Mediators of Prolonged Hematopoietic Progenitor Cell Mobilization After Severe Trauma

Abstract

Background:

This study investigated the molecular mediators of prolonged hematopoietic progenitor cell mobilization a trauma and chronic stress and the role of propranolol in modifying this response.

Methods:

Sprague–Dawley rats were randomized to lung contusion (LC), LC plus hemorrhagic shock (LCHS), or LCHS with daily restraint stress (LCHS/CS). Propranolol was administered daily. Bone marrow (BM) and lung expression of high mobility group box 1 (HMGB1), granulocyte colony-stimulating factor (G-CSF), neutrophil elastase, stromal cell–derived factor 1 (SDF-1)/CXR4, and vascular cell adhesion protein 1 (VCAM-1)/very late antigen-4 were measured by real-time polymerase chain reaction.

Results:

Bone marrow HMGB1, G-CSF, and neutrophil elastase expression were significantly elevated two- to four-fold after LCHS/CS, and all were decreased with the use of propranolol. SDF-1 and VCAM-1 were both significantly decreased after LCHS/CS.

Conclusions:

The increased expression of HMGB1 and G-CSF and decreased expression of BM anchoring molecules, SDF-1 and VCAM-1, after LCHS/CS, likely mediates prolonged hematopoietic progenitor cell mobilization. Propranolol’s ability to reduce HMGB1, G-CSF, and neutrophil elastase expression suggests that the mobilization of hematopoietic progenitor cells was driven by persistent hypercatecholaminemia.

Introduction

Under physiological conditions, hematopoietic progenitor cells (HPCs) reside in the bone marrow (BM), and there are small numbers of circulating HPCs in the peripheral blood.¹ These HPCs normally traffic between the BM, blood, and peripheral organs. After trauma, there is robust HPC mobilization from the BM to the peripheral blood.^1–4 HPC mobilization is a mechanism that allows migration of progenitor cells to sites of injury to participate in repair of injured tissue, which plays an important role in the functional recovery of peripheral organs.^3,4 Studies have shown that lung contusion (LC) and hemorrhagic shock followed by chronic stress causes prolonged mobilization HPCs into the peripheral blood, which is associated with decreased BM cellularity and reduced growth of erythroid lineage colonies.^2,5 It is the persistent stress and hypercatecholaminemia that drives prolonged HPC mobilization that is associated with abnormal wound healing.^2,5–8 In addition, the administration of propranolol after injury and chronic stress has been shown to be effective in improving BM function with reduced progenitor cell mobilization and improved hemoglobin levels.^7,8 Despite the reduction of HPC mobilization with the use of propranolol after severe trauma, there was no worsening of lung injury healing.⁸

High mobility group box 1 (HMGB1) is a nuclear protein that acts as a stress signal for cytokine production when secreted by immune cells.⁹ HMGB1 is a damage-associated molecular pattern molecule that acts as a mediator of injury and plays a role in inflammation, cell migration, and stem cell recruitment. HMGB1 has been shown to play a role in catecholamine-induced mobilization of HPC via the secretion of granulocyte colony-stimulating factor (G-CSF) from BM macrophages.^10,11 G-CSF initiates proliferation and differentiation of mature granulocytes, but it is a potent inducer of HPC mobilization from the BM to the peripheral blood. G-CSF release has been shown to be mediated by norepinephrine, beta-adrenergic stimulation, and hemorrhagic shock.^8,11–13

Although HMGB1 and G-CSF play a role in the recruitment and mobilization of HPC from the BM, little is known about the mechanisms responsible for HPC retention in the BM and how it may be disrupted after trauma. Stromal cell–derived factor 1 (SDF-1) and its receptor CXCR4 function to retain cells within the BM under normal conditions.^12,14 Very late antigen-4 (VLA-4) and its receptor VCAM-1 also participate in HPC retention in the BM via their binding with endothelial and stromal surfaces.^12,14 After trauma and inflammation, neutrophils degranulate and release various proteases, including matrix metalloproteinase (MMP)-9, MMP-2, and neutrophil elastase.^15,16 MMP-9 and MMP-2 can cleave the extracellular matrix and SDF-1, whereas neutrophil elastase can cleave both SDF-1 and VCAM-1.^3,12,14–20 Tissue inhibitors of metalloproteinases (TIMP) are major regulators of MMP-9 and MMP-2.²¹

The aim of this study was to examine the molecular mechanisms involved in prolonged HPC mobilization in an established rodent model of LC, hemorrhagic shock, and chronic stress. We hypothesize that sustained HPC mobilization is because of the chronic stress/proinflammatory state that regulates proteolysis and disrupts HPC retention and that the pathways controlling HPC mobilization and retention can be mitigated with propranolol.

Materials and methods

Animals

Male Sprague–Dawley rats (Charles River, Raleigh, NC), weighing 300–400 g, were housed in pairs and fed ad libitum with Teklad diet (Harlan Laboratories Inc, Tampa, FL) and water. Light and dark cycles were 12 h each of light and dark cycles. Female animals were excluded because of estrous cycle and its possible impact on hemorrhagic shock. All animal care was conducted in accordance with the University of Florida Institutional Animal Care and Use Committee and comply with Animal Research: Reporting of In Vivo Experiments guidelines.

Rats (n = 6–8 per group) were randomly assigned to one of seven groups. The experimental groups consisted of (1) naïve controls, (2) LC, (3) LC followed by hemorrhagic shock (LCHS), (3) LCHS followed by daily chronic restraint stress (LCHS/CS), (4) LC followed by propranolol (BB) use daily (LC + BB), (5) LCHS + BB, and (6) LCHS/CS + BB. Propranolol (Sigma, St. Louis, MO) was administered intraperitoneal at 10 mg/kg 10 min after resuscitation and then daily. After 7 d, all animals were sacrificed.

Whole blood was collected in a heparinized syringe after cardiac puncture. Plasma was separated by centrifugation at 3000× g for 10 min, then aliquoted and stored at −80°C. The left femoral epiphysis was removed, and BM was eluted with 1 mL of Iscove’s Modified Dulbecco’s Medium (Lonza, Walkersville, MD) containing 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA). Bone marrow was stored at −80°C for protein and RNA extraction. Both the left lung and the injured right lung were excised, then rinsed in 1× phosphate-buffed saline and stored at −80°C.

Trauma rodent model

On Day 1, rats were anesthetized with 50 mg/kg pentobarbital (Lundbeck Inc, Deerfield, IL) administered intraperitoneal, and then LC was performed with a percussive nail gun applied over a 12 mm metal plate applied to right lateral chest wall (PowerShot Model 5700M, Saddle Brook, NJ). This established model simulates a clinically relevant reproducible pulmonary contusion.^5–8

After LC, animals randomized to LCHS or LCHS/CS were placed on a heating pad to maintain normothermia, and the right internal jugular vein and right femoral artery were cannulated using heparinized saline (10 units/mL). The femoral artery catheter was transduced to a BP-2 Digital Blood Pressure Monitor (Columbus Instruments, Columbus, OH). Hemorrhagic shock was generated after blood removal to reach a mean arterial pressure of 30–35 mm Hg for 45 min. Shed blood was then reinfused at 1 mL/min immediately after the shock period.

Chronic restraint stress was incorporated to simulate stressors associated with the intensive care unit environment for human trauma patients.²² For animals randomized to LCHS/CS, CS began 1 d after LCHS and was performed by placing the rodents in a restraint cylinder (Kent Scientific Corporation, Torrington, CT) for 2 h daily until Day 7. Although restrained, every 30 min, they were noise stressed by enduring a 2-min alarm (80–85 dB) while being repositioned to prevent acclimation. All non-CS rodents were subjected to daily fasting while restraint stress was being performed.

Messenger RNA analysis

RNA was extracted from the BM and right lung tissue using the PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA). The nucleic acid purity was assessed (using 260/280 ratio), and concentration of RNA was determined by Epoch7 microplate reader and Gen5 software (BioTek Instruments, Inc, Winooski, VT). Complementary DNA was made from 2 µg RNA using the High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Vilnius, Lithuania) and protocol. The following analytes were examined by quantitative polymerase chain reaction (PCR) using Brilliant II Sybr Green QPCR Master Mix with Low Rox (Agilent Technologies, Cedar Creek, TX) and Stratagene Mx3005P cycler and MxPro software (Agilent Technologies). Bone marrow HMGB1, G-CSF, MMP-9, MMP-2, neutrophil elastase, SDF-1, CXCR4, VCAM-1, VLA-4, TIMP1, and TIMP2 expression were analyzed. Also, G-CSF, CXCR4, and VLA-4 expressions were analyzed in the lung.

Primers for these analytes were designed using Oligo-Perfect Primer Designer (ThermoFisher Scientific, Waltham, MA) and based on gene sequences acquired from NCBI GeneDataBank (National Center for Biotechnology Information, Bethesda, MD). Information regarding primer sequences and real-time PCR conditions are present in Table.

Table –

RT-PCR primers and conditions used for messenger RNA expression analysis.

Gene	NCBI RefSeq: NM_	Primer	Primer sequence	Amplified region	Amplicon length (bp)	Annealing temperature (°C)
HMGB1	012963.2	Forward Reverse	5′-CTAGCCCTGTCCTGGTGGTA-3′ 5′-TGTGCACCAACAAGAACCTG-3′	876–980	104	56
G-CSF	017104.2	Forward Reverse	5′-CCTTGGAGCAAGTGAGGAAG-3′ 5′-TTGGGGATACCCAGAGAGTG-3′	152–275	123	56
MMP-9	031055.1	Forward Reverse	5′-CACTGTAACTGGGGGCAACT-3′ 5′-AGAGTACTGCTTGCCCAGGA-3′	1008–1080	72	56
MMP-2	031054.2	Forward Reverse	5′-TGACGATGAGCTGTGGACTC-3′ 5′-CTGCTGTATTCCCGACCATT-3′	915–1028	113	56
Neutrophil elastase	001106767.1	Forward Reverse	5′-CAGTCAGTGCAAGTGGTGCT-3′ 5′-GGTCAAAGCCGTTCTCAAAG-3′	252–351	99	56
SDF-1	001033882.1	Forward Reverse	5′-AAGCCCAAAGAAAGGTGGTT-3′ 5′-TCGGTTCCAGGAAGCTAAGA-3′	1168–1278	110	56
VCAM-1	012889.1	Forward Reverse	5′-AAGGGGCTACATCCACACTG-3′ 5′-CTCCAGTTTCCTTCGCTGAC-3′	1142–1236	94	56
CXCR4	022205.3	Forward Reverse	5′-CTCCAAGCTGTCACACTCCA-3′ 5′-TCCCCACGTAATACGGTAGC-3′	735–841	106	56
VLA-4	001107737.1	Forward Reverse	5′-GGACACGGCCTGTAGTGATT-3′ 5′-CCACGCACACAGACAGAAGT-3′	1632–1736	104	56
TIMP1	053819.1	Forward Reverse	5′-GGTTCCCTGGCATAATCTGA-3′ 5′-ATGGCTGAACAGGGAAACAC-3′	410–508	98	56
TIMP2	021989.2	Forward Reverse	5′-GCATCACCCAGAAGAAGAGC-3′ 5′-GTCCATCCAGAGGCACTCAT-3′	535–653	118	56
Beta-actin	031144.3	Forward Reverse	5′-AGCCATGTACGTAGCCATCC-3′ 5′-ACCCTCATAGATGGGCACAG-3′	468–582	114	56

Enzyme-linked immunosorbent assay analysis

Enzyme-linked immunosorbent assay (ELISA) for MMP-9 and MMP-2 in BM supernatant and plasma was performed using Quantikine ELISA kits (R&D Systems, Minneapolis, MN). Plates were read using Epoch7 96-well plate reader and the kinetic function on Gen5 software. Because of high cost and low availability of commercially prepared ELISA kits for neutrophil elastase in our species, an enzymatic assay of elastase (EC 3.4.1.36) (Sigma–Aldrich, St. Louis, MO) cleavage function was adapted for use in a 96-well plate with the following modifications: 10 µL of sample (BM supernatant or plasma), 120 µL of Trizma Buffer (12.1 mg/mL, pH 8.0 at 25°C) (Sigma–Aldrich), and 20 µL of N-succinyl-(Ala)₃-p-nitroanilide substrate (2 mg/mL; Sigma–Aldrich) were used per well. The pathlength for 150 µL in the 96-well Flat Bottom plate (Becton Dickinson Labware, Franklin Lakes, NJ) was experimentally determined and used to correct the millimolar coefficient (for 1 cm pathlength) provided in the protocol defined as units per milliliter. All samples were run in duplicate.

Statistical analysis

All data were statistically analyzed by analysis of variance in GraphPad Prism version 6.0 (GraphPad Software, Inc, San Diego, CA). Statistical significance is defined as *P < 0.05 versus naïve controls and **P < 0.05 versus treated counterpart (LC + BB, LCHS + BB, LCHS/CS + BB). All values were expressed as mean ± standard deviation.

Results

Signaling proteins: HMGB1 and G-CSF

Seven days after injury, HMGB1 messenger RNA expression in the BM was significantly elevated relative to naïve control after LC, LCHS, and LCHS/CS (Fig. 1A). The use of propranolol after LCHS/CS significantly reduced HMGB1 expression when compared with LCHS/CS alone (1.1 ± 0.5** versus 4.0 ± 2.1).

Fig. 1 –

Bone marrow expression of HMGB1 (A) and G-CSF (B) with and without propranolol after severe trauma and chronic stress. Lung G-CSF expression (C) with and with without propranolol after severe trauma and chronic stress. BM = bone marrow; CS = chronic stress; HS = hemorrhagic shock. *P < 0.05 versus naïve, **P < 0.05 versus untreated counterpart.

Bone marrow expression of G-CSF was also significantly elevated in all groups relative to control on Day 7 (LC 2.2 ± 0.9*, LCHS 2.6 ± 1.4*, LCHS/CS 3.0 ± 1.9*; Fig. 1B). The use of propranolol after LC, LCHS, and LCHS/CS significantly reduced BM expression of G-CSF (Fig. 1B).

At the site of injury, lung G-CSF expression was only significantly elevated after LCHS/CS (4.4 ± 3.1* versus 1; Fig. 1C). Propranolol use after LCHS/CS + BB did decrease lung expression of G-CSF but was not statistically significant.

Proteases: MMP-9, MMP-2, neutrophil elastase

Bone marrow MMP-9 and MMP-2 expressions were not significantly altered after LC, LCHS, or LCHS/CS when compared with naïve controls (Figs. 2A and 3A). After propranolol use, BM MMP-2 expression decreased significantly after LCHS + BB (0.3 ± 0.1**) and LCHS/CS + BB (0.2 ± 0.1**) when compared with untreated groups (Figs. 2A and 3A).

Fig. 2 –

Bone marrow MMP-9 (A) expression with and without propranolol after severe trauma and chronic stress. Bone marrow MMP-9 concentration (B) and plasma MMP-9 concentration (C) with and without propranolol after severe trauma and chronic stress. BM = bone marrow; LC = lung contusion; HS = hemorrhagic shock; CS = chronic stress. *P < 0.05 versus naïve, **P < 0.05 versus untreated counterpart.

Fig. 3 –

Bone marrow MMP-2 (A) expression, BM MMP-2 concentration (B), and plasma MMP-2 concentration (C) with and without propranolol after severe trauma and chronic stress. BM = bone marrow; LC = lung contusion; HS = hemorrhagic shock; CS = chronic stress. *P < 0.05 versus naïve, **P < 0.05 versus untreated counterpart.

Figure 2B demonstrates significantly elevated BM MMP-9 concentration after LC and LCHS/CS when compared with naïve (244 ± 38*, 292 ± 77* versus 145 ± 53 ng/mL). The use of propranolol significantly reduced BM MMP-9 levels in all groups. Figure 3B shows that propranolol use significantly decreased BM MMP-2 levels after LCHS and LCHS/CS.

Figures 2C and 3C demonstrate little change in the plasma protein concentrations of MMP-9 and MMP-2 across all groups relative to naïve (11 ± 4 and 1326 ± 246 ng/mL, respectively). The use of propranolol after LC, LCHS, and LCHS/CS had little impact on plasma protein levels of MMP-9 and MMP-2.

Figure 4A illustrates significantly increased BM neutrophil elastase expression after LCHS and LCHS/CS (1.4 ± 0.4* and 2.1 ± 0.6*) relative to naïve. The use of propranolol after LCHS/CS significantly decreased neutrophil elastase when compared with LCHS/CS alone (1.2 ± 0.4** versus 2.1 ± 0.6). Bone marrow neutrophil elastase levels were significantly increased from naïve control after LCHS/CS and propranolol use after LCHS/CS significantly decreased neutrophil elastase levels (Fig. 4B). The plasma neutrophil elastase levels (Fig. 4C) were significantly elevated after LC, LCHS, and LCHS/CS when compared with naïve (2.6 ± 1.0*, 3.5 ± 1.6*, and 3.2 ± 0.8* versus 1.1 ± 1.3 units/L). The use of propranolol after LC, LCHS, and LCHS/CS significantly decreased plasma neutrophil elastase levels in all groups to that of naïve levels.

Fig. 4 –

Bone marrow neutrophil elastase (A) expression, BM neutrophil elastase concentration (B), and plasma neutrophil concentration (C) with and without propranolol after severe trauma and chronic stress. BM = bone marrow; LC = lung contusion; HS = hemorrhagic shock; CS = chronic stress. *P < 0.05 versus naïve, **P < 0.05 versus untreated counterpart.

Bone marrow adhesion molecules: SDF-1/CXR4, and VCAM-1/VLA-4

Bone marrow expression of SDF-1 was significantly decreased after LCHS and LCHS/CS (0.5 ± 0.2* and 0.3 ± 0.4*), as depicted in Figure 5A. The use of propranolol after LC significantly reduced BM expression of SDF-1 when compared with naïve (Fig. 5A). CXCR4 BM expression (Fig. 5B) was significantly elevated two- to three-fold after LC, LCHS, and LCHS/CS when compared with naïve (3.3 ± 1.7*, 1.9 ± 0.6+, and 2.6 ± 1.3 versus 1.0). The use of propranolol only significantly reduced CXCR4 expression after LCHS/CS (Fig. 5B). At the site of injury, CXCR4 expression was significantly elevated after LCHS/CS, and the use of propranolol significantly reduced CXCR4 expression (1.3 ± 0.2 versus 0.8 ± 0.3**; Fig. 5C).

Fig. 5 –

Bone marrow expression of SDF-1 (A) and CXCR4 (B) with and without propranolol after severe trauma and chronic stress. Lung CXCR4 expression (C) with and without propranolol after severe trauma and chronic stress. BM = bone marrow; LC = lung contusion; HS = hemorrhagic shock; CS = chronic stress. *P < 0.05 versus naïve, **P < 0.05 versus untreated counterpart.

VCAM-1 BM expression was only suppressed after LCHS/ CS, and the use of propranolol after LC, LCHS, and LCHS/CS did not significantly alter VCAM-1 expression in any group (Fig. 6A). Bone marrow VLA-4 expression was only significantly elevated after LCHS/CS (Fig. 6B). The use of propranolol after LCHS/CS significantly reduced VLA-4 expression when compared with LCHS/CS alone (1.1 ± 0.4** versus 1.7 ± 0.3). At the site of injury, lung expression of VLA-4 was elevated fourfold when compared with naïve (Fig. 6C). The use of propranolol after LCHS/CS significantly reduced lung expression of VLA-4 (1.8 ± 0.9** versus 3.9 ± 2.5; Fig. 6C).

Fig. 6 –

Bone marrow expression of VCAM-1 (A) and VLA-4 (B) with and without propranolol after severe trauma and chronic stress. Lung VLA-4 expression (C) with and without propranolol after severe trauma and chronic stress. BM = bone marrow; LC = lung contusion; HS = hemorrhagic shock; CS = chronic stress. *P < 0.05 versus naïve, **P < 0.05 versus untreated counterpart.

Protease inhibitors: TIMP1 and TIMP2

Bone marrow expression of TIMP1 (Fig. 7A) was significantly elevated after LC, LCHS, and LCHS/CS when compared with naïve. The use of propranolol significantly reduced TIMP1 expression after LC and LCHS/CS when compared with LC and LCHS/CS alone (1.0 ± 0.5** and 1.0 ± 0.4** versus 1.8 ± 0.3 and 1.7 ± 0.4).

Fig. 7 –

Bone marrow expression of TIMP1 (A) and TIMP2 (B) with and without propranolol after severe trauma and chronic stress. BM = bone marrow; LC = lung contusion; HS = hemorrhagic shock; CS = chronic stress. *P < 0.05 versus naïve, **P < 0.05 versus untreated counterpart.

TIMP2 BM expression relative to naïve was significantly increased after LCHS and LCHS/CS (Fig. 7B). The use of propranolol after LCHS and LCHS/CS significantly reduced TIMP2 expression (1.0 ± 0.5** and 1.0 ± 0.6** versus 1.7 ± 0.6 and 1.8 ± 0.5; Fig. 7B).

Discussion

After mild blunt traumatic injury, HPCs mobilize from the BM to the peripheral blood and to site of injury to participate in wound healing and tissue repair.^2–4 However, severe blunt trauma can be accompanied by hemorrhagic shock and chronic stress with a prolonged stay in the intensive care unit. Previously, we have shown that after trauma, HPC mobilization is prolonged, and this contributes to decreased BM cellularity, decreased erythroid progenitor cell growth, BM dysfunction, and anemia.^6–8 In the present study, we sought to characterize the molecular mediators involved in HPC mobilization in mild blunt traumatic injury, LC, and compare this to severe blunt traumatic injury accompanied by hemorrhagic shock and chronic stress. Additional experiments evaluated the use of propranolol, a nonselective beta-blocker, which competitively inhibits the effects of catecholamines at beta-adrenergic receptors. Our results suggest that HMGB1, G-CSF, and neutrophil elastase may each play a role in mobilizing HPCs from the BM and decreased expression of BM anchoring molecules, SDF-1, and VCAM-1 likely mediate prolonged HPC mobilization (Fig. 8). In addition, the ability of propranolol to reduce HMGB1, G-CSF, and neutrophil elastase expression after LCHS/CS suggests that the proinflammatory-mediated mobilization of HPCs is driven by persistent hypercatecholaminemia (Fig. 8).

Fig. 8 –

Conceptual diagram of HPC mobilization from BM to peripheral blood after trauma. The neuroendocrine stress response upregulates HMGB1 and the G-CSF/SDF-1 axis. SDF-1 interacts with the CXCR4 receptor on BM leukocytes and together with the neuroendocrine response and G-CSF activation, there is upregulation of matrix metalloproteinases (MMP) and neutrophil elastase (NE) in BM leukocytes. MMPs and NE impact the anchoring of HPC and the VLA-4/VCAM-1 axis, thus increasing HPC mobilization. Propranolol reduces the neuroendocrine stress response and decreases the expression of HMGB1, G-CSF, and NE to reduce HPC mobilization.

Severe traumatic injury results in sustained release of catecholamines that leads to a wide variety of hemodynamic, metabolic, and immune changes.²³ Katayama et al.¹³ found that norepinephrine signaling regulates HPC egress from the BM through a mechanism that involves G-CSF and SDF-1. In this study, we found that BM HMGB1 and G-CSF expression increased steadily 7 d after LC, LCHS, and LCHS/CS. Previously, we demonstrated that there is a steady increase in HPCs found in peripheral blood after LC, LCHS, and LCHS/CS.^2,6,8 Given epinephrine and norepinephrine act through shared adrenergic receptors, previous work demonstrating epinephrine-driven HMGB1 expression supports this relationship between chronic stress and elevated HMGB1 expression.^10,11 After LC alone, increased HMGB1 and G-CSF expression demonstrate the role of the BM in healing injured lung tissue. The addition of hemorrhagic shock as well as chronic stress also increased HMGB1 and G-CSF expression when compared with naïve animals, and this correlated with previously shown prolonged HPC mobilization.² In addition, we show that propranolol administration significantly decreased expression of HMGB1 after LCHS/CS and of G-CSF after LC, LCHS, and LCHS/CS. The correlative trend between HMGB1 and G-CSF supports a causal relationship, given the role of HMGB1 as a danger signal for local cellular stress that triggers the formation of the inflammasome and production of proinflammatory cytokines, of which includes G-CSF.⁹ Taken together, these findings indicate that catecholamines are responsible for HMGB1 and G-CSF release that leads to prolonged HPC mobilization. The significant elevation of lung G-CSF expression after LCHS/CS may play a role in delayed injured lung healing described previously.⁸

Prolonged elevation of G-CSF likely activates maturing neutrophils in the BM, which can lead to increased production and degranulation of acute inflammatory proteases. These proteases function to cleave the extracellular matrix and BM stromal anchoring molecules after acute inflammation.¹⁴ By analyzing various neutrophil-derived acute inflammatory proteases, we found that BM expressions of MMP-9 and MMP-2 were not significantly altered relative to naïve controls. However, there was a significant increase in MMP-9 concentration after LC and LCHS/CS, whereas MMP-2 elevation was only significant after LCHS. These indistinct changes suggest that MMP-9 and MMP-2 are not directly regulated by HMGB1 and G-CSF after trauma.

Plasma neutrophil elastase concentration mirrored HMGB1 and G-SCCF expressions with significant increases in each injury model. In contrast, only the most injured animals undergoing LCHS/CS had significant increases in BM neutrophil elastase expression and concentration. As opposed to MPP-9 and MMP-2, neutrophil elastase appears to be a principal mediator of HPC mobilization after LCHS/CS. Serine protease inhibitors, known as serpins, have also been implicated in HPC mobilization.¹² MMPs and their tissue inhibitors (TIMPs) both play a role in the remodeling of injured tissue.²⁴ This study demonstrated that BM expressions of TIMP1 and TIMP2 were significantly elevated after injury and stress, and this expression was decreased with the use of propranolol. TIMP1 expression has been shown to be increased by G-CSF treatment, and there was a correlation with increased fibrosis in injured tissue in a murine model of myocardial infarction.²⁵ The increased expression of TIMP1 and TIMP2 may play a role in the delayed healing and increased fibrosis of injured lung tissue previously shown after LCHS/CS.⁸

Adhesion molecules that anchor HPCs to the BM stroma are essential as these cells mature in the BM. The chemokine, SDF-1, and its receptor CXCR4 have crucial roles in regulating HPC trafficking, homing, and maintenance. SDF-1 has been shown to cleaved by both MMPs and neutrophil elastase, whereas VCAM-1 and its receptor VLA-4 are substrates to neutrophil elastase only.¹² In our study, BM expression of SDF-1 was significantly inhibited after LCHS and LCHS/CS, whereas VCAM-1 expression was only reduced after LCHS/CS. Numerous studies support the mechanism of G-CSF induced HPC mobilization via inhibition of the SDF-1/CXCR4 axis.^12,14 Prolonged treatment with G-CSF has been shown to cause progressive decline in SDF-1 expression in the BM with a concurrent drop in CXCR4.¹⁴ After LCHS/CS, there is significant inhibition of SDF-1 expression, and Loftus et al.² previously demonstrated that after LCHS/CS, there was greatest degree of HPC mobilization. This is supported by Mendez-Ferrer et al.²⁶ who demonstrated that the level of SDF-1 is inversely correlated with the degree of mobilization. Bone marrow expressions of SDF-1 and VCAM-1 were not altered by the use of propranolol, indicating that their expression is not directly mediated by catecholamines. However, the use of propranolol after LCHS/CS did significantly reduce BM CXCR4 and VLA-4 expression. This suggests that pharmacologically inhibiting catecholamines blunts HPC mobilization by G-CSF, most likely by regulating expression of anchoring receptors. We have also previously shown the LCHS/CS induced hepatocyte growth factor/c-met signaling potentially contributing to mobilization by inhibiting the ability of HPC to home in response to SDF-1.²

Similar to the principle of leukocyte recruitment to inflammation sites, HPCs mobilize from the BM and home to injured tissue. In the injured tissue, these cells participate directly and indirectly in tissue repair.^3,4 As HPCs are mobilized to the peripheral blood after LCHS/CS, the increased lung expression of CXCR4 and VLA-4 may direct them to the site of injury. This supports the previous finding that HPCs mobilize to the site of injury as evidenced by the growth of HPC progenitors and fluorescent labeling of these cells.^4,8

The clinical translation of these rodent findings depends on effectively recapitulating neuroendocrine activation and inflammation experienced by severely injured trauma patients who are admitted to the intensive care unit. In this regard, several limitations of this study will be discussed. Rodent models have poor track record for translation in human studies because of the complex pathophysiology of human disease processes, and previous animal models often study early time points that may be suited to mortality studies but not the mechanistic understanding of the pathophysiology. This unique rodent model has been developed to recapitulate severe traumatic injury followed by an intensive care unit stay. In addition to injury and hemorrhagic shock, the addition of daily chronic stress to simulate an intensive care unit environment with stress, alarms, restrain, and repositioning has demonstrated that these animals do not immediately recover from their injuries.^8,23 In addition, female rodents were excluded because of the known influence of sex hormones on outcomes after hemorrhagic shock.²⁷ Although human BM samples have been obtained from trauma patients for research studies, BM samples are more difficult to obtain because of the timing of the orthopedic intervention, the ability to obtain patient/health care surrogate consent, and the cessation of patient enrollment during the pandemic. Therefore, based on previous work, the authors believe that the experimental methods presented here suffice to evaluate mechanistic changes.^8,23 Future research should examine the efficacy of other potentially useful methods for recapitulating chronic intensive care unit stress, such as dysregulation of day and night sleep–wake cycles, representing a methodological limitation. This work also provides insight into the regulation of HPC mobilization, but the corollary role of cell surface molecules remains unknown. In addition, we demonstrated that TIMP and neutrophil elastase expression correlated with HMGB1 and G-CSF expression, but the precise mechanism remains unclear. Further study of protein levels of SDF-1, VCAM-1, and lung MMPs as well as outcomes of HPC mobilization, such as lung histologic healing and hemoglobin levels, would further our understanding.

Conclusion

In summary, this study identified HMGB1, G-CSF, and neutrophil elastase as mediators of HPC mobilization after injury and chronic stress. This HPC mobilization is due in part to increased proteolysis as well as decreased expression of BM anchors, SDF-1, and VCAM-1. In addition, we found increased expression of CXCR4 and VLA-4 injured tissue, which likely contributes to homing of HPCs to injured tissue. Propranolol’s ability to reduce HMGB1, G-CSF, and neutrophil elastase expression suggests that the mobilization of HPCs was driven by persistent hypercatecholaminemia. In summary, although our understanding of the molecular mechanisms contributing to prolonged HPC mobilization after injury and stress has improved, there remain many unanswered questions. Future research on HPC regulation and trafficking and the role of sympathetic tone may identify potential therapy targets to enhance HPC homing and potential healing of injured tissues.