Phillip A. Letourneau

Bone marrow derived mesenchymal stem cells inhibit inflammation and preserve vascular endothelial integrity in the lungs after hemorrhagic shock.

Hemorrhagic shock (HS) and trauma is currently the leading cause of death in young adults worldwide. Morbidity and mortality after HS and trauma is often the result of multi-organ failure such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), conditions with few therapeutic options. Bone marrow derived mesenchymal stem cells (MSCs) are a multipotent stem cell population that has shown therapeutic promise in numerous pre-clinical and clinical models of disease. In this paper, in vitro studies with pulmonary endothelial cells (PECs) reveal that conditioned media (CM) from MSCs and MSC-PEC co-cultures inhibits PEC permeability by preserving adherens junctions (VE-cadherin and β-catenin). Leukocyte adhesion and adhesion molecule expression (VCAM-1 and ICAM-1) are inhibited in PECs treated with CM from MSC-PEC co-cultures. Further support for the modulatory effects of MSCs on pulmonary endothelial function and inflammation is demonstrated in our in vivo studies on HS in the rat. In a rat “fixed volume” model of mild HS, we show that MSCs administered IV potently inhibit systemic levels of inflammatory cytokines and chemokines in the serum of treated animals. In vivo MSCs also inhibit pulmonary endothelial permeability and lung edema with concurrent preservation of the vascular endothelial barrier proteins: VE-cadherin, Claudin-1, and Occludin-1. Leukocyte infiltrates (CD68 and MPO positive cells) are also decreased in lungs with MSC treatment. Taken together, these data suggest that MSCs, acting directly and through soluble factors, are potent stabilizers of the vascular endothelium and inflammation. These data are the first to demonstrate the therapeutic potential of MSCs in HS and have implications for the potential use of MSCs as a cellular therapy in HS-induced lung injury.

Mesenchymal Stem Cells Regulate Blood-Brain Barrier Integrity Through TIMP3 Release After Traumatic Brain Injury

The matrix metalloproteinase inhibitor TIMP3 mediates the beneficial effects of mesenchymal stem cells on the blood-brain barrier of the injured mouse brain. Mesenchymal Stem Cells Spill Their Secrets Traumatic brain injury (TBI) is the leading cause of death and disability in children and young adults worldwide and is considered a “silent epidemic” in the United States in both civilian and military populations. Pathological cerebral edema and blood-brain barrier (BBB) permeability are the leading causes of death acutely after TBI with very few therapeutic options. It has been established in animal models that intravenously administered adult bone marrow–derived mesenchymal stem cells (MSCs) are able to ameliorate BBB permeability in mice after TBI. In new work, Menge et al. identify the mechanism responsible for this beneficial effect and identify the mediator as a soluble factor produced by MSCs called TIMP3. In a mouse model of TBI, Menge et al. show that down-regulation of TIMP3 expression in intravenously administered human MSCs abrogates their protective effects on the BBB and endothelial cell stability after TBI. Furthermore, the authors demonstrate that administering intravenous recombinant human TIMP3 alone to mice after TBI can fully recapitulate the protective effects of MSCs on vascular stability and BBB integrity, indicating that TIMP3 may be a key factor regulating integrity of the BBB. Although much more work needs to be done, TIMP3 could be a useful cell-free therapeutic for treating the breakdown of BBB integrity and cerebral edema that occurs after TBI. Mesenchymal stem cells (MSCs) may be useful for treating a variety of disease states associated with vascular instability including traumatic brain injury (TBI). A soluble factor, tissue inhibitor of matrix metalloproteinase-3 (TIMP3), produced by MSCs is shown to recapitulate the beneficial effects of MSCs on endothelial function and to ameliorate the effects of a compromised blood-brain barrier (BBB) due to TBI. Intravenous administration of recombinant TIMP3 inhibited BBB permeability caused by TBI, whereas attenuation of TIMP3 expression in intravenously administered MSCs blocked the beneficial effects of the MSCs on BBB permeability and stability. MSCs increased circulating concentrations of soluble TIMP3, which blocked vascular endothelial growth factor-A–induced breakdown of endothelial cell adherens junctions in vitro and in vivo. These findings elucidate a potential molecular mechanism for the beneficial effects of MSCs on the BBB after TBI and demonstrate a role for TIMP3 in the regulation of BBB integrity.

Multiple Levels of Degradation Diminish Hemostatic Potential of Thawed Plasma

BACKGROUND Severe bleeding after injury requires transfusion of blood products, including fresh frozen plasma (FFP). Many centers are keeping thawed plasma (TP) ready for massively transfused patients. According to the American Association of Blood Banks Standards, TP is approved for transfusion up to 5 days after thawing, when stored at 1°C to 6°C. However, there are no clinical data analyzing the effects of the approved 5-day storage on plasma. We hypothesize that the hemostatic potential (HP) of freshly thawed (FFP-0) was superior to plasma stored for 5 days (FFP-5). METHODS FFP from 30 single donors were thawed at 37°C and kept at 1°C to 6°C for 5 days. HP was evaluated at day 0 and 5 by measuring kinetics of thrombin generation (TG), kinetics of clot formation by thromboelastography, clotting factors and inhibitors, and cell-derived microparticles (MPs) by flow cytometry. RESULTS When comparing FFP-5 to FFP-0, FFP-5 exhibited only 40% of the potential of FFP-0 for TG (6.2 nM/min vs. 14.3 nM/min, p<0.0001), a slower clotting response via thromboelastography (reaction time: 4.3 minutes vs. 3.2 minutes, p<0.0001) and a longer delay in reaching maximum thrombus generation (5.7 minutes vs. 4.6 minutes, p<0.01). Diminished HP was accompanied by a significant decline in multiple coagulation proteins, including FV, VII, VIII, von Willebrand factor, and free Protein S, by up to 30%, and a decrease of 50% in MP counts. CONCLUSION The HP and clot forming ability of TP significantly declined with storage. Hence, freshly TP may have a greater ability to restore hemostasis and correct coagulopathy compared with FFP-5. The clinical consequences for transfused patients deserve further exploration.

Location of Pulmonary Metastasis in Pediatric Osteosarcoma is Predictive of Outcome

BACKGROUND The 3-year survival after pulmonary metastasectomy for osteosarcoma (OS) is approximately 30%. Resection of metastatic disease can prolong life in pediatric patients with OS. Our objective is to assess the outcome of pediatric patients with pulmonary metastases located centrally as compared with peripheral lesions. METHODS A retrospective review of patients 0 to 21 years old with a diagnosis of OS with pulmonary metastases on computed tomographic scan between 1985 and 2000 was completed. Demographics, metastasis location, survival, morbidity, and mortality were evaluated. RESULTS Of 115 patients who had pulmonary metastasis secondary to OS, there were 96 wedge resections and 13 lobectomy/pneumonectomies in 84 patients. The morbidity of wedge resection was 9% and lobectomy/pneumonectomy was 8%. There were no deaths from surgery. The median survival for patients undergoing lobectomy compared with wedge resection was 0.61 and 1.14 years, respectively, but did not reach statistical significance. The median overall survival for the entire cohort was 0.75 years. The median overall survival after initial detection of metastatic disease was 1.06 years among the patients with peripheral disease, compared with 0.38 years in patients with central disease (P = .008). CONCLUSION Patients with central pulmonary metastases in OS have a very poor prognosis, even after operative treatment, compared with those with peripheral disease. Patients with central lesions may benefit from other nonsurgical treatment options.

Resection of pulmonary metastases in pediatric patients with Ewing sarcoma improves survival

BACKGROUND Ewing sarcoma (ES) is the second most common bone tumor in children, and survival of those with metastatic ES has not improved. Previous studies have shown a survival benefit to whole lung irradiation in patients with pulmonary metastases and may be given either before, after, or instead of surgical pulmonary metastasectomy (PM). The contribution of surgery compared with irradiation in ES has not previously been studied. METHODS A retrospective review of patients younger than 21 years (median age, 16 years) treated at a single institution (1990-2006) was performed. Kaplan-Meier survival curves were compared using log-rank test and a multivariate Cox proportional hazards model. P ≤ .05 was regarded as significant. RESULTS Eighty patients with ES were identified. Of these, 31 (39%) had pulmonary metastases. Nine patients had incomplete details of their full treatment regimen, but the following groups could be defined from the remainder: resection alone (n = 5), radiation alone (n = 3), radiation and resection (n = 3), or chemotherapy alone (n = 11). There were 24 deaths overall, with a median overall survival (OS) of 2.7 (95% confidence interval [CI], 1.7-5.2) years. Patients who had PM had the best OS (80%), whereas those who underwent radiation to the lung without PM compared with chemotherapy only for pulmonary metastasis both had similar OS of 0% at 5 years (P = .002). Patients who had radiation followed by PM for lung metastasis had a 5-year OS of 65%. Patients with PM had a longer OS compared with those without lung resection (P < .0001). CONCLUSION These data suggest a possible benefit for ES patients who undergo surgical resection of lung metastases.

Strategies for modulating the inflammatory response after decompression from abdominal compartment syndrome

BackgroundManagement of the open abdomen is an increasingly common part of surgical practice. The purpose of this review is to examine the scientific background for the use of temporary abdominal closure (TAC) in the open abdomen as a way to modulate the local and systemic inflammatory response, with an emphasis on decompression after abdominal compartment syndrome (ACS).MethodsA review of the relevant English language literature was conducted. Priority was placed on articles published within the last 5 years.Results/ConclusionRecent data from our group and others have begun to lay the foundation for the concept of TAC as a method to modulate the local and/or systemic inflammatory response in patients with an open abdomen resulting from ACS.

Aged plasma transfusion increases mortality in a rat model of uncontrolled hemorrhage.

INTRODUCTION Recent data have associated improved survival after hemorrhagic shock with the early use of plasma-based resuscitation. Our laboratory has shown that FFP5 has decreased hemostatic potential compared with freshly thawed plasma (FFP0). We hypothesized that FFP5 would increase bleeding and mortality compared with FFP0 in a rodent bioassay model of uncontrolled liver hemorrhage. METHODS Hemostatic potential of plasma was assessed with the Calibrated Automated Thrombogram (CAT) assay. Rats underwent isovolemic hemodilution by 15% of blood volume with the two human plasma groups (FFP0 and FFP5) and two controls (sham and lactated Ringers). A liver injury was created by excising a portion of liver resulting in uncontrolled hemorrhage. Rats that lived for 30 minutes after liver injury were resuscitated to their baseline blood pressure and followed for 6 hours. Hemostasis was assessed by thromboelastography. RESULTS Hemostatic potential of FFP5 decreased significantly in all areas measured in the CAT assay as compared with FFP0 (p < 0.01). In the FFP5 group, overall survival was 54%, compared with 100% in the FFP0 and sham group (p = 0.03). For animals that survived 30 minutes and were resuscitated, there was no difference in bleeding and/or coagulopathy between groups. Irrespective of treatment, animals that died after resuscitation demonstrated increased intraperitoneal fluid volume (14.85 mL ± 1.9 mL vs. 7.02 mL ± 0.3 mL, p < 0.001). CONCLUSION In this model of mild preinjury hemodilution with plasma, rats that received FFP5 had decreased survival after uncontrolled hemorrhage from hepatic injury. There were no differences in coagulation function or intraperitoneal fluid volume between the two plasma groups.

Human Bone Marrow Derived Mesenchymal Stem Cells Regulate Leukocyte-Endothelial Interactions and Activation of Transcription Factor NF-Kappa B.

Bone marrow derived mesenchymal stem cells (MSCs) have been shown to demonstrate benefit in multiple disease models characterized by inflammation such as sepsis and acute lung injury. Mechanistically we hypothesized that MSCs exhibit these properties through inhibition of leukocyte activation and modulation of leukocyte-endothelial interactions; key interlinked processes involved in the deleterious effects of injury and inflammation. In this paper we found that MSCs co-cultured with a monocytoid line, U937, inhibit U937 binding to pulmonary endothelial cells (PECs) stimulated with the inflammatory cytokine TNFα. Furthermore, we show that these effects on functional adhesion are not due to changes in inflammatory adhesion molecule expression on U937s. No changes were found in CD62L, CD29, CD11b and CD18 expression on U937s co-cultured with MSCs. To determine if the effects of MSCs on leukocyte-endothelial interactions are due to the effects of MSCs on leukocyte activation, we investigated whether MSCs affect functional activation of the transcription factor NF-Kappa B. We found that MSCs significantly inhibit transcriptional activation of NF-kappa B in U937s. We also found that MSCs inhibit DNA binding of NF-kappa B subunits p50 and p65 to putative NF-kappa B DNA binding sites. Concomitant with a decrease in NF-kappa B activation was a significant increase in IL-10, an anti-inflammatory cytokine known to inhibit activation of NF-kappa B. Taken together, these findings show that MSCs have potent effects on leukocyte-endothelial interactions which may be due to the direct effects of MSCs on IL-10 and NF-kB. These findings suggest a potential therapeutic role for MSCs in diseases characterized by inflammation such as acute lung injury or multi-organ failure induced by traumatic injury.

Biphasic Regulation of Myosin Light Chain Phosphorylation by p21-activated Kinase Modulates Intestinal Smooth Muscle Contractility

Background: Myosin light chain (MLC) phosphorylation is the driving force of cell contractility. Results: p21-activated kinase (PAK) can either activate or inhibit myosin light chain phosphorylation, depending on the level of PAK activation. Conclusion: Inhibition of intestinal motility by supraphysiological mechanical stretch is attributable to increased PAK activity. Significance: The biphasic regulation of PAK may provide an explanation for the conflicting observations on PAK-mediated MLC phosphorylation. Supraphysiological mechanical stretching in smooth muscle results in decreased contractile activity. However, the mechanism is unclear. Previous studies indicated that intestinal motility dysfunction after edema development is associated with increased smooth muscle stress and decreased myosin light chain (MLC) phosphorylation in vivo, providing an ideal model for studying mechanical stress-mediated decrease in smooth muscle contraction. Primary human intestinal smooth muscle cells (hISMCs) were subjected to either control cyclical stretch (CCS) or edema (increasing) cyclical stretch (ECS), mimicking the biophysical forces in non-edematous and edematous intestinal smooth muscle in vivo. ECS induced significant decreases in phosphorylation of MLC and MLC phosphatase targeting subunit (MYPT1) and a significant increase in p21-activated kinase (PAK) activity compared with CCS. PAK regulated MLC phosphorylation in an activity-dependent biphasic manner. PAK activation increased MLC and MYPT1 phosphorylation in CCS but decreased MLC and MYPT1 phosphorylation in hISMCs subjected to ECS. PAK inhibition had the opposite results. siRNA studies showed that PAK1 plays a critical role in regulating MLC phosphorylation in hISMCs. PAK1 enhanced MLC phosphorylation via phosphorylating MYPT1 on Thr-696, whereas PAK1 inhibited MLC phosphorylation via decreasing MYPT1 on both Thr-696 and Thr-853. Importantly, in vivo data indicated that PAK activity increased in edematous tissue, and inhibition of PAK in edematous intestine improved intestinal motility. We conclude that PAK1 positively regulates MLC phosphorylation in intestinal smooth muscle through increasing inhibitory phosphorylation of MYPT1 under physiologic conditions, whereas PAK1 negatively regulates MLC phosphorylation via inhibiting MYPT1 phosphorylation when PAK activity is increased under pathologic conditions.

Fresh frozen plasma increases adhesion molecule expression on human pulmonary endothelial cells.

BACKGROUND Current blood banking practices allow fresh frozen plasma (FFP) to be thawed and then stored for 5 d between 1 and 6 °C. We hypothesized that aged plasma (d 5 FFP) would be pro-inflammatory to the endothelium compared with fresh plasma (d 0 FFP). MATERIALS AND METHODS Human pulmonary endothelial cells (PECs) were treated with (1) media, (2) media + lipopolysaccharide (LPS), (3) lactated Ringers (LR), (4) LR + LPS, (5) d 0 FFP, (6) d 0 FFP + LPS, (7) d 5 FFP, and (8) d 5 FFP + LPS. After a 24 h incubation, the PECs were stained with antibodies for I-CAM, V-CAM, P-selectin, and E-selectin. The cells were subsequently analyzed by flow cytometry. RESULTS In both PEC groups treated with FFP and stimulated with LPS, I-CAM, V-CAM, P-selectin, and E-selectin were significantly up-regulated compared with LR when stimulated by LPS. CONCLUSION FFP at both ages significantly increased expression of four different adhesion molecules compared with LR in PECs. This may represent a possible mechanism for increased leukocyte binding on the endothelium as a result of FFP transfusion.