Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Apr 1.
Published in final edited form as: J Trauma Acute Care Surg. 2023 Dec 28;96(4):548–556. doi: 10.1097/TA.0000000000004225

Exosomal miRNA Following Severe Trauma: Role in Bone Marrow Dysfunction

Jennifer A Munley a, Micah L Willis a, Gwendolyn S Gillies a, Kolenkode B Kannan a, Valerie E Polcz a, Jeremy A Balch a, Evan L Barrios a, Shannon M Wallet b, Letitia E Bible a, Philip A Efron a, Robert Maile a, Alicia M Mohr a
PMCID: PMC10978306  NIHMSID: NIHMS1946131  PMID: 38151766

Abstract

Introduction:

Severe trauma disrupts bone marrow function and is associated with persistent anemia and altered hematopoiesis. Previously, plasma-derived exosomes isolated after trauma have been shown to suppress in vitro bone marrow function. However, the cargo contained in these vesicles has not been examined. We hypothesized that trauma plasma-derived exosomes exhibit microRNA (miR) changes that impact bone marrow function after severe injury.

Methods:

Plasma was collected from a prospective, cohort study of trauma patients (n = 15; 7 males, 8 females) with hip and/or femur fractures and an injury severity score (ISS) ≥ 15; elective total hip arthroplasty (THA) patients (n = 8; 4 males, 4 females) served as operative controls. Exosomes were isolated from plasma with the Invitrogen Total Exosome Isolation Kit and RNA was isolated using a miRNeasy Mini Kit. Direct quantification of miRNA was performed by NanoString Technologies on a human miRNA gene panel and analyzed with nSolver with significance defined as p<0.05.

Results:

There were no differences in age or sex distribution between trauma and THA groups; the average ISS was 23. Trauma plasma-derived exosomes had 60 miR identities that were significantly downregulated and 3 miR upregulated when compared to THA (p<0.05). Twelve of the downregulated miR have a direct role in hematopoiesis regulation. Further, male trauma plasma-derived exosomes demonstrated downregulation of 150 miR compared to male THA (p<0.05). Female trauma plasma-derived exosomes demonstrated downregulation of only four miR and upregulation of two miR compared to female THA (p<0.05).

Conclusions:

We observed downregulation of 12 miRNA linked to hematopoiesis along with sexual dimorphism in miR expression from plasma-derived exosomes following severe trauma. Understanding sexually dimorphic miR expression provides new insight into sex-based changes in postinjury systemic inflammation, immune system dysregulation, and bone marrow dysfunction and will aid us in more precise future potential therapeutic strategies.

Level of Evidence:

II

Study Type:

Prognostic and Epidemiological

Keywords: exosomes, trauma, injury, microRNA

Social Media Summary:

Severe trauma in males and females results in plasma exosome cargo changes which are more pronounced in males than females. Altered miRNA in these exosomes are associated with normal hematopoiesis and inflammation.

Hashtags: #exosomes #trauma #injury

Author Social Media Handles: @jen_munley; @gee_gills; @ValeriePolcz; @balchja; @LBibleMD; @md_mohr

Affiliated Institution Social Media Handles: @UFSurgery, @UFSurgResidents

BACKGROUND

Severe trauma results in a hyperadrenergic state and a cascade of inflammation which can result in multiorgan failure, notably including altered bone marrow function (1, 2). Bone marrow dysfunction after trauma can manifest as persistent anemia despite appropriate resuscitation, which can last for months after injury (3). Suppression of hematopoietic progenitor growth, increased hematopoietic progenitor cell mobilization, altered myelopoiesis and persistent anemia are hallmarks of postinjury bone marrow dysfunction (2, 4). Despite this knowledge, the multifactorial nature of postinjury bone marrow dysfunction requires that underlying mechanisms remain to be fully studied.

Exosomes are a subgroup of extracellular vesicles formed from endosomes and are present in all bodily fluids (5, 6). Exosomes contain proteins, lipids, and nucleotides and facilitate cell-to-cell communication (5, 6). Among exosomal cargo are microRNAs (miRNA), which are small, non-coding ribonucleic acids that serve as post-transcriptional regulators of gene expression (6, 7). A small number of studies have evaluated extracellular vesicles after trauma, which includes exosomes, microvesicles/microparticles, and apoptotic bodies (5). These studies have demonstrated how extracellular vesicles play a role in postinjury inflammation and coagulopathy, and that the cell origin of these vesicles changes after injury (8–12). Studies of hematopoietic malignancies have emphasized the role of exosomes in bone marrow alterations (13, 14).

Even fewer studies have specifically investigated exosomes after multicompartmental injury. A rodent model of trauma and hemorrhagic shock found that exosomes from mesenteric lymph nodes triggered inflammatory responses in monocytes and macrophages (15). In another rodent model of blast, fracture, soft tissue crush injury, tourniquet and amputation demonstrated that exosomes contain significant amounts of inflammatory cytokines (16). One study of polytrauma patients demonstrated a significant change in the surface antigens on exosomes and showed that this correlated with specific injuries (17). Recently, plasma-derived exosomes from trauma patients have been implicated both to bone marrow hematopoietic progenitor cell growth suppression as well as alterations in bone marrow stroma expression of factors associated with inflammation and mobilization of hematopoietic progenitor cells (18). Moreover, one group showed unique bone marrow immune responses between males and females in a murine model of trauma-hemorrhage, with proestrus females having augmented cell differentiation in granulopoiesis and robust cytokine responses (19). However, changes in the miRNA of circulating plasma exosomes after severe injury remains unknown and could be critical to further understanding the underlying mechanisms affecting bone marrow dysfunction after trauma and may be sex-specific.

Given the known effects of plasma exosomes on bone marrow dysfunction after severe trauma, we sought to evaluate changes to exosome cargo after injury in trauma patients compared to operative controls (elective patients undergoing total hip arthroplasty) with regard to pathways involved in hematopoiesis in a pilot study. Specifically, we aimed to characterize expression of microRNA after severe injury in cohorts of males and females compared to operative controls. We hypothesized that plasma-derived exosomes undergo significant changes in miRNA implicated in hematopoiesis after injury compared to operative controls and that these changes would be sex-specific between males and females.

METHODS

Study Population

A single center, prospective observational cohort study was conducted between 2021–2023 at a Level 1 trauma center comparing two populations: severely injured trauma patients (n = 15) and patients undergoing elective total hip arthroplasty (THA) (n = 8). These sample sizes were chosen based on a previous power analysis assuming a greater than 80% incidence of changes at baseline, a 30% change would require 8 participants per group. Groups contained both males and females; primary comparisons evaluated cohorts to reveal changes in miRNA and secondary outcomes were changes in miRNA expression by sex. Patients undergoing THA were utilized as an operative control to account for anesthesia exposure, blood loss, and orthopedic manipulation. Inclusion criteria for patients undergoing THA included the following age ≥ 18 years, undergoing THA for non-infectious reasons, and ability to obtain informed consent. Exclusion criteria for this group included pregnancy, imprisonment, chronic corticosteroid or immunosuppression use, history of chemotherapy or radiation in last six months, history of bone marrow transplantation, end stage renal disease, and pre-existing hematological diseases. Trauma patients were screened on admission for inclusion; criteria included age ≥ 18 years, ability to obtain informed consent, blunt or penetrating trauma resulting in femur or pelvic fractures requiring open surgical intervention, injury severity score (ISS) greater than or equal to 15, and in hemorrhagic shock on admission as defined by systolic blood pressure < 90mmHg, mean arterial pressure ≤ 65mmHg, base deficit ≥ 5mEq, or lactic acid ≥ 2mmol/L. Exclusion criteria for trauma patients were the same as the THA group but with the addition of patients not expected to survive greater than 48 hours. This study was approved by the Institutional Review Board (IRB 201601386) and is registered at ClinicalTrials.gov (NCT02577731). All subjects gave informed consent to participate in the study. Reporting of this study conforms to the Strengthening the Reporting of Observational studies in Epidemiology (STROBE) guidelines (Supplementary Digital Content 1).

Clinical Data and Blood Collection

Clinical data including baseline characteristics such as age, sex, race, medical comorbidities were collected to characterize the demographics of patient cohorts. Management and outcome parameters included injury severity score, blood product transfusion, intensive care unit (ICU) length of stay, hospital length of stay, and mortality.

Peripheral blood was collected at the time of surgical intervention via singular venipuncture in heparinized blood collection tubes (Becton-Dickinson & Co., Franklin Lakes, NJ, USA). Blood was aliquoted and centrifuged for plasma at 800g for ten minutes; plasma was then stored in a −80°C until further processing.

Isolation, Characterization, and Quantitation of Plasma Exosomes

Plasma exosomes were isolated using the Invitrogen Total Exosome Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instructions similar to previously described (18). A positive control was added to the plasma prior to exosome isolation (osa-miR-414). The size and concentration of exosomes for each sample were evaluated with the Malvern NanoSight NS300 with nanoparticle tracking analysis (NTA) (Malvern Panalytical, Worcestershire, United Kingdom). The suspension of plasma exosomes from each sample was diluted 1:1000 in filtered PBS to obtain 20–100 particles per field. Next, 1mL of the diluted exosome suspension was drawn up and transferred to an O-ring top plate NTA chamber carefully to avoid the introduction of air bubbles. Particles were analyzed using a 488nm laser captured for 60 seconds per run for five total runs per sample; size and concentration of exosome particles were averaged from these five runs.

Plasma exosomes were then imaged with electron microscopy. Exosomes were examined by transmission electron microscopy (TEM) negative stain. Glow discharged, carbon-coated Formvar copper grids, 400 mesh were floated onto 5μL aliquot suspensions at dilution of 1:1000 filtered PBS for five minutes. Excess solution was drawn off with filter paper, and grids were post-stained with 0.05% aqueous uranyl acetate for 30 seconds. Stain was removed with filter paper, air-dried for ten minutes, and examined with a FEI TecnaiG2 Spirit Twin TEM and digital images acquired with a Gatan UltraScan 2k x 2k camera and Digital Micrograph software.

Isolation of miRNA from Plasma Exosomes and miRNA Analysis

RNA was isolated from exosomes similar to previously described (20). Briefly, exosomes were disrupted with the QIAzol Lysis reagent (Qiagen, Hilden, Germany) and RNA isolated with the QIAzol Lysis reagent with the miRNeasy kit (Qiagen, Hilden, Germany) according to manufacturer’s protocol. The quantity and quality of mRNA in each sample was determined with an Epoch Spectrophotometer (Agilent, Santa Clara, CA) at A260/A280. A total of 100ng of total exosome mRNA per sample was used for the human NanoString nCounter miRNA Expression Assay (NanoString Technologies, Seattle, WA) according to manufacturer’s protocol. miRNAs were then hybridized to probes at 65°C for 30 hours. The nCounter-generated relative fluorescent intensities were analyzed using nSolver version 4.0 analysis platform software to generate appropriate data normalization as well as fold-changes, resulting ratios and differential expression per manufacturer’s protocol. nSolver Advanced Analysis and Ingenuity Pathway Analysis (IPA) (Qiagen, Hilden, Germany) was performed along with robust R statistics to identify pathway-specific responses (21). To reduce bias, samples were blinded to the researcher performing assays and analysis.

Statistical Analysis

Statistical analysis of patient characteristics was performed with GraphPad Prism version 9.4.1 (GraphPad Software, La Jolla, CA). Comparisons of patient characteristics and were analyzed using Fisher’s exact test for categorical variables. For continuous variables, Welch’s t test for data with a normal distribution or Mann-Whitney test for data without a normal distribution with *p<0.05 considered statistically significant; data are presented as mean ± standard deviation. Data from each sample were normalized using built-in positive controls to control for technique in hybridization, housekeeping genes to account for varying sample degradation, and built-in negative and spin-in miRNA controls to account for background signal similar to previous (20). Samples were grouped using the mean of the normalized samples and the significance was calculated using an unpaired t-test in R; p-values adjusted for multiple comparisons were determined. IPA analysis, specifically ‘Causal Network Analysis’ and ‘Downstream Effects Analysis’, were implemented with Bayesian Z-scores calculated with adjusted p-values to assess the match of observed and prediction up/down regulation patterns (22). Significantly altered genes (p<0.01) are uploaded to NCBI Gene Expression Omnibus (GEO) (GSE243485).

RESULTS

Patient Characteristics

Patient characteristics for trauma and total hip arthroplasty patient cohorts are listed in Supplemental Digital Content 2. All patients in the trauma cohort were injured by blunt mechanisms; most of these were due to motor vehicle accidents (80%). The average injury severity score in the trauma cohort was 23; subgroup analysis by sex revealed similar average ISS in males (23) and females (23) within this group. There were no significant differences between patients in the THA cohort or trauma cohort in terms of age, sex, race, and medical comorbidities (Supplemental Digital Content 2). Despite efforts to enroll a diverse group of patients, majority of patients in both groups were Caucasian (87%) potentially due to local demographics. Trauma patients received more packed red blood cells than THA (p=0.002), but received similar amounts of cryoprecipitate, plasma, and platelets (Supplemental Digital Content 2). Trauma patients had a lower pre-operative hemoglobin compared to THA counterparts, but both cohorts had similar higher estimated blood loss (Supplemental Digital Content 2). Trauma patients had a significantly longer stay in the intensive care unit and also hospital length of stay compared to patients receiving elective total hip arthroplasty (Supplemental Digital Content 2). On average, trauma patients went to the operating room for fixation of femur or pelvic fractures on hospital day three (±2.7 days) at which time peripheral blood was collected; six of the fifteen trauma patients received blood products prior to this blood collection. The average hemoglobin on discharge among trauma patients was 9.2±1.2 g/dL. One female trauma patient died during admission as a result of multiorgan failure.

In comparing males only in the THA and trauma cohorts, there were no significant differences between groups in terms of age, race, or medical comorbidities. Male trauma patients received more packed red blood cells and plasma than THA patients, although these did not reach statistical significance. Male trauma patients had similar intraoperative estimated blood loss to their male THA counterparts, but had a significantly lower pre-operative hemoglobin (10.3±2.0 vs. 15.4±0.7; p=0.006). Male trauma patients had a significantly longer ICU length of stay (6.1±4.7 vs. 0 days; p=0.01) and hospital length of stay (11.9±6.0 vs. 0 days; p=0.006) than male patients undergoing elective THA. Regarding females in the THA and trauma groups, there were no significant differences between cohorts in terms of age, race, medical comorbidities, or administration of platelets, plasma or cryoprecipitate. Despite similar estimated blood loss intraoperatively, female trauma patients had a lower pe-operative hemoglobin (9.2±0.9 g/dL vs. 13.7±1.1 g/dL; p=0.004) and received more packed red blood cells than their female counterparts in the THA cohort (9.0±13.1 vs. 0 units; p=0.02) during their hospitalization. Female trauma patients had a longer ICU length of stay (10.5±8.6 vs. 0 days; p=0.004) and hospital length of stay (15.3±9.3 vs. 0.5±0.6 days; p=0.004) than females undergoing THA.

In comparing male and female trauma patients, male trauma patients tended to have hypertension compared to female trauma patients (p=0.03). Otherwise, there were no significant differences between groups in terms of age, race, or other medical comorbidities. Male and female trauma patients also had similar amounts of blood product administration, ICU length of stay, hospital length of stay, and ISS. Male and female patients in the trauma cohort also had similar pre-operative hemoglobin, estimated blood loss intraoperatively, and discharge hemoblogin.

Plasma Exosome Quantitation and Imaging

After isolation of exosomes from plasma, particles were evaluated on the NS300 for size and concentration. The mean size of exosomes in the THA cohort was 138 ± 17 nm which was not significantly different from that of trauma patients (153 ± 24 nm). Similarly, there was no significant difference in the concentration of exosomes isolated from the plasma of THA or trauma patients. A sample graph illustrating the concentration of particles per milliliter from nanoparticle tracking analysis is shown in Figure 1A. Transmission electron microscopy was performed to confirm the presence of exosomes isolated from plasma; a sample image is shown in Figure 1B.

Figure 1.

Figure 1

Open in a new tab

A-B. A) Sample nanosight particle tracking analysis showing concentration of particles by size (nm) and B) sample electron microscopy image of exosomes; size bar indicates 100nm.

Trauma induces a unique set of miRNA cargo in exosomes derived from plasma

Comparison of plasma exosomes from trauma and THA patients revealed significantly different expression of 63 miRNAs in the trauma cohort compared to the THA cohort (p<0.05). Of these miRNAs, 60 were decreased in expression in trauma patient plasma exosomes while only three had increased expression compared to THA patient plasma exosome miRNAs. Notably, twelve which were downregulated (hsa-let-7g-5p, hsa-miR-451a, hsa-miR-125b-5p, hsa-miR-935, hsa-miR-16-5p, hsa-let-7b-5p, hsa-miR-223-3p, hsa-miR-202-3p, hsa-miR-92a-3p, hsa-miR-146b-5p, hsa-miR-1915-3p, hsa-miR-125a-5p) have been associated with hematopoiesis or macrophage function (Table 1). A heat map of the miRNA differentially expressed in the plasma exosomes of trauma patients relative to THA patients, separated by subject, can be found in Figure 2, where each lane represents a per-row normalized expression levels of total miRNAs with significant miRNAs (p<0.05) stratified at the top. Significantly different miRNAs when comparing trauma patient plasma exosomes to THA patient plasma exosomes are illustrated by the volcano plot in Figure 3A. When evaluating the most significant miRNAs between cohorts (p<0.01), we identified 19 miRNAs which had the lowest expression and 3 with the highest expression in trauma patient plasma exosomes compared to THA patient plasma exosomes. (Fig. 3B). Of these miRNAs, six which were downregulated (hsa-let-7g-5p, hsa-miR-451a, hsa-miR-125b-5p, hsa-miR-935, hsa-miR-16-5p, hsa-let-7b-5p) play a role in hematopoiesis (Table 1). nSolver Advanced Analysis and Ingenuity Pathway Analysis revealed putative immune mRNA gene targets of these plasma exosome miRNA which were significantly different in trauma patients compared to THA patients (p<0.05) (Supplemental Digital Content 3). Together these data suggest that severe injury induces significant changes in the miRNA cargo of plasma exosomes.

Figure 2.

Figure 2.

Open in a new tab

Heat map of exosomal miRNA expression log2-transformed trauma patients compared to THA patients; only significant differentially expressed miRNAs shown (p<0.05).

Figure 3.

Figure 3

Open in a new tab

A-B. A) log2-transformed differential fold change in miRNA expression with associated p-value significance after data normalization, and B) fold change of significantly different miRNA in trauma patients versus THA patients (p<0.01).

Trauma induces unique miRNA cargo in plasma exosomes of males

Subgroup analysis of male trauma patient plasma exosomes versus male THA patient plasma exosomes revealed unique changes in 153 miRNAs (p<0.05) (Fig. 4A). Of these, only 3 miRNAs were expressed more in plasma exosomes of male trauma patients when compared to male THA patients. The remaining 150 miRNAs were downregulated in the trauma cohort of males only; of these, fifteen are involved in hematopoiesis (hsa-miR-376a-2-5p, hsa-miR-181c-5p, hsa-let-7g-5p, hsa-miR-210-3p, hsa-miR-494-3p, hsa-miR-935, hsa-miR-299-5p, hsa-miR-451a, hsa-miR-146b-5p, hsa-miR-34a-5p, hsa-miR-144-3p, hsa-miR-125a-5p, hsa-miR-202-3p, hsa-miR-92a-3p, hsa-miR-124-3p) (Supplemental Digital Content 5).

Figure 4.

Figure 4

Open in a new tab

A-D. Male trauma versus male THA patient plasma exosome miRNA A) log2-transformed differential fold change in miRNA expression with associated p-value significance after data normalization and B) fold change of significantly different miRNA in male trauma versus male THA patients (p<0.01). Female trauma versus female THA patient plasma exosome miRNA A) log2-transformed differential fold change in miRNA expression with associated p-value significance after data normalization and B) fold change of significantly different miRNA in female trauma versus female THA patients (p<0.05).

The most significantly differentially expressed miRNA in male trauma patients compared to male THA subjects are shown in Figure 4B (p<0.01). Overall, 25 miRNAs were identified as the most differentially expressed plasma exosome miRNA in males in the trauma compared to males in the THA cohorts, with five associated with hematopoiesis (hsa-miR-376a-2-5p, hsa-miR-181c-5p, hsa-let-7g-5p, hsa-miR-210-3p, hsa-miR-494-3p) (p<0.01) (Supplemental Digital Content 5). IPA revealed mRNA gene targets of trauma-induced exosome changes in miRNA cargo, with a frequency of distribution of immune genes known to be impacted by miRNA (p<0.05) (Supplemental Digital Content 4). These data demonstrate the unique exosome miRNA cargo changes in males after injury.

Females are resistant to miRNA cargo alterations in plasma exosomes after severe injury

Female trauma patients only demonstrated six miRNAs with significantly altered expression after injury compared to female THA patients (Fig. 4C). As shown in Figure 4D, two had increased expression of miRNAs whereas four had decreased expression in female trauma patients compared to female THA patients (p<0.05). Two of the miRNAs (hsa-miR-125b-5p, hsa-let-7b-5p) with decreased expression in the plasma exosomes of female trauma patients are associated with hematopoiesis (p<0.05) (Supplemental Digital Content 6). Utilization of IPA to investigate putative immune mRNA gene targets of these trauma-induced exosome-bound miRNA identities showed the numerous targets of these miRNA (Supplemental Digital Content 4). These data suggest that females are more resistant to plasma exosome miRNA cargo changes than males after severe injury.

Males have significant downregulation of key miRNA than females after severe trauma

Analysis of male trauma patients compared to female trauma patients revealed that 69 miRNAs were differentially expressed in males compared to females after injury (Fig. 5A). All of these miRNAs were downregulated in male compared to female trauma patients. The most significantly downregulated miRNAs in male trauma patients compared to female trauma patients can be found in Figure 5B. Four of these miRNAs (hsa-miR-210-3p, hsa-miR-494-3p, hsa-miR-376a-2-5p, hsa-miR-181c-5p) have been associated with hematopoiesis or hematopoietic cell function (Supplemental Digital Content 7). Utilization of IPA to investigate putative immune mRNA gene targets of these trauma-induced exosome-bound miRNA identities showed the numerous targets of these miRNA, with thousands of targets identified. Together this demonstrates how the plasma exosome cargo of males after severe injury are more affected that that in female trauma patients.

Figure 5.

Figure 5

Open in a new tab

A-B. Male trauma versus female trauma patient plasma exosome miRNA A) log2-transformed differential fold change in miRNA expression with associated p-value significance after data normalization and B) fold change of significantly different miRNA in male trauma versus female patients (p<0.01).

DISCUSSION

In this pilot study, isolation of plasma exosomes after severe injury with subsequent evaluation of miRNA revealed drastic changes in the cargo of circulating exosomes in trauma patients compared to operative controls. Overall, 63 miRNAs were identified as significantly differentially expressed in trauma patients compared to operative controls, with 60 of them downregulated. Subgroup analysis comparing male trauma patients to male patients undergoing THA significant changes in expression of 153 miRNAs with majority (150) of them downregulated. When comparing female trauma patients to females who underwent THA, only 6 miRNAs were different. Further analysis of male trauma patients compared to female trauma patient showed that males had 69 downregulated miRNAs in a direct comparison of the sexes after injury. Investigation of altered miRNA showed that many are associated with hematopoiesis. Together, this suggests that not only the cargo of circulating plasma exosomes after trauma are drastically different, but that males subjected to injury are more susceptible to plasma exosome cargo alterations postinjury.

Analysis of exosome samples with nanoparticle tracking analysis revealed particle diameters consistent with exosomes (23). We did not find significant differences in plasma exosome concentrations between the trauma or operative control cohorts. These findings of similar concentrations of plasma exosomes between trauma patients and controls are similar to others (17, 18). Together with electron microscopy, this confirmed the successful isolation of exosomes from plasma.

This study demonstrated changes in plasma exosome miRNA after injury compared to operative controls. While there is a paucity of data evaluating the cargo of exosomes after multicompartmental injury, one study showed that plasma exosomes demonstrate changes to surface antigens after trauma, correlating different antigens with abdominal trauma, chest trauma or traumatic brain injury (TBI) (17). Studies of plasma exosome miRNA in patients after TBI found alterations in miRNA compared to healthy controls, although different miRNA that in our trauma patient cohort (24). Despite this, miRNA such as hsa-miR-9-5p, hsa-miR-873-5p, and hsa-449a which were downregulated in this trauma cohort have been associated with mitigation of neuroinflammation in spinal cord injury or traumatic brain injury (25–27). Other animal studies of TBI also showed unique miRNA changes after TBI (28, 29). This confirms that injury is associated with changes in plasma exosome miRNA which may influence a number of inflammatory and other pathways postinjury.

Plasma exosomes have recently been shown to play a role in bone marrow dysfunction after severe trauma (18). Similar to other studies, our trauma patients were all anemic on discharge (3). In our initial analysis comparing all trauma patients and patients undergoing THA, we identified twelve downregulated miRNA which have been associated with hematopoiesis or hematopoietic cell function in other studies (Supplemental Digital Content 2) (30–40). In particular, hsa-miR-451a is highly expressed during erythroid development and is activated by GATA1 for erythroid maturation; hsa-miR-16-5p is also upregulated in late stage erythropoiesis (31, 41). In addition, hsa-miR-223-3p is involved in late-stage hematopoiesis in myeloid lineages (34). Finally, both hsa-miR-146b-5p and hsa-miR-1915-3p are associated with the fate of hematopoietic progenitor cells (37, 38). Together, these data suggest that these changes in miRNA of plasma exosomes could be associated with altered hematopoiesis and play a role in bone marrow dysfunction after trauma. While there are no current studies in the literature evaluating the manipulation of plasma exosomes to mitigate these postinjury effects on bone marrow dysfunction, others have explored the use of exosome inhibitors in clinical trials in oncology (42). Thus, this represents a potential therapeutic option and further studies to alter or inhibit postinjury exosomes effects warrants further study.

Subgroup analysis of only male trauma patients compared to male patients undergoing THA showed a large number of downregulated miRNA. Of these, fifteen downregulated miRNA play a role in hematopoietic cells (Supplemental Digital Content 5) (30, 31, 33, 35–37, 39, 40, 43–50). For example, hsa-miR-210-3p is upregulated during erythroid differentiation, whereas hsa-miR-376a-2-5p is involved in early stages of erythropoiesis (43, 45). In addition, hsa-miR-144-3p has been shown to play a role in the regulation of erythropoiesis (49). However, when only evaluating the cargo of plasma exosomes from female trauma patients compared to female patients in the THA cohort, only six miRNA were significantly different than operative controls, suggesting resilience to exosomal miRNA changes postinjury. Of these, only two miRNA which were downregulated have been shown to play a role in hematopoiesis (Supplemental Digital Content 6) (30, 32). Direct comparison of male versus female trauma patients within the trauma cohort demonstrated that males had a large number of significantly less expressed miRNA compared to females in plasma exosomes. From this, four have been linked to hematopoiesis or associated cell activation (30, 32, 43, 44). Together, this highlights that males may be more significantly affected than female trauma patients after severe injury in terms of exosome miRNA expression.

Multiple previous works indicate that females have preserved immune function and even improved mortality compared to males after severe trauma (51, 52). These differences in outcomes have been attributed to estrogen levels in females (51, 52). Despite this evidence, there is a gap in the literature evaluating bone marrow function postinjury in males and females. While our study did not investigate underlying mechanisms behind the observed sexual dimorphism in plasma exosome miRNA changes after trauma, future studies should attempt to elucidate if bone marrow dysfunction is in fact different between males and females and also underlying mechanisms behind these differences.

This study has limitations. This proof of concept study consisted of a small number of patients with varying injury severity; future studies should consist of a larger patient population to allow for additional analyses including correlation with clinical outcomes. Of the fifteen trauma patients from which plasma was isolated, six received blood products prior to blood collection, which undoubtedly affected the plasma exosomes isolated for this study (53). Many methods allow for the isolation of exosomes, and the use of a precipitation kit results in a high yield; however, it has been shown that this method does not result in the purest suspension of exosomes, as samples may contain other proteins (54). However, particle size observed with nanoparticle tracking analysis demonstrated the appropriate diameter for exosomes and electron microscopy performed confirmed the presence of exosomes in these samples (23). While our demographic characteristics were similar between groups, our sample size was small and we were unable to recruit a diverse group of patients. Future studies should include a larger number of patients from different age groups and of different race to understand how this affects exosomes postinjury. This investigation also evaluated all plasma exosomes isolated and did not characterize their origin with flow cytometry, which should be considered in future studies (17).

In summary, this data demonstrates how severe trauma induces drastic differences in expression of microRNA in circulating plasma exosomes compared to patients undergoing similar procedures. In addition, these miRNA alterations were found to be different between males and females. Females are more resilient to these alterations, which warrants further investigation into the sexual dimorphisms underlying differences in miRNA expression between males and females after multicompartmental injury. This novel study should guide additional studies containing larger patient populations to investigate direct links between exosome miRNA changes and bone marrow dysfunction after trauma and include analyses into how age, injury severity, and other factors influence miRNA expression and the duration of such alterations. Future studies should also focus on how the observed changes in miRNA of exosomes after trauma correlate with clinical outcomes, including transfusions requirements and anemia recovery.

Supplementary Material

Supplemental Data File (.doc, .tif, pdf, etc.)_1

Supplemental Digital Content 1. STROBE checklist for observational cohort studies.

Supplemental Data File (.doc, .tif, pdf, etc.)_2

Supplemental Digital Content 3. miRNA and mRNA interactions predicted in miRNA differentially expressed in trauma compared to THA patients (p<0.05). Only select mRNA targets listed if the number of targets for a miRNA was greater than or equal to 30.

Supplemental Data File (.doc, .tif, pdf, etc.)_3

Supplemental Digital Content 4. Number of plasma exosome miRNA targets identified by Ingenuity Pathway Analysis using knowledge from the literature (p<0.05) differentially expressed in A) male trauma patients compared to male THA patients and B) female trauma patients compared to female THA patients.

Supplemental Data File (.doc, .tif, pdf, etc.)_4

Supplemental Digital Content 2. Patient characteristics. COPD = chronic obstructive pulmonary disease; CHF = congestive heart failure; Cr = creatinine; ICU = intensive care unit; RBC = red blood cells.

Supplemental Data File (.doc, .tif, pdf, etc.)_5

Supplemental Digital Content 5. miRNAs with downregulated expression in the plasma exosomes of male trauma patients compared to those from male patients undergoing elective total hip arthroplasty implicated in hematopoiesis or hematopoietic cell function (p<0.05).

Supplemental Data File (.doc, .tif, pdf, etc.)_6

Supplemental Digital Content 6. miRNAs with downregulated expression in the plasma exosomes of female trauma patients compared to those from female patients undergoing elective total hip arthroplasty implicated in hematopoiesis or hematopoietic cell function (p<0.05).

Supplemental Data File (.doc, .tif, pdf, etc.)_7

Supplemental Digital Content 7. miRNAs with downregulated expression in the plasma exosomes of male trauma patients compared to those from female trauma patients implicated in hematopoiesis or hematopoietic cell function (p<0.05).

ACKNOWLEDGEMENTS

The authors would like to thank the University of Florida Orthopedic Surgery Division for their collaboration and assistance with enrollment of patients undergoing total hip arthroplasty. The authors would also like to acknowledge the University of Florida Interdisciplinary Center for Biotechnology Research Cytometry (Research Resource Identifier SCR_019119) for the use of the Malvern NanoSight NS300 with Nanoparticle Tracking Analysis and Electron Microscopy (Research Resource Identifier SCR_019146) for performing Transmission Electron Microscopy.

Funding:

This research was supported by the National Institutes of Health. AMM was supported by NIH NIGMS R01 GM105893. JAM, VEP, JAB, and ELB were supported by postgraduate training grant NIH NIGMS T32 GM-008721 in burns, trauma, and perioperative injury. GSG was supported by postgraduate training grant NIH NHLBI T32 HL-160491 interdisciplinary training for vascular surgeon scientists. MLW was supported by NIH NIGMS F31 GM-149109 and NIH NIEH F32 ES007126.

Footnotes

Conflicts of Interest: The authors declare no conflicts of interest. All JTACS Disclosure forms have been supplied and are provided as supplemental digital content.

These findings have been presented at the 82nd Annual Meeting of the AAST in Anaheim, California on September 22, 2023.

REFERENCES

  • 1.Sauaia A, Moore FA, Moore EE. Postinjury Inflammation and Organ Dysfunction. Crit Care Clin. 2017;33(1):167–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Munley JA, Kelly LS, Mohr AM. Adrenergic Modulation of Erythropoiesis After Trauma. Front Physiol. 2022;13:859103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Kelly LS, Munley JA, Kannan KB, Pons EE, Coldwell PS, Bible LE, et al. Anemia Recovery after Trauma: A Longitudinal Study. Surg Infect (Larchmt). 2023;24(1):39–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Kelly LS, Darden DB, Fenner BP, Efron PA, Mohr AM. The Hematopoietic Stem/Progenitor Cell Response to Hemorrhage, Injury, and Sepsis: A Review of Pathophysiology. Shock. 2021;56(1):30–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Alsaadi N, Srinivasan AJ, Seshadri A, Shiel M, Neal MD, Scott MJ. The emerging therapeutic potential of extracellular vesicles in trauma. J Leukoc Biol. 2022;111(1):93–111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Walsh SA, Hoyt BW, Rowe CJ, Dey D, Davis TA. Alarming Cargo: The Role of Exosomes in Trauma-Induced Inflammation. Biomolecules. 2021;11(4). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Krol J, Loedige I, Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet. 2010;11(9):597–610. [DOI] [PubMed] [Google Scholar]
  • 8.Kuravi SJ, Yates CM, Foster M, Harrison P, Hazeldine J, Hampson P, et al. Changes in the pattern of plasma extracellular vesicles after severe trauma. PLoS One. 2017;12(8):e0183640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Weber B, Franz N, Marzi I, Henrich D, Leppik L. Extracellular vesicles as mediators and markers of acute organ injury: current concepts. Eur J Trauma Emerg Surg. 2022;48(3):1525–44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Matijevic N, Wang YW, Wade CE, Holcomb JB, Cotton BA, Schreiber MA, et al. Cellular microparticle and thrombogram phenotypes in the Prospective Observational Multicenter Major Trauma Transfusion (PROMMTT) study: correlation with coagulopathy. Thromb Res. 2014;134(3):652–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Seibold T, Schonfelder J, Weeber F, Lechel A, Armacki M, Waldenmaier M, et al. Small Extracellular Vesicles Propagate the Inflammatory Response After Trauma. Adv Sci (Weinh). 2021;8(24):e2102381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Lopez E, Srivastava AK, Burchfield J, Wang YW, Cardenas JC, Togarrati PP, et al. Platelet-derived- Extracellular Vesicles Promote Hemostasis and Prevent the Development of Hemorrhagic Shock. Sci Rep. 2019;9(1):17676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Chen T, Moscvin M, Bianchi G. Exosomes in the Pathogenesis and Treatment of Multiple Myeloma in the Context of the Bone Marrow Microenvironment. Front Oncol. 2020;10:608815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Boyiadzis M, Whiteside TL. Exosomes in acute myeloid leukemia inhibit hematopoiesis. Curr Opin Hematol. 2018;25(4):279–84. [DOI] [PubMed] [Google Scholar]
  • 15.Kojima M, Gimenes-Junior JA, Langness S, Morishita K, Lavoie-Gagne O, Eliceiri B, et al. Exosomes, not protein or lipids, in mesenteric lymph activate inflammation: Unlocking the mystery of post-shock multiple organ failure. J Trauma Acute Care Surg. 2017;82(1):42–50. [DOI] [PubMed] [Google Scholar]
  • 16.Walsh SA, Davis TA. Key early proinflammatory signaling molecules encapsulated within circulating exosomes following traumatic injury. J Inflamm (Lond). 2022;19(1):6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Weber B, Henrich D, Schindler CR, Marzi I, Leppik L. Release of exosomes in polytraumatized patients: The injury pattern is reflected by the surface epitopes. Front Immunol. 2023;14:1107150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Munley JA, Kelly LS, Gillies GS, Kannan KB, Pons EE, Bible LE, et al. Effects of Trauma Plasma-Derived Exosomes on Hematopoietic Progenitor Cells. Shock. 2023;59(4):591–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Schneider CP, Schwacha MG, Chaudry IH. Impact of sex and age on bone marrow immune responses in a murine model of trauma-hemorrhage. J Appl Physiol (1985). 2007;102(1):113–21. [DOI] [PubMed] [Google Scholar]
  • 20.Desai CS, Khan A, Bellio MA, Willis ML, Mahung C, Ma X, et al. Characterization of extracellular vesicle miRNA identified in peripheral blood of chronic pancreatitis patients. Mol Cell Biochem. 2021;476(12):4331–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Team RC. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2017. [Google Scholar]
  • 22.Kramer A, Green J, Pollard J, Jr., Tugendreich S. Causal analysis approaches in Ingenuity Pathway Analysis. Bioinformatics. 2014;30(4):523–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Chan BD, Wong WY, Lee MM, Cho WC, Yee BK, Kwan YW, et al. Exosomes in Inflammation and Inflammatory Disease. Proteomics. 2019;19(8):e1800149. [DOI] [PubMed] [Google Scholar]
  • 24.Lai N, Wu D, Liang T, Pan P, Yuan G, Li X, et al. Systemic exosomal miR-193b-3p delivery attenuates neuroinflammation in early brain injury after subarachnoid hemorrhage in mice. J Neuroinflammation. 2020;17(1):74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.He X, Zhang J, Guo Y, Yang X, Huang Y, Hao D. Exosomal miR-9-5p derived from BMSCs alleviates apoptosis, inflammation and endoplasmic reticulum stress in spinal cord injury by regulating the HDAC5/FGF2 axis. Mol Immunol. 2022;145:97–108. [DOI] [PubMed] [Google Scholar]
  • 26.Long X, Yao X, Jiang Q, Yang Y, He X, Tian W, et al. Astrocyte-derived exosomes enriched with miR-873a-5p inhibit neuroinflammation via microglia phenotype modulation after traumatic brain injury. J Neuroinflammation. 2020;17(1):89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.He C, Xiao J, Ye Y, Huang S, Zhong Y, Liu L, et al. Long non-coding RNA-small nucleolar RNA host gene 7 regulates inflammatory responses following spinal cord injury by regulating the microRNA-449a/TNF-alpha-induced protein 3-interacting protein 2 axis. Bioengineered. 2022;13(4):10215–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Yang C, Gao C, Liu N, Zhu Y, Zhu X, Su X, et al. The effect of traumatic brain injury on bone healing from a novel exosome centered perspective in a mice model. J Orthop Translat. 2021;30:70–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Wang P, Ma H, Zhang Y, Zeng R, Yu J, Liu R, et al. Plasma Exosome-derived MicroRNAs as Novel Biomarkers of Traumatic Brain Injury in Rats. Int J Med Sci. 2020;17(4):437–48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Lessard S, Beaudoin M, Orkin SH, Bauer DE, Lettre G. 14q32 and let-7 microRNAs regulate transcriptional networks in fetal and adult human erythroblasts. Hum Mol Genet. 2018;27(8):1411–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Masaki S, Ohtsuka R, Abe Y, Muta K, Umemura T. Expression patterns of microRNAs 155 and 451 during normal human erythropoiesis. Biochem Biophys Res Commun. 2007;364(3):509–14. [DOI] [PubMed] [Google Scholar]
  • 32.Duroux-Richard I, Roubert C, Ammari M, Presumey J, Grun JR, Haupl T, et al. miR-125b controls monocyte adaptation to inflammation through mitochondrial metabolism and dynamics. Blood. 2016;128(26):3125–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Zhang B, Du Y, He Y, Liu Y, Zhang G, Yang C, et al. INT-HA induces M2-like macrophage differentiation of human monocytes via TLR4-miR-935 pathway. Cancer Immunol Immunother. 2019;68(2):189–200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Chen CZ, Li L, Lodish HF, Bartel DP. MicroRNAs modulate hematopoietic lineage differentiation. Science. 2004;303(5654):83–6. [DOI] [PubMed] [Google Scholar]
  • 35.Yu G, Sun W, Wang W, Le C, Liang D, Shuai L. Overexpression of microRNA-202–3p in bone marrow mesenchymal stem cells improves cerebral ischemia-reperfusion injury by promoting angiogenesis and inhibiting inflammation. Aging (Albany NY). 2021;13(8):11877–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Liu F, Peng W, Chen J, Xu Z, Jiang R, Shao Q, et al. Exosomes Derived From Alveolar Epithelial Cells Promote Alveolar Macrophage Activation Mediated by miR-92a-3p in Sepsis-Induced Acute Lung Injury. Front Cell Infect Microbiol. 2021;11:646546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Zhai PF, Wang F, Su R, Lin HS, Jiang CL, Yang GH, et al. The regulatory roles of microRNA-146b-5p and its target platelet-derived growth factor receptor alpha (PDGFRA) in erythropoiesis and megakaryocytopoiesis. J Biol Chem. 2014;289(33):22600–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Qu M, Zou X, Fang F, Wang S, Xu L, Zeng Q, et al. Platelet-derived microparticles enhance megakaryocyte differentiation and platelet generation via miR-1915–3p. Nat Commun. 2020;11(1):4964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Wang W, Guo ZH. Downregulation of lncRNA NEAT1 Ameliorates LPS-Induced Inflammatory Responses by Promoting Macrophage M2 Polarization via miR-125a-5p/TRAF6/TAK1 Axis. Inflammation. 2020;43(4):1548–60. [DOI] [PubMed] [Google Scholar]
  • 40.Bhatlekar S, Manne BK, Basak I, Edelstein LC, Tugolukova E, Stoller ML, et al. miR-125a-5p regulates megakaryocyte proplatelet formation via the actin-bundling protein L-plastin. Blood. 2020;136(15):1760–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Merkerova M, Belickova M, Bruchova H. Differential expression of microRNAs in hematopoietic cell lineages. Eur J Haematol. 2008;81(4):304–10. [DOI] [PubMed] [Google Scholar]
  • 42.Kim JH, Lee CH, Baek MC. Dissecting exosome inhibitors: therapeutic insights into small-molecule chemicals against cancer. Exp Mol Med. 2022;54(11):1833–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Wang F, Yu J, Yang GH, Wang XS, Zhang JW. Regulation of erythroid differentiation by miR-376a and its targets. Cell Res. 2011;21(8):1196–209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Xue Q, Guo ZY, Li W, Wen WH, Meng YL, Jia LT, et al. Human activated CD4(+) T lymphocytes increase IL-2 expression by downregulating microRNA-181c. Mol Immunol. 2011;48(4):592–9. [DOI] [PubMed] [Google Scholar]
  • 45.Hu C, Yan Y, Fu C, Ding J, Li T, Wang S, et al. Effects of miR‑210‑3p on the erythroid differentiation of K562 cells under hypoxia. Mol Med Rep. 2021;24(2). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Yang Z, Yan L, Cao H, Gu Y, Zhou P, Shi M, et al. Erythropoietin Protects against Diffuse Alveolar Hemorrhage in Mice by Regulating Macrophage Polarization through the EPOR/JAK2/STAT3 Axis. J Immunol. 2021;206(8):1752–64. [DOI] [PubMed] [Google Scholar]
  • 47.Tenedini E, Roncaglia E, Ferrari F, Orlandi C, Bianchi E, Bicciato S, et al. Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoiesis: role of hsa-mir-299–5p in CD34+ progenitor cells commitment. Cell Death Dis. 2010;1(2):e28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Nowicki M, Szemraj J, Wierzbowska A, Pluta A, Grzybowska-Izydorczyk O, Nowicka A, et al. Alterations in microRNA Expression during Hematopoietic Stem Cell Mobilization. Biology (Basel). 2021;10(7). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Wu CW, Cao X, Berger CK, Foote PH, Mahoney DW, Simonson JA, et al. Novel Approach to Fecal Occult Blood Testing by Assay of Erythrocyte-Specific microRNA Markers. Dig Dis Sci. 2017;62(8):1985–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Liu Y, Huang X, Timani KA, Broxmeyer HE, He JJ. MicroRNA-124 Targets Tip110 Expression and Regulates Hematopoiesis. Stem Cells Dev. 2015;24(17):2009–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Bosch F, Angele MK, Chaudry IH. Gender differences in trauma, shock and sepsis. Mil Med Res. 2018;5(1):35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Choudhry MA, Bland KI, Chaudry IH. Trauma and immune response--effect of gender differences. Injury. 2007;38(12):1382–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Fang X, Li J, Hao X, Zhang W, Zhong J, Zhu T, et al. Exosomes From Packed Red Cells Induce Human Mast Cell Activation and the Production of Multiple Inflammatory Mediators. Front Immunol. 2021;12:677905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Chen J, Li P, Zhang T, Xu Z, Huang X, Wang R, et al. Review on Strategies and Technologies for Exosome Isolation and Purification. Front Bioeng Biotechnol. 2021;9:811971. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Data File (.doc, .tif, pdf, etc.)_1

Supplemental Digital Content 1. STROBE checklist for observational cohort studies.

NIHMS1946131-supplement-Supplemental_Data_File___doc___tif__pdf__etc___1.docx (30.2KB, docx)
Supplemental Data File (.doc, .tif, pdf, etc.)_2

Supplemental Digital Content 3. miRNA and mRNA interactions predicted in miRNA differentially expressed in trauma compared to THA patients (p<0.05). Only select mRNA targets listed if the number of targets for a miRNA was greater than or equal to 30.

NIHMS1946131-supplement-Supplemental_Data_File___doc___tif__pdf__etc___2.docx (17.9KB, docx)
Supplemental Data File (.doc, .tif, pdf, etc.)_3

Supplemental Digital Content 4. Number of plasma exosome miRNA targets identified by Ingenuity Pathway Analysis using knowledge from the literature (p<0.05) differentially expressed in A) male trauma patients compared to male THA patients and B) female trauma patients compared to female THA patients.

NIHMS1946131-supplement-Supplemental_Data_File___doc___tif__pdf__etc___3.tif (523.8KB, tif)
Supplemental Data File (.doc, .tif, pdf, etc.)_4

Supplemental Digital Content 2. Patient characteristics. COPD = chronic obstructive pulmonary disease; CHF = congestive heart failure; Cr = creatinine; ICU = intensive care unit; RBC = red blood cells.

NIHMS1946131-supplement-Supplemental_Data_File___doc___tif__pdf__etc___4.docx (19.7KB, docx)
Supplemental Data File (.doc, .tif, pdf, etc.)_5

Supplemental Digital Content 5. miRNAs with downregulated expression in the plasma exosomes of male trauma patients compared to those from male patients undergoing elective total hip arthroplasty implicated in hematopoiesis or hematopoietic cell function (p<0.05).

NIHMS1946131-supplement-Supplemental_Data_File___doc___tif__pdf__etc___5.docx (17.4KB, docx)
Supplemental Data File (.doc, .tif, pdf, etc.)_6

Supplemental Digital Content 6. miRNAs with downregulated expression in the plasma exosomes of female trauma patients compared to those from female patients undergoing elective total hip arthroplasty implicated in hematopoiesis or hematopoietic cell function (p<0.05).

NIHMS1946131-supplement-Supplemental_Data_File___doc___tif__pdf__etc___6.docx (16.2KB, docx)
Supplemental Data File (.doc, .tif, pdf, etc.)_7

Supplemental Digital Content 7. miRNAs with downregulated expression in the plasma exosomes of male trauma patients compared to those from female trauma patients implicated in hematopoiesis or hematopoietic cell function (p<0.05).

NIHMS1946131-supplement-Supplemental_Data_File___doc___tif__pdf__etc___7.docx (16.3KB, docx)

RESOURCES