Effect of Sphingomyelinase-Treated LDLs on HUVECs.

Low-density lipoproteins (LDLs) exert a key role in the transport of esterified cholesterol to tissues. Among the atherogenic modifications of LDLs, the oxidative modification has been mainly investigated as a major risk factor for accelerating atherogenesis. Since LDL sphingolipids are also emerging as important regulators of the atherogenic process, increasing attention is devoted to the effects of sphingomyelinase (SMase) on LDL structural and atherogenic properties. The aims of the study were to investigate the effect of SMase treatment on the physical-chemical properties of LDLs. Moreover, we evaluated cell viability, apoptosis, and oxidative and inflammatory status in human umbilical vein endothelial cells (HUVECs) treated with either ox-LDLs or SMase-treated LDLs (SMase-LDLs). Both treatments were associated with the accrual of the intracellular ROS and upregulation of the antioxidant Paraoxonase 2 (PON2), while only SMase-LDLs induced an increase of superoxide dismutase 2 (SOD2), suggesting the activation of a feedback loop to restrain the detrimental effects of ROS. The increased caspase-3 activity and reduced viability observed in cells treated with SMase-LDLs and ox-LDLs suggest a pro-apoptotic effect of these modified lipoproteins on endothelial cells. Moreover, a strong proinflammatory effect of SMase-LDLs compared to ox-LDLs was confirmed by an increased activation of NF-κB and consequent increased expression of its downstream cytokines IL-8 and IL-6 in HUVECs.

MiR-422a promotes adipogenesis via MeCP2 downregulation in human bone marrow mesenchymal stem cells.

Methyl-CpG binding protein 2 (MeCP2) is a ubiquitous transcriptional regulator. The study of this protein has been mainly focused on the central nervous system because alterations of its expression are associated with neurological disorders such as Rett syndrome. However, young patients with Rett syndrome also suffer from osteoporosis, suggesting a role of MeCP2 in the differentiation of human bone marrow mesenchymal stromal cells (hBMSCs), the precursors of osteoblasts and adipocytes. Here, we report an in vitro downregulation of MeCP2 in hBMSCs undergoing adipogenic differentiation (AD) and in adipocytes of human and rat bone marrow tissue samples. This modulation does not depend on MeCP2 DNA methylation nor on mRNA levels but on differentially expressed miRNAs during AD. MiRNA profiling revealed that miR-422a and miR-483-5p are upregulated in hBMSC-derived adipocytes compared to their precursors. MiR-483-5p, but not miR-422a, is also up-regulated in hBMSC-derived osteoblasts, suggesting a specific role of the latter in the adipogenic process. Experimental modulation of intracellular levels of miR-422a and miR-483-5p affected MeCP2 expression through direct interaction with its 3' UTR elements, and the adipogenic process. Accordingly, the knockdown of MeCP2 in hBMSCs through MeCP2-targeting shRNA lentiviral vectors increased the levels of adipogenesis-related genes. Finally, since adipocytes released a higher amount of miR-422a in culture medium compared to hBMSCs we analyzed the levels of circulating miR-422a in patients with osteoporosis-a condition characterized by increased marrow adiposity-demonstrating that its levels are negatively correlated with T- and Z-scores. Overall, our findings suggest that miR-422a has a role in hBMSC adipogenesis by downregulating MeCP2 and its circulating levels are associated with bone mass loss in primary osteoporosis.

Sex/gender-related differences in inflammaging.

Geroscience puts mechanisms of aging as a driver of the most common age-related diseases and dysfunctions. Under this perspective, addressing the basic mechanisms of aging will produce a better understanding than addressing each disease pathophysiology individually. Worldwide, despite greater functional impairment, life expectancy is higher in women than in men. Gender differences in the prevalence of multimorbidity lead mandatory to the understanding of the mechanisms underlying gender-related differences in multimorbidity patterns and disability-free life expectancy. Extensive literature suggested that inflammaging is at the crossroad of aging and age-related diseases. In this review, we highlight the main evidence on sex/gender differences in the mechanisms that foster inflammaging, i.e. the age-dependent triggering of innate immunity, modifications of adaptive immunity, and accrual of senescent cells, underpinning some biomarkers of inflammaging that show sex-related differences. In the framework of the "gender medicine perspective", we will also discuss how sex/gender differences in inflammaging can affect sex differences in COVID-19 severe outcomes.

Genome-Wide Methylation Changes Associated with Replicative Senescence and Differentiation in Endothelial and Bone Marrow Mesenchymal Stromal Cells.

Bone marrow mesenchymal stromal cells (BMSCs) are multipotent cells able to self-renew and differentiate, depending on the microenvironment, into adipocytes and osteoblasts. These cells have a limited number of replications and enter replicative senescence during in vitro expansion. The role of DNA methylation (DNAm) assumes importance in cell function and commitment; however, its exact contribution to BMSC differentiation and replicative senescence is still unclear. We performed a genome-wide DNAm analysis on BMSCs cultured in vitro at early passages and induced to differentiate into adipocytes and osteoblasts, and on replicative senescent BMSCs and HUVECs, to identify DNAm patterns of senescence and differentiation. We also compared BMSCs and HUVECs in replicative senescence and found that, in both cellular systems, genome-wide hypomethylation was accompanied by a higher-than-expected overlap of differentially methylated positions (DMPs) and concordance in terms of direction of the change. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on lineage-independent senescence-associated DMPs revealed 16 common pathways, including Insulin resistance, Molecule adhesion, and Wnt/ß-catenin signaling. In both adipogenesis and osteogenesis, we observed a general demethylation of CpG sites compared with undifferentiated BMSCs with a higher number of DMPs in osteogenesis. KEGG analysis resulted in 30 pathways enriched in osteoblasts and only 2 in adipocytes when compared to undifferentiated cells. When comparing differentiated BMSCs with senescent ones, osteogenesis exhibited a greater overlap with senescence in terms of number of DMPs and direction of methylation change compared to adipogenesis. In conclusion, this study may be useful for future research on general mechanisms that occur in replicative senescence and furthermore to identify trajectories of BMSC differentiation and common aspects of differentiated and senescent cells.

Replicative Senescence-Associated LINE1 Methylation and LINE1-Alu Expression Levels in Human Endothelial Cells.

One of the main challenges of current research on aging is to identify the complex epigenetic mechanisms involved in the acquisition of the cellular senescent phenotype. Despite some evidence suggested that epigenetic changes of DNA repetitive elements, including transposable elements (TE) sequences, are associated with replicative senescence of fibroblasts, data on different types of cells are scarce. We previously analysed genome-wide DNA methylation of young and replicative senescent human endothelial cells (HUVECs), highlighting increased levels of demethylated sequences in senescent cells. Here, we aligned the most significantly demethylated single CpG sites to the reference genome and annotated their localization inside TE sequences and found a significant hypomethylation of sequences belonging to the Long-Interspersed Element-1 (LINE-1 or L1) subfamilies L1M, L1P, and L1HS. To verify the hypothesis that L1 demethylation could be associated with increased transcription/activation of L1s and/or Alu elements (non-autonomous retroelements that usually depend on L1 sequences for reverse transcription and retrotransposition), we quantified the RNA expression levels of both L1 (generic L1 elements or site-specific L1PA2 on chromosome 14) and Alu elements in young and senescent HUVECs and human dermal fibroblasts (NHDFs). The RNA expression of Alu and L1 sequences was significantly increased in both senescent HUVECs and NHDFs, whereas the RNA transcript of L1PA2 on chromosome 14 was not significantly modulated in senescent cells. Moreover, we found an increased amount of TE DNA copies in the cytoplasm of senescent HUVECs and NHDFs. Our results support the hypothesis that TE, which are significantly increased in senescent cells, could be retrotranscribed to DNA sequences.

Safety and efficacy of direct oral anticoagulants in geriatric patients with non-valvular atrial fibrillation: A single-center retrospective study.

INTRODUCTION: Direct oral anticoagulants (DOACs) are widely employed for antithrombotic prophylaxis in patients with atrial fibrillation (AF). However, there is still uncertainty about their risk-benefit profile in older patients. Here, we evaluated the efficacy, safety, and dose appropriateness of DOACs in a real-world population of outpatients with non-valvular AF, with a specific focus on subjects aged over 80 years and/or with reduced renal function. MATERIALS AND METHODS: Single-center retrospective study including patients who had been prescribed a DOAC between May 2014 and May 2021 for long-term anticoagulation in non-valvular AF. Patients anticoagulated for <4 weeks were excluded. The primary efficacy outcome was a composite of cardiovascular (CV) death, stroke, or systemic embolism. The primary safety outcome was major bleeding. RESULTS: A total of 1154 patients (median age 84 yrs., range 57-100 yrs.), among which 862 were 80 years and older, were included. In the subgroup of subjects ≥80 yrs., a subtherapeutic dose of DOAC was associated with an increased incidence of CV mortality, stroke, or systemic embolism (multivariable Cox regression, HR = 2.09, 95 % CI: 1.09-4.02), with no benefit in terms of prevalence of bleeding events (21.5 % vs. 18.6 %, p = 0.428), and the incidence of adverse safety and efficacy outcomes was not increased in patients with a reduced renal function (eGFR ≤30 mL/min). Plasma concentration of DOACs, assessed in a subset of 367 patients, did not increase with advanced age (≥ 80 yrs., two-way ANOVA, p = 0.656) nor with declining eGFR (≤30 mL/min, two-way ANOVA, p = 0.643) and was not associated with adverse safety and efficacy outcomes. CONCLUSIONS: Data from our study support the use of DOACs in populations of older adults and remark on the risks associated with inappropriate prescriptions in terms of CV mortality and adverse events.

Vitamin C intake potentially lowers total cholesterol to improve endothelial function in diabetic patients at increased risk of cardiovascular disease: A systematic review of randomized controlled trials.

Background: Vitamin C is one of the most consumed dietary compounds and contains abundant antioxidant properties that could be essential in improving metabolic function. Thus, the current systematic review analyzed evidence on the beneficial effects of vitamin C intake on cardiovascular disease (CVD)-related outcomes in patients with diabetes or metabolic syndrome. Methods: To identify relevant randomized control trials (RCTs), a systematic search was run using prominent search engines like PubMed and Google Scholar, from beginning up to March 2022. The modified Black and Downs checklist was used to assess the quality of evidence. Results: Findings summarized in the current review favor the beneficial effects of vitamin C intake on improving basic metabolic parameters and lowering total cholesterol levels to reduce CVD-risk in subjects with type 2 diabetes or related metabolic diseases. Moreover, vitamin C intake could also reduce the predominant markers of inflammation and oxidative stress like C-reactive protein, interleukin-6, and malondialdehyde. Importantly, these positive outcomes were consistent with improved endothelial function or increased blood flow in these subjects. Predominantly effective doses were 1,000 mg/daily for 4 weeks up to 12 months. The included RCTs presented with the high quality of evidence. Conclusion: Clinical evidence on the beneficial effects of vitamin C intake or its impact on improving prominent markers of inflammation and oxidative stress in patients with diabetes is still limited. Thus, more RCTs are required to solidify these findings, which is essential to better manage diabetic patients at increased risk of developing CVD.

Prognostic value of soluble ST2, high-sensitivity cardiac troponin, and NT-proBNP in type 2 diabetes: a 15-year retrospective study.

BACKGROUND: Patients with type 2 diabetes (T2DM) present an increased risk of cardiovascular (CV) disease and excess CV-related mortality. Beyond the established role of brain natriuretic peptide (BNP) and cardiac troponins (cTn), other non-cardiac-specific biomarkers are emerging as predictors of CV outcomes in T2DM. METHODS: Serum levels of soluble suppression of tumorigenesis 2 (sST2), high-sensitivity (hs)-cTnI, and N-terminal (NT)-proBNP were assessed in 568 patients with T2DM and 115 healthy controls (CTR). Their association with all-cause mortality and the development of diabetic complications was tested in T2DM patients over a median follow-up of 16.8 years using Cox models and logistic regressions. RESULTS: sST2 followed an increasing trend from CTR to uncomplicated T2DM patients (T2DM-NC) to patients with at least one complication (T2DM-C), while hs-cTnI was significantly higher in T2DM-C compared to CTR but not to T2DM-NC. A graded association was found between sST2 (HR 2.76 [95% CI 1.20-6.33] for ≥ 32.0 ng/mL and 2.00 [1.02-3.94] for 16.5-32.0 ng/mL compared to < 16.5 ng/mL, C-statistic = 0.729), NT-proBNP (HR 2.04 [1.90-4.55] for ≥ 337 ng/L and 1.48 [1.05-2.10] for 89-337 ng/L compared to < 89 ng/L, C-statistic = 0.741), and 15-year mortality in T2DM, whereas increased mortality was observed in patients with hs-cTnI ≥ 7.8 ng/L (HR 1.63 [1.01-2.62]). A 'cardiac score' based on the combination of sST2, hs-cTnI, and NT-proBNP was significantly associated with all-cause mortality (HR 1.35 [1.19-1.53], C-statistic = 0.739) and development of CV events. CONCLUSIONS: sST2, hs-cTnI, and NT-proBNP are associated with 15-year mortality and onset of CV events in T2DM. The long-term prognostic value of sST2 and its ability to track variables related to insulin resistance and associated metabolic disorders support its implementation into routine clinical practice.

Randomized, Double-Blind, Placebo-Controlled Trial to Test the Effects of a Nutraceutical Combination Monacolin K-Free on the Lipid and Inflammatory Profile of Subjects with Hypercholesterolemia.

BACKGROUND: Nutraceutical combinations (NCs) against hypercholesterolemia are increasing in the marketplace. However, the availability of NCs without monacolin K is scarce even though the statin-intolerant population needs it. METHODS: This study is a parallel-group, randomized, placebo-controlled, double-blind trial. We evaluated the effects of the NC containing phytosterols, bergamot, olive fruits, and vitamin K2 on lipid profile and inflammatory biomarkers in 118 subjects (mean age ± SD, 57.9 ± 8.8 years; 49 men and 69 women) with hypercholesterolemia (mean total cholesterol ± SD, 227.4 ± 20.8 mg/dL) without clinical history of cardiovascular diseases. At baseline and 6 and 12 weeks of treatment, we evaluated lipid profile (total, LDL and HDL cholesterol, and triglycerides), safety (liver, kidney, and muscle parameters), and inflammatory biomarkers such as hs-CRP, leukocytes, interleukin-32, and interleukin-38 and inflammatory-microRNAs (miRs) miR-21, miR-126, and miR-146a. RESULTS: Compared to the placebo, at 6 and 12 weeks, NC did not significantly reduce total cholesterol (p = 0.083), LDL cholesterol (p = 0.150), and triglycerides (p = 0.822). No changes were found in hs-CRP (p = 0.179), interleukin-32 (p = 0.587), interleukin-38 (p = 0.930), miR-21 (p = 0.275), miR-126 (p = 0.718), miR-146a (p = 0.206), myoglobin (p = 0.164), and creatine kinase (p = 0.376). Among the two reported, only one adverse event was probably related to the nutraceutical treatment. CONCLUSIONS: The evaluated nutraceutical combination did not change serum lipid profile and inflammatory parameters, at least not with the daily dose applied in the present study.

Curcumin, Polydatin and Quercetin Synergistic Activity Protects from High-Glucose-Induced Inflammation and Oxidative Stress.

Chronic hyperglycemia, the diagnostic biomarker of Type 2 Diabetes Mellitus (T2DM), is a condition that fosters oxidative stress and proinflammatory signals, both involved in the promotion of cellular senescence. Senescent cells acquire a proinflammatory secretory phenotype, called SASP, exacerbating and perpetuating the detrimental effects of hyperglycemia. Bioactive compounds can exert antioxidant and anti-inflammatory properties. However, the synergistic anti-inflammatory and antioxidant effects of the most extensively investigated natural compounds have not been confirmed yet in senescent cells and in hyperglycemic conditions. Here, we exposed young and replicative senescent HUVEC (yHUVEC and sHUVEC) to a high-glucose (HG) condition (45 mM) and treated them with Polydatin (POL), Curcumin (CUR) and Quercetin (QRC), alone or in combination (MIX), to mirror the anti-inflammatory component OxiDefTM contained in the novel nutraceutical GlicefenTM (Mivell, Italy). In both yHUVEC and sHUVEC, the MIX significantly decreased the expression levels of inflammatory markers, such as MCP-1, IL-1ß and IL-8, and ROS production. Importantly, in sHUVEC, a synergistic effect of the MIX was observed, suggesting its senomorphic activity. Moreover, the MIX was able to reduce the expression level of RAGE, a receptor involved in the activation of proinflammatory signaling. Overall, our data suggest that the consumption of nutraceuticals containing different natural compounds could be an adjuvant supplement to counteract proinflammatory and pro-oxidative signals induced by both hyperglycemic and senescence conditions.

Circulating levels of AGEs and soluble RAGE isoforms are associated with all-cause mortality and development of cardiovascular complications in type 2 diabetes: a retrospective cohort study.

BACKGROUND: Advanced glycation end-products (AGEs) and their interaction with the receptor for advanced glycation end-products (RAGE) play a pivotal role in the development and progression of type 2 diabetes. In this retrospective cohort study, we explored the association of circulating levels of soluble RAGE (sRAGE) isoforms, i.e., endogenous secretory esRAGE and cleaved cRAGE, AGEs and their respective ratios with 15-year all-cause mortality in type 2 diabetes. METHODS: Baseline AGEs and sRAGE isoforms concentration were measured by ELISA in 362 patients with type 2 diabetes and in 125 age- and gender-matched healthy control subjects (CTR). Independent predictors of mortality were determined using Cox proportional-hazards models and used to build and validate a nomogram for all-cause mortality prediction in type 2 diabetes. RESULTS: AGEs, total sRAGE, cRAGE and the AGEs/sRAGE and AGEs/esRAGE ratios were significantly increased in patients with type 2 diabetes compared to CTR (p < 0.001). In CTR subjects, but not in type 2 diabetes patients, a significant negative correlation between cRAGE and age was confirmed (p = 0.003), whereas the AGEs/sRAGE (p = 0.032) and AGEs/cRAGE (p = 0.006) ratios were positively associated with age. At an average follow-up of 15 years (4,982 person-years), 130 deaths were observed. The increase in the AGEs/cRAGE ratio was accompanied by a higher risk of all-cause mortality in patients with type 2 diabetes (HR per each SD increment = 1.30, 95% CI 1.15-1.47; p < 0.001). Moreover, sRAGE was associated with the development of major adverse cardiovascular events (MACE) in type 2 diabetes patients without previous MACE (OR for each SD increase: 1.48, 95% CI 1.11-1.89). A nomogram based on age, sex, HbA1c, systolic blood pressure, and the AGEs/cRAGE ratio was built to predict 5-, 10- and 15-year survival in type 2 diabetes. Patients were categorized into quartiles of the monogram scores and Kaplan-Meier survival curves confirmed the prognostic accuracy of the model (log-rank p = 6.5 × 10- 13). CONCLUSIONS: The ratio between AGEs and the cRAGE isoform is predictive of 15-year survival in patients with type 2 diabetes. Our data support the assessment of circulating AGEs and soluble RAGE isoforms in patients with type 2 diabetes as predictors of MACE and all-cause mortality.

The Association between Single Nucleotide Polymorphisms, including miR-499a Genetic Variants, and Dyslipidemia in Subjects Treated with Pharmacological or Phytochemical Lipid-Lowering Agents.

Disorders of lipoprotein metabolism are among the major risk factors for cardiovascular disease (CVD) development. Single nucleotide polymorphisms (SNPs) have been associated with the individual variability in blood lipid profile and response to lipid-lowering treatments. Here, we genotyped 34 selected SNPs located in coding genes related to lipid metabolism, inflammation, coagulation, and a polymorphism in the MIR499 gene-a microRNA previously linked to CVD-to evaluate the association with lipid trait in subjects with moderate dyslipidemia not on lipid-lowering treatment (Treatment-naïve (TN) cohort, n = 125) and in patients treated with statins (STAT cohort, n = 302). We also explored the association between SNPs and the effect of a novel phytochemical lipid-lowering treatment in the TN cohort. We found that 6 SNPs (in the MIR499, TNFA, CETP, SOD2, and VEGFA genes) were associated with lipid traits in the TN cohort, while no association was found with the response to twelve-week phytochemical treatment. In the STAT cohort, nine SNPs (in the MIR499, CETP, CYP2C9, IL6, ABCC2, PON1, IL10, and VEGFA genes) were associated with lipid traits, three of which were in common with the TN cohort. Interestingly, in both cohorts, the presence of the rs3746444 MIR499 SNP was associated with a more favorable blood lipid profile. Our findings could add information to better understand the individual genetic variability in maintaining a low atherogenic lipid profile and the response to different lipid-lowering therapies.

Ciliary neurotrophic factor is increased in the plasma of patients with obesity and its levels correlate with diabetes and inflammation indices.

To establish whether obesity involves activation of endogenous ciliary neurotrophic factor (CNTF) signalling, we evaluated its plasma levels in patients with obesity and correlated its values with the major clinical and haematological indices of obesity, insulin resistance and systemic inflammation. This study involved 118 subjects: 39 healthy controls (19 men), 39 subjects with obesity (19 men) and 40 subjects with obesity and diabetes (20 men). Plasma CNTF and CNTF receptor α (CNTFRα) were measured using commercial ELISA kits. The results showed that plasma CNTF was significantly higher in males and females with obesity with and without diabetes than in healthy subjects. Women consistently exhibited higher levels of circulating CNTF. In both genders, CNTF levels correlated significantly and positively with obesity (BMI, WHR, leptin), diabetes (fasting insulin, HOMA index and HbA1c) and inflammation (IL-6 and hsCRP) indices. Circulating CNTFRα and the CNTF/CNTFRα molar ratio tended to be higher in the patient groups than in controls. In conclusion, endogenous CNTF signalling is activated in human obesity and may help counteract some adverse effects of obesity. Studies involving a higher number of selected patients may reveal circulating CNTF and/or CNTFRα as potential novel diagnostic and/or prognostic markers of obesity, diabetes and associated diseases.

TAS1R3 and TAS2R38 Polymorphisms Affect Sweet Taste Perception: An Observational Study on Healthy and Obese Subjects.

BACKGROUND: The inter-individual differences in taste perception find a possible rationale in genetic variations. We verified whether the presence of four different single nucleotide polymorphisms (SNPs) in genes encoding for bitter (TAS2R38; 145G > C; 785T > C) and sweet (TAS1R3; -1572C > T; -1266C > T) taste receptors influenced the recognition of the basic tastes. Furthermore, we tested if the allelic distribution of such SNPs varied according to BMI and whether the associations between SNPs and taste recognition were influenced by the presence of overweight/obesity. METHODS: DNA of 85 overweight/obese patients and 57 normal weight volunteers was used to investigate the SNPs. For the taste test, filter paper strips were applied. Each of the basic tastes (sweet, sour, salty, bitter) plus pure rapeseed oil, and water were tested. RESULTS: Individuals carrying the AV/AV diplotype of the TAS2R38 gene (A49P G/G and V262 T/T) were less sensitive to sweet taste recognition. These alterations remained significant after adjustment for gender and BMI. Moreover, a significant decrease in overall taste recognition associated with BMI and age was found. There was no significant difference in allelic distribution for the investigated polymorphisms between normal and overweight/obese patients. CONCLUSIONS: Our findings suggest that overall taste recognition depends on age and BMI. In the total population, the inter-individual ability to identify the sweet taste at different concentrations was related to the presence of at least one genetic variant for the bitter receptor gene but not to the BMI.

Routine laboratory parameters, including complete blood count, predict COVID-19 in-hospital mortality in geriatric patients.

To reduce the mortality of COVID-19 older patients, clear criteria to predict in-hospital mortality are urgently needed. Here, we aimed to evaluate the performance of selected routine laboratory biomarkers in improving the prediction of in-hospital mortality in 641 consecutive COVID-19 geriatric patients (mean age 86.6 ± 6.8) who were hospitalized at the INRCA hospital (Ancona, Italy). Thirty-four percent of the enrolled patients were deceased during the in-hospital stay. The percentage of severely frail patients, assessed with the Clinical Frailty Scale, was significantly increased in deceased patients compared to the survived ones. The age-adjusted Charlson comorbidity index (CCI) score was not significantly associated with an increased risk of death. Among the routine parameters, neutrophilia, eosinopenia, lymphopenia, neutrophil-to-lymphocyte ratio (NLR), C-reactive protein, procalcitonin, IL-6, and NT-proBNP showed the highest predictive values. The fully adjusted Cox regressions models confirmed that high neutrophil %, NLR, derived NLR (dNLR), platelet-to-lymphocyte ratio (PLR), and low lymphocyte count, eosinophil %, and lymphocyte-to-monocyte ratio (LMR) were the best predictors of in-hospital mortality, independently from age, gender, and other potential confounders. Overall, our results strongly support the use of routine parameters, including complete blood count, in geriatric patients to predict COVID-19 in-hospital mortality, independent from baseline comorbidities and frailty.

Circulating biomarkers of inflammaging as potential predictors of COVID-19 severe outcomes.

The COVID-19 pandemic caused by SARS-CoV-2 infection has been of unprecedented clinical and socio-economic worldwide relevance. The case fatality rate for COVID-19 grows exponentially with age and the presence of comorbidities. In the older patients, COVID-19 manifests predominantly as a systemic disease associated with immunological, inflammatory, and procoagulant responses. Timely diagnosis and risk stratification are crucial steps to define appropriate therapies and reduce mortality, especially in the older patients. Chronically and systemically activated innate immune responses and impaired antiviral responses have been recognized as the results of a progressive remodeling of the immune system during aging, which can be described by the words 'immunosenescence' and 'inflammaging'. These age-related features of the immune system were highlighted in patients affected by COVID-19 with the poorest clinical outcomes, suggesting that the mechanisms underpinning immunosenescence and inflammaging could be relevant for COVID-19 pathogenesis and progression. Increasing evidence suggests that senescent myeloid and endothelial cells are characterized by the acquisition of a senescence-associated pro-inflammatory phenotype (SASP), which is considered as the main culprit of both immunosenescence and inflammaging. Here, we reviewed this evidence and highlighted several circulating biomarkers of inflammaging that could provide additional prognostic information to stratify COVID-19 patients based on the risk of severe outcomes.

Senescent macrophages in the human adipose tissue as a source of inflammaging.

Obesity is a major risk factor for type 2 diabetes and a trigger of chronic and systemic inflammation. Recent evidence suggests that an increased burden of senescent cells (SCs) in the adipose tissue of obese/diabetic animal models might underlie such pro-inflammatory phenotype. However, the role of macrophages as candidate SCs, their phenotype, the distribution of SCs among fat depots, and clinical relevance are debated. The senescence marker ß-galactosidase and the macrophage marker CD68 were scored in visceral (vWAT) and subcutaneous (scWAT) adipose tissue from obese patients (n=17) undergoing bariatric surgery and control patients (n=4) subjected to cholecystectomy. A correlation was made between the number of SCs and BMI, serum insulin, and the insulin resistance (IR) index HOMA. The monocyte cell line (THP-1) was cultured in vitro in high glucose milieu (60 mM D-glucose) and subsequently co-cultured with human adipocytes (hMADS) to investigate the reciprocal inflammatory activation. In obese patients, a significantly higher number of SCs was observed in vWAT compared to scWAT; about 70% of these cells expressed the macrophage marker CD68; and the number of SCs in vWAT, but not in scWAT, positively correlated with BMI, HOMA-IR, and insulin. THP-1 cultured in vitro in high glucose milieu acquired a senescent-like phenotype (HgSMs), characterized by a polarization toward a mixed M1/M2-like secretory phenotype. Co-culturing HgSMs with hMADS elicited pro-inflammatory cytokine expression in both cell types, and defective insulin signaling in hMADS. In morbid obesity, expansion of visceral adipose depots involves an increased burden of macrophages with senescent-like phenotype that may promote a pro-inflammatory profile and impair insulin signaling in adipocytes, supporting a framework where senescent macrophages fuel obesity-induced systemic inflammation and possibly contribute to the development of IR.

Circulating miR-320b and miR-483-5p levels are associated with COVID-19 in-hospital mortality.

The stratification of mortality risk in COVID-19 patients remains extremely challenging for physicians, especially in older patients. Innovative minimally invasive molecular biomarkers are needed to improve the prediction of mortality risk and better customize patient management. In this study, aimed at identifying circulating miRNAs associated with the risk of COVID-19 in-hospital mortality, we analyzed serum samples of 12 COVID-19 patients by small RNA-seq and validated the findings in an independent cohort of 116 COVID-19 patients by qRT-PCR. Thirty-four significantly deregulated miRNAs, 25 downregulated and 9 upregulated in deceased COVID-19 patients compared to survivors, were identified in the discovery cohort. Based on the highest fold-changes and on the highest expression levels, 5 of these 34 miRNAs were selected for the analysis in the validation cohort. MiR-320b and miR-483-5p were confirmed to be significantly hyper-expressed in deceased patients compared to survived ones. Kaplan-Meier and Cox regression models, adjusted for relevant confounders, confirmed that patients with the 20% highest miR-320b and miR-483-5p serum levels had three-fold increased risk to die during in-hospital stay for COVID-19. In conclusion, high levels of circulating miR-320b and miR-483-5p can be useful as minimally invasive biomarkers to stratify older COVID-19 patients with an increased risk of in-hospital mortality.

Prognostic relevance of normocytic anemia in elderly patients affected by cardiovascular disease.

BACKGROUND: Anemia associated with cardiovascular diseases (CVD) is a common condition in older persons. Prevalence and prognostic role of anemia were extensively studied in patients with myocardial infarction (MI) or congestive heart failure (CHF) whereas limited data were available on patients with atrial fibrillation (AF). This study was conducted to assess the clinical prevalence and prognostic relevance of anemia in elderly patients affected by AF and other CVDs. METHODS: A total of 866 elderly patients (430 men and 436 women, age: 65-98 years, mean age: 85 ± 10 years) were enrolled. Among these patients, 267 patients had acute non-ST-segment elevation MI (NSTEMI), 176 patients had acute CHF, 194 patients had acute AF and 229 patients were aged-matched healthy persons (CTR). All parameters were measured at the hospital admission and cardiovascular mortality was assessed during twenty-four months of follow-up. RESULTS: The prevalence of anemia was higher in NSTEMI, CHF and AF patients compared to CTR subjects (50% vs. 15%, P < 0.05), with normocytic anemia being the most prevalent type (90%). Adjusted mortality risk was higher in anemic patient versus non-anemic patient in all the groups of patients [NSTEMI: hazard ratio (HR) = 1.81, 95% CI: 1.06-2.13; CHF: HR = 2.49, 95% CI: 1.31-4.75; AF: HR = 1.98, 95% CI: 1.01-3.88]. Decreased hemoglobin levels ( P = 0.001) and high reticulocyte index (P = 0.023) were associated with higher mortality in CVD patients. CONCLUSIONS: The significant associations between CVD and anemia and the prognostic relevance of anemia for elderly patients with CVD were confirmed in this study. The presence of anemia in AF patients is associated with a two-fold increased mortality risk compared with non-anemic AF patients. Low hemoglobin and high reticulocyte count independently predict mortality in elderly patients with CVD.