Abstract

   Time spent sitting is positively correlated with endothelial
   dysfunction and cardiovascular disease risk. The underlying molecular
   mechanisms are unknown. MicroRNAs contained in extracellular vesicles
   (EVs) reflect cell/tissue status and mediate intercellular
   communication. We explored the association between sitting patterns and
   microRNAs isolated from endothelial cell (EC)-derived EVs. Using extant
   actigraphy based sitting behavior data on a cohort of 518
   postmenopausal overweight/obese women, we grouped the woman as
   Interrupted Sitters (IS; N = 18) or Super Sitters (SS; N = 53) if they
   were in the shortest or longest sitting pattern quartile, respectively.
   The cargo microRNA in EC-EVs from the IS and SS women were compared.
   MicroRNA data were weighted by age, physical functioning, MVPA, device
   wear days, device wear time, waist circumference, and body mass index.
   Screening of CVD-related microRNAs demonstrated that miR-199a-5p,
   let-7d-5p, miR-140-5p, miR-142-3p, miR-133b level were significantly
   elevated in SS compared to IS groups. Group differences in let-7d-5p,
   miR-133b, and miR-142-3p were validated in expanded groups. Pathway
   enrichment analyses show that mucin-type O-glycan biosynthesis and
   cardiomyocyte adrenergic signaling (P < 0.001) are downstream of the
   three validated microRNAs. This proof-of-concept study supports the
   possibility that CVD-related microRNAs in EC-EVs may be molecular
   transducers of sitting pattern-associated CVD risk in overweight
   postmenopausal women.

   Subject terms: Epidemiology, Translational research, Cell biology

Introduction

   In U.S. adults aged 45 years old or older, excessive sedentary behavior
   and prolonged sitting patterns are associated with obesity,
   cardiometabolic disorders, increased cardiovascular events, cancer, and
   all-cause mortality^[46]1–[47]3. Sedentary behavior is characterized by
   energy expenditure lower than 1.5 metabolic equivalent (MET), including
   lying, reclining, and sitting^[48]4. On average, older adults (age ≥ 45
   years) spend 65% of their waking hours in sedentary behavior, 33% in
   light physical activity (100–2019 counts per minute, cpm), and 2% in
   moderate-to-vigorous physical activity (MVPA, ≥ 1952 cpm), as measured
   with accelerometry^[49]5,[50]6. Sitting is a non-movement posture form
   of sedentary behavior^[51]4. Using device-based measures of sitting
   behavior, we recently showed that longer total sitting time and mean
   sitting bout duration (i.e., sitting pattern) were associated with
   cardiometabolic and cancer risk biomarkers among 518 overweight and
   obese postmenopausal women^[52]7. In this population, the women had a
   mean (SD) daily sitting time of 9.1 (1.6) hours and a mean (SD) sitting
   bout duration of 39.2 (15.5) minutes. A nationwide study involving 4757
   participants revealed women at mean age 47.7 spent an average 8.51
   (1.29) hours in sedentary behavior and 5.46 (1.55) minutes in sedentary
   bouts^[53]8. Sitting and sedentary time are associated with CVD risk,
   including endothelial dysfunction.

   Endothelial dysfunction is an early event during vascular injury, which
   impairs vasodilation and promotes development of plaque and
   inflammation in the vascular wall^[54]9. Several laboratory studies
   show that endothelial function is rapidly impaired by prolonged
   sitting^[55]10,[56]11. Shortening bouts of prolonged sitting with low
   energy expenditure active breaks acutely prevents endothelial function
   decline in young, active adults and improves endothelial function in
   older, sedentary overweight/obese women^[57]10,[58]12,[59]13. Although
   the association between prolonged sitting and endothelial dysfunction
   are becoming clearer, the molecular mechanisms that link physiological
   alterations and pathological progression remain unknown.

   In this regard, microRNAs (miRs) in endothelial cells (ECs) are one of
   the molecule classes that changes with cellular status in response to
   aging, oxidized low-density lipoprotein, hyperglycemia, hypertension,
   and magnitudes of antegrade blood flow^[60]14,[61]15. MiRs are small
   non-coding RNAs of approximately 22 nucleotides, which alter biological
   functions by silencing genes via translational repression and mRNA
   degradation^[62]16. Growing evidence reveals that cargo molecules,
   including miRs, in extracellular vesicles (EVs) are stable markers for
   clinical diagnosis, prognosis, and monitoring treatment
   response^[63]17. In addition to regulating intra-cellular functions,
   miRs transported in EVs mediate tissue cross-talk and inter-cellular
   communication^[64]18. They are found in biofluids and can be biomarkers
   of the physiologic function and disease status of their originating
   cell type^[65]18. For example, EC-derived miR-92a secreted in
   CD144-enriched microparticles mediates endothelial dysfunction and thus
   predisposes chronic kidney disease patients to cardiovascular disease
   (CVD) progression^[66]19. Twelve miRs isolated from human plasma EVs
   have been identified to potentially impact muscle remodeling and growth
   resulting from acute exercise^[67]20.

   Given that miRs may be biomarkers and molecular transducers of health
   outcomes and that the vascular endothelium is a primary tissue affected
   by prolonged sitting, we hypothesized that miR expression in EC-derived
   EVs (EC-EVs) are influenced by prolonged sitting pattern. Specifically,
   we examined whether miRs from isolated plasma EC-EVs used in a targeted
   screen of CVD-miRs are associated with sitting pattern among
   overweight/obese postmenopausal women with either short or long mean
   sitting bout duration. We further explored functional pathways through
   which the identified miRs may link the physiological perturbations and
   disease risk associated with prolonged sitting pattern.

Results

   From an original cohort of 518 postmenopausal, overweight or obese,
   sedentary women classified by a validated machine-learned algorithm
   based on accelerometer measures, 18 were categorized as Interrupted
   Sitters (IS) and 53 as Super Sitters (SS). The two groups were
   classified using quartile cross-tabulation of individual sitting
   patterns and MVPA, including the lowest and highest quartiles of mean
   sitting bout duration, and the lowest quartile MVPA (< 7 min/day,
   Supplementary Table [68]S1). Data and plasma from these individuals
   were used to screen 84 CVD-related miRs (Supplementary Table [69]S2) in
   the first stage of this study.

   Table [70]1 shows participant demographic, activity, and
   cardiometabolic risk biomarker characteristics of the IS and SS groups.
   Although IS and SS are similar to each other in many respects, the SS
   group had significantly lower physical functioning, MVPA, and walking
   time, and greater total sitting time and mean sitting bout duration,
   compared to the IS group. SS women sat for an average of 11.0 ± 1.2 h
   per day in bouts averaging 64.0 min, while IS women sat for an average
   of 7.4 ± 1.1 h per day in bouts averaging 25.5 min.

Table 1.

   Demographics, activity-related measures, and cardiometabolic risk
   biomarkers of Interrupted Sitters (IS) and Super Sitters (SS).
   Total (n = 71) IS (n = 18) SS (n = 53) P-value
   Age, mean (sd) 65.9 (6.6) 64.2 (5.0) 66.5 (7.0) 0.12
   Race, n (%)^a 0.13
   White 58 (81.7) 14 (87.5) 44 (83)
   Black 3 (4.2) 0 (0) 3 (5.7)
   Native American 1 (1.4) 0 (0) 1 (1.9)
   Asian 0 (0) 0 (0) 0 (0)
   Pacific Islander 2 (2.8) 2 (12.5) 0 (0)
   Other/Unknown 0 (0) 0 (0) 0 (0)
   Mixed 2 (2.8) 0 (0) 2 (3.8)
   Hispanic ethnicity, n (%) 0.42
   Hispanic 24 (33.8) 1 (5.6) 9 (17)
   Non-Hispanic 47 (66.2) 17 (94.4) 44 (83)
   Marital Status, n (%) 0.12
   Married/Living together 34 (47.9) 12 (66.7) 22 (41.5)
   Single/Divorced/Widowed/Separated 37 (52.1) 6 (33.3) 31 (58.5)
   Highest education level, n (%) 0.51
   Up to high school completion 7 (9.9) 3 (16.7) 4 (7.5)
   Some college or vocation training 36 (50.7) 9 (50.0) 27 (50.9)
   College graduate 28 (39.4) 6 (33.3) 22 (41.5)
   Physical functioning, mean (sd) 61.2 (28.9) 74.9 (20.7) 56.5 (30.0)
   0.01*
   Activity-related measures, mean (sd)
   Total sitting time; min/day 603.4 (120.8) 442.6 (68.8) 658.0 (73.8)
    < 0.01*
   Mean sitting bout duration; min/day 54.3 (26.9) 25.5 (3.2) 64.0 (24.3)
    < 0.01*
   Moderate-to-vigorous activity; min/day 3.5 (1.8) 4.5 (1.6) 3.2 (1.8)
   0.01*
   Walking time; min/day 32.7 (25.8) 53.2 (30.7) 25.7 (19.9) < 0.01*
   Cardiometabolic biomarkers, mean (sd)
   Body mass index; kg/m^2 32.8 (5.0) 31.3 (3.2) 33.3 (5.4) 0.06
   Waist circumference; cm^b 101.4 (15.9) 99.0 (8.5) 102.1 (17.7) 0.34
   Fasting glucose; mg/dL^c 116.3 (43.8) 120.4 (50.3) 115.0 (41.8) 0.68
   Fasting insulin; pg/mL 671.9 (443.4) 644.4 (417.4) 681.3 (455.4) 0.75
   HOMA-IR 5.6 (4.9) 5.3 (3.7) 5.8 (5.2) 0.68
   HOMA2-IR 2.7 (1.9) 2.5 (1.5) 2.7 (2.0) 0.69
   Parent study, n (%) < 0.01*
   CoM 21 (29.6) 2 (11.1) 19 (35.8)
   RFH 42 (59.1) 13 (72.2) 29 (54.7)
   MENU 8 (11.3) 3 (16.7) 5 (9.4)
   [71]Open in a new tab

   CoM, Community of Mine; RFH, Reach for Health; MENU, Metabolic,
   Exercise and Nutrition at University of California, San Diego; HDL,
   high-density lipoproteins; HOMA-IR, homeostatic model assessment of
   insulin resistance; LDL, low-density lipoproteins. P-values computed
   using Chi-square tests for categorical variables and t-tests for
   continuous variables.

   *P < 0.05.

   ^aMissing race data from 3 participants in RfH and 2 participants in
   CoM.

   ^bMissing waist circumference data from 2 participants in RfH.

   ^c4 participants in IS and 8 participants in SS had fasting
   glucose > 125 mg/dL.

   In preparation for analysis of the clinical samples, we examined the
   specificity of CD144 for EC-EVs (vs. EVs from other cell types that
   predominate in the vasculature) and the biochemical and ultrastructural
   characteristics of the CD144 + EC-EVs. CD144, also known VE-cadherin,
   is a junctional protein specifically expressed on ECs for controlling
   vascular permeability. Dot-blot assay of EVs purified from conditioned
   media of ECs, Smooth muscle cells (SMCs), and peripheral blood
   mononuclear cells (PBMCs) showed CD144 protein signal on EC-EVs but not
   SMC- and PBMC-EVs (Supplementary Fig. [72]S1A). CD63 protein, a common
   EV marker, was expressed on EVs from all three cell types. Transmission
   electronic microscopy (TEM) images with immunogold labeling show the
   ultrastructure of EVs and markers of EV (CD81, CD63, LAMP1) and EC
   (CD144) on EVs purified from EC-conditioned media and plasma
   (Supplementary Fig. [73]S1B). Fluorescence flow cytometry analysis
   demonstrated a subpopulation of plasma CD63^+ EVs co-stained with
   anti-CD144 antibody and the lipid membrane dye CFSE (Supplementary Fig.
   [74]S1C). Approximately 71.1% of CD63^+ EVs were CD144^+CFSE^+.
   Together, these experiments demonstrate that CD144 can be used to
   specifically identify EC-EVs.

   To address the fraction and stability of miRs in intact EC-EVs, we
   conducted protection assays treating purified EC-EVs from
   EC-conditioned media with PBS, proteinase K (PK), Triton, and/or RNase.
   Levels of MiR-126, a known EC-enriched miR, were reduced 93% by the
   Triton + RNase treatment compared to the Mock treatment (PBS),
   suggesting that EV-contained miRs are protected from degradation in the
   circulation (Supplementary Fig. [75]S1D). Treatment with RNase alone or
   with PK resulted in 24% and 31% reductions in miR-126 levels,
   respectively, suggesting that EC-EVs are intact and protect the
   majority of miRs from RNase degradation in the conditioned media.

   Having developed a protocol to isolate EC-EVs from plasma, EC-EVs were
   isolated from IS or SS plasma samples using anti-CD144
   immunoprecipitation and subsequent total RNA extraction. CVD-related
   miRs were screened for differential expression in IS and SS groups
   using an array of 84 CVD-related miRs. RNA samples were pooled in sets
   of 3 (a single SS pool consisted of only 2 samples), as shown in
   Supplementary Fig. [76]S2. Obtained miR expression values across the
   samples were adjusted by balancing on key covariates using Inverse
   Probability of Treatment Weighting (IPTW). MiR-133b, miR-140-5p,
   miR-142-3p, let-7d-5p, miR-199a-5p were selected for technical
   validation using a two-step process. The top 10 miRs were identified by
   standardized absolute ATE ranking. Then, in that set of 10 miRs, the 5
   miRs with the largest standard deviation were selected (Table [77]2) to
   ensure the low SD did not artificially inflate the ATE, and hence lead
   to a high ATE rank in the first step. Supplementary Table [78]S3 shows
   standardized absolute ATE rankings of the other 74 miRs on the array.

Table 2.

   Top 10 miRs ranked by standardized average treatment effect (ATE) &
   standard deviation (SD) of miR levels from IS and SS groups.
   Mean difference in relative expr Relative expr, SD^a Standardized ATE
   rank Standardized ATE SD rank
   miR-133b* 11.30 5.15 7 4.38 31
   miR-140-5p* 5.70 2.26 8 4.37 43
   miR-142-3p* 5.15 2.21 3 5.29 45
   let-7d-5p* 4.19 1.94 2 5.33 53
   miR-199a-5p* 3.25 1.53 1 5.44 58
   miR-146a-5p 2.96 1.51 5 4.82 60
   miR-103a-3p 1.80 0.80 10 4.17 70
   miR-424-5p 1.15 0.61 6 4.50 75
   let-7e-5p 0.52 0.28 9 4.20 80
   miR-155-5p 0.76 0.27 4 5.02 81
   [79]Open in a new tab

   ATE, average treatment effect: weighted mean difference of miR levels
   between SS and IS; Standardized ATE, ATE divided by bootstrapped SD of
   ATE; Expr, expression. SD, standard deviation.

   *Top 5 miRs based on combined standardized ATE ranking then, SD
   ranking.

   ^aUnweighted SD across IS and SS.

   We next conducted technical validation studies of the 5 prioritized
   miRs, miR-133b, miR-140-5p, miR-142-3p, let-7d-5p, miR-199a-5p, using
   individual EC-EV RNA samples. In addition, we augmented the IS and SS
   groups for Hispanic ethnicity to enable exploration of potential
   differences by ethnicity in miR expression associated with IS and SS
   sitting patterns. All women who self-identified as Hispanic and who
   were in the 2nd-lowest quartile of MVPA (7–15.5 min/day) and 1st and
   4th quartiles of mean sitting bout duration (N = 7 from each) were
   added to the original IS and SS groups to create the amended groups
   IS + (n = 25; 8 Hispanic) and SS + (n = 60; 16 Hispanic), respectively
   (Supplementary Table [80]S4). These amended groups were used to
   validate the 5 miRs selected in the screening stage of analyses.

   Supplementary Table [81]S4 shows the quartile characterization of mean
   sitting bout duration and MVPA for the IS + and SS + groups.
   Supplementary Table [82]S5 shows the age and activity-related
   characteristics. Differential expression was confirmed for let-7d-5p,
   miR-133b, and miR-142-3p using unadjusted data, with greater miR
   expression in EC-EVs from SS + compared to IS + individuals (Fig.
   [83]1). Differential expression of let-7d-5p, miR-133b, and miR-142-3p
   between IS + and SS + was sustained after IPTW (Table [84]3). We
   examined correlations among miRs using unadjusted data (Supplementary
   Table [85]S6). Let-7d-5p and miR-142-3p were positively correlated
   (Pearson’s r: 0.426) across all IS + and SS + individuals. After group
   stratification, we found a stronger correlation between let-7d-5p and
   miR-142-3p in SS + (Pearson’s r: 0.434) than IS + (Pearson’s r: 0.176,
   Fig. [86]2).

Figure 1.

   Figure 1
   [87]Open in a new tab

   Differential expression of individually validated miRs from IS + and
   SS + (unadjusted data). The highest ranking 5 cardiovascular disease
   (CVD)-related EC-EV miRs initially identified in pooled RNA sample sets
   from IS and SS were validated by qPCR in individual RNA samples. IS + :
   Interrupted Sitter group with enhanced ethnic diversity (N = 25);
   SS + : Super Sitter group with enhanced ethnic diversity (N = 60).
   Statistical significance between IS + and SS + groups was examined by
   unadjusted t-test. Horizonal bars indicate group means.

Table 3.

   Validation analysis of target miRs differentiating IS + and
   SS + groups.
              ATE   95%CI            Standardized ATE
   miR-133b   0.168 (0.108, 0.228)*       5.462
   let-7d-5p  0.002 (0.001, 0.004)*       3.323
   miR-142-3p 0.006 (0.001, 0.011)*       2.196
   miR-140-5p 0.056 (− 0.026, 0.138)      1.332
   miR-199-5p 0.010 (0.000, 0.0206)       1.973
   [88]Open in a new tab

   ATE, average treatment effect: weighted mean difference of miR levels
   between SS + and IS + ; Standardized ATE, ATE divided by bootstrapped
   SD of ATE.

   *Statistically significant, zero not included in 95% CI.

Figure 2.

   Figure 2
   [89]Open in a new tab

   Correlation between miR-142-3p and let-7d-5p. Measurements of
   miR-142-3p and let-7d-5p from individual participants in IS + and
   SS + were plotted. Data from participants in IS + and SS + groups are
   shown in blue and red dots, respectively. Blue, red, and black lines
   indicate linear correlations in IS + , SS + , and combined groups,
   respectively.

   To explore possible miR-mediated biological mechanisms that underlie
   associations between prolonged sitting pattern and cardiometabolic risk
   in older women, we conducted bioinformatics-based functional pathway
   analyses on the 3 validated miRs (let-7d-5p, miR-133b, and miR-142-3p).
   Two approaches were used: TargetScan to predict miR target genes based
   on potential sequence alignments between the miRs and target mRNAs; and
   TarBase to identify miR target genes supported by experimental evidence
   in the literature. Pathway analysis using TargetScan identified 7
   functional pathways as potential targets of let-7d-5p, miR-133b, and/or
   miR-142-3p: mucin type O-Glycan biosynthesis, adrenergic signaling in
   cardiomyocytes, signaling pathways regulating pluripotency of stem
   cells, valine, leucine, and isoleucine biosynthesis, biosynthesis of
   amino acids, oocyte meiosis, and adherens junction (Table [90]4).
   Pathway analysis using TarBase identified 20 functional pathways as
   potential targets of let-7d-5p, miR-133b, and/or miR-142-3p (Table
   [91]4, Supplementary Tables [92]S7 and [93]S8). Direct and indirect
   target genes of let-7d-5p and miR-143-3p that are components of the
   adherens junction pathway are shown in Supplementary Fig. [94]S3. This
   pathway is a potential mechanism underlying prolonged sitting among
   overweight/obese postmenopausal women.

Table 4.

   Functional pathway analysis of predicted sequence and literature-based
   miR target genes.
   KEGG pathway^a Mucin type O-Glycan biosynthesis Adrenergic signaling in
   cardiomyocytes Signaling pathways regulating pluripotency of stem cells
   Valine, leucine and isoleucine biosynthesis Biosynthesis of amino acids
   Oocyte meiosis Adherens junction
   P-value 2.42E−08 4.34E−04 3.70E−03 4.51E−03 3.24E−02 4.05E−02 4.48E−02
   MicroRNAs associated with prolonged sitting

   miR-133b

   let-7d-5p
   miR-133b let-7d-5p let-7d-5p

   let-7d-5p

   miR-142-3p

   miR-133b

   let-7d-5p

   miR-142-3p
   miR-142-3p
   Predicted target genes (direct target) GALNT1 PPP2CA ACVR1C BCAT1 ARG2
   CDC25C RAC1
   GALNT8 PPP2R2D HAND1 BCAT1 CPEB1 WASL
   PPP2CB HOXB1 TKTL2 ITPR2
   TPM4 SKIL PPP2CA
   SMARCAD1 PPP2CB
   WNT9A SMC1A
   KEGG Pathway^b Adherens junction Lysine degradation TGF-β signaling
   pathway Hippo signaling pathway Viral carcinogenesis Cell cycle
   Pathways in cancer
   P-value 5.60E−07 9.75E−06 2.66E−05 9.22E−05 1.09E−04 1.19E−04 1.68E−04
   MicroRNAs associated with prolonged sitting

   let-7d-5p

   miR-142-3p

   let-7d-5p

   miR-142-3p

   let-7d-5p

   miR-142-3p

   let-7d-5p

   miR-142-3p

   miR-133b

   let-7d-5p

   miR-142-3p
   let-7d-5p

   let-7d-5p

   miR-142-3p
   Literature-supported target genes (direct target) ACTG1 CREBBP ACTB
   CDK4 CCNA2 AR
   CREBBP E2F4 ACTG1 NRAS CCND1 ARHGEF1
   CTNNB1 E2F5^c BIRC2 YWHAE CCNE2 BRAF
   FGFR1 MAPK1 CCND1 CCNA2 CDK4 CASP3
   IGF1R MYC CTNNB1 ATF6B CHEK1 CDK4
   MAPK1 PPP2CA DVL3 YWHAG CREBBP CCND1
   PTPN6 PPP2CB FZD1 DDX3X E2F2 CCNE2
   PTPRJ PPP2R1B FZD3 CHEK1 E2F3 CXCR4
   RAC1 RPS6KB2 GLI2 TP53 E2F4 DVL3
   SMAD2 SMAD2 MYC CASP3 MDM2 E2F2^c
   SMAD3 SMAD3 PPP2CA CCND1 MYC E2F3
   SMAD4 SMAD4 PPP2CB CCNE2 SMAD2 FADD
   TGFBR1^c SMAD7 PPP2R1B SKP2 SMAD3 FZD3
   TGFBR2 TGFBR1^c PARD6B CCR5 SKP2 GLI2
   TGFBR2 SMAD2 PRKACA TP53 HIF1A
   SMAD3 DDB1 YWHAE HSP90AA1
   SMAD4 RAC1 YWHAG IGF1R
   SMAD7 CDKN1A ITGAV
   TGFBR1 MAPK1 MAPK8
   TGFBR2 CREBBP MSH6
   YWHAE JAK1 NRAS
   YWHAG MDM2^c PLCG1
   RUNX1
   SMAD2
   SMAD3
   SKP2
   TGFBR1^c
   TP53
   Literature-supported target genes (non-direct target) ACTB SETD1B
   ACVR1B PPP1CA PKM ESPL1 ADCY1
   ACP1 PLOD2 ACVR2A YAP1 HLA-B CDC6 ETS1
   CSNK2A1 KMT2D^c BAMBI AMOT DDX2X PCNA GNG12
   CSNK2A2 SUV420H1 PPP2R1A LIMD1 EP300 CCNB1 LPAR2
   CTNND1 DOT1L^c BMP8A CSNK1E GTF2B ORC2 GNB1
   FARP2 KMT2C BMPR2 TEAD4 MRPS18B DBF4 ARNT2
   FER SETD1A MOB1A HPN STAG2 GNB2
   FYN EHMT1 FHF1 HIST1H2BG WEE1 MYC
   INSR KMT2B WNT9A RBPJ E2F5 RASGRP4
   NLK WHSC1L1 FZD7 HIST1H2BL CDC45 PDGFB
   PTPRF ASH1L PPP2R2C GTF2A1 CDC27 PRKACA
   PVRL2 WHSC1 PPP2R2A SRF CDKN1A EGLN3
   SSX2IP KMT2A PPP2R1A GTF2E1 PRKDC RAC1
   TJP1 ALDH9A1 CSNK1E HIST1H2BB PLK1
   VCL PLOD1 BMP8A HIST1H4D ANAPC13
   WASF1 SMAD7 ATF2 MCM3
   WASF2 LATS1 GTF2H1 CDC25A
   WASL BMPR2 CREB5
   YES1 PIK3R2
   [95]Open in a new tab

   ^aKEGG pathways were identified based on TargetScan predicted miR
   target genes.

   ^bKEGG pathway analysis based on Tarbase v7.0

   ^cGenes targeted by 2 miRs.

Discussion

   In this exploratory, proof of concept study, sitting patterns
   consisting of longer mean bout duration (SS + ; 62.8 ± 23.4 min) were
   associated with significant elevation of let-7d-5p, miR-133b, and
   miR-142-3p in circulating EC-EVs, compared to patterns with more
   interrupted, shorter sitting bouts (IS + ; 25.9 ± 2.8 min) among 85
   overweight/obese postmenopausal women. Pathway analyses of the putative
   and literature supported gene targets of these three miRs suggest that
   they may alter multiple biological pathways relevant to sitting
   time-associated disease risk. To our knowledge, this is the first study
   to demonstrate a link between device-measured sitting pattern
   differences and EC-originating, circulating miRs in any population. It
   is also the first to identify candidate molecular mediators of health
   impacted by sitting pattern.

   Adults who are older than 60 years are the largest sedentary population
   in the United States^[96]21. Give the association of sedentary time and
   CVD risk, this risk may be augmented in postmenopausal women who have
   higher CVD risk due to steep estrogen decline during menopause^[97]22.
   Although older women spend less time on sedentary behavior than
   age-matched men, CVD risk driven by biological differences and
   lifestyle factors are notable among this high-risk population^[98]21.
   Dose–response associations of CVD risk with sitting time and patterns
   was demonstrated in a large cohort study among older women (age range
   63–97 years)^[99]2. Postmenopausal women have increased risk for
   overweight/obesity and insulin resistance, which further elevates CVD
   risk^[100]23. Clinical and epidemiological evidence indicate that
   research studies among postmenopausal women is urgently needed to
   enhance healthy aging and quality of life for this special population.
   The women in the SS group on average sat 3.6 h longer per day and spent
   38.5 min longer at each sitting bout than the women in the IS group
   (Table [101]1). Daily behavior is composed in an isotemporal framework
   wherein reduction in sitting time is simultaneously associated with an
   increase in other non-sitting behaviors. There was a 1.3-min difference
   in daily MVPA and 27.5-min difference in daily walking time between IS
   and SS women, suggesting that “interrupted sitter” overweight/obese
   postmenopausal women replace sitting behavior with light physical
   activity (i.e., walking time) rather than MVPA (Table [102]1). Daily
   MVPA in the combined groups was very low (3.5 ± 1.8 min/day;
   mean ± SD). Although the 1.3-min group difference in daily MVPA was
   statistically significant, it is unlikely to be associated with a
   biologically or clinically significant impact.

   During prolonged sitting, particularly in uninterrupted bouts, blood
   flow and skeletal muscle contractions are reduced in the lower
   extremities, collectively contributing to prolonged sitting-associated
   endothelial dysfunction^[103]12,[104]24. Expression of miRs in ECs is
   dynamic and dose-responsive to reflect endothelial homeostasis or
   dysfunction in response to external stimuli and conditions such as
   those associated with prolonged sitting^[105]25,[106]26. The three miRs
   identified in this study are linked with CVD risk factors and EC
   biology. Overexpression of miR-133b in human retinal EC exposed to a
   hyperglycemic condition prohibited proliferation and facilitated
   apoptosis^[107]27. Overexpression of miR-142-3p in primary human aortic
   endothelial cells prevented high-glucose-induced
   endothelial-to-mesenchymal transition, a process involved in cardiac
   fibrosis^[108]28. Systemic administration of let-7d mimetics into
   diabetic ApoE^-/- mice decreased inflammatory genes, suggesting a
   protective role of let-7d in diabetes-associated
   atheroscleorsis^[109]29. In an atherosclerotic mouse model with chronic
   inflammation, miR-133b and miR-142-3p facilitated vulnerable plaque
   formation and induced EC apoptosis, respectively^[110]30,[111]31.
   Let-7d overexpression was shown to inhibit endothelial migration,
   proliferation, and angiogenesis in vitro^[112]32. These studies
   demonstrate that potential EC regulatory function and CVD-causal
   effects of the three differentially expressed miRs identified in the
   present study are associated with the prolonged sitting pattern.

   Extracellular miRs released from cells and tissues are potential
   biomarkers and/or mediators of acute myocardial infarction, chronic
   heart failure, diabetes, and other CVDs to mediate cellular
   communication when they are taken up by other
   cells^[113]17,[114]33–[115]35. Body fluid levels of extracellular miRs,
   without tissue(s) origin identity, are associated with modifiable
   lifestyle factors, including screen time (a sedentary
   behavior-associated activity), exercise, and diet^[116]36–[117]38.
   Among 80 primary school children in Belgium (COGNition and Air
   pollution in Children study), each additional screen time hour per week
   was associated with a 3.44% higher level of miR-222 and 1.84% higher
   level of miR-146a in saliva^[118]36. Regular exercise for 20 weeks
   significantly increased miR-142-3p, miR-221-3p, miR-126-3p, miR-146-5p,
   and miR-27b-3p, and decreased miR-486-5p, let-7b-5p, miR-29c-3p,
   let-7e-5p, miR-93-5p, miR-7-5p, miR-25-3p, miR-92a-3p, and miR-29b-3p
   in serum from 20 participants enrolled in the HERITAGE Family
   study^[119]37. Nine miRs, including miR-10b, miR-155, miR-200b,
   miR-296-5p, miR-375, miR-92a, miR-145, miR-204, and miR-211, responded
   to dietary zinc deprivation and repletion among 10 men from the General
   Clinical Research Center at University of Florida^[120]38. We found
   that older women who spent an average of 62.8 ± 23.4 uninterrupted
   minutes in a sitting posture (mean sitting bout duration) have a higher
   level of let-7d-5p, miR-133b, and miR142-5p in circulating EC-EVs
   compared to those who sat 25.9 ± 2.8 min.

   By targeting EVs with specific tissue origin, this study provides a
   better resolution of physiological-to-pathological changes in
   endothelial dysfunction. It identifies potential sitting
   behavior-associated miR target signaling pathways in ECs as well as
   recipient cells and tissues that mediate sitting-associated disease
   risk. Functional pathway enrichment analyses of the top three
   differentially expressed, technically validated miRs identified target
   genes and biological pathways. Prolonged sitting patterns among
   postmenopausal women potentially affect (1) cellular and vascular
   function through regulating mucin-type O-glycosylation and adherens
   junction pathways, (2) cardiomyocyte function via modulation of
   adrenergic signaling, and (3) branched chain amino acid (BCAA; valine,
   leucine, and isoleucine) metabolism. Mucin-type O-glycosylation is a
   glycosylation type that adds an N-acetylgalactosamine moiety to serine
   and threonine residues in proteins and these glycosylation
   modifications are critical for vascular integrity, especially during
   blood vessel development^[121]39. Adherens junctions are the major
   structural components that create cell-to-cell barriers and enable
   endothelial cells to control vascular permeability^[122]40. Abnormal
   β-adrenergic signaling usually found in aged hearts with cardiac
   dysfunction^[123]41. Elevation of circulating BCAA concentrations is
   associated with insulin resistance, onset of type 2 diabetes and
   cardiovascular events, and mitochondrial dysfunction^[124]41–[125]43,
   and BCAA concentrations are decreased with weight loss and insulin
   sensitization^[126]42,[127]43.

   In this exploratory study, we identified three EC-derived, circulating
   miRs that bridge device-measured sitting patterns to biological
   effector genes and pathways. Several limitations warrant
   acknowledgement when interpreting our findings. First, the small sample