Pampee P. Young

Damage control hematology: The impact of a trauma exsanguination protocol on survival and blood product utilization

BACKGROUND The importance of early and aggressive management of trauma- related coagulopathy remains poorly understood. We hypothesized that a trauma exsanguination protocol (TEP) that systematically provides specified numbers and types of blood components immediately upon initiation of resuscitation would improve survival and reduce overall blood product consumption among the most severely injured patients. METHODS We recently implemented a TEP, which involves the immediate and continued release of blood products from the blood bank in a predefined ratio of 10 units of packed red blood cells (PRBC) to 4 units of fresh frozen plasma to 2 units of platelets. All TEP activations from February 1, 2006 to July 31, 2007 were retrospectively evaluated. A comparison cohort (pre-TEP) was selected from all trauma admissions between August 1, 2004 and January 31, 2006 that (1) underwent immediate surgery by the trauma team and (2) received greater than 10 units of PRBC in the first 24 hours. Multivariable analysis was performed to compare mortality and overall blood product consumption between the two groups. RESULTS Two hundred eleven patients met inclusion criteria (117 pre-TEP, 94 TEP). Age, sex, and Injury Severity Score were similar between the groups, whereas physiologic severity (by weighted Revised Trauma Score) and predicted survival (by trauma-related Injury Severity Score, TRISS) were worse in the TEP group (p values of 0.037 and 0.028, respectively). After controlling for age, sex, mechanism of injury, TRISS and 24-hour blood product usage, there was a 74% reduction in the odds of mortality among patients in the TEP group (p = 0.001). Overall blood product consumption adjusted for age, sex, mechanism of injury, and TRISS was also significantly reduced in the TEP group (p = 0.015). CONCLUSIONS We have demonstrated that an exsanguination protocol, delivered in an aggressive and predefined manner, significantly reduces the odds of mortality as well as overall blood product consumption.

VEGF and PlGF promote adult vasculogenesis by enhancing EPC recruitment and vessel formation at the site of tumor neovascularization

There are growing data to suggest that tissue hypoxia represents a critical force that drives adult vasculogenesis. Vascular endothelial growth factor (VEGF) expression is dramatically up‐regulated by hypoxia and results in enhanced neovascularization. Although the role of VEGF in angiogenesis has been well characterized, its role in adult vasculogenesis remains poorly understood. We used two distinct murine bone marrow transplantation (BMT) models to demonstrate that increased VEGF levels at the site of tumor growth promoted vasculogenesis in vivo. This effect of VEGF was downstream of its effect to enhance either mobilization or survival of circulating endothelial progenitor cells (EPCs). Both VEGFR1 (flt1) and VEGFR2 (flk1) are expressed on culture expanded human EPCs. Previous studies suggest that the effect of VEGF on endothelial cell migration is primarily mediated via VEGFR2; however, VEGF‐induced EPC migration in vitro was mediated by both receptors, suggesting that VEGF‐VEGFR1 interactions in EPCs are distinct from differentiated endothelial cells. We used specific blocking antibodies to these receptors to demonstrate that VEGFR1 plays an important role in human EPC recruitment to tumors. These findings were further supported by our finding that tumor‐associated placental growth factor (PlGF), a VEGFR1‐specific agonist, increased tumor vasculogenesis in a murine BMT model. We further showed that both VEGF receptors were necessary for the formation of functional vessels derived from exogenously administered human ex vivo expanded EPCs. Our data suggest local VEGF and/or PlGF expression promote vasculogenesis; VEGF plays a role in EPC recruitment and subsequent formation of functional vessels.—Li, B., Sharpe, E. E., Maupin, A., Teleron, A. A., Pyle, A., Carmeliet, P., Young, P. P. VEGF and PlGF promote adult vasculogenesis by enhancing EPC recruitment and vessel formation at the site of tumor neovascularization. FASEB J. 20, E664–E676 (2006)

Optimizing outcomes in damage control resuscitation: identifying blood product ratios associated with improved survival.

BACKGROUND Despite recent attention and impressive results with damage control resuscitation, the appropriate ratio of blood products to be transfused has yet to be defined. The purpose of this study was to evaluate whether suggested blood product ratios yield superior survival rates. MATERIALS After IRB approval, a retrospective evaluation was performed on all trauma exsanguination protocol (TEP, n = 118) activations from February 1, 2006 to July 31, 2007. A comparison cohort (pre-TEP, n = 140) was selected from all trauma admissions between August 1, 2004 and January 31, 2006 that (1) underwent immediate surgery by the trauma team and (2) received greater than 10 units of PRBC in the first 24 hours. We then compared those who received FFP:RBC (2:3) and platelet:RBC (1:5) ratios with those who did not reach these ratios. Multivariate analysis was performed for independent predictors of mortality. RESULTS A total of 259 patients were available for study. Patients receiving FFP:RBC at a ratio of 2:3 or greater (n = 64) had a significant reduction in 30-day mortality compared with those who received less than a 2:3 ratio (n = 195); 41% versus 62%, p = 0.008. Patients receiving platelets:RBC at a ratio of 1:5 or greater (n = 63) had a lower 30-day mortality when compared with those with who received less than this ratio (n = 196); (38% vs. 61%, p = 0.001). Regression model demonstrated that a ratio of FFP to PRBC is an independent predictor of 30-day mortality, controlling for age and TRISS (OR 1.78, 95% CI 1.01-3.14). CONCLUSIONS Increased FFP:PRBC and PLT:PRBC ratios during a period of massive transfusion improved survival after major trauma. Massive transfusion protocols should be designed to achieve these ratios to provide maximal benefit.

Predefined massive transfusion protocols are associated with a reduction in organ failure and postinjury complications.

INTRODUCTION Massive transfusion (MT) protocols have been shown to improve survival in severely injured patients. However, others have noted that these higher fresh frozen plasma (FFP):red blood cell (RBC) ratios are associated with increased risk of organ failure. The purpose of this study was to determine whether MT protocols are associated with increased organ failure and complications. METHODS Our institutions exsanguination protocol (TEP) involves the immediate delivery of products in a 3:2 ratio of RBC:FFP and 5:1 for RBC:platelets. All patients receiving TEP between February 2006 and January 2008 were compared with a cohort (pre-TEP) of all patients from February 2004 to January 2006 that (1) went immediately to the operating room and (2) received MT (>or=10 units of RBC in first 24 hours). RESULTS Two hundred sixty-four patients met inclusion (125 in the TEP group, 141 in the pre-TEP). Demographics and Injury Severity Score were similar. TEP received more intraoperative FFP and platelets but less in first 24 hours (p < 0.01). There was no difference in renal failure or systemic inflammatory response syndrome, but pneumonia, pulmonary failure, open abdomens, and abdominal compartment syndrome were lower in TEP. In addition, severe sepsis or septic shock and multiorgan failure were both lower in the TEP patients (9% vs. 20%, p = 0.011 and 16% vs. 37%, p < 0.001, respectively). CONCLUSIONS Although MT has been associated with higher organ failure and complication rates, this risk appears to be reduced when blood products are delivered early in the resuscitation through a predefined protocol. Our institutions TEP was associated with a reduction in multiorgan failure and infectious complications, as well as an increase in ventilator-free days. In addition, implementation of this protocol was followed by a dramatic reduction in development of abdominal compartment syndrome and the incidence of open abdomens.

Effects of Red-Cell Storage Duration on Patients Undergoing Cardiac Surgery

BACKGROUND Some observational studies have reported that transfusion of red-cell units that have been stored for more than 2 to 3 weeks is associated with serious, even fatal, adverse events. Patients undergoing cardiac surgery may be especially vulnerable to the adverse effects of transfusion. METHODS We conducted a randomized trial at multiple sites from 2010 to 2014. Participants 12 years of age or older who were undergoing complex cardiac surgery and were likely to undergo transfusion of red cells were randomly assigned to receive leukocyte-reduced red cells stored for 10 days or less (shorter-term storage group) or for 21 days or more (longer-term storage group) for all intraoperative and postoperative transfusions. The primary outcome was the change in Multiple Organ Dysfunction Score (MODS; range, 0 to 24, with higher scores indicating more severe organ dysfunction) from the preoperative score to the highest composite score through day 7 or the time of death or discharge. RESULTS The median storage time of red-cell units provided to the 1098 participants who received red-cell transfusion was 7 days in the shorter-term storage group and 28 days in the longer-term storage group. The mean change in MODS was an increase of 8.5 and 8.7 points, respectively (95% confidence interval for the difference, -0.6 to 0.3; P=0.44). The 7-day mortality was 2.8% in the shorter-term storage group and 2.0% in the longer-term storage group (P=0.43); 28-day mortality was 4.4% and 5.3%, respectively (P=0.57). Adverse events did not differ significantly between groups except that hyperbilirubinemia was more common in the longer-term storage group. CONCLUSIONS The duration of red-cell storage was not associated with significant differences in the change in MODS. We did not find that the transfusion of red cells stored for 10 days or less was superior to the transfusion of red cells stored for 21 days or more among patients 12 years of age or older who were undergoing complex cardiac surgery. (Funded by the National Heart, Lung, and Blood Institute; RECESS ClinicalTrials.gov number, NCT00991341.).

The Wnt modulator sFRP2 enhances mesenchymal stem cell engraftment, granulation tissue formation and myocardial repair.

Cell-based therapies, using multipotent mesenchymal stem cells (MSCs) for organ regeneration, are being pursued for cardiac disease, orthopedic injuries and biomaterial fabrication. The molecular pathways that regulate MSC-mediated regeneration or enhance their therapeutic efficacy are, however, poorly understood. We compared MSCs isolated from MRL/MpJ mice, known to demonstrate enhanced regenerative capacity, to those from C57BL/6 (WT) mice. Compared with WT-MSCs, MRL-MSCs demonstrated increased proliferation, in vivo engraftment, experimental granulation tissue reconstitution, and tissue vascularity in a murine model of repair stimulation. The MRL-MSCs also reduced infarct size and improved function in a murine myocardial infarct model compared with WT-MSCs. Genomic and functional analysis indicated a downregulation of the canonical Wnt pathway in MRL-MSCs characterized by significant up-regulation of specific secreted frizzled-related proteins (sFRPs). Specific knockdown of sFRP2 by shRNA in MRL-MSCs decreased their proliferation and their engraftment in and the vascular density of MRL-MSC-generated experimental granulation tissue. These results led us to generate WT-MSCs overexpressing sFRP2 (sFRP2-MSCs) by retroviral transduction. sFRP2-MSCs maintained their ability for multilineage differentiation in vitro and, when implanted in vivo, recapitulated the MRL phenotype. Peri-infarct intramyocardial injection of sFRP2-MSCs resulted in enhanced engraftment, vascular density, reduced infarct size, and increased cardiac function after myocardial injury in mice. These findings implicate sFRP2 as a key molecule for the biogenesis of a superior regenerative phenotype in MSCs.

VEGF increases engraftment of bone marrow-derived endothelial progenitor cells (EPCs) into vasculature of newborn murine recipients

Recent evidence suggests that bone marrow-derived angioblasts or endothelial progenitor cells circulate in peripheral blood and can incorporate at sites of pathologic neovascularization or during the ovarian cycle. However, the incorporation of endothelial progenitor cells into vessels of nonischemic tissues in adult animals has not been observed. We hypothesized that the vascular microenvironment differs between newborn and adult animals, and that donor endothelial cell progenitors would engraft in rapidly growing normal tissues during the neonatal period. After nonablative administration of bone marrow cells either at birth or at 4 weeks of age, donor-derived endothelial cells were found only in the neovasculature of the newborn recipients. Both the incorporation of donor endothelial cells into the newborn neovasculature as well as tissue vascularity were significantly increased by coadministering vascular endothelial growth factor with bone marrow cells. These findings suggest that bone marrow-derived endothelial progenitor cells can contribute to neovascularization during the newborn period and are responsive to vascular endothelial growth factor.

Pyrvinium, a Potent Small Molecule Wnt Inhibitor, Promotes Wound Repair and Post-MI Cardiac Remodeling

Wnt signaling plays an important role in developmental and stem cell biology. To test the hypothesis that temporary inhibition of Wnt signaling will enhance granulation tissue and promote angiogenesis in tissue repair, we employed a recently characterized small molecule Wnt inhibitor. Pyrvinium is an FDA-approved drug that we identified as a Wnt inhibitor in a chemical screen for small molecules that stabilize β-catenin and inhibit Axin degradation. Our subsequent characterization of pyrvinium has revealed that its critical cellular target in the Wnt pathway is Casein Kinase 1α. Daily administration of pyrvinium directly into polyvinyl alcohol (PVA) sponges implanted subcutaneously in mice generated better organized and vascularized granulation tissue; this compound also increased the proliferative index of the tissue within the sponges. To evaluate its effect in myocardial repair, we induced a myocardial infarction (MI) by coronary artery ligation and administered a single intramyocardial dose of pyrvinium. Mice were evaluated by echocardiography at 7 and 30 days post-MI and treatment; post mortem hearts were evaluated by histology at 30 days. Pyrvinium reduced adverse cardiac remodeling demonstrated by decreased left ventricular internal diameter in diastole (LVIDD) as compared to a control compound. Increased Ki-67+ cells were observed in peri-infarct and distal myocardium of pyrvinium-treated animals. These results need to be further followed-up to determine if therapeutic inhibition of canonical Wnt may avert adverse remodeling after ischemic injury and its impact on myocardial repair and regeneration.

Massive transfusion protocols for patients with substantial hemorrhage.

Transfusion medicine for the resuscitation of patients with massive hemorrhage has recently advanced from reactive, supportive treatment with crystalloid and red blood cell therapy to use of standardized massive transfusion protocols (MTPs). Through MTPs, medical facilities are able to standardize the most effective posthemorrhage treatments and execute them rapidly while reducing potential waste of blood products. Damage control resuscitation is an example of an MTP, where patients are (1) allowed more permissive hypotension, (2) spared large volumes of crystalloid/colloid therapy (through low volume resuscitation), and (3) transfused with blood products preemptively using a balanced ratio of plasma and platelets to red blood cells. This focused approach improves the timely availability of blood components during resuscitation. However, the use of MTPs remains controversial. This review describes published experiences with MTPs and illustrates the potential value of several MTPs currently utilized by academic transfusion services.

sFRP2 suppression of bone morphogenic protein (BMP) and Wnt signaling mediates mesenchymal stem cell (MSC) self-renewal promoting engraftment and myocardial repair.

Transplantation of mesenchymal stem cells (MSCs) is a promising therapy for ischemic injury; however, inadequate survival of implanted cells in host tissue is a substantial impediment in the progress of cellular therapy. Secreted Frizzled-related protein 2 (sFRP2) has recently been highlighted as a key mediator of MSC-driven myocardial and wound repair. Notably, sFRP2 mediates significant enhancement of MSC engraftment in vivo. We hypothesized that sFRP2 improves MSC engraftment by modulating self-renewal through increasing stem cell survival and by inhibiting differentiation. In previous studies we demonstrated that sFRP2-expressing MSCs exhibited an increased proliferation rate. In the current study, we show that sFRP2 also decreased MSC apoptosis and inhibited both osteogenic and chondrogenic lineage commitment. sFRP2 activity occurred through the inhibition of both Wnt and bone morphogenic protein (BMP) signaling pathways. sFRP2-mediated inhibition of BMP signaling, as assessed by levels of pSMAD 1/5/8, was independent of its effects on the Wnt pathway. We further hypothesized that sFRP2 inhibition of MSC lineage commitment may reduce heterotopic osteogenic differentiation within the injured myocardium, a reported adverse side effect. Indeed, we found that sFRP2-MSC-treated hearts and wound tissue had less ectopic calcification. This work provides important new insight into the mechanisms by which sFRP2 increases MSC self-renewal leading to superior tissue engraftment and enhanced wound healing.