Daily propranolol prevents prolonged mobilization of hematopoietic progenitor cells in a rat model of lung contusion, hemorrhagic shock, and chronic stress

doi:10.1016/j.surg.2015.06.031

Surgery

Volume 158, Issue 3, September 2015, Pages 595-601

Presented at the 10th Annual Academic Surgical Congress in Las Vegas, NV, February 3–5, 2015.

https://doi.org/10.1016/j.surg.2015.06.031 Get rights and content

Introduction

Propranolol has been shown previously to decrease the mobilization of hematopoietic progenitor cells (HPCs) after acute injury in rodent models; however, this acute injury model does not reflect the prolonged period of critical illness after severe trauma. Using our novel lung contusion/hemorrhagic shock/chronic restraint stress model, we hypothesize that daily administration of propranolol will decrease prolonged mobilization of HPCs without worsening lung healing.

Methods

Male Sprague–Dawley rats underwent 6 days of restraint stress after undergoing lung contusion or lung contusion/hemorrhagic shock. Restraint stress consisted of a daily 2-hour period of restraint interrupted every 30 minutes by alarms and repositioning. Each day after the period of restraint stress, the rats received intraperitoneal propranolol (10 mg/kg). On day 7, peripheral blood was analyzed for granulocyte-colony stimulating factor (G-CSF) and stromal cell-derived factor 1 via enzyme-linked immunosorbent assay and for mobilization of HPCs using c-kit and CD71 flow cytometry. The lungs were examined histologically to grade injury.

Results

Seven days after lung contusion and lung contusion/hemorrhagic shock, the addition of chronic restraint stress significantly increased the mobilization of HPC, which was associated with persistently increased levels of G-CSF and increased lung injury scores. The addition of propranolol to lung contusion/chronic restraint stress and lung contusion/hemorrhagic shock/chronic restraint stress models greatly decreased HPC mobilization and restored G-CSF levels to that of naïve animals without worsening lung injury scores.

Conclusion

The daily administration of propranolol after both lung contusion and lung contusion/hemorrhagic shock subjected to chronic restraint stress decreased the prolonged mobilization of HPC from the bone marrow and decreased plasma G-CSF levels. Despite the decrease in mobilization of HPC, lung healing did not worsen. Alleviating chronic stress with propranolol may be a future therapeutic target to improve healing after severe injury.

Section snippets

Animals

Male Sprague–Dawley rats (5–8/group) were housed in pairs with free access to food (Teklad22/5 Rodent Diet W-8640; Harlan Teklad, Madison, WI) and water during daily night day cycles of 12 hours each. The animals were housed and handled in accordance with Institutional Animal Care and Use Committee regulations.

Experimental design

The rats were allocated randomly into 1 of 7 groups to identify the effect of Prop in mitigating the effect of chronic stress after lung contusion and lung contusion combined with

The impact of Prop on HPC mobilization

With flow cytometry, HPC that have mobilized into the peripheral blood were defined as those cells expressing both CD71 and CD117. In naïve animals, only 0.5% of cells are circulating HPC. The addition of CRS to LC and LCHS models significantly increased the mobilization of HPCs (Fig 1). This observation demonstrates that the addition of CRS significantly prolonged the mobilization of HPC compared with the models of acute injury of LC and LCHS alone.

When LC/CRS animals received daily Prop after

Discussion

The trafficking of HPC from the bone marrow to the peripheral blood and eventually to injured tissue is believed to be beneficial and aids in the host defense and is integral to wound healing.9, 27 Severe traumatic injury and shock, however, are associated with significant increases in norepinephrine levels and subsequently increased mobilization of HPC.15, 17 Previously, the administration of Prop in acute injury rodent models has been shown to mitigate the release of HPC from the bone marrow.

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Cited by (22)

The role of bone marrow microRNA (miR) in erythropoietic dysfunction after severe trauma
2021, Surgery (United States)
Previous data has shown that severe traumatic injury is associated with bone marrow dysfunction, which manifests as persistent injury-associated anemia. This study sought to identify whether the expression of erythropoiesis-related microRNAs were altered in the bone marrow of trauma patients to determine if these microRNAs play a role in persistent injury-associated anemia.
Bone marrow was collected from severely injured trauma patients who underwent fracture fixation as well as patients who underwent elective hip replacement. There were 27 trauma patients and 10 controls analyzed. Total RNA and microRNA were isolated from CD34-positive cells using the RNeasy Plus Mini kit, and genome-wide microRNA expression patterns were assayed. Genes with significant expression differences were found using BRB-ArrayTools with a significance of P < .01.
There were marked differences in expression of 108 microRNAs in the trauma group when compared with hip replacement patients. Four of these microRNAs play a role in regulating erythropoiesis: microRNA-150, microRNA-223, microRNA15a, and microRNA-24. These microRNAs were all upregulated significantly, with trauma/hip replacement fold changes of 1.7, 1.8, 1.2, and 1.2 respectively, and all act to suppress or regulate erythropoiesis.
Assessment of the bone marrow microRNA profile in trauma patients compared to those undergoing elective hip replacement revealed the differential expression of microRNA-150, microRNA-223, microRNA-15a, and microRNA-24. These microRNAs all play a role in decreased erythroid progenitor cell growth and provide important insight to the erythropoietic dysfunction seen after trauma.
Mediators of Prolonged Hematopoietic Progenitor Cell Mobilization After Severe Trauma
2021, Journal of Surgical Research
Citation Excerpt :
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
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.
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.
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.
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.
The effects of selective beta-adrenergic blockade on bone marrow dysfunction following severe trauma and chronic stress
2020, American Journal of Surgery
Citation Excerpt :
In particular, bone marrow dysfunction following trauma is associated with decreased erythroid progenitor colony growth, prolonged hematopoietic progenitor cell mobilization to the peripheral blood, erythroid to myeloid reprioritization, impaired functional iron homeostasis, and hematopoietic progenitor cell sequestration at the site of injury.3,6,11–16 In previous studies using a rodent trauma model, as well as a prospective trial in trauma patients, the use of daily propranolol, a non-selective beta adrenergic blocker, was found to increase bone marrow erythroid progenitor colony growth, reduce hematopoietic progenitor cell mobilization, improve iron homeostasis and redirect bone marrow hematopoietic commitment toward erythropoiesis.3–6,11,14,15 The exact mechanism of improved bone marrow function following trauma with the use of a non-selective beta blockade remains unknown.
Propranolol has been shown to improve erythroid progenitor cell growth and anemia following trauma and this study sought to investigate the mechanisms involved by evaluating the effects of selective beta blockade.
Male Sprague-Dawley rats were subjected to lung contusion, hemorrhagic shock and chronic stress (LCHS/CS) ± daily selective beta-1, beta-2, or beta-3 blockade (B1B, B2B, B3B). Bone marrow cellularity and growth of erythroid progenitor colonies, hemoglobin, plasma granulocyte colony-stimulating factor (G-CSF), hematopoietic progenitor cell mobilization, and daily weight were assessed.
Selective beta-2 and beta-3 blockade improved bone marrow cellularity, erythroid progenitor colony growth and hemoglobin levels, while decreasing plasma G-CSF, progenitor cell mobilization and weight loss following LCHS/CS.
Attenuating the neuroendocrine stress response with the use of selective beta-2 and 3 adrenergic blockade may be an alternative to improve bone marrow erythroid function following trauma.
Impact of Injury Severity on the Inflammatory State and Severe Anemia
2020, Journal of Surgical Research
Severe traumatic injury is a major cause of morbidity and mortality. Our goal was to analyze blunt traumatic injury by injury severity score (ISS) and compare with elective hip repair, as a transient injury, and healthy control with the hypothesis that more severe injury would lead to an increase in neuroendocrine activation, systemic inflammation, and worse anemia.
A prospective observational cohort study was performed at a level 1 trauma center, comparing blunt trauma patients (n = 37), elective hip replacement patients (n = 26), and healthy controls (n = 8). Bone marrow and plasma were assessed for hyperadrenergic state, erythropoiesis, and systemic inflammation. Trauma patient's ISS ranged from 4 to 41 and were broken down into quartiles for analysis. The ISS quartiles were 4-13, 14-20, 21-26, and 27-41.
Plasma norepinephrine, interleukin-6, tumor necrosis factor-alpha, and hepcidin increased progressively as ISS increased. Hemoglobin significantly decreased as ISS increased and packed red blood cell (pRBC) transfusion increased as ISS increased. Elective hip replacement patients had an appropriate increase in the bone marrow expression of erythropoietin and the erythropoietin receptor, which was absent in all trauma patient groups.
Increased neuroendocrine activation, systemic inflammation, and anemia correlated with worsening injury severity, lower age, and increased pRBC transfusions. Elective hip replacement patients have only minimal systemic inflammation with an appropriate bone marrow response to anemia. This study demonstrates a link between injury severity, neuroendocrine activation, systemic inflammation, and the bone marrow response to anemia.
Effect of Beta-Blockade on the Expression of Regulatory MicroRNA after Severe Trauma and Chronic Stress
2020, Journal of the American College of Surgeons
Beta-blockade administration after lung contusion, hemorrhagic shock, and chronic stress has been shown to improve bone marrow function, decrease hypercatecholaminemia, and reduce inflammation. MicroRNAs (miR) are critical biologic regulators that can downregulate gene expression by causing messenger RNA degradation or inhibition of translation. This study sought to expand our understanding of the molecular mechanisms underlying the reduced inflammatory response after the administration of beta-blockade (BB) in our rodent trauma model.
Male Sprague-Dawley rats aged 8 to 9 weeks were randomized to lung contusion, hemorrhagic shock with daily restraint stress (LCHS/CS) or LCHS/CS plus propranolol (LCHS/CS+BB). Restraint stress occurred 2 hours daily after LCHS. Propranolol (10 mg/kg) was given daily until day 7. Total RNA and miR were isolated from bone marrow and genome-wide miR expression patterns were assayed. Bone marrow cytokine expression was determined with quantitative polymerase chain reaction.
LCHS/CS led to significantly increased bone marrow expression of interleukin (IL) 1β, tumor necrosis factor-α, IL-6, nitric oxide, and plasma C-reactive protein. There were marked differences in expression of 45 miRs in the LCHS/CS+BB group compared with the LCHS/CS group when using a p value <0.001. Rno-miR-27a and miR-25 were upregulated 7- to 8-fold in the rodents who underwent LCHS/CS+BB compared with LCHS/CS alone, and this correlated with reduced bone marrow expression of IL-1β, tumor necrosis factor-α, IL-6, nitric oxide, and reduced plasma C-reactive protein in the LCHS/CS+BB group.
The genomic and miR expression patterns in bone marrow after LCHS/CS differed significantly compared with rodents that received propranolol after LCHS/CS. The use of BB after severe trauma can help mitigate persistent inflammation by upregulating Rno-miR-27a and miR-25 and reducing inflammatory cytokines in those who remain critically ill.
Systemic Regulation of Bone Marrow Stromal Cytokines After Severe Trauma
2019, Journal of Surgical Research
Traumatic injury generates a prolonged hypercatecholamine state that is associated with reduced growth of bone marrow erythroid progenitors mediated by the bone marrow stroma. The bone marrow stroma is made up of many cells including fibroblasts, which respond to inflammatory stimuli and alter the cytokine profile. We hypothesized that trauma plasma would increase bone marrow stromal fibroblast expression of interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), stem cell factor (SCF), and activation of nuclear factor kappa-light-chain-enhancer of activated B cells and correlate with injury severity and anemia.
Plasma from 15 trauma patients was cultured with bone marrow fibroblast cells and compared with that from healthy volunteers. At 6, 24, and 48 h, the expression of IL-6, G-CSF, EPO, SCF, and the activation of nuclear factor kappa-light-chain-enhancer of activated B cells were measured using quantitative polymerase chain reaction. The influence of trauma plasma on cytokine expression was further stratified by injury severity score (ISS).
The average hemoglobin significantly decreased from admission to discharge (10.7 ± 2.5 to 9.2 ± 1.1 g/dL, P < 0.04). The discharge hemoglobin significantly decreased by 14% from the admission hemoglobin. After 48 h, trauma plasma significantly increased IL-6, G-CSF, and EPO bone marrow fibroblast expression when compared with normal plasma. When stratified by ISS, IL-6, G-CSF, and EPO, bone marrow fibroblast expression was highest in the trauma plasma ISS 27-41 group and was significantly elevated compared with normal plasma. When SCF expression was stratified by ISS, there was a significant increase in expression in ISS 27-41. Higher ISS was also associated with a larger decrease in hemoglobin despite no difference in total blood transfusions.
Severe trauma can systemically increase IL-6, G-CSF, and EPO expression in bone marrow stroma. Increased hematopoietic cytokine expression after traumatic injury correlated with a hypercatecholamine state, anemia, and injury severity.

View all citing articles on Scopus

: Supported by the National Institutes of Health grants R01 NIH GM105893-01A1 and T32 GM069330.

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Trauma/Critical CareDaily propranolol prevents prolonged mobilization of hematopoietic progenitor cells in a rat model of lung contusion, hemorrhagic shock, and chronic stress

Introduction

Methods

Results

Conclusion

Section snippets

Animals

Experimental design

The impact of Prop on HPC mobilization

Discussion

Cell

J Am Coll Surg

Immunol Allergy Clin North Am

Adv Neuroimmunol

Brain Behav Immun

J Surg Res

J Surg Res

Exp Hematol

Lancet

Surgery

Surgery

G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4

Nat Immunol

Regulatory systems in bone marrow for hematopoietic stem/progenitor cells mobilization and homing

Biomed Res Int

Mobilization of hematopoietic stem/progenitor cells: general principles and molecular mechanisms

Methods Mol Biol

Novel trafficking routes for hematopoietic stem and progenitor cells

Ann NY Acad Sci

Bone marrow-derived progenitor cells in pulmonary fibrosis

J Clin Invest

Role of the SDF-1/CXCR4 axis in the pathogenesis of lung injury and fibrosis

Am J Respir Cell Mol Biol

Participation of bone marrow derived cells in cutaneous wound healing

J Cell Physiol

Mobilization of bone marrow cells to the site of injury is necessary for wound healing

J Trauma

Stromal cell-derived factor-1 enhances wound healing through recruiting bone marrow-derived mesenchymal stem cells to the wound area and promoting neovascularization

Cells Tissues Organs

Do all β-blockers attenuate the excess hematopoietic progenitor cell mobilization from the bone marrow following trauma hemorrhagic shock?

J Trauma Acute Care Surg

Trauma/Critical Care
Daily propranolol prevents prolonged mobilization of hematopoietic progenitor cells in a rat model of lung contusion, hemorrhagic shock, and chronic stress