Walter D. Alzate

Impact of enhanced mobilization of bone marrow derived cells to site of injury.

BACKGROUND Bone marrow derived cells (BMDC) and mesenchymal stem cells (MSC) are necessary for healing of injured tissues. Intravenous granulocyte-colony stimulating factor (G-CSF) is known to induce mobilization of BMDC to peripheral blood and the tissue levels of the stromal cell derived factor-1 (SDF-1) to be key in their homing to sites of injury. We hypothesized that injection of SDF-1 to the site of injury and/or systemic administration of G-CSF increases homing of BMDC and improves healing of traumatic injury. We also postulated that increased homing of MSC alone to sites of injury would also improve tissue healing. METHODS Male Sprague-Dawley rats were subjected to unilateral lung contusion (LC) and assigned to the following groups: LC + injection of SDF-1 (LC + SDF-1) in the contused lung, pretreatment with systemic G-CSF for 5 days followed by either LC alone (LC + G-CSF) or by LC + injection of SDF-1 (LC + SDF-1/G-CSF). Rats in the MSC group were subjected to LC followed by systemic injection of MSC (LC + MSC). Unmanipulated controls and LC + local injection of saline (LC + saline) served as controls. Lung injury was assessed on days 1 and 5 postinjury using a histologic Lung Injury Score. BMDC and MSC homing were assessed on day 1 by hematopoietic progenitor cell (CFU-GEMM, BFU-E, and CFU-E) colony growth and immunofluorescence tracking of tagged MSC in the injured lung, respectively. RESULTS Both LC + SDF-1 and LC + G-CSF had increased hematopoietic progenitor cell colony growth in the injured lung, and their combination (LC + SDF-1/G-CSF) was additive when compared with LC + saline (18 ± 3, 24 ± 3, 32 ± 3; 21 ± 3, 36 ± 10, 36 ± 3; 31 ± 4, 44 ± 10, 53 ± 5 vs. 6 ± 2, 11 ± 3, 17 ± 4; *p < 0.05). Tagged MSC were tracked predominantly in the contused lung versus the non-contused lung (7 ± 3 vs. 3 ± 2, N° MSC/HPF; *p < 0.05). Lung Injury Score on day 5 after injury was significantly lower in the LC + SDF-1, LC + G-CSF, LC + SDF-1/G-CSF and LC + MSC groups versus LC + saline (1 ± 0.6, 0.7 ± 0.5, 1 ± 0.9, 1.1 ± 0.9 vs. 3.1 ± 0.8; *p < 0.05). CONCLUSION Local SDF-1 and/or systemic G-CSF can effectively increase BMDC homing to sites of traumatic injury in an additive way and improve wound healing. This process appears to be mediated predominantly through MSC. Additional investigations are needed to identify the optimal adjuncts to improve wound healing following severe traumatic injury.

Hematopoietic Progenitor Cell Mobilization Is Mediated Through β-2 and β-3 Receptors After Injury

BACKGROUND Hematopoietic progenitor cells (HPCs) are mobilized into the peripheral blood (PB) and then sequestered in injured tissue after trauma. Nonselective beta-adrenergic blockade (BB) has been shown to cause a decrease in mobilization of HPCs to the periphery and to injured tissue. Given the vast physiologic effects of nonselective BB, the aim of this study is to delineate the role of selective BB in HPC growth and mobilization. METHODS Rats underwent daily intraperitoneal injections of propranolol (Prop), atenolol (B1), butoxamine (B2), or SR59230A (B3) for 3 days to induce BB. All groups then underwent lung contusion (LC). HPC presence was assessed by GEMM, BFU-E, and CFU-E colony growth both in injured lung and bone marrow (BM). Flow cytometry, using c-kit and CD71, was used to determine mobilization into PB. RESULTS LC alone decreased BM HPC growth in all erythroid cell types and increased their number in injured lung (all *p < 0.05). beta-Blockade with Prop, B2, and B3 blockades restored BM HPC growth to control levels and decreased HPCs recovered in the injured lung. Similarly, Prop, B2, and B3 blockade prevented HPC mobilization to PB. B1 blockade with atenolol had no impact on HPC growth and mobilization following LC. CONCLUSIONS Nonselective BB reduced suppression of HPC growth in BM after injury and prevented the mobilization and subsequent sequestration of HPCs in injured tissue. Our data have shown that this effect is mediated through the B2 and B3 receptors. Therefore, after trauma, treatment with selective B2 or B3 blocker may attenuate the BM suppression associated with tissue injury.

Early propranolol administration to severely injured patients can improve bone marrow dysfunction.

BACKGROUND Bone marrow (BM) dysfunction is common in severely injured trauma patients, resulting from elevated catecholamines and plasma granulocyte colony-stimulating factor (G-CSF) as well as prolonged mobilization of hematopoietic progenitor cells (HPCs). We have previously shown that propranolol (&bgr;-blocker [BB]) reduces HPC mobilization in a rodent model of injury and hemorrhagic shock. We hypothesize that BB would prevent BM dysfunction in humans following severe injury. METHODS Forty-five severely injured trauma patients were studied in a prospective, randomized pilot trial. Twenty-five patients received BB, and 20 served as untreated controls. The dose of propranolol was adjusted to decrease the heart rate by 10% to 20% from baseline. Blood was analyzed for the presence of HPC (blast-forming unit erythroid cells [BFU-E] and colony-forming unit erythroid cells) and G-CSF. Demographic data, Injury Severity Score (ISS), hemoglobin, reticulocyte number, and outcome data were obtained. RESULTS The mean age of the study population was 33 years; 87% were male, with a mean ISS of 29. There is a significant increase in BFU-E in peripheral blood immediately following traumatic injury, and this mobilization persists for 30 days. The use of BB significantly decreases BFU-E and colony-forming unit erythroid cells at all time points. G-CSF is significantly elevated in both groups on admission; the use of BB decreases G-CSF levels by 51% as compared with 37% for controls. The average hemoglobin is nearly 1 g higher on the day of discharge with propranolol treatment (BB, 9.9 ± 0.4 g/dL vs. no BB, 9.1 ± 0.6 g/dL). CONCLUSION Following severe trauma, early treatment with propranolol following resuscitation is safe. The use of propranolol blunts early tachycardia, reduces HPC mobilization, and results in a faster return to baseline of the G-CSF peak seen after injury. There is also a trend toward faster recovery and resolution of anemia. Propranolol may be the first therapeutic agent to show improved BM function after severe injury. LEVEL OF EVIDENCE Therapeutic study, level III.

Beta-Blockade Prevents Hematopoietic Progenitor Cell Suppression after Hemorrhagic Shock

BACKGROUND Severe injury is accompanied by sympathetic stimulation that induces bone marrow (BM) dysfunction by both suppression of hematopoietic progenitor cell (HPC) growth and loss of cells via HPC mobilization to the peripheral circulation and sites of injury. Previous work demonstrated that beta-blockade (BB) given prior to tissue injury both reduces HPC mobilization and restores HPC colony growth within the BM. This study examined the effect and timing of BB on BM function in a hemorrhagic shock (HS) model. METHODS Male Sprague-Dawley rats underwent HS via blood withdrawal, maintaining the mean arterial blood pressure at 30-40 mm Hg for 45 min, after which the extracted blood was reinfused. Propranolol (10 mg/kg) was given either prior to or immediately after HS. Blood pressure, heart rate, BM cellularity, and death were recorded. Bone marrow HPC growth was assessed by counting colony-forming unit-granulocyte-, erythrocyte-, monocyte-, megakaryocyte (CFU-GEMM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-erythroid (CFU-E) cells. RESULTS Administration of BB prior to injury restored HPC growth to that of naïve animals (CFU-GEMM 59 ± 11 vs. 61 ± 4, BFU-E 68 ± 9 vs. 73 ± 3, and CFU-E 81 ± 35 vs. 78 ± 14 colonies/plate). Beta-blockade given after HS increased the growth of CFU-GEMM, BFU-E, and CFU-E significantly and improved BM cellularity compared with HS alone. The mortality rate was not increased in the groups receiving BB. CONCLUSION Administration of propranolol either prior to injury or immediately after resuscitation significantly reduced post-shock BM suppression. After HS, BB may improve BM cellularity by decreasing HPC mobilization. Therefore, the early use of BB post-injury may play an important role in attenuating the BM dysfunction accompanying HS.

Does beta blockade postinjury prevent bone marrow suppression

BACKGROUND Trauma-induced hypercatecholaminemia negatively impacts bone marrow (BM) function by suppressing BM hematopoietic progenitor cell (HPC) growth and increasing HPC egress to injured tissue. Beta blockade (BB) given before tissue injury alone has been shown to reduce both HPC mobilization and restore HPC colony growth within the BM. In a clinically relevant model, this study examines the effect of BB given after both tissue injury and hemorrhagic shock (HS). METHODS Male Sprague-Dawley rats underwent lung contusion (LC) with a blast wave percussion. HS was achieved after LC by maintaining the mean arterial blood pressure 30 mm Hg to 35 mm Hg for 45 minutes. Propranolol (10 mg/kg) was given once the mean arterial blood pressure>80 mm Hg and subsequent doses were given daily (LC/HS/BB). One-day and 7-day postinjury, analysis of BM and lung tissue for the growth of HPCs, hematologic parameters, and histology of lung injury were performed. RESULTS LC/HS significantly worsens BM CFU-E growth suppression (15±8 vs. 35±2) and increases CFU-E growth in injured tissue when compared with LC at 1 day and 7 days (33±5 vs. 22±9). The use of BB after LC/HS ameliorated BM suppression, the degree of anemia and HPC growth in the injured lung at 1 day and 7 days postinjury. Lung injury score shows that there was no worsening of lung healing with BB (LC/HS/BB 3.2±2 vs. LC/HS 3.8±0.8). CONCLUSION In an injury and shock model, administration of propranolol immediately after resuscitation significantly reduced BM suppression, and the protective effect is maintained at 7 days with daily BB. Although BB appears to improve BM function by decreasing HPC mobilization to injured tissue, there was no worsening of lung healing. Therefore, the use of propranolol after trauma and resuscitation may minimize long-term BM suppression after injury with no adverse impact on healing.

Role of bone marrow and mesenchymal stem cells in healing after traumatic injury.

BACKGROUND The role of bone marrow-derived cells (BMDCs) and mesenchymal stem cells (MSC) in healing of traumatic-induced injury remains poorly understood. Mesenteric lymph duct ligation (LDL) results in decreased BMDC mobilization and impaired healing. We hypothesized that LDL-mediated impaired healing would be abrogated by reinjection of BMDC or MSC. METHODS Sprague-Dawley rats were subjected to LDL + lung contusion (LC+LDL) with or without injection of BMDCs or MSCs. Unmanipulated control (UC) and lung contusion alone (LC) served as controls. BMDC and MSC homing was assessed by hematopoietic progenitor cell (HPC [granulocyte-, erythrocyte-, monocyte-, and megakaryocyte colony-forming units; erythroid burst-forming units; and erythroid colony-forming units]) colony growth and immunofluorescent microscopic tracking of tagged MSC, respectively. Histologic lung injury score (LIS) was used to grade injury. Data are mean ± SD. *P < .05/Student t test. RESULTS Lung HPC growth was decreased in LC+LDL versus LC alone (HPC colonies: 2 ± 2, 4 ± 3, 4 ± 2 vs. 11 ± 2, 20 ± 6, 22 ± 9. *P < .05). LC+LDL had greater degree of lung injury on days 5 and 7 LC alone (LIS: 5 ± 1, 4 ± 1 vs. 3 ± 1, 1 ± 0.4. *P < .05). BMDC injection into rats with LC + LDL increased lung HPC growth to LC level (HPC colonies: 12 ± 2, 19 ± 5, 17 ± 4 vs 11 ± 2, 20 ± 6, 22 ± 9. P > .05). Injected MSCs into LC+LDL rats homed preferentially to contused versus noncontused lung (MSC/high-powered field: 6 ± 4 vs. 2 ± 2 *P < .05). Either BMDC or MSC injection into LC+LDL rats returned lung injury to LC level on day 7 (LIS: 1 ± 0.4 and 1 ± 1 vs. 1 ± 0.4. P > .05). CONCLUSION LDL-mediated impaired tissue healing is abrogated by either whole BMDC or MSC injection. This highlights the critical role of BMDC and MSC on healing of trauma-induced injury.

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

BACKGROUND Severe injury results in increased mobilization of hematopoietic progenitor cells (HPC) from the bone marrow (BM) to sites of injury, which may contribute to persistent BM dysfunction after trauma. Norepinephrine is a known inducer of HPC mobilization, and nonselective &bgr;-blockade with propranolol has been shown to decrease mobilization after trauma and hemorrhagic shock (HS). This study will determine the role of selective &bgr;-adrenergic receptor blockade in HPC mobilization in a combined model of lung contusion (LC) and HS. METHODS Male Sprague-Dawley rats were subjected to LC, followed by 45 minutes of HS. Animals were then randomized to receive atenolol (LCHS + &bgr;1B), butoxamine (LCHS + &bgr;2B), or SR59230A (LCHS + &bgr;3B) immediately after resuscitation and daily for 6 days. Control groups were composed of naive animals. BM cellularity, %HPCs in peripheral blood, and plasma granulocyte-colony stimulating factor levels were assessed at 3 hours and 7 days. Systemic plasma-mediated effects were evaluated in vitro by assessment of BM HPC growth. Injured lung tissue was graded histologically by a blinded reader. RESULTS The use of &bgr;2B or &bgr;3B following LCHS restored BM cellularity and significantly decreased HPC mobilization. In contrast, &bgr;1B had no effect on HPC mobilization. Only &bgr;3B significantly reduced plasma G-CSF levels. When evaluating the plasma systemic effects, both &bgr;2B and &bgr;3B significantly improved BM HPC growth as compared with LCHS alone. The use of &bgr;2 and &bgr;3 blockade did not affect lung injury scores. CONCLUSION Both &bgr;2 and &bgr;3 blockade can prevent excess HPC mobilization and BM dysfunction when given after trauma and HS, and the effects seem to be mediated systemically, without adverse effects on subsequent healing. Only treatment with &bgr;3 blockade reduced plasma G-CSF levels, suggesting different mechanisms for adrenergic-induced G-CSF release and mobilization of HPCs. This study adds to the evidence that therapeutic strategies that reduce the exaggerated sympathetic stimulation after severe injury are beneficial and reduce BM dysfunction.

Beta Blockade Protection of Bone Marrow Following Injury: A Critical Link between Heart Rate and Immunomodulation.

Introduction Severe trauma induces a profound elevation of catecholamines that is associated with bone marrow (BM) hematopoietic progenitor cell (HPC) colony growth suppression, excessive BM HPC mobilization, and a persistent anemia. Previously, propranolol (BB) use after injury and shock has been shown to prevent this BM dysfunction and improve hemoglobin levels. This study seeks to further investigate the optimal therapeutic dose and timing of BB administration following injury and shock. Methods Male Sprague-Dawley rats were subjected to a combined lung contusion (LC), hemorrhagic shock (HS) model ± BB. In our dose response experiments, animals received BB at 1, 2.5, 5, or 10 mg/kg immediately following resuscitation. In our therapeutic window experiments, following LCHS rats were given BB immediately, 1 hour, or 3 hours following resuscitation. BM and peripheral blood (PB) were collected in all animals to measure cellularity, BM HPC growth, circulating HPCs, and plasma G-CSF levels. Results Propranolol at 5 and 10 mg/kg significantly reduced HPC mobilization, restored BM cellularity and BM HPC growth, and decreased plasma G-CSF levels. Propranolol at 5 and 10 mg/kg also significantly decreased heart rate. When BB was administered beyond 1 hour after LCHS, its protective effects on cellularity, BM HPC growth, HPC mobilization, and plasma G-CSF levels were greatly diminished. Conclusion Early Buse following injury and shock at a dose of at least 5mg/kg is required to maintain BM cellularity and HPC growth, prevent HPC mobilization, and reduce plasma G-CSF levels. This suggests that propranolol exerts its BM protective effect in a dose and time dependent fashion in a rodent model. Finally, heart rate may be a valuable clinical marker to assess effective dosing of propranolol.

Is the sympathetic system involved in shock-induced gut and lung injury?

BACKGROUND &bgr;-blockade (BB) has been shown to prevent bone marrow (BM) dysfunction after trauma and hemorrhagic shock (HS). The impact of the sympathetic system and the role of BB on shock-induced distant organ injury is not known. This study will determine if BB has systemic effects and can diminish gut and lung injury after trauma and HS. METHODS Male Sprague-Dawley rats were subjected to lung contusion (LC) followed by 45 minute of HS. Animals (n = 6 per group) were then randomized to either receive propranolol (LCHS + BB) immediately after resuscitation or not (LCHS). Gut permeability was evaluated in by diffusion of Mr 4,000 of fluorescein dextran (FD4) from a segment of small bowel into peripheral blood. Villous injury and lung injury were graded histologically by a blinded reader. Plasma-mediated effects of BB were evaluated in vitro by an assessment of BM progenitor growth. RESULTS Animals undergoing LCHS had significantly higher plasma levels of FD4 compared with control animals (mean [SEM], 2.8 [0.4] µg/mL vs. 0.8 [0.2] µg/mL). However, animals receiving BB had a significant reduction in plasma FD4 compared with the LCHS group. With the use of BB after LCHS, both ileal and lung injury scores were similar to control. In addition, BM progenitor growth was inhibited by the addition of LCHS plasma, and LCHS + BB plasma showed no inhibition of BM progenitor growth. CONCLUSION Propranolol can protect against the detrimental effects of trauma and HS on gut permeability, villous, and lung injury. The effects of BB are likely systemic and appear to be mediated through plasma. BB likely blunts the exaggerated sympathetic response after shock and injury. Propranolol’s reduction of both BM dysfunction and distant organ injury further demonstrates the importance of the sympathetic nervous system and its role in potentiating end organ dysfunction after severe trauma.

Transfusion begets anemia: the effect of aged blood on hematopoiesis.

BACKGROUND Following trauma, transfusion of aged stored blood is often necessary yet associated with increased morbidity and mortality. Despite blood replacement, many patients have a prolonged anemia requiring further transfusions. The effects of aged blood on bone marrow (BM) hematopoiesis have not been studied, and we hypothesized that stored blood suppresses BM function. METHODS Blood from Sprague-Dawley rats was stored for 1, 14, or 28 days with the industry preservative citrate-phosphate-dextrose-adenine-1 (CPDA-1). For in vitro studies, 5% supernatant was incubated with normal rat BM and cultured for erythroid (CFU-E) and granulocyte-macrophage (CFU-GM) colony-forming units. Data were compared with cultures of BM alone, 5% control plasma (negative control), and 12% CPDA-1. For in vivo studies, rats were transfused with stored supernatants (5% estimated blood volume (EBV) over 30 minutes). BM from each recipient was cultured for CFU-E and CFU-GM at 3 hours after transfusion. Data were compared with cultures of BM alone. Difference between groups determined by analysis of variance and Tukey’s multiple comparison test. RESULTS In vitro exposure to CPDA-1, control plasma, or 1-day supernatant (D1) had no effect on BM growth compared with BM alone. In vitro exposure to 14-day (D14) and 28-day (D28) supernatant significantly suppressed CFU-E by 60% and CFU-GM growth by 71% (both p < 0.05) compared with D1 or medial alone. There were no differences between D14 and D28. In vivo exposure to D14 reduced BM CFU-E and CFU-GM growth by 55% (both p < 0.05) compared with D1 supernatant. CONCLUSION Plasma from aged blood adversely affects CFU-E and CFU-GM growth in rats. The effect is not mediated by CPDA-1. Transfusion of aged stored blood may contribute to BM dysfunction in critically ill patients, resulting in persistent anemia and the need for further transfusion. This BM dysfunction may also partly explain the observed increased susceptibility to infection.