ORIGINAL RESEARCH published: 29 July 2021 doi: 10.3389/fcell.2021.716435 Functional and Cognitive Decline Is Associated With Increased Endothelial Cell Inflammation and Platelet Activation: Liquid Biopsy of Microvesicles in Community- Dwelling Octogenarians Edited by: Gemma Chiva-Blanch 1,2,3* , Alba Vilella-Figuerola 1 , Teresa Padró 1,4 , Francesc Formiga 5,6 , Luca Liberale, Assumpta Ferrer 7 and Lina Badimon 1,4* University of Zurich, Switzerland 1 Cardiovascular Program ICCC, Institut de Recerca Hospital Santa Creu i Sant Pau-IIB Sant Pau, Barcelona, Spain, Reviewed by: 2 Endocrinology and Nutrition Department, Institut d’Investigacions Biomèdiques August Pi Sunyer, Hospital Clínic, Kieran O’dea, Barcelona, Spain, 3 Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Imperial College London, Carlos III, Madrid, Spain, 4 Centro de Investigación Biomédica en Red Cardiovascular, Instituto de Salud Carlos III, Madrid, United Kingdom Spain, 5 Geriatric Unit, Internal Medicine Service, Hospital Universitari de Bellvitge, Barcelona, Spain, 6 Bellvitge Biomedical Håkan Wallen, Research Institute, IDIBELL, L’Hospitalet de Llobregat, Barcelona, Spain, 7 Primary Healthcare Centre “El Plà” CAP-I, Sant Karolinska Institutet (KI), Sweden Feliu de Llobregat, Barcelona, Spain *Correspondence: Gemma Chiva-Blanch gchiva@clinic.cat Increased life expectancy is usually associated with comorbidities, such as cardio and Lina Badimon cerebrovascular disease causing impaired functionality. A common underlying cause of LBadimon@santpau.cat these comorbidities is vascular inflammation and injury. Elevated levels of circulating Specialty section: microvesicles (cMV), as a product of a hemostatic and inflammatory cell activation, This article was submitted to could be direct mapping of an imbalanced hemostasis. In this manuscript, we aimed Signaling, a section of the journal to investigate by liquid biopsy whether successful aging can be discriminated by Frontiers in Cell and Developmental cMV levels and phenotype. To this purpose, we included 135 community-dwelling Biology octogenarians in a cross-sectional study. Successful aging was defined as good Received: 28 May 2021 functional (Barthel Index > 90 points, and Lawton index score > 7/4 points for Accepted: 12 July 2021 Published: 29 July 2021 women and men, respectively) and cognitive status (Spanish version of the Mini- Citation: Mental State Examination -MEC- > 24 points) and no need for institutionalization. Chiva-Blanch G, Total, annexin V positive (AV+ ), and AV− cMV from different cell origins from the Vilella-Figuerola A, Padró T, Formiga F, Ferrer A and Badimon L vascular compartment were phenotypically characterized and quantified from fasting (2021) Functional and Cognitive plasma samples by flow cytometry. Successful aging was associated with lower Decline Is Associated With Increased plasma concentrations of total and AV+ CD141+ /CD41+ -CD61+ , and PAC1+ /AV+ , Endothelial Cell Inflammation and Platelet Activation: Liquid Biopsy CD141+ /AV+ , and CD36+ /AV− cMV. From these phenotypes, ROC curve analyses of Microvesicles in Community- revealed that CD141+ /AV+ and CD141+ /CD41+ -CD61+ /AV+ endothelial- and platelet- Dwelling Octogenarians. Front. Cell Dev. Biol. 9:716435. derived cMV discriminate successful and non-successful aging with an AUC (95%CI) of doi: 10.3389/fcell.2021.716435 0.655 (0.551, 0.758), P = 0.005, and 0.638 (0.535, 0.741), P = 0.013, respectively. In Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 July 2021 | Volume 9 | Article 716435 Chiva-Blanch et al. Microvesicles and Cognitive Decline conclusion, successful aging is associated with low levels of cMV released by endothelial cells and platelets, indicating lower endothelial cell inflammation and platelet activation. Our results contribute to the understanding of the link between unsuccessful aging, cognitive decline and vascular cell inflammatory disturbances. Keywords: circulating microvesicles, octogenarians, successful aging, cognitive decline, inflammation, platelet activation, thrombomodulin, endothelial cells INTRODUCTION to inflammation and coagulation, which were directly associated with cognitive status, cardiovascular event presentation (Cubedo Successful aging can be defined as growing old in optimum et al., 2015) and increased cardiovascular mortality at 5 years conditions (von Faber et al., 2001), considering the fact that follow-up (Cubedo et al., 2017). Considering the biological link increased life expectancy is usually associated with comorbidities, between aging, vascular damage, cognitive decline and functional impaired functionality (Mitra et al., 2020), and diminished performance, and the worldwide importance of successful quality of life (Formiga et al., 2011). The 2018 Aging Report aging in the society, the aim of our study is to investigate from the European Union confirms that the European population whether successful aging is related to a lower activation of is continuously aging in a significant manner (European cells in the vascular compartment through the quantification of Commission [EC], 2018). Although life expectancy may be their MV release. currently affected by the COVID pandemic (Trias-Llimós and Bilal, 2020), hopefully in a passing manner, the old age-related dependency is expected to increase in more than 20% in 2070 MATERIALS AND METHODS (European Commission [EC], 2018). Non-successful aging is associated with higher risk of Subjects malnutrition and falls, frailty, and with higher co-morbidity, In this cross-sectional study, we included 135 community- and more specifically with cardiovascular disease (CVD) and dwelling octogenarians from the OCTABAIX (Oldest cerebrovascular disease (Formiga et al., 2011; Beauchet et al., CharacTheristic and Assessments-Baix Llobregat) cohort 2020), with vascular damage as a common underlying cause. (Ferrer et al., 2010; Badia et al., 2015) at baseline (registered at In fact, it is of paramount importance to reduce the incidence clinicaltrials.gov with the reference NCT01141166). Inclusion of major vascular events such as coronary heart disease or criteria, as per protocol, were to be born in 1924 and not being stroke, in order to prevent cognitive and functional decline, or institutionalized (24 h of professional care available). Therefore, even disability. all subjects were 85 years old at the year of inclusion (2009). Circulating microvesicles (cMV) are membrane blebs ranged Exclusion criteria in this sub-study was having a cardiovascular 0.1–1 µm extruded from almost all types of cells after activation, event within the last year. In addition, samples with repeated injury or apoptosis. cMV contain bioactive molecules that can freeze/thaw cycles and hemolyzed samples were also excluded modulate the phenotype of target recipient cells by paracrine (Supplementary Figure 1). All samples were processed within regulation (Gnecchi et al., 2006; Badimon et al., 2019). Indeed, October-November 2019. cMV can bind and activate endothelial cells, that by recruiting Functional, cognitive and nutritional status were assessed platelets and leukocytes and inducing smooth muscle cell with questionnaires currently used in geriatric practice. Barthel proliferation, may overall enhance vascular inflammation and Index was performed to measure the functional capacity for damage (Daniel et al., 2006; Dalli et al., 2013; Hulsmans and basic activities of daily living; Lawton Index was administered Holvoet, 2013). Elevated concentrations of cMV have been to evaluate the ability to carry out instrumental activities; and observed after a stroke (Chiva-Blanch et al., 2016) or coronary the Spanish version of the Mini-Mental State Examination heart disease (Chiva-Blanch et al., 2017a), and are believed to (MEC) was performed to assess cognitive impairment. In the be both the cause and consequence of atherosclerosis. We have three test, the higher the score, the higher the functionality recently observed that frail older adults also show elevated levels and independence. Subjects with Barthel Index scores > 90 of cMV from platelets and leukocytes (Arauna et al., 2020). were classified as functional independent. Women with Lawton Therefore, specific cMV phenotypes from blood and vascular Index scores > 7 and men with scores > 4, respectively, cells associated with non-successful aging, may be candidate (Graf, 2008), were considered able for instrumental activities, biomarkers of age-related cognitive and functional impairment. and subjects above 24 points in the MEC score showed These may help in the research of prognostic biomarkers minimal cognitive impairment. Nutritional status was evaluated indicators of overall functional and cognitive decline in the with the mini-nutritional assessment (MNA), and scores below future, and may help in the identification of subjects requiring 23.5 were classified as malnutrition or at risk. Successful specific strategies to prevent vascular dysfunction at the onset of aging was defined as good functional (Barthel Index > 90 cognitive and functional decline. points, and Lawton Index scores > 7 for women and >4 Previously, we observed that older octogenarians with non- men) and cognitive status (MEC > 24 points), and no need successful aging had altered levels in plasma of proteins related for institutionalization (Formiga et al., 2011). Non-successful Frontiers in Cell and Developmental Biology | www.frontiersin.org 2 July 2021 | Volume 9 | Article 716435 Chiva-Blanch et al. Microvesicles and Cognitive Decline aging was further categorized in having one, two or three Therefore, to ensure sufficient power for the other outcomes scores below good functional and/or cognitive status (Barthel and to ensure sufficient statistical power to control for potential Index ≤ 90 points, Lawton Index scores ≤ 7 for women confounders, the sample size was nearly doubled according to and ≤4 men, and/or MEC ≤ 24 points). Medical data sample availability (Supplementary Figure 1). were primarily obtained from general practice records, and Statistical analyses were performed using the SPSS Statistical interviews were performed at primary care services. Subjects Analysis System (version 23.0). Results are expressed as signed an informed consent before inclusion in the study, mean ± SD, mean + SEM, or n (%) when indicated. and all procedures were conducted in accordance with the Normality of variables was assessed with the Shapiro–Wilk Declaration of Helsinki. test, and all variables followed a non-parametric distribution. Therefore, non-parametric analyses were conducted. To analyze Blood Sampling differences according to functional and cognitive status or Fasting venous blood was withdrawn using a 20-gage needle, successful aging, Mann–Whitney test for two independent and blood was collected into EDTA-treated tubes. Blood cells samples (for the comparison between successful and non- were removed by centrifugation (1,500 × g, 15 min) at room successful aging), or the Kruskal–Wallis test for K independent temperature (RT). Plasma was carefully aspirated, and 250 µL samples (for the comparison between different degrees of non- aliquots of EDTA-anticoagulated plasma were immediately successful aging) were carried out. Multiple comparisons were frozen and stored at −80◦ C until processing. performed with the Bonferrroni post hoc test. Multivariable models for successful aging were performed with logistic Laboratory Measurements regression models (conventional and stepwise) with cMV from Biochemical and hematological parameters were quantified by different cell origins and phenotypes adjusting by potential standardized methods. confounders in two different models. The first model, was adjusted by previous CVD (yes/no), psychotropics (yes/no) and Isolation, Phenotyping and MNA > 23.5, according to stepwise regression. The second model was further adjusted by sex, dyslipidemia (yes/no), statins Quantification of Circulating (yes/no), and antiplatelet agents (yes/no), factors known to Microvesicles modulate MV release (Badimon et al., 2019), and with existing Circulating microvesicles were quantified as previously (Chiva- significant differences between groups. Receiver operating Blanch et al., 2019, 2020; Arauna et al., 2020) and currently characteristic (ROC)-curve estimations were performed, and described in Supplementary Material. Briefly, cMV were isolated their corresponding C statistics [area under the curve (AUC) with from 250 µL plasma by differential centrifugation and were their 95% CI] were calculated for cMV phenotypes associated triple-label stained with Annexin V (AV, which has high with successful aging. A 2-tailed P value of <0.05 was considered affinity for phosphatidylserine) and two specific monoclonal statistically significant. antibodies (Supplementary Table 1) labeled with fluorescein isothiocyanate (FITC) or phycoerythrin (PE), prior flow cytometric analyses in a FACSCanto II (BD Biosciences). Flow RESULTS cytometry controls are shown in Supplementary Figure 2. cMV were phenotypically characterized for bioactive and/or Subjects Characteristics biomarker molecules from their parental cells and were Table 1 depicts the clinical characteristics of the subjects defined as total cMV (AV± ), AV+ , or AV− , according according to their aging success. Subjects with a successful aging to phosphatidylserine exposure (Supplementary Figure 3). were women in a lower percentage, showed higher levels of Phosphatidylserine exposure promotes thrombin generation and HDL cholesterol and the renal function was more preserved thrombus formation. Therefore, AV+ cMV present higher than in non-healthy aged subjects. No differences in the levels prothrombotic activity than platelets (Sinauridze et al., 2007). of circulating leukocytes, platelets, hemoglobin, transaminases, albumin, total protein, thyroid stimulating hormone or tiroxin Statistical Analyses 4 were observed between subjects with and without successful Sample size was determined with the ENE 3.0 statistical program aging (not shown). (GlaxoSmithKline, Brentford, United Kingdom) assuming a loss As expected, and as per definition, subjects with a successful of 0% participants because samples were already collected. aging scored higher in the Barthel and Lawton indexes, and We estimated that to recognize as statistically significant a MEC and MNA scores compared to non-successfully aged difference in the number of PAC-1+ /AV+ cMV of 5 units subjects. Consequently, a higher proportion of subjects with (MV)/µL plasma with a conservative SD of 6, we needed 19 successful aging were classified as functionally independent and 28 subjects per group (successful and non-successful aging), (Barthel Index > 90), able to conduct instrumental activities accepting an alpha risk of 0.05 and a beta risk of 0.2 in a (Lawton Index > 7 or 4 for women and men, respectively), with two-sided test, and considering the proportion of successful high cognitive performance (MEC score > 24), and/or adequately aging around 0.30 of the population (Formiga et al., 2011). nourished (MNA scores ≥ 24). Although PAC-1+ /AV+ was used to determine sample size, Subjects who reported non-successful aging had a higher all cMV phenotypes tested were considered primary outcomes. incidence of previous CVD (heart failure and stroke). Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 July 2021 | Volume 9 | Article 716435 Chiva-Blanch et al. Microvesicles and Cognitive Decline TABLE 1 | Characteristics of the 85-year old subjects included in the study according to successful aging. Non-successful aging (n = 87) Successful aging (n = 48) P1 P2 First degree (n = 28) Second degree (n = 23) Third degree (n = 36) Females [n (%)] 22 (78.6) 18 (78.3) 23 (63.9) 24 (50.0) 0.009 0.033 Systolic blood pressure (mmHg) 136.68 ± 16.57 137.52 ± 16.59 135.14 ± 20.74 137.56 ± 16.68 0.591 0.932 Diastolic blood pressure (mmHg) 72.43 ± 10.84 71.04 ± 9.33 70.64 ± 11 71.4 ± 11.17 0.874 0.938 BMI (Kg/m2 ) 28.76 ± 5.48 28.22 ± 4.83 29.85 ± 3.99 28.58 ± 4.12 0.226 0.498 Glucose (mmol/L) 5.37 ± 1.48 5.56 ± 1.85 5.81 ± 1.82 5.83 ± 0.98 0.498 0.567 HbA1C (%) 6.35 ± 1.58 6.01 ± 1.06 6.08 ± 0.91 5.89 ± 0.56 0.393 0.303 Triglycerides (mmol/L) 1.35 ± 0.56 1.56 ± 0.64 1.32 ± 0.57 1.24 ± 0.6 0.056 0.216 Total cholesterol (mmol/L) 5.12 ± 1.14 5.09 ± 1.26 4.66 ± 0.75 5.24 ± 0.85 0.069 0.053 HDL cholesterol (mmol/L) 1.48 ± 0.34 1.32 ± 0.4 1.39 ± 0.3 1.62 ± 0.45 0.002 0.009 LDL cholesterol (mmol/L) 3.03 ± 1.03 3.08 ± 1.17 2.67 ± 0.67 3.06 ± 0.73 0.218 0.165 Tobacco consumption [n (%)] 0.151 0.183 Current smokers 1 (3.6) 1 (4.34) 0 (0) 0 (0) Former smokers 3 (10.7) 3 (13.0) 10 (27.8) 15 (31.2) Creatinin (mg/dL) 77.82 ± 19.26 100.35 ± 62.2 101.86 ± 31.45 82.75 ± 20.91 0.019 0.008 Glomerular filtration (mL/min) 57.42 ± 7.02 52.94 ± 14.6 51.04 ± 10.37 58.22 ± 5.72 0.005 0.002 Functional, cognitive and nutritional indexes BARTHEL 93.04 ± 6.43 72.61 ± 24.58 62.36 ± 27.03 98.54 ± 2.3 <0.0001 <0.0001 BARTHEL > 90 16 (57.1) 2 (8.7) 0 (0) 48 (100) <0.0001 <0.0001 LAWTON 6.07 ± 1.72 3.78 ± 2.13 1.89 ± 1.7 6.9 ± 1.37 <0.0001 <0.0001 LAWTON > 7 (women) 14 (63.4) 3 (16.7) 0 (0) 24 (100) <0.0001 <0.0001 LAWTON > 4 (men) 4 (66.7) 0 (0) 0 (0) 24 (100) <0.0001 <0.0001 MEC 28.18 ± 4.78 26.3 ± 5.47 16.08 ± 5.49 31.29 ± 2.76 <0.0001 <0.0001 MEC > 24 22 (78.6) 18 (78.2) 0 (0) 48 (100) <0.0001 <0.0001 MNA 25.38 ± 1.88 20.78 ± 4.04 22.29 ± 2.9 26.74 ± 2.95 <0.0001 <0.0001 MNA > 23.5 24 (85.7) 7 (30.4) 13 (36.1) 43 (89.6) <0.0001 <0.0001 Dyslipidemia [n (%)] 13 (46.4) 11 (47.8) 14 (38.9) 8 (16.7) 0.002 0.014 Diabetes [n (%)] 10 (35.7) 6 (26.1) 19 (52.8) 12 (25.0) 0.126 0.075 Years of evolution 13 ± 8.33 16 ± 8.29 13.89 ± 5.47 8.58 ± 4.4 0.027 0.075 History of CVD [n (%)] 11 (39.3) 15 (65.2) 32 (88.9) 12 (25.0) <0.0001 <0.0001 Myocardial infarction 3 (10.7) 5 (21.7) 10 (27.8) 6 (12.5) 0.234 0.205 Heart failure 4 (14.3) 12 (52.2) 8 (22.2) 2 (4.2) 0.001 <0.0001 Peripheral vascular disease 1 (3.6) 1 (4.3) 5 (13.9) 1 (2.1) 0.16 0.125 Stroke 4 (14.3) 9 (39.1) 16 (44.4) 4 (8.3) 0.001 <0.0001 Atrial fibrillation 4 (14.3) 5 (21.7) 7 (19.4) 3 (6.25) 0.052 0.221 Chronic renal failure [n (%)] 0 (0) 3 (13.0) 3 (8.3) 3 (6.25) 0.885 0.298 Medication [n (%)] Psychotropic 9 (32.1) 16 (69.6) 21 (58.3) 12 (25.0) 0.002 <0.0001 Cardiac dysrhythmia medication 3 (10.7) 5 (21.7) 6 (16.7) 6 (12.5) 0.574 0.671 Statins 14 (50) 10 (43.5) 18 (50) 11 (22.9) 0.004 0.034 ACE Inhibitors 16 (57.1) 10 (43.5) 17 (47.2) 14 (29.2) 0.023 0.098 Angiotensin-II receptor blockers 2 (7.1) 4 (17.4) 14 (38.9) 8 (16.7) 0.386 0.012 β- Blockers 8 (28.6) 5 (21.7) 11 (30.6) 6 (12.5) 0.044 0.193 α- Blockers 0 (0) 0 (0) 8 (22.2) 7 (14.6) 0.34 0.009 Calcium channel blockers 4 (14.3) 8 (34.8) 14 (38.9) 6 (12.5) 0.023 0.013 Diuretics 18 (64.3) 16 (69.6) 22 (61.1) 22 (45.8) 0.037 0.19 Antiplatelet agents 10 (35.7) 9 (39.1) 21 (58.3) 9 (18.7) 0.009 0.003 Oral anticoagulant therapy 3 (10.7) 4 (17.4) 8 (22.2) 4 (8.3) 0.154 0.289 Oral Antidiabetic agents 8 (28.6) 4 (17.4) 8 (22.2) 7 (14.6) 0.243 0.498 Insulin 2 (7.1) 4 (17.4) 4 (11.1) 0 (0) 0.015 0.048 P1 for the comparison between successful and non-successful aging (Mann–Whitney test or Chi-squared test), and P2 , for the comparison between successful aging and different degrees of non-successful aging (Kruskal–Wallis test or Chi-squared test). Successful aging was defined as good functional (Barthel Index > 90 points, and Lawton Index scores > 7 for women and >4 men) and cognitive status (MEC > 24 points), and no need for institutionalization. Non-successful aging was further categorized in having one (first degree), two (second degree), or three scores (third degree) below good functional and/or cognitive status (Barthel Index ≤ 90 points, Lawton Index scores ≤ 7 for women and ≤4 men, and/or MEC ≤ 24 points). Glomerular filtration was calculated with the Modification of Diet in Renal Disease formula (MDRD). BMI indicates body mass index; HDL, high density lipoprotein; LDL, low density lipoprotein; MNA, mini-nutritional assessment; MEC, Spanish version of the Mini-Mental State Examination; CVD, cardiovascular disease; and ACE, angiotensin-converting-enzyme. Frontiers in Cell and Developmental Biology | www.frontiersin.org 4 July 2021 | Volume 9 | Article 716435 Chiva-Blanch et al. Microvesicles and Cognitive Decline Consequently, these subjects were in more proportion Individuals with a Lawton index score > 7 for women and under statins, β- blockers, calcium channel blockers, and >4 for men (n = 69), indicating ability for instrumental activities, antiplatelet therapies. In addition, a higher percentage of presented lower levels of platelet-derived total and AV+ GPVI+ subjects who reported non-successful aging were under and CD36+ /GPVI+ cMV compared to subjects with a significant psychotropic medication. degree of disability (n = 66), as illustrated in Supplementary Figure 5 (P ≤ 0.042, all). In the same line, octogenarians with a MEC score < 24 MV- Functional, Cognitive and Nutritional (n = 47), indicating cognitive impairment, showed higher Status concentrations of platelet-derived total and AV+ CD36+ /GPVI+ Subjects with a Barthel index score > 90 (n = 66), indicating and GPVI+ /AV+ cMV; and CD9+ /AV− and CD36+ /AV− cMV functional independence, showed lower levels of platelet-, compared to subjects with MEC scores ≥ 24 (n = 88), as depicted endothelial-, immune cell-, and erythrocyte- derived total, in Supplementary Figure 6 (P ≤ 0.034, all). AV+ or AV− cMV compared to dependent subjects (Barthel Supplementary Figure 7 shows that subjects with or at risk of score ≤ 90, n = 69; P ≤ 0.049, all), as can be observed in malnutrition (MNA ≤ 23.5; n = 48) show higher concentrations Supplementary Figure 4. of platelet-derived total, AV+ and AV− cMV compared to FIGURE 1 | cMV levels according to successful aging in the 135 octogenarians included in the study. Results are shown as mean + SEM of concentrations of: (A) total microvesicles; (B) AV+ microvesicles; and (C) AV− microvesicles. Successful aging (n = 48) was defined as good functional (Barthel Index > 90 points, and Lawton Index scores > 7 for women and >4 men) and cognitive status (MEC > 24 points), and no need for institutionalization. Non-successful aging (NSA, n = 87) was further categorized in having one (NSA-1, n = 28), two (NSA-2, n = 23), or three scores (NSA-3, n = 36) below good functional and/or cognitive status (Barthel Index ≤ 90 points, Lawton Index scores ≤ 7 for women and ≤4 men, and/or MEC ≤ 24 points). AV indicates annexin V and cMV, circulating microvesicles. Used markers for MV phenotyping are shown in Supplementary Table 1. Columns with different superscript letters indicate significant differences (P < 0.05, Bonferroni post hoc test). Frontiers in Cell and Developmental Biology | www.frontiersin.org 5 July 2021 | Volume 9 | Article 716435 Chiva-Blanch et al. Microvesicles and Cognitive Decline subjects with MNA scores ≥ 24 (n = 87), P ≤ 0.047, all, indicating TABLE 2 | Association between cMV from different phenotypes and successful aging. platelet hyperreactivity in this situation. On the other hand, subjects with MNA ≤ 23.5 showed lower concentrations of B (95% CI) P immune cell-derived total CD9+ and CD9+ /AV+ , and higher concentrations of CD40+ /AV+ and CD16+ /AV− cMV compared Total cMV to subjects adequately nourished (P ≤ 0.043, all), suggesting CD141+ /CD41+ -CD61+ altered immune functionality in subjects with malnutrition. Unadjusted 0.006 (0002, 0.010) 0.003 No significant associations were found between all other Multivariate-adjusted1 0.003 (0.000, 0.007) 0.038 MV phenotypes determined and functional, cognitive and Multivariate-adjusted2 0.003 (0.000, 0.007) 0.039 nutritional status. GPVI+ Unadjusted 0.008 (0.000, 0.016) 0.056 Multivariate-adjusted1 Not included in the stepwise model MV- Successful Aging Multivariate-adjusted2 0.006 (0.000, 0.012) 0.088 Endothelial-derived CD31+ /CD41a− (P = 0.044), and AV+ cMV CD31+ /CD41a− /AV− (P = 0.028); platelet-derived CD141+ PAC-1+ /AV+ /CD41+ -CD61+ (P = 0.009), CD141+ /CD41+ -CD61+ /AV− Unadjusted 0.048 (0.009, 0.086) 0.016 (P = 0.004), CD36+ /GPVI+ (P = 0.024), and P2RY12+ (P = Multivariate-adjusted1 0.044 (0.014, 0.073) 0.004 0.032); as well as CD141+ (P = 0.033), CD36+ /AV− (P = 0.029), Multivariate-adjusted2 0.046 (0.017, 0.076) 0.002 and CD40+ (P = 0.004) cMV concentrations were lower in CD141+ /AV+ subjects who reported a successful aging. Moreover, AV+ cMV Unadjusted 0.004 (0.001, 0.007) 0.014 from platelet origin [CD41+ -CD61+ /CD141+ /AV+ (P = 0.009) Multivariate-adjusted1 0.002 (0.000, 0.005) 0.035 and CD36+ /GPVI+ /AV+ (P = 0.045)], and CD141+ /AV+ (P = Multivariate-adjusted2 0.002 (0.000, 0.005) 0.047 0.006) cMV, were also lower in subjects who reported a successful CD141+ /CD41+ -CD61+ /AV+ aging, compared to those subjects non-successfully aged. As Unadjusted 0.006 (0.002, 0.010) 0.003 depicted in Figure 1, when non-successful aging was divided Multivariate-adjusted1 0.003 (0.000, 0.007) 0.045 in three degrees of severity, increasing concentrations of total Multivariate-adjusted2 0.003 (0.000, 0.007) 0.043 (Figure 1A), AV+ (Figure 1B), and AV− (Figure 1C) cMV from GPVI+ /AV+ platelet origin are observed, according to the severity of cognitive Unadjusted 0.020 (−0.010, 0.051) 0.191 and functional impairment. Multivariate-adjusted1 Not included in the stepwise model As shown in Table 2, after multivariate-adjustments by sex, Multivariate-adjusted2 0.020 (−0.005, 0.045) 0.109 dyslipidemia, previous CVD, psychotropics, statins, antiplatelet CD36+ /GPVI+ /AV+ agents, and MNA > 23.5, total and AV+ CD141+ /CD41+ - Unadjusted 0.025 (−0.006, 0.056) 0.114 CD61+ , and PAC1+ /AV+ , CD141+ /AV+ , and CD36+ /AV− Multivariate-adjusted1 Not included in the stepwise model cMV remained inversely associated with successful aging. Multivariate-adjusted2 0.019 (−0.006, 0.043) 0.129 From these phenotypes, ROC curve analyses revealed that AV− cMV CD141+ /AV+ and CD141+ /CD41+ -CD61+ /AV+ endothelial- CD141+ /CD41+ -CD61+ /AV− and platelet-derived cMV modestly but significantly discriminate Unadjusted 0.056 (−0.005, 0.117) 0.071 successful and non-successful aging with an AUC (95%CI) of Multivariate-adjusted1 Not included in the stepwise model 0.655 (0.551, 0.758), P = 0.005, and 0.638 (0.535, 0.741) P = 0.013, Multivariate-adjusted2 0.022 (−0.027, 0.071) 0.378 respectively, (Figure 2), considering non-successful aging as the PAC-1+ /CD62P+ /AV− state of the variable. However, when both phenotypes were Unadjusted 0.307 (0.064, 0.549) 0.014 considered together in the ROC analyses, there was no significant Multivariate-adjusted1 Not included in the stepwise model improvement for the discrimination between successful and non- Multivariate-adjusted2 0.195 (0.000, 0.389) 0.050 successful aging AUC (95%CI): 0.656 (0.554, 0.759), P = 0.005. No CD36+ /AV− associations were found between successful aging and all other Unadjusted 0.01 (0.005, 0.017) 0.001 MV phenotypes determined (Supplementary Table 2). Multivariate-adjusted1 0.011 (0.006, 0.015) <0.0001 Multivariate-adjusted2 0.011 (0.006, 0.016) <0.0001 Results are expressed as B coefficient of regression with its 95% confidence interval DISCUSSION [B (95% CI)]. P from the regression model unadjusted, and 1 adjusted by previous CVD (yes/no), psychotropics (yes/no) and MNA > 23.5, according to stepwise In this cross-sectional study, we have reported an inverse regression; and 2 adjusted by sex, previous CVD (yes/no), psychotropics (yes/no) and MNA > 23.5, dyslipidemia (yes/no), statins (yes/no), and antiplatelet agents association between successful aging and elevated concentrations (yes/no). AV indicates annexin V and cMV, circulating microvesicles. Used markers of cMV from platelets and endothelial cells in dwelling- for MV phenotyping are shown in Supplementary Table 1. Successful aging was community octogenarians. To our knowledge, this is the first defined as good functional (Barthel Index > 90 points, and Lawton Index scores > 7 time that the link between successful aging and cell activation, for women and >4 men) and cognitive status (MEC > 24 points), and no need for institutionalization. Non-successful aging was categorized in having one, two through the quantification of cMV release at the bloodstream, or three scores below good functional and/or cognitive status (Barthel Index ≤ 90 has been reported. points, Lawton Index scores ≤ 7 for women and ≤4 men, and/or MEC ≤ 24 points). Frontiers in Cell and Developmental Biology | www.frontiersin.org 6 July 2021 | Volume 9 | Article 716435 Chiva-Blanch et al. Microvesicles and Cognitive Decline thrombomodulin (CD141), both exposing AV or not, are also in lower concentrations in successfully aged octogenarians. This might be important on the grounds that, besides playing an essential role in coagulation, thrombomodulin has been shown to participate in the regulation of inflammatory processes (Conway, 2012). Consistent with these results, subjects who suffered a stroke show elevated concentrations of cMV from platelets, endothelial cells, erythrocytes and leukocytes with a prothrombotic phenotype at the long term (Chiva-Blanch et al., 2016). Patients with Alzheimer disease and cognitive decline show increased levels of neural-derived MV of specific phenotypes (Kapogiannis et al., 2019; Eren et al., 2020), which are able to identify Alzheimer-related cognitive decline again at the long term. Additionally, we have previously observed in a Chilean cohort that frail older adults present elevated concentrations of total, AV+ and AV− cMV derived from FIGURE 2 | ROC Receiver operating characteristic (ROC)-Curve analysis for monocytes, natural killers and platelets (Arauna et al., 2020), successful aging discrimination. ROC curve analyses revealed that which contributes explaining, at least in part, the increased risk of CD141+ /AV+ (discontinued black line), and CD141+ /CD41+ -CD61+ /AV+ CVD in these subjects. In the same line, in another subset of the (gray line) endothelial- and platelet-derived circulating microvesicles (cMV) OCTABAIX cohort, subjects with previous CVD and cognitive discriminate successful and non-successful aging with an AUC (95%CI) of 0.655 (0.551, 0.758), P = 0.005, and 0.638 (0.535, 0.741) P = 0.013, decline presented higher concentrations of the antifibrinolytic respectively. Black continuous line is the reference line of the ROC curve proteins alpha-2-antiplasmin (A2AP) and coagulation factor (AUC = 0.5). AV indicates annexin V. Used markers for MV phenotyping are XIII-B-chain (FXIIIB) compared to non-CVD subjects with shown in Supplementary Table 1. preserved cognition and functionality (Cubedo et al., 2015). This was accompanied with a coordinated imbalance in several proteins related to inflammation and coagulation (Cubedo et al., Elevated concentrations of cMV from platelets and 2017), pinpointing the association between cytokine-triggered endothelial cells have been found in subjects after a major inflammation and vascular microthrombosis in unhealthy aging, cardiovascular event such as an acute myocardial infarction with CVD and cognitive impairment as underlying causes. In (Chiva-Blanch et al., 2017b, 2019) or stroke (Lee et al., 2012; this setting, it is plausible, therefore, that elevated concentrations Chiva-Blanch et al., 2016). These major adverse cardiovascular of cMV from platelets and endothelial cells with both AV+ and events are known to cause an important decline in functional AV− phenotypes contribute to the pathological state associated and cognitive performance, or even disability because of the lack with non-successful aging (cognitive impairment, CVD and of oxygen supply. In fact, and as expected, subjects with previous diminished quality of life). CVD had significantly lower functional, cognitive and nutritional In our study subjects, malnutrition scores were associated scores compared to subjects free of major adverse cardiovascular with high platelet MV release and low immune cell-derived MV events at 85 years old (data not shown). Vascular dysfunction is blebbing, suggesting that malnutrition might be associated with closely associated with dementia, especially in the oldest (van der platelet hyperreactivity and immune dysfunction. However, data Flier et al., 2018). In fact, cerebral amyloid angiopathy, large and on dietary intake and physical activity was not available, and lacunar infarcts, micro and large hemorrhages, microaneurysms, therefore this association requires further research. perivascular space dilation, and arteriosclerosis predict vascular The study of the effects of (non-successful) aging is primarily cognitive impairment (Skrobot et al., 2016), and such situations focused on tissues and cells, and the effects of aging in circulating have also been linked to increased MV release from platelets, factors is largely unexplored. After multivariate adjustments, endothelial cells and leukocytes (Badimon et al., 2019; Castillo and although B coefficients are low, we have observed that et al., 2019). Given the interrelationship between CVD and octogenarians successfully aged also show lower concentrations cMV, and CVD and functional and cognitive performance, of cMV from platelets and endothelial cells non-exposing it seems biologically plausible that lower scores of functional phosphatidylserine in their surface, thus with lower potential and cognitive tests might be associated with high levels of prothrombotic effect. This observation fits the inflammaging cMV. However, if elevated cMV are the result of cognitive theory (Santoro et al., 2020), which attributes to subclinical dysfunction (vascular cognitive impairment), or one of its causes and chronic low-grade inflammation, that may be perpetuated still remains unknown. by sustained elevated concentrations of proinflammatory cMV, We have observed that subjects with low functional and an underlying cause of aging. In fact, senescent cells have cognitive performance, quantified by Barthel and Lawton been shown to have a proinflammatory secretome (Eitan et al., Indexes and MEC score, show increased levels of cMV 2017; Takasugi, 2018) with increased release of MV and overall with prothrombotic phenotype (AV+ ) derived from platelets, extracellular vesicles (Willis et al., 2020) with proinflammatory and cMV not exposing PS (AV− ) from platelets, endothelial phenotypes (Alibhai et al., 2020; Salminen et al., 2020), cells, leukocytes and erythrocytes. In addition, cMV carrying contributing to age-related comorbidities (Misawa et al., 2020). Frontiers in Cell and Developmental Biology | www.frontiersin.org 7 July 2021 | Volume 9 | Article 716435 Chiva-Blanch et al. Microvesicles and Cognitive Decline However, these studies have been performed in in vitro or in as previously mentioned, this is the first study associating low in vivo models of aging (Russell et al., 2020; Salminen et al., 2020), concentrations of platelet- and endothelial cell-derived cMV and human data is lacking. with successful aging, which opens new future lines of research, In this study, successful aging has been defined as good such as, for instance, the evaluation of the potential of cMV functional (Barthel Index > 90 points, and Lawton Index in adulthood as predictive biomarker of successful aging, from scores ≤ 7 for women and ≤4 men) and cognitive status a holistic and comprehensive perspective, in the late elderly, (MEC > 24 points) and no need for institutionalization (Formiga or the identification of subjects requiring specific strategies et al., 2011). However, this definition of successful aging has been to prevent vascular dysfunction and the onset of cognitive criticized (Laplante, 2014), because wellbeing and perception and functional decline, in order to modulate the rate of non- of successful aging may not be limited to elders with full successful aging. functionality and independence (Golja et al., 2020), or “healthy” from a strictly biomedical/clinical perspective, and may not consider the individual feeling, social aspects (Ding et al., 2020), DATA AVAILABILITY STATEMENT depression, quality of life (Jang, 2020), or the “disability paradox” (Albrecht and Devlieger, 1999). It is of course more likely that The raw data supporting the conclusions of this article will be disabled elders do not report successful aging, yet still some made available by the authors, without undue reservation. elders with some degrees of disability may report wellness (Mitra et al., 2020), especially those enclosed in aging services systems facilitating their risk reduction of chronic diseases associated with ETHICS STATEMENT aging and promoting social inclusion. In this setting, successful aging may be better defined and understood including qualities This study involving human participants was reviewed and and capacities appreciated in the elderly. approved by CEIm of the Jordi Gol i Gurina Foundation. The This study is not exempt of limitations. It is a cross- patients/participants provided their written informed consent to sectional study of associative nature and therefore hypothesis- participate in this study. generating. Further studies are needed to evaluate the causality or consequence of elevated concentrations of cMV in functional and cognitive performance. In addition, data on age of CVD AUTHOR CONTRIBUTIONS onset, and data on other comorbidities such as neurodegenerative GC-B, TP, and LB: conceptualization. GC-B and AV-F: diseases or cancer are lacking. However, it is unlikely that methodology. FF and AF: validation. GC-B: formal analysis. LB, subjects with advanced Alzheimer disease or with a tumoral FF, and AF: investigation, all, resources. TP: data curation. GC-B: process, for instance, or within the acute phase of a major writing—original draft preparation, writing—review and editing, adverse cardiovascular event are not being institutionalized. all. LB: funding acquisition. All authors have read and agreed to Samples used in this study belong to a biobank, and have the published version of the manuscript. been carefully stored under −80◦ C for 10 years. This might provoke an unknown impact on MV stability. However, samples were carefully processed. They were collected and immediately frozen in aliquots in 2009, and were processed FUNDING and analyzed within 3 months in 2019. All samples were This work was supported by grants received from the Spanish handled following the same procedure and all samples were Ministry of Science, Innovation and Universities (PID 2019- frozen and thawed only for one specific analysis, in this case 107160RB-I00); from Institute of Health Carlos III (CIBERCV the MV analysis. Therefore, the potential effects of long-term CB16/11/00411 and Red TerCel RD16/0011/0018) cofounded by effects of storage at −80◦ C in MV stability is comparable FEDER; ERA-CVD JTC2020-023/AC209-00054. and homogeneous between samples. Finally, the lower limit of detection of the cytometer used for cMV quantification is between 0.2 and 0.3 µm. Therefore, information on ACKNOWLEDGMENTS extracellular vesicles from smaller sizes are missing and might be of relevance. We thank the Fundación Investigación Cardiovascular- In summary, our study shows an inverse association between Fundación Jesus Serra for the continuous support. endothelial- and platelet-derived cMV release at the bloodstream and higher scores in functional and cognitive tests reflecting successful aging. Our results contribute in the accumulating SUPPLEMENTARY MATERIAL evidence of the link between unsuccessful aging, cognitive decline and vascular inflammation and damage, determined The Supplementary Material for this article can be found here as elevated circulating levels of MV from platelets and online at: https://www.frontiersin.org/articles/10.3389/fcell.2021. endothelial cells at the bloodstream. 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M., Skoog, I., Schneider, J. A., Pantoni, L., Mok, V., and Chen, C. L. H. (2018). Vascular cognitive impairment. Nat. Rev. Dis. Primers 4:18003. Copyright © 2021 Chiva-Blanch, Vilella-Figuerola, Padró, Formiga, Ferrer and von Faber, M., Bootsma-van der Wiel, A., van Exel, E., Gussekloo, J., Lagaay, A. M., Badimon. This is an open-access article distributed under the terms of the van Dongen, E., et al. (2001). Successful aging in the oldest old. Arch. Intern. Creative Commons Attribution License (CC BY). The use, distribution or Med. 161, 2694–2700. reproduction in other forums is permitted, provided the original author(s) Willis, C. M., Nicaise, A. M., Bongarzone, E. R., Givogri, M., Reiter, C. R., Heintz, and the copyright owner(s) are credited and that the original publication O., et al. (2020). Astrocyte Support for Oligodendrocyte Differentiation can in this journal is cited, in accordance with accepted academic practice. No be Conveyed via Extracellular Vesicles but Diminishes with Age. Sci. Rep. use, distribution or reproduction is permitted which does not comply with 10:828. these terms. Frontiers in Cell and Developmental Biology | www.frontiersin.org 10 July 2021 | Volume 9 | Article 716435