Daniel J. Watkins

Heparin-Binding Epidermal Growth Factor-Like Growth Factor and Mesenchymal Stem Cells Act Synergistically to Prevent Experimental Necrotizing Enterocolitis

BACKGROUND We have shown that administration of heparin-binding EGF (epidermal growth factor)-like growth factor (HB-EGF) protects the intestines from experimental necrotizing enterocolitis (NEC). We have also demonstrated that systemically administered mesenchymal stem cells (MSC) can engraft into injured intestines. This study investigated the effects of HB-EGF on MSC in vitro, and whether MSC and HB-EGF can act synergistically to prevent NEC in vivo. STUDY DESIGN In vitro, the effect of HB-EGF on MSC proliferation, migration, and apoptosis was determined. In vivo, rat pups received MSC either intraperitoneally (IP) or intravenously (IV). Pups were assigned to 1 of 7 groups: Group 1, breast-fed; Group 2, experimental NEC; Group 3, NEC+HB-EGF; Group 4, NEC+MSC IP; Group 5, NEC+HB-EGF+MSC IP; Group 6, NEC+MSC IV; or Group 7, NEC+HB-EGF+MSC IV. Mesechymal stem cell engraftment, histologic injury, intestinal permeability, and mortality were determined. RESULTS Heparin-binding EGF-like growth factor promoted MSC proliferation and migration, and decreased MSC apoptosis in vitro. In vivo, MSC administered IV had increased engraftment into NEC-injured intestine compared with MSC administered IP (p < 0.05). Heparin binding EGF-like growth factor increased engraftment of IP-administered MSC (p < 0.01) and IV-administered MSC (p < 0.05). Pups in Groups 3 to 7 had a decreased incidence of NEC compared with nontreated pups (Group 2), with the lowest incidence in pups treated with HB-EGF+MSC IV (p < 0.01). Pups in Group 7 had a significantly decreased incidence of intestinal dilation and perforation, and had the lowest intestinal permeability, compared with other treatment groups (p < 0.01). Pups in all experimental groups had significantly improved survival compared with pups exposed to NEC, with the best survival in Group 7 (p < 0.05). CONCLUSIONS Heparin-binding EGF-like growth factor and MSC act synergistically to reduce injury and improve survival in experimental NEC.

The role of the intestinal microcirculation in necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) continues to be a devastating inflammatory disease of the newborn intestine. Despite advances in management, morbidity and mortality remain high. While it is clear that intestinal ischemia plays a large role in disease pathogenesis, attempts to link NEC to intestinal macrovascular derangement have been largely unsuccessful. More recently, there has been a concerted effort to characterize the pathologic changes of the intestinal microcirculation in response to intestinal injury, including NEC. This microcirculatory regulation is controlled by a balance of vasoconstrictor and vasodilator forces. Vasoconstriction is mediated primarily by endothelin-1 (ET-1), while vasodilation is mediated primarily by nitric oxide (NO). These chemical mediators have been implicated in many aspects of intestinal ischemic injury and NEC, with the balance shifting toward increased vasoconstriction associated with intestinal injury. With a proper understanding of these antagonistic forces, potential therapeutic avenues may result from improving this pathologic microcirculatory dysregulation.

Enteric nervous system abnormalities are present in human necrotizing enterocolitis: potential neurotransplantation therapy

IntroductionIntestinal dysmotility following human necrotizing enterocolitis suggests that the enteric nervous system is injured during the disease. We examined human intestinal specimens to characterize the enteric nervous system injury that occurs in necrotizing enterocolitis, and then used an animal model of experimental necrotizing enterocolitis to determine whether transplantation of neural stem cells can protect the enteric nervous system from injury.MethodsHuman intestinal specimens resected from patients with necrotizing enterocolitis (n = 18), from control patients with bowel atresia (n = 8), and from necrotizing enterocolitis and control patients undergoing stoma closure several months later (n = 14 and n = 6 respectively) were subjected to histologic examination, immunohistochemistry, and real-time reverse-transcription polymerase chain reaction to examine the myenteric plexus structure and neurotransmitter expression. In addition, experimental necrotizing enterocolitis was induced in newborn rat pups and neurotransplantation was performed by administration of fluorescently labeled neural stem cells, with subsequent visualization of transplanted cells and determination of intestinal integrity and intestinal motility.ResultsThere was significant enteric nervous system damage with increased enteric nervous system apoptosis, and decreased neuronal nitric oxide synthase expression in myenteric ganglia from human intestine resected for necrotizing enterocolitis compared with control intestine. Structural and functional abnormalities persisted months later at the time of stoma closure. Similar abnormalities were identified in rat pups exposed to experimental necrotizing enterocolitis. Pups receiving neural stem cell transplantation had improved enteric nervous system and intestinal integrity, differentiation of transplanted neural stem cells into functional neurons, significantly improved intestinal transit, and significantly decreased mortality compared with control pups.ConclusionsSignificant injury to the enteric nervous system occurs in both human and experimental necrotizing enterocolitis. Neural stem cell transplantation may represent a novel future therapy for patients with necrotizing enterocolitis.

Synergistic Effects of HB-EGF and Mesenchymal Stem Cells in a Murine Model of Intestinal Ischemia/Reperfusion Injury

BACKGROUND We have previously demonstrated that heparin-binding EGF-like growth factor (HB-EGF) administration protects the intestines from ischemia/reperfusion (I/R) injury in vivo. We have also shown that HB-EGF promotes mesenchymal stem cell (MSC) proliferation and migration in vitro. The goals of the current study were to examine the effects of HB-EGF and both bone marrow (BM)- and amniotic fluid (AF)-derived MSC on intestinal I/R injury in vivo. MATERIALS AND METHODS MSC were isolated from pan-EGFP mice, expanded, and purified. Pluripotency was confirmed by induced differentiation. Mice were subjected to terminal ileum I/R and received either: (1) no therapy; (2) HB-EGF; (3) BM-MSC; (4) HB-EGF+BM-MSC; (5) AF-MSC; or (6) HB-EGF+AF-MSC. MSC engraftment, histologic injury, and intestinal permeability were quantified. RESULTS There was increased MSC engraftment into injured compared to uninjured intestine for all experimental groups, with significantly increased engraftment for AF-MSC+HB-EGF compared to AF-MSC alone. Administration of HB-EGF and MSC improved intestinal histology and intestinal permeability after I/R injury. The greatest improvement was with combined administration of HB-EGF+AF-MSC. CONCLUSIONS Both HB-EGF alone and MSC alone can protect the intestines from I/R injury, with synergistic efficacy occurring when HB-EGF and AF-MSC are administered together.

Heparin-binding EGF-like growth factor (HB-EGF) protects the intestines from radiation therapy-induced intestinal injury.

PURPOSE Radiation therapy (RT) often induces enteritis by inhibiting proliferation and inducing apoptosis. Heparin-binding EGF-like growth factor (HB-EGF) has been shown to protect the intestine in several animal injury models. The objective of this study was to examine whether HB-EGF affects RT-induced intestinal injury. METHODS HB-EGF or PBS was administered intraperitoneally to mice daily for 3 days, followed by total body irradiation (TBI). Three days after TBI, intestinal segments were harvested, and BrdU immunohistochemistry was performed to identify proliferating crypts (n=25). Four days after TBI, intestinal segments were harvested and assessed for histologic injury (n=34), and FITC-dextran was administered via gavage with serum FITC-dextran levels quantified to determine gut barrier function (n=18). RESULTS Compared to non-HB-EGF-treated irradiated mice, administration of HB-EGF to irradiated mice led to a significantly increased percentage of proliferative crypts (72.6% vs. 50.5%, p=0.001), a significantly decreased percent of histologic sections with severe histologic injury (13.7% vs. 20.3%, p=0.005), and significantly reduced intestinal permeability (18.8 μg/mL vs. 22.6 μg/mL, p=0.02). CONCLUSIONS These results suggest that administration of HB-EGF protects the intestines from injury after exposure to radiation therapy. Administration of HB-EGF may represent a novel therapy for the prevention of radiation enteritis in the future.

HB-EGF augments the ability of mesenchymal stem cells to attenuate intestinal injury

BACKGROUND We have previously demonstrated that heparin-binding EGF-like growth factor (HB-EGF) and mesenchymal stem cell (MSC) administration protect the intestines from ischemia/reperfusion (I/R) injury in vivo, with amniotic fluid-derived MSC (AF-MSC) being more efficacious than bone marrow-derived MSC (BM-MSC). The goal of the current study was to determine whether the protective effects of HB-EGF were from direct effects on MSC or via alternative mechanisms. METHODS Murine MSC were transfected with an HB-EGF plasmid or control plasmid by electroporation. Mice were subjected to segmental intestinal I/R injury and received either BM-MSC or AF-MSC either with or without exogenous HB-EGF, or BM-MSC or AF-MSC that endogenously over-expressed HB-EGF. MSC engraftment, intestinal histologic injury, and intestinal permeability were quantified. RESULTS There was increased MSC engraftment into injured compared to uninjured intestine. HB-EGF increased AF-MSC engraftment into injured intestine. Administration of HB-EGF and MSC improved intestinal histology and intestinal permeability after I/R injury, with AF-MSC being most efficacious. The effect of HB-EGF on MSC was similar when the growth factor was administered exogenously, or when it was overexpressed endogenously. CONCLUSIONS The effect of HB-EGF on AF-MSC was similar with both exogenous administration and endogenous overexpression of the growth factor, implying that HB-EGF has a direct effect on AF-MSC. This information may assist in guiding potential future AF-MSC-based therapies for patients at risk of intestinal ischemic injuries.

Heparin-binding epidermal growth factor-like growth factor protects mesenchymal stem cells

BACKGROUND We have previously demonstrated that mesenchymal stem cell (MSC) administration protects the intestines from injury in a mouse model of intestinal ischemia/reperfusion injury. We have also shown that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent intestinal cytoprotective agent in vivo that can protect the intestines by way of its effects on stem cells. The goal of the present study was to examine the effects of HB-EGF on both amniotic fluid (AF)- and bone marrow (BM)-derived MSCs in vitro. METHODS MSCs were isolated from the AF and BM of pan-EGFP mice, grown in MSC-specific culture medium, and purified by sequential passages according to their adherence properties. Pluripotency was confirmed by induced differentiation. After incubation of MSCs with HB-EGF, proliferation was quantified using the CyQuant cell proliferation assay kit under normoxic and anoxic conditions. Chemotaxis was quantified using the CHEMICON QCM cell migration kit, and apoptosis was determined using caspase-3 immunohistochemistry after exposure of the MSCs to anoxic stress. RESULTS AF-MSCs and BM-MSCs showed significantly increased proliferation and migration in response to HB-EGF. HB-EGF significantly protected AF-MSCs and BM-MSCs from anoxia-induced apoptosis. The proliferative and anti-apoptotic effects of HB-EGF were even more pronounced in AF-MSCs than in BM-MSCs. CONCLUSIONS These results have demonstrated that HB-EGF acts as a mitogenic and chemotactic agent for MSCs that protects MSCs from injury. These findings could have important implications for future experiments designed to use MSCs to protect the intestines from injury.

A technique for systemic mesenchymal stem cell transplantation in newborn rat pups

ABSTRACT Mesenchymal stem cells (MSC) have the potential to aid tissue regeneration. Intravenous (IV) MSC administration is currently being assessed following tissue injury. However, few studies have been performed to establish a safe and effective method of IV MSC infusion for newborns. We have established a safe, nontraumatic and effective technique for systemic MSC transplantation in newborn rats. Yellow-fluorescent-protein (YFP)-labeled MSC were characterized using MSC markers and their differentiation potential was confirmed. Rat pups were delivered by C-section on gestational day 21. The umbilical vein (UV) was cannulated and used for IV injection of MSC or saline control, which was performed under ultrasonographic imaging. An additional control group consisted of UV MSC injection in adult mice. Mean operating time, success rate of cannulation and death rate were recorded. YFP-MSC quantification in multiple organs was performed. Mean operating time was 3.9 ± 1.1 min. The success of UV MSC injection was 92.8%. The immediate and 24 hr delayed death rate for rat pups was significantly lower than that of adult mice (p < .05). No pups receiving saline injection died. After locating the patent foramen ovale (PFO) of newborn pups by ultrasonographic imaging, extra pulse-waves and wave-shape changes were detected when MSC were injected. The number of YFP-MSC was 15.8 ± 4.1 cells per visual field (CPVF) in the lungs, 2.9 ± 1.2 CPVF in the heart, and 19.8 ± 5.0 CPVF in the intestines. We conclude that IV MSC infusion through the UV is a convenient, safe, and effective method for systemic MSC transplantation in prematurely delivered newborn rats.

Heparin-binding epidermal growth factor-like growth factor attenuates acute lung injury and multiorgan dysfunction after scald burn.

BACKGROUND Impaired gut barrier function and acute lung injury (ALI) are significant components of the multiorgan dysfunction syndrome that accompanies severe burns. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to reduce inflammation, preserve gut barrier function, and protect the lungs from acute injury in several models of intestinal injury; however, comparable effects of HB-EGF after burn injury have never been investigated. The present studies were based on the hypothesis that HB-EGF would reduce the severity of ALI and multiorgan dysfunction after scald burns in mice. MATERIALS AND METHODS Mice were randomized to sham, burn (25% of total body surface area with full thickness dorsal scald), and burn + HB-EGF groups. The HB-EGF group was pretreated with two enteral doses of HB-EGF (1200 μg/kg/dose). Mice were resuscitated after injury and sacrificed at 8 h later. Their lungs were harvested for determination of pulmonary myeloperoxidase activity, wet:dry ratios, and terminal deoxynucleotidyl transferase dUTP nick end label and cleaved caspase 3 immunohistochemistry. Lung function was assessed using the SCIREQ Flexivent. Splenic apoptosis was quantified by Western blot for cleaved caspase 3, and intestinal permeability was measured using the everted gut sac method. RESULTS Mice subjected to scald burn injury had increased lung myeloperoxidase levels, increased pulmonary and splenic apoptosis, elevated airway resistance and bronchial reactivity, and increased intestinal permeability compared with sham mice. These abnormalities were significantly attenuated in mice that were subjected to scald burn injury but treated with enteral HB-EGF. CONCLUSIONS These data suggest that HB-EGF protects mice from ALI after scald burn and attenuates the severity of postburn multiorgan dysfunction.

An unusual case of foreign body pulmonary embolus: case report and review of penetrating trauma at a pediatric trauma center

PurposePenetrating thoracic trauma is relatively rare in the pediatric population. Embolization of foreign bodies from penetrating trauma is very uncommon. We present a case of a 6-year-old boy with a penetrating foreign body from a projectile dislodged from a lawn mower. Imaging demonstrated a foreign body that embolized to the left pulmonary artery, which was successfully treated non-operatively.MethodsWe reviewed the penetrating thoracic trauma patients in the trauma registry at our institution between 1/1/03 and 12/31/12. Data collected included demographic data, procedures performed, complications and outcome.ResultsSixty-five patients were identified with a diagnosis of penetrating thoracic trauma. Fourteen of the patients had low velocity penetrating trauma and 51 had high velocity injuries. Patients with high velocity injuries were more likely to be older and less likely to be Caucasian. There were no statistically significant differences between patients with low vs. high velocity injuries regarding severity scores or length of stay. There were no statistically significant differences in procedures required between patients with low and high velocity injuries.ConclusionsPenetrating thoracic trauma is rare in children. The case presented here represents the only report of cardiac foreign body embolus we could identify in a pediatric patient.