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.

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.

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.

ß-blockade protection of bone marrow following trauma: the role of G-CSF.

BACKGROUND: Following severe trauma, there is a profound elevation of catecholamine that is associated with a persistent anemic state. We have previously shown that ß-blockade (ßB) prevents erythroid growth suppression and decreases hematopoietic progenitor cell (HPC) mobilization following injury. Under normal conditions, granulocyte colony stimulating factor (G-CSF) triggers the activation of matrix metalloprotease-9 (MMP-9), leading to the egress of progenitor cells from the bone marrow (BM). When sustained, this depletion of BM cellularity may contribute to BM failure. This study seeks to determine if G-CSF plays a role in the ßB protection of BM following trauma. METHODS: Male Sprague-Dawley rats were subjected to either unilateral lung contusion (LC) ± ßB, hemorrhagic shock (HS) ± ßB, or both LC/HS ± ßB. Propranolol (ßB) was given immediately following resuscitation. Animals were sacrificed at 3 and 24 h and HPC mobilization was assessed by evaluating BM cellularity and flow cytometric analysis of peripheral blood for HPCs. The concentration of G-CSF and MMP-9 was measured in plasma by ELISA. RESULTS: BM cellularity is decreased at 3 h following LC, HS, and LC/HS. HS and LC/HS resulted in significant HPC mobilization in the peripheral blood. The addition of ßB restored BM cellularity and reduced HPC mobilization. Three h following HS and LC/HS, plasma G-CSF levels more than double, however LC alone showed no change in G-CSF. ßB significantly decreased G-CSF in both HS and LC/HS. Similarly, MMP-9 is elevated following LC/HS, and ßB prevents this elevation (390 ± 100 pg/mL versus 275 ± 80 pg/mL). CONCLUSION: ßB protection of the BM following shock and injury may be due to reduced HPC mobilization and maintenance of BM cellularity. Following shock, there is an increase in plasma G-CSF and MMP-9, which is abrogated by ßB and suggests a possible mechanism how ßB decreases HPC mobilization thus preserving BM cellularity. In contrast, ßB protection of BM following LC is not mediated by G-CSF. Therefore, the mechanism of progenitor cell mobilization from the BM is dependent on the type of injury.

Hematopoietic progenitor cell mobilization is mediated through beta-2 and beta-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.