fibrils and cause fibril disassembly [2+ ions and by the presence of three distinct protein regions within alpha-synuclein [Our observations show that brain alpha-synuclein pathology is similar in LBD assembly . Our NMRynuclein , 52. Usiynuclein , 57.The observation of the distinctive cleavage patterns in the different PDD EV by NMR correlated with the presence of proteases, such as caspases, in the EV proteomes, that have been previously implicated in facilitating alpha-synuclein aggregation in vitro , and likOur results with EV using protein aggregation assay also strongly implicate alpha-synuclein fibrillisation upon EV membrane binding. Intact EV membranes are essential for the process of EV-mediated aggregation of alpha-synuclein . It is uGene Ontology Biological Process term enrichment analysis of differentially expressed proteins identified in the LBD and control EV revealed apparent downregulation of GABAergic synaptic activity and upregulation of pro-inflammatory glycolytic metabolism in DLB EV. We have previously described altered GABAergic neuronal function in LBD visual cortex, and the current study indicates that functional changes in GABAergic neurons may be an inherent feature of LBD . The accGBA mutation status, and these ceramide alterations are prominent within EV. These ceramide changes may be related to the ER stress caused by GBA protein misfolding and general loss of endolysosomal homeostasis and lysosomal degradation capacity, and consequently cause abnormal pathogenic alpha-synuclein to be associated with EV. This elevation of ceramide in CSF EV and the highly alpha-synuclein aggregation-promoting properties of EV may provide a diagnostic and therapeutic marker of LBD.In summary, our findings indicate that abnormal ceramide levels are a feature of LBD independent of Supplementary file1 (DOCX 5287 KB)Supplementary file2 (XLSX 214 KB)Below is the link to the electronic supplementary material.


Aberrant deployment of the immune response is a hallmark pathogenic feature of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related disease (COVID-19), possibly accounting for high morbidity and mortality, especially in patients with comorbidities, including immune-mediated disorders. Immunisation with SARS-COV-2 vaccines successfully instructs the immune system to limit viral spread into tissues, mitigate COVID-19 manifestations and prevent its most detrimental inflammatory complications in the general population. Patients with immune-mediated diseases have been excluded from vaccine registration trials, foreclosing the acquisition of specific efficacy and safety data. In this review, we aimed to summarise and critically discuss evidence from real-world studies addressing this issue to provide a comprehensive view of the impact of vaccination practices in patients with allergy, autoimmunity or immunodeficiency. We analysed clinical and laboratory data from 34 studies involving more than 13,000 subjects with various immune disorders who were vaccinated with mRNA- DNA- or inactivated viral particle-based vaccines. These data globally support the safe and effective use of SARS-CoV-2 vaccines in patients with immune-mediated diseases, although patient-tailored strategies to determine vaccination timing, vaccine choice and background therapy management are warranted to optimise vaccination outcomes. More data are needed regarding patients with primary immunodeficiencies.  History of the Peloponnesian War when describing the unique features of people surviving the V century BC Athens’ epidemic. This first notion was outlined as the ability of the host to effectively resist the most detrimental consequences of an infection after a first exposure to its aetiological agent [An embryonic concept of immunological memory had already been introduced by Thucydides in his al agent . In lateal agent . Growingal agent . As in the case of ancient Athenians, we are currently facing a pandemic crisis caused by a novel infectious agent, namely severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes a multi-organ disorder characterised by systemic inflammation, prominent respiratory symptoms and relatively high rates of morbidity and mortality, conventionally referred to as coronavirus disease 2019 (COVID-19). In contrast to Thucydides’ contemporaries, we have been fortunate enough to have the scientific and industrial capacity to develop, test and clinically validate effective vaccination tools against SARS-CoV-2 in a relatively short time. Nonetheless, this age also poses novel challenges to the widespread practice of vaccination. Vaccine hesitancy is a growing issue for public health, especially in this pandemic setting. This reticent attitude may be possibly explained by a combination of factors, including (a) ever increasing expectations from Science and Medicine , (b) a generalised progressive loss of trust in public institutions, and (c) misinformation ,5.The challenge to overcome vaccine hesitancy due to fears of potential detrimental effects of vaccination is enhanced for people living with chronic dysfunctions in the physiology of the immune response, such autoimmune/rheumatic or allergic diseases and/or primary immunodeficiencies. These subjects might in fact be at increased risk of severe or complicated COVID-19 ,7,8 due With the beginning of mass-vaccination campaigns in multiple countries, real-world data about efficacy and safety of SARS-CoV-2 vaccines in patients with immune-mediated diseases have progressively been published . In thisWe interrogated the PubMed database for peer-reviewed articles published until 15 July 2021 matching the following search criteria: “COVID-19 vaccine AND autoimmune diseases”, “COVID-19 vaccine AND rheumatic diseases”, “SARS-CoV-2 vaccine AND autoimmune diseases”, “SARS-CoV-2 vaccine AND rheumatic diseases”, “COVID-19 vaccine AND allergy”, “COVID-19 vaccine AND hypersensitivity”, “SARS-CoV-2 vaccine AND allergy”, “COVID-19 vaccine AND mast-cell disease”, “SARS-CoV-2 vaccine AND mast-cell disease”, “SARS-CoV-2 vaccine AND hypersensitivity”, “COVID-19 vaccine AND primary immunodeficiency”, “SARS-CoV-2 vaccine AND primary immunodeficiency”. For aggregate analysis, we considered studies with original data only and excluded those describing single patient cases and reports of de novo autoimmune manifestations. We also excluded articles reporting duplicated data and those updated with larger samples by the same authors studying the same cohorts. For a comprehensive analysis of safety data, articles describing patients with incomplete vaccination schedules were considered separately. Works focusing on adverse events only, without data from the context cohorts, were excluded. Articles describing surrogate laboratory or clinical efficacy data in patients who did not complete their vaccination schedule or who received unscheduled supplementary vaccine doses were also excluded.n = 21) and safety after one (n = 22) and two (n = 13) doses of SARS-CoV-2 vaccines. Most studies (33/34) included patients vaccinated with the BNT162b2  or the m-1372  mRNA vaccines. Only 731 patients receiving recombinant adenoviral vector vaccines  and 51 receiving inactivated virus vaccines  have been described in 7/34 studies [Thus far, several real-world observational studies, describing the clinical features and vaccination outcomes of patients with immune-mediated diseases, have been published. As of 15 July 2021, 34 studies involving more than 13,000 patients have provided data regarding efficacy/immunogenicity after complete vaccinal cycles . The majority of data came from subjects with an autoimmune/rheumatic disorder as primary diagnosis (84%). In 16 of 25 studies with available data, the average age of the participants was between 40 and 55 years  . Clinical and laboratory outcomes were highly heterogeneous, reflecting the lack of consolidated data about the characteristics of the immune response in COVID-19 and anti-SARS-CoV-2 vaccination. Similar to studies investigating COVID-19 prevalence in patients with immune mediated-diseases ,68,69,70A total of 24 studies reporting on more than 11,000 patients with autoimmune/rheumatic diseases have been published so far. Beside one large study focused on elderly patients with inflammatory bowel diseases , most stAdverse events were relatively frequent after the first, second or both doses in patients with rheumatic diseases, consistent with data from the general population . Among sFive studies (three with data from subjects who completed the vaccination schedule) compared the rates of vaccination-related adverse events between patients and healthy controls, yielding conflicting results. Specifically, one study reported higher frequencies of post-vaccination symptoms in patients than in controls after the first vaccination dose , while tOnly two studies attempted exploratory analyses to investigate potential factors associating with adverse events, and consistently reported a higher prevalence of post-vaccinal symptoms in women and younger subjects ,51, in lData regarding recombinant adenoviral vector vaccines are limited to four studies ,35,36,38In the light of concerns about the potential of vaccines to reactivate or exacerbate inflammation in patients with autoimmune disorders, disease flares were crucial parameters within safety analyses in many studies. Overall, disease flares have been reported in up to 8% of patients with autoimmune/rheumatic disorders . HoweverOf more than 8000 patients with available follow up data for an average time of 4–6 weeks after vaccination, less than 0.1% had evidence of incident COVID-19 . AlthougPatients receiving B-cell depleting treatments accounted for more than 10% of patients with available data  and consInstead, anti-SARS-CoV-2 T-cell responses appeared relatively preserved even in patients treated with B-cell depleting agents, although the limited number of studied subjects (13/290 receiving B-cell depletion) prevents definite conclusions . Beside B-cell depletion, the use of glucocorticoids, mycophenolate, fingolimod, abatacept and multi-drug immunosuppressive therapies was also associated with lower response rates according to multiple studies ,52,60,61Despite evidence of increased risk of COVID-19-related morbidity and mortality in patients with primary immunodeficiencies (PID) , who werMore than 2100 patients with allergies with or without other immune-mediated diseases receiving anti-SARS-CoV-2 vaccination have so far been described . RespiraNo data have been published about the clinical and biological efficacy of anti-SARS-CoV-2 vaccination in patients with allergic disorders without other known immunological comorbidities. This knowledge gap might be explained by the common assumption that immunisation capacity in patients with allergic disorders should be similar to that of the general population, which however is supported by limited data only . FurtherAccording to the currently published literature , patientstricto sensu accounting for these manifestations [Local reactions to the first dose apparently did not associate with more severe reactions to the second dose in line with evidence from registration trials , and sugstations . Nonethestations . A largestations  reportedThis result should, however, be interpreted considering the use of a variety of prophylactic strategies to minimise the risk of hypersensitivity reactions in patients with allergies. From a therapeutic point of view, the use of antihistamines and/or antileukotrienes has been reported in patients with mast-cell disorders ,40. InstReal-world data support the safe and effective use of SARS-CoV-2 vaccines in patients with immune-mediated disorders. More extensive evidence of immunogenicity and safety has been acquired for mRNA-based vaccines compared to recombinant adenoviral vector vaccines. Taken together with efficacy signals observed in the general population, these data support the preferential use of mRNA vaccines in patients with dysregulated immune response. Despite relatively low rates of adverse events or immunisation failures among people with autoimmune/rheumatic disease, allergy or immunodeficiency, disease- and treatment-related factors might have relevant clinical implications, at least for selected patient subsets. In particular, drugs interfering with antigen presentation and B-cell biology, might impair effective humoral, but possibly not cellular immune response to vaccines, while treatments affecting other, possibly redundant inflammatory pathways, probably have less significant impacts on immunisation ,91. Furt


Cardiovascular diseases (CVDs) represent a major global health problem, due to their continued high incidences and mortality. The last few decades have witnessed new advances in clinical research which led to increased survival and recovery in CVD patients. Nevertheless, elusive and multifactorial pathophysiological mechanisms of CVD development perplexed researchers in identifying efficacious therapeutic interventions. Search for novel and effective strategies for diagnosis, prevention, and intervention for CVD has shifted research focus on extracellular vesicles (EVs) in recent years. By transporting molecular cargo from donor to recipient cells, EVs modulate gene expression and influence the phenotype of recipient cells, thus EVs prove to be an imperative component of intercellular signaling. Elucidation of the role of EVs in intercellular communications under physiological conditions implied the enormous potential of EVs in monitoring and treatment of CVD. The EVs secreted from the myriad of cells in the cardiovascular system such as cardiomyocytes, cardiac fibroblasts, cardiac progenitor cells, endothelial cells, inflammatory cells may facilitate the communication in physiological and pathological conditions. Understanding EVs-mediated cellular communication may delineate the mechanism of origin and progression of cardiovascular diseases. The current review summarizes exosome-mediated paracrine signaling leading to cardiovascular disease. The mechanistic role of exosomes in cardiovascular disease will provide novel avenues in designing diagnosis and therapeutic interventions. Described initially as debris with no functional significance or potential involvement in the clearance of damaged cellular components, extracellular vesicles (EVs) represent a heterogenous population of vesicles. Accumulating evidence suggesting the involvement of EVs in intercellular communication by virtue of the transfer of bioactive molecules invoked scientific interest in the field of EVs , 2. IncrCVDs represent a major health concern resulting in significant mortality and morbidity in developed and developing countries –14. Advevia the recruitment of immune cells. The EVs-mediated transfer of effector molecules suggested a functional correlation between plaque-derived EVs and plaque development in atherosclerosis  phenotype transformation, and plaque formation . In respclerosis . Atherosclerosis .Elevated levels of Pro-atherogenic inducers, oxidized LDL (ox-LDL), and homocysteine (Hcy) were reported as risk factors for atherosclerosis and cardiovascular morbidities. In rat aortic endothelial cells (RAECs), ox-LDL and Hcy induced the dose-dependent release of HSP70-containing exosomes. The exosome-dependent HSP70 release triggered activation of monocytes, resulting in monocyte adhesion to ECs and hence Pro-atherogenic, pro-inflammatory effects in the onset of atherosclerosis . EndotheDiverse physiological effects were exerted by the platelet-derived exosomes on the inflammation process in response to pathogenic stimuli. Platelet-derived EVs reported being involved in the mitogen-induced proliferation of SMCs in atherosclerosis . Similarvia prevention of two important factors of atherothrombosis i.e., foam cell formation and activation of platelets in response to damaged blood vessels. The endothelial damage is characterized by the formation of foam cells followed by activation of platelets and recruitment of immune cells i.e., macrophages and T lymphocytes. CD36, a type II scavenger receptor, through microparticle binding and intracellular signaling leads to platelet aggregation and atherothrombosis. Platelet-derived exosomes increase protein ubiquitination and enhance proteasome degradation of CD36. This leads to reduced platelet aggregation and adhesion to collagen matrix which attenuates occlusive thrombosis in a FeCl3-induced arterial injury model  increased the proportion of Th17 cells in-vitro and in-vivo. Regulated by Akt and NF-kB pathways, IL-6 contained in CD137-Exo activated Th17 cell differentiation. Lipopolysaccharide (LPS)-induced inflammation in ECs showed IL-17-mediated apoptosis, inhibited cell viability, and increased lactose dehydrogenase (LDH) release. CD137-Exo promoted Th17 cell differentiation via the NF-kB pathway-mediated IL-6 expression and atherosclerosis progression (CD137 is a tumor necrosis factor receptor (TNFR) superfamily member which ligates to its ligand, CD137 L for activation of CD137 signaling. Ten-eleven translocation 2 (TET2), is a DNA demethylase, a member of the methylcytosine dioxygenase family that oxidizes 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmc). It plays a crucial role in gene activation through DNA demethylation mediated by the conversion of 5hmc to unmethylated cytosine. TET2 has been reported to be involved in the regulation of SMCs plasticity by inactivating dedifferentiation genes and activating contractile marker genes of SMCs. Activation of CD137 signaling in ECs plays an important role in the induction of immune and inflammatory responses in atherosclerosis. The activation of CD137 inflammatory signaling in endothelial cells (ECs) downregulates TET2 expression in EC-derived exosomes. This CD137 signaling-mediated exosomal downregulation of TET2 expression resulted in a pro-proliferation, phenotype switch of VSMCs, and intimal hyperplasia after arterial injury. The phenotypic switching and intimal hyperplasia of VSMCs induced by activation of CD137 signaling attenuated by overexpression of TET2 expression in EC-derived exosomes. This corroborated that activation of endothelial CD137 signaling promoted neointimal formation by reducing the expression of TET2 in ECs and ECs-derived exosomes, leading to the reduced exosome-mediated TET2 transfer to VSMCs . T helpegression .via downregulation of ATP-binding cassette transporter (ABCA1 and ABCG1)–mediated cholesterol efflux facilitated macrophage foam cell generation. HFD-VAT exosome-induced increased lipid accumulation in macrophages was associated with decreased cellular levels of LXR-α, a nuclear receptor known to mediate the transcription of ABCA1 and ABCG1. Thus, exosomes exerted the dual effects of repression of ABC transporter expression as well as cholesterol efflux. In vitro, HFD-VAT exosomes induced M1 phenotype transition and proinflammatory cytokine (TNFα and IL-6) secretion in macrophages. Intravenous administration of HFD-VAT exosomes to hyperlipidemic apolipoprotein E–deficient mice leads to exacerbated atherosclerosis, suggesting their proatherogenic effects by modulation of foam cell formation and M1 phenotype polarization of macrophages  rophages . In the rophages . Though rophages –45, furtvia downregulation of superoxide dismutase 2 (SOD2) expression. Intravenous administration of miR-146a enriched exosomes derived from oxLDL-treated THP-1 cells to a murine model of atherosclerosis exhibited exacerbation of atherosclerosis. This suggested exosomal miR-146a-induced promotion of atherosclerosis via cellular oxidative stress and NETs formation  levels, and subsequently the release of NET by neutrophils. In vivo studies involving the atherosclerotic murine model showed accelerated progression of atherosclerosis upon administration of ox-LDL-exosome  generation and Neutrophil extracellular traps (NETs) released ormation , 48. In--exosome . The intH. pylori PMSS1 infection and the progression of atherosclerosis revealed exosomal CagA-mediated pathogenic progression. H. pylori PMSS1 is a CagA-positive strain that can translocate CagA into host cells. Through Type-IV secretion system (T4SS) translocation into host cells, CagA is a major virulence factor that is involved in upper gastrointestinal diseases. CagA-positive H. pylori infection did not initiate but aggravated the progression of atherosclerosis via exosome CagA-mediated macrophage foam cell formation. H. pylori-infected gastric epithelial cells-derived exosomes (Hp-GES-Exo) promoted macrophage-derived foam cell formation and lesion development via exosomal CagA in-vitro and in vivo. Mechanistically, exosomal CagA downregulated the expression of transcriptional factors PPARγ and LXRα and led to the suppression of transcription of cholesterol efflux transporters. Thus, the study elucidated another mechanism i.e., the effect of H. pylori infection on the progression of atherosclerosis (via modulation of the proatherogenic phenotype of VSMCs. Exosomes from nicotine-stimulated macrophages (NM-Exo) promoted VSMCs migration and proliferation. NM-Exo was found to be enriched in miR-21-3p which modulated the proatherogenic phenotype of VSMCs by regulation of phosphatase and tension homolog (PTEN). The study suggested nicotine-stimulated macrophages facilitated atherosclerosis progression by enhanced VSMCs migration and proliferation via exosomal miR-21-3p- mediated regulation of PTEN  and activated NRF2 signaling, which inhibited ROS accumulation and subsequently DCs maturation. In vivo experiments suggested an association between atherosclerosis progression and decreased MALAT1 content in VECs-exosomes. Thus, loss of MALAT1 expression in ox-LDL-treated VECs- derived exosomes lead to the maturation of DCs in the development of atherosclerosis  has been implicated in the development of atherosclerosis. Involved in tumorigenesis, Metastasis- associated lung adenocarcinoma transcript-1 (MALAT1) is a long non-coding RNA (lncRNA), and its downregulation promotes atherosclerosis. clerosis . Exosomeclerosis .in-vitro study reported the role of exosome-mediated lncRNA ZEB1-AS1 and its underlying mechanisms in atherogenesis in HUVECs. In ox-LDL-stimulated HUVECs, exosomal lncRNA ZEB1-AS1 facilitates cell injury by miR-590-5p/ETS1 Axis through the TGF-β/Smad Pathway , potent proinflammatory lipid mediators, can be induced by exosomes –64. EssaHF is a progressive condition marked by the inefficiency of the heart to pump enough blood to support the physiological demand of the body. It is characterized by signs and symptoms involving multiple organ systems such as the heart, liver, kidney, and lungs. The abnormal cardiac remodeling in HF includes cardiomyocyte hypertrophy, fibrosis, aberrant angiogenesis, and infiltration of inflammatory cells . Based oAs a systemic vascular disease with the underlying pro-inflammatory state has been implicated in development in HF, in an observational study comprising dyspnoea patients, EV markers have been investigated in patients with or without HF. The study reported the association of extracellular vesicle levels of systemic vascular markers such as CD14, SerpinG1, and SerpinF2 with the development of HF in patients suspected of acute HF . A recenin-vitro study investigating the effect of TGF- β on the development of HF suggested the onset of HF phenotype in cardiomyocytes via TGF- β-treated cardiac fibroblast-derived exosomes. Cardiomyocytes co-cultured with exosomes from TGF-β treated fibroblasts and fibroblasts from HF patients exhibited differentially expressed genes and transcriptional changes associated with hypertrophic cardiac cells. In concordance with the previous animal studies reporting fibroblasts exosome-induced cardiac hypertrophy  and 2 (AT2R). The CFs derived exosomes upregulated the expression of renin, angiotensinogen, AT1R, and AT2R while downregulated expression of angiotensin-converting enzyme 2 and increased production of Ang II in cultured cardiomyocytes thus overall exacerbated Ang II-induced cardiac hypertrophy. Ang II-induced release of CFs derived exosomes, therefore, suggested a paracrine mechanism of Ang II-mediated cardiac hypertrophy  pathway activation  co-cultured with fibroblasts or fibroblast conditioned media (FCM) demonstrated enhanced hypertrophy. The cardiomyocyte hypertrophy and CM regulated fibroblast adhesion/ proliferation was attributed to the paracrine interaction between CMs and fibroblasts  and subsequent increased angiotensin II (Ang II) levels have been implicated in pathological cardiac hypertrophy. Treatment of cultured cardiomyocytes fibroblasts (CFs) with Ang II enhanced the release of exosomes by activation of Ang II receptor type 1   . Hypoxicinducer) , 99. Theinducer)  and showinducer) . Increasinducer) .via the increased proliferation of HUVECs , thus upregulating CXCR4 and CXCL12 signaling , improving the environmental conditions in the damaged myocardium , which regulates cell cycle checkpoints, thus protecting against senescence  or heat shock protein 70 (Hsp70) from BM derived-stem cells , 95. In intained , 99. miRnescence , 105.via remote ischemic preconditioning (RIPC) mechanism  were secreted by cardiomyocytes cultured in hypoxic conditions  and PDLIM5 (PDZ and LIM domain 5)  derived miR-22 enriched exosomes resulted in reduced apoptosis of cardiomyocytes. Delivery of miR-22 to fibroblasts under hypoxic conditions leads to reduced cardiomyocyte apoptosis and fibrosis with increased angiogenicity of cardiomyocytes post-MI , 95. Theood flow , especiaood flow . Anotheromain 5) , 101. Thomain 5) . Exosomaertrophy , 92, 95.ertrophy , 99. Theertrophy , 95. MMPertrophy , 107 to ertrophy . SDF1 isertrophy , 109. MMertrophy .Exosomes can also have anti-inflammatory impacts by transferring miRNA-222 into recipient cardiac cells to lower endothelial ICAM-1 expression . In the via myosin heavy chain regulation during hypertrophy , the miRNA inside them can behave differently. For example, in peripartum cardiomyopathy, prolactin-induced miRNA-146a results in increased apoptosis and decreased proliferation , 96. Exovia repeated RIC. miRNA-29a inhibits ADAM12 and ADAM19, which are disintegrin proteases. miRNA-29a also inhibits the pro-fibrotic TGF-β-dependent signaling pathway, by inhibiting TGF-β and TAB1 (TGF-β-activated kinase 1-binding protein 1)  into the cell  . High le3II/LC3I . Blockin3II/LC3I , 101. mi3II/LC3I , 95. On 3II/LC3I . miR-1443II/LC3I , 99. Inc miR-144 , 101. Th miR-144 , 109. MSthe cell . GATA-4 the cell . PDGF-D rization . miRNAs via reprogramming and initiating exosome-mediated crosstalk between cardiac and BM cells  or Angiotensin II (Ang II)-treated cardiomyocytes (CM-Ang II) showed increased expression of miR-208a. Elevated expression of miR-208a was observed in cardiac tissues of murine models of MI and DOX-induced cardiomyopathy. Transfer of miR-208a from CM-Hypo or CM-Ang II to cardiac fibroblasts via exosomes promoted cardiac fibroblast proliferation and myofibroblast differentiation. Intramyocardial administration of exosomes derived from CM-Hypo or CM-Ang II led to the elevated cardiac miR-208a expression, decreased cardiac function, increased cardiac fibrosis in rats. Inhibition of miR-208a in post-MI rats attenuated cardiac fibrosis, improved cardiac function suggesting the role of cardiomyocyte-derived exosomal miR-208a in the progression of fibrosis post-MI. The pro-fibrotic effects of miR-208a are mediated by targeting Dyrk2, which via phosphorylation of nuclear factor of activated T-cells (NFAT) prevents its nuclear transfer for induction of fibrosis . fibrosis . Similaracute MI .in-vitro study demonstrated CPC-derived exosomes in the enhancement of endothelial cell migration via an extracellular matrix metalloproteinase inducer (EMMPRIN)-mediated mechanism. The exosomes released by CPCs upon transplantation resulted in increased capillary density and promoted angiogenesis (in-vitro treatment of cardiomyoblasts (H9C2) cells with CPCs-exosomes protected from oxidative stress by inhibition of caspase 3/7 activation, in-vivo delivery of CPCs-exosomes in myocardial ischemia/reperfusion murine model resulted in inhibition of cardiomyocyte apoptosis  has been increasingly investigated as a crucial cardiac regenerative mechanism post-ischemic injury. CPCs are a heterogeneous population of cells capable of differentiating into different cell types such as cardiomyocytes, SMCs, and ECs. Various studies reported improvement of LV function and reduction in scar size after intra-myocardial transplantation of CPCs after MI injury in preclinical models –121. An ogenesis . Anotherpoptosis .via the miR29a-mediated IGF-1 expression. This suggested exosomal circHIPK3 as a potential target in cardiomyocytes to control CMVECs dysfunction under oxidative conditions  are a class of endogenous non-coding RNAs that regulate gene expression in eukaryotes. Exosomal circHIPK3 derived from hypoxia-pretreated cardiomyocytes regulates oxidative damage in cardiac microvascular endothelial cells (CMVECs) nditions . Dou et unctions .Circulating exosomes from patients with ST-segment-elevation MI were enriched with sphingolipid species such as ceramides, dihydroceramides, and sphingomyelins. Significantly increased Ceramide, dihydroceramide, and sphingomyelin exosome content correlated with cardiac troponin, leucocyte count, and lower left ventricular ejection fraction .in-vitro model of hypoxic cardiomyocytes, Wang & Zhang showed the effect of exosomal lncRNA AK139128 on cardiac fibroblasts (CFs) proliferation, migration, and invasion. Exosomal lncRNA AK139128 derived from hypoxic cardiomyocytes stimulated CF apoptosis in-vitro and exacerbated MI in a rat model by promotion of CFs apoptosis and inhibition of cell proliferation , hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RCM), and arrhythmogenic cardiomyopathy , 131. Thin-vivo effect of DCM serum-derived exosome or exosomal cargo contributing to gene expression alteration, it explained the exosome-mediated, renin-angiotensin-aldosterone system (RAAS) and adrenergic system independent, pathological hypertrophy in cardiomyocytes (via a paracrine mechanism. In-vitro Ang II treatment of CFs enhanced exosome secretion by activation of Ang II receptor types 1 (AT1R) and 2 (AT2R). CFs derived exosomes enhanced Ang II production by cardiomyocytes by upregulation of renin, angiotensinogen, AT1R, and AT2R and downregulation of angiotensin-converting enzyme 2 expressions, thus resulting in cardiomyocyte remodeling. Proteomic analysis revealed the association of exosomal proteins with PI3K/Akt and MAPK pathways, hypertrophic cardiomyopathy, and dilated cardiomyopathy. The CF-derived exosomes activated mitogen-activated protein kinases (MAPKs) and Akt and thus upregulated RAS in cardiomyocytes. Treatment with GW4869 and DMA resulted in a reduction in Ang II-induced exosome secretion from CFs and pathological remodeling in cardiomyocytes. Thus, the study demonstrated Ang II-mediated stimulation of CFs exosome secretion and CFs exosome-mediated cardiomyocytes remodeling leading to hypertrophy and HF , is characterized by an increase in myocardial mass and volume. It is generally associated with the enlarged left ventricle and contractile dysfunction. Thirty five percentage of DCM cases are attributed to genetic causes, and acquired causes include hypertension, infection, inflammation, and endocrine disturbances. The limited literature on the role of exosomes in the pathogenesis of DCM warrants further investigation of the interplay between various cell-derived exosomes and its effect on cardiomyocyte and thus left ventricular remodeling characteristics of DCM. Serum-derived exosomes from pediatric DCM upregulated the expression of ANP and BNP in primary cardiomyocytes. The inhibition of exosome uptake by cytochalasin D ameliorated alteration in gene expression, further corroborated exosome-mediated cardiomyocyte hypertrophy. Though the study did not investigate the myocytes . Other smyocytes . Bang etmyocytes . Loss ofmyocytes . Cardiacmyocytes . Angiotey and HF . Other sy and HF –138. ExoDiabetes mellitus is a metabolic disease clinically identified as high blood glucose levels either due to lack of (Type I) or resistance to insulin . In the in-vivo diabetes mouse model, resulted in impaired Hsp20 mediated improvement in cardiac function. The study suggested Hsp20 transgenic cardiomyocytes-derived exosomes and their paracrine effect in Hsp20 mediated cardioprotection against diabetes-induced cardiac injury , a regulator of mitochondrial ATP production, miR-122 affected cardiac energy homeostasis and thus led to the development of metabolic cardiomyopathy  to cardiomyocyte (CM) contributed to pathogenesis to DbCM. The diabetic model of endothelial-specific Mst1 transgenic mice showed deteriorated cardiac function and aggravated insulin resistance. Uptake of CMECs-derived exosomal Mst1 by CMs inhibited autophagy and enhanced apoptosis. Under diabetic conditions, increased binding of Mst1 and Daxx resulted in inhibited glucose uptake by the disruption in the glucose transporter type 4 (GLUT4) membrane translocation through decreasing the interaction between Daxx and GLUT4. The study suggested pathogenic effects of diabetic CMECs-derived exosomal Mst1 on CM via autophagy inhibition, apoptosis promotion, and suppression of glucose metabolism leading to diabetic cardiomyopathy  under hyperglycemic conditions regulates the senescence of VSMCs. circRNA 0077930 from hyperglycemia-stimulated vascular endothelial cell exosomes induced VSMCs senescence by down-regulation of miR-622 expression and up-regulation of Kras, p21, p53, and p16 expression .Stat3 knockout mice heart showed an increased level of miR-146a. This suggested apart from ECs, fibroblasts as a potential source of 16K PRL induced secretion of miR-146a enriched exosomes in PPCM. Furthermore, significantly higher plasma levels of exosomal miR-146a were observed in patients with acute PPCM compared to healthy postpartum controls and patients with dilated cardiomyopathy. Blockade of PRL using the standard treatment for HF (beta-blocker and ACE inhibitor) and bromocriptine lead to the recovery of PPCM patients with normalization of circulating exosomal miR-146a levels. The study suggested the potential utility of miR-146a as a specific biomarker for PPCM (Peripartum cardiomyopathy (PPCM) is pregnancy-associated cardiomyopathy characterized by left ventricular dysfunction leading to HF during the late peripartum period and/or few months of postpartum , 156. Cafor PPCM .in-vitro study investigating mechanistic involvement of platelet-derived exosomes in endothelial cell apoptosis and development of septic vascular dysfunction, authors described the redox reactive role of platelet-derived exosomes in vascular dysfunction in sepsis. The study reported that the release of platelet-derived exosomes in sepsis was triggered by increased nitric oxide (NO) generation and exposure to lipopolysaccharide (LPS). The platelet-derived septic exosomes showed high content of NADPH oxidase, nitric oxide synthases (NOS), and NADPH oxidase regulatory protein disulfide isomerase (PDI). Further, these septic-like platelet-derived exosomes and NO promoted generation of active ROS/RNS by NADPH oxidase and NO synthase type II leading to caspase-3 activation and subsequently apoptosis of target ECs  death. It is associated with multiple organ dysfunctions with sepsis-induced cardiomyopathy being the leading contributor to septic death , 160. Thrget ECs . The sturget ECs . In anotrget ECs . The dowexosomes . Some ofexosomes , 169. miexosomes –172. Thuvia the transfer of miR-155 promoted pyroptosis in the heart. Exosomal miR-155 targeted and suppressed transcription of Forkhead Transcription Factor 3a (FoxO3a). Overexpression of FoxO3a or inhibition of miR-155 in uremic mice ameliorated pyroptosis and improved UCM, as evidenced by reduced heart weight, size, myocardial hypertrophy, interstitial fibrosis area with improved cardiac function. In-vitro, blockage of macrophage-derived exosomal miR-155 using GW4869 attenuated pyroptosis and improved UCM in cardiomyocytes indicated by reduced pro-inflammatory markers and apoptosis. This was the first report addressing the pathological contribution of pyroptosis mediated by macrophage-derived exosomal miR-155 in the development of UCM (Described as a complication of chronic kidney disease (CKD), uremic cardiomyopathy (UCM) is characterized by hypertension, cardiac hypertrophy, inflammation, and fibrosis. It accounts for approximately 50% of deaths due to CKD –175. Undt of UCM .Pulmonary arterial hypertension (PAH) is a rare disease characterized by progressive vascular remodeling of the pulmonary artery and increased pulmonary vascular resistance due to vasoconstriction and subsequently right ventricular (RV) failure . The remIncreased levels of EVs and their association with pulmonary vascular resistance and vascular dysfunction had been reported in patients with PAH. To explain the mechanistic role of EVs in PAH, Aliotta et al.  studied The pathological vascular remodeling associated with PAH has been attributed to an imbalance between cell proliferation and apoptosis. Translationally controlled tumor protein (TCTP) is a pro-survival and anti-apoptotic mediator that has been implicated in microtubule stabilization and secretion of proteins through exosomes , 186. TrValvular heart disease (VHD) is a leading cause of cardiovascular mortality and is associated with anatomical or functional abnormalities of cardiac valves. If untreated, VHD leads to progressive cardiac dysfunction. Stenosis and regurgitation/insufficiency represent major structural and functional aberration associated with VHD. Stenosis is characterized by the inadequate outflow of blood due to the narrowing of the Valvular orifice or failure of the valve to open completely. The inability of the valve in preventing backflow of blood due to impaired valve closure is termed regurgitation/insufficiency. VHD can be caused by congenital disorders or by acquired pathological conditions .Exosomal miRNAs have been implicated in the regulation of the formation of the cardiac valve during development. Aberrant miRNAs mediated up/down-regulation of target genes involved in valve formation may lead to the pathogenesis of VHD. During the cardiac valve formation, endocardial cells undergo endothelial to mesenchymal transition and constitute a major part of cardiac jelly in the atrioventricular canal . Regulatin-vitro osteogenic environment containing elevated levels of calcium and phosphate leads to an increase in the expression of osteogenic markers such as Msx2, Runx2, PiT-1, TNAP, and Alpl. Similar to MVs derived from calcifying chondrocytes and VSMCs, the pro-calcified VICs derived EVs showed up-regulation of Annexins I, II, III, IV, V, VI, VII, and XI. The in-vitro study demonstrated EVs secretion with elevated calcium and annexin VI from rat VICs cultured with high calcium and phosphate and suggested a role in calcified aortic valve disease evidenced by co-localization of annexin VI with EVs in the aortic valve  lead to calcified aortic valve disease due to extracellular matrix remodeling and mineral deposition. VICs can differentiate into either activated myofibroblast-like VICs or osteoblast-like VICs in hyperphosphatemic and pro-inflammatory conditions. The intercellular communication between VICs and Valvular endothelial cells (VECs) plays a vital role in valve homeostasis , 194. Thic valve . Carrionic valve .Despite enormous research on paracrine effects of exosomes in intercellular communication, understanding of the function of secreted exosomes and exosome-mediated interaction among various cardiac cells in the pathological condition is still in the nascent stage. The cardiovascular function depends on orchestrated communication between cardiomyocytes and other cardiac cell types. Molecular cargo transferred by exosomes modulates cardiac homeostasis in physiological conditions. In pathological conditions, alterations in proteins, nucleic acids, and other cargo molecules transported by exosomes lead to the onset and progression of CVDs. Considering the major role of exosome-mediated paracrine signaling in the modulation of cardiac function in physiological and pathological conditions, further investigations should focus on sources of exosomes, their effect on neighboring cells, and alteration in cargo composition in cardiac pathology. The delineation of molecular mechanisms emphasizing these biological processes will help in the discovery of exosome-based diagnostic and potential therapeutic tools against cardiovascular pathologies.AJ, AP, KG, and RS prepared the manuscript. AJ, AP, and VP wrote the main parts of the article and produced graphics. KG, RS, and VP reviewed and edited the manuscript. AJ and VP drafted the final version of the manuscript. All authors read and approved the final manuscript.This work received support from Canadian Institutes of Health Research , Natural Sciences and Engineering Research Council of Canada , and Libin Cardiovascular Institute, Cumming School of Medicine .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.