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Obstructive Lung Disease Induced by Mesenchymal Specific Inhibition of Vascular Endothelial Growth Factor (VEGF)
258
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
migrate more quickly than AFb (FFb 180.7 vs AFb 67.55, p¼0.004). IL-10-/-FFb migration is delayed compared to FFb (FFb 180.7 vs IL10-/-FFb 116.7, p¼0.03). FFb migration is also delayed with HA synthase inhibition (FFb 180.7 vs FFb+4MU 31.3, p¼0.002). IL-10 supplementation accelerates migration in AFb (AFb 67.6 vs AFb+IL-10 89.4, p¼0.0002), but the effect is decreased with inhibition of HA synthesis (AFb+IL-10 89.4 vs AFb+IL-10+4MU 40.3, p¼0.0001). FFb invade more efficiently than AFb (FFb 8.4 cells/HPF vs AFb 3.6 cells/HPF, p¼0.04). IL-10-/-FFb invade less efficiently than FFb (FFb 8.4 cells/ HPF vs IL-10-/-FFb 3.7 cells/HPF). Inhibition of HA synthesis impedes FFB invasion (FFb 8.4 cells/HPF vs FFb+4-MU 4.4 cells/HPF). Treatment with IL-10 increases invasion in AFb (AFb 3.6 cells/HPF vs AFb+IL-10 6.3 cells/HPF, p¼0.01). This IL-10 mediated increase is abrogated by HA synthase inhibition (AFb+IL-10 6.3 cells/HPF vs AFb+IL-10+4-MU 1.4 cells/HPF). Conclusions: There are distinct functional profile differences between FFb and AFb. Enhanced migration and invasion in FFb is dependent on IL-10 and HA synthesis. IL-10 mediated HA production can recapitulate this part of the fetal functional phenotype in AFb. Accelerated migration and invasion may facilitate scarless healing in the fetus and provide a potential mechanism for IL-10’s ability to recapitulate the fetal regenerative phenotype in post-natal wounds. 30.2. Donor-Specific NK Cell Anergy in Prenatal Allogeneic Chimeras is Transiently Reversed During Viral Illness, but Does not Result in Loss of Engraftment. K. O. Ryken, L. E. Turner, R. Wadhwani, A. M. Alhajjat, A. F. Shaaban; University of Iowa Carver College of Medicine, Iowa City, IA Introduction: The clinical potential of in utero hematopoietic cellular transplantation (IUHCT) lies in its unique ability to correct congenital diseases before the patient experiences a single day of illness. Longterm engraftment following prenatal transplantation requires functional anergy in host natural killer cells that are phenotypically hostile toward the donor. It is unclear if this anergy is dependent upon the cellular activation state, which could allow for its reversal during a viral illness. Loss of anergy could potentially result in an abrupt and devastating rejection of previously engrafted donor cells, permitting relapse of disease in the patient. Therefore, we examined the effect of viral illness upon NK cell tolerance in prenatal allogeneic chimeras, and hypothesized that the activation of hostile NK cells would lead to acute rejection of stably engrafted cells. Methods: Using an established BALB/ c-to-B6 murine model of IUHCT, NK cell activation was achieved in vivo with lymphocytic choriomeningitis virus infection (LCMV-Armstrong variant). The responsiveness of donor-specific hostile NK cells was determined using multi-parameter flow cytometry for intracellular cytokine expression. Reversal of donor-specific tolerance was then correlated with the stability of engraftment over time. Results: Hostile NK cells from LCMV-infected chimeric mice demonstrated robust interferon responses. A significant reversal of donor-specific anergy in NK cells was uniformly observed in LCMV-infected mice compared to saline-injected control (IFN-g ratio of 1.3 +/- 0.2 vs. 0.43 +/- 0.06 respectively, p<0.0001). Consistent with this loss of anergy, chimerism levels fell dramatically early during LCMV infection while remaining stable in saline control. This drop in chimerism was transient, however, and engraftment persisted long-term in all mice. Chimerism quickly returned to baseline levels following resolution of the viral illness. Conclusions: NK cell tolerance in prenatal allogeneic chimeras is dependent upon the cellular activation state. However, engraftment proved to be durable despite potent NK cell activation during viral infection. A thorough understanding of the mechanisms regulating donor-specific NK cell tolerance can lead to the implementation of improved strategies for IUHCT. 30.3. Obstructive Lung Disease Induced by Mesenchymal Specific Inhibition of Vascular Endothelial Growth Factor (VEGF). D. E. Levin, H. Xiaogang, S. Navarro, O. Garcia, J. Lee, B. Driscoll, T. C. Grikscheit; Children’s Hospital Los Angeles, Los Angeles, CA
Introduction: Cronic obstructive pulmonary disease (COPD), asthma and bronchiectasis comprise a category of respiratory disease referred to as obstructive lung disease (OLD). COPD is the fifth leading cause of death in the USA and a common comorbidity among surgical patients. VEGF is an endothelial cell signal protein that is a key mediator of angiogenesis and vasculogenesis. Chronic blockade of the VEGF receptor (VEGFR) in an animal model has been shown to induce alveolar cell apoptosis resulting in emphysema and cigarette smoke has been demonstrated to interrupt the VEGF-VEGFR signaling complex in an animal model of COPD. Previous studies have shown that whole body and organ specific growth of neonatal mice is attenuated by mesenchymal specific inhibition of VEGF. Therefore, we hypothesized that induction of mesenchymal specific VEGF inhibition would result in a novel model of OLD for therapeutic investigation. Methods: Triple transgenic mice expressing the soluble VEGF receptor, sFlt-1 specifically in the mesenchyme (Dermo-1(Cre)-rtTA(flox/flox)-tet(0)-sflt-1) were generated by selectively crossing transgenic mice. Mothers of the resulting pups (transgenic N¼2 and littermate controls N¼2) were fed doxycycline chow at birth for transgene activation via breastmilk. Once weaned, all mice were fed doxycycline chow until sacrifice after pulmonary function testing (PFT) at 3 months. PFTs were obtained with plethysmography via tracheostomy and forced pulmonary maneuvers. Pressure-volume loop (PV loop), static compliance (Cst), force expiratory volume in100ms (FEV0.1), forced vital capacity(FVC) and FEV0.1/FVC ratio, peak expiratory flow (PEF) and Inspiratory capacity (IC) were compared. Values were corrected for weight. A p value of <0.05 was considered significant. Paraffin sections were H&E stained for histologic comparison. Results: PV loop of mutant animals was shifted left and upward compared to controls (figure). Cst was increased in mutants v. control (2.91 60.65 ml/cmH20/kg v. 2.37 60.31 ml/cmH20/kg, p<0.05). Both FEV0.1 and FVC were reduced in mutants v. controls (0.58 60.10ml v. 0.94 60.14 ml and 0.59ml 60.10ml v. 0.97 60.16ml, p<0.05 respectively). There was no difference in FEV0.1/FVC between the two groups (99.2% 6 0.9% v. 96.8% 6 2.8%, p ¼ NS). PEF was decreased in mutants (11.93 6 1.86ml/s v. 16.50 6 2.31ml/s, p<0.05). IC was greatly increased in the mutant group (42.7 6 2.68 ml/kg v. 29.0 6 1.5 ml/kg, p<0.001). Mutant airway spaces appear enlarged on H&E. Conclusions: Mesenchymal specific inhibition of VEGF in mice results in OLD. This transgenic mouse represents an inducible, novel model of OLD and supports a role for VEGF in the pathogenesis of OLD.
FIG. Pressure-volume curves comparing Dermo-1(Cre)-rtTA(flox/flox)-tet(0)-sfit-1 transgenic mice with littermate controls for (A) male and females averaged together and (B) males (N¼2) and females (N¼2) plotted individually. 30.4. Fgfr-Akt-Beta-Catenin and Tgfbeta-Smad3 Signaling Pathways are Associated With Expansion and Epithelial-Mesenchyme Trans-Differentiation of Cd133pos Cd49fpos Cells in Murine Model of Biliary Atresia. N. Mavila,1 D. James,1 P. Shivakumar,2 S. Utley,1 A. Wu,1 K. Mak,1 C. Vendyres,1 J. Bezerra,2 K. Wang1; 1Children’s Hospital Los Angeles, Los Angeles, CA; 2Cincinnati Children’s Hospital Medical Center, Cincinnati, OH Introduction: Biliary atresia (BA) is a progressive obliterative extrahepatic cholangiopathy of infants. Despite successful surgical
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
migrate more quickly than AFb (FFb 180.7 vs AFb 67.55, p¼0.004). IL-10-/-FFb migration is delayed compared to FFb (FFb 180.7 vs IL10-/-FFb 116.7, p¼0.03). FFb migration is also delayed with HA synthase inhibition (FFb 180.7 vs FFb+4MU 31.3, p¼0.002). IL-10 supplementation accelerates migration in AFb (AFb 67.6 vs AFb+IL-10 89.4, p¼0.0002), but the effect is decreased with inhibition of HA synthesis (AFb+IL-10 89.4 vs AFb+IL-10+4MU 40.3, p¼0.0001). FFb invade more efficiently than AFb (FFb 8.4 cells/HPF vs AFb 3.6 cells/HPF, p¼0.04). IL-10-/-FFb invade less efficiently than FFb (FFb 8.4 cells/ HPF vs IL-10-/-FFb 3.7 cells/HPF). Inhibition of HA synthesis impedes FFB invasion (FFb 8.4 cells/HPF vs FFb+4-MU 4.4 cells/HPF). Treatment with IL-10 increases invasion in AFb (AFb 3.6 cells/HPF vs AFb+IL-10 6.3 cells/HPF, p¼0.01). This IL-10 mediated increase is abrogated by HA synthase inhibition (AFb+IL-10 6.3 cells/HPF vs AFb+IL-10+4-MU 1.4 cells/HPF). Conclusions: There are distinct functional profile differences between FFb and AFb. Enhanced migration and invasion in FFb is dependent on IL-10 and HA synthesis. IL-10 mediated HA production can recapitulate this part of the fetal functional phenotype in AFb. Accelerated migration and invasion may facilitate scarless healing in the fetus and provide a potential mechanism for IL-10’s ability to recapitulate the fetal regenerative phenotype in post-natal wounds. 30.2. Donor-Specific NK Cell Anergy in Prenatal Allogeneic Chimeras is Transiently Reversed During Viral Illness, but Does not Result in Loss of Engraftment. K. O. Ryken, L. E. Turner, R. Wadhwani, A. M. Alhajjat, A. F. Shaaban; University of Iowa Carver College of Medicine, Iowa City, IA Introduction: The clinical potential of in utero hematopoietic cellular transplantation (IUHCT) lies in its unique ability to correct congenital diseases before the patient experiences a single day of illness. Longterm engraftment following prenatal transplantation requires functional anergy in host natural killer cells that are phenotypically hostile toward the donor. It is unclear if this anergy is dependent upon the cellular activation state, which could allow for its reversal during a viral illness. Loss of anergy could potentially result in an abrupt and devastating rejection of previously engrafted donor cells, permitting relapse of disease in the patient. Therefore, we examined the effect of viral illness upon NK cell tolerance in prenatal allogeneic chimeras, and hypothesized that the activation of hostile NK cells would lead to acute rejection of stably engrafted cells. Methods: Using an established BALB/ c-to-B6 murine model of IUHCT, NK cell activation was achieved in vivo with lymphocytic choriomeningitis virus infection (LCMV-Armstrong variant). The responsiveness of donor-specific hostile NK cells was determined using multi-parameter flow cytometry for intracellular cytokine expression. Reversal of donor-specific tolerance was then correlated with the stability of engraftment over time. Results: Hostile NK cells from LCMV-infected chimeric mice demonstrated robust interferon responses. A significant reversal of donor-specific anergy in NK cells was uniformly observed in LCMV-infected mice compared to saline-injected control (IFN-g ratio of 1.3 +/- 0.2 vs. 0.43 +/- 0.06 respectively, p<0.0001). Consistent with this loss of anergy, chimerism levels fell dramatically early during LCMV infection while remaining stable in saline control. This drop in chimerism was transient, however, and engraftment persisted long-term in all mice. Chimerism quickly returned to baseline levels following resolution of the viral illness. Conclusions: NK cell tolerance in prenatal allogeneic chimeras is dependent upon the cellular activation state. However, engraftment proved to be durable despite potent NK cell activation during viral infection. A thorough understanding of the mechanisms regulating donor-specific NK cell tolerance can lead to the implementation of improved strategies for IUHCT. 30.3. Obstructive Lung Disease Induced by Mesenchymal Specific Inhibition of Vascular Endothelial Growth Factor (VEGF). D. E. Levin, H. Xiaogang, S. Navarro, O. Garcia, J. Lee, B. Driscoll, T. C. Grikscheit; Children’s Hospital Los Angeles, Los Angeles, CA
Introduction: Cronic obstructive pulmonary disease (COPD), asthma and bronchiectasis comprise a category of respiratory disease referred to as obstructive lung disease (OLD). COPD is the fifth leading cause of death in the USA and a common comorbidity among surgical patients. VEGF is an endothelial cell signal protein that is a key mediator of angiogenesis and vasculogenesis. Chronic blockade of the VEGF receptor (VEGFR) in an animal model has been shown to induce alveolar cell apoptosis resulting in emphysema and cigarette smoke has been demonstrated to interrupt the VEGF-VEGFR signaling complex in an animal model of COPD. Previous studies have shown that whole body and organ specific growth of neonatal mice is attenuated by mesenchymal specific inhibition of VEGF. Therefore, we hypothesized that induction of mesenchymal specific VEGF inhibition would result in a novel model of OLD for therapeutic investigation. Methods: Triple transgenic mice expressing the soluble VEGF receptor, sFlt-1 specifically in the mesenchyme (Dermo-1(Cre)-rtTA(flox/flox)-tet(0)-sflt-1) were generated by selectively crossing transgenic mice. Mothers of the resulting pups (transgenic N¼2 and littermate controls N¼2) were fed doxycycline chow at birth for transgene activation via breastmilk. Once weaned, all mice were fed doxycycline chow until sacrifice after pulmonary function testing (PFT) at 3 months. PFTs were obtained with plethysmography via tracheostomy and forced pulmonary maneuvers. Pressure-volume loop (PV loop), static compliance (Cst), force expiratory volume in100ms (FEV0.1), forced vital capacity(FVC) and FEV0.1/FVC ratio, peak expiratory flow (PEF) and Inspiratory capacity (IC) were compared. Values were corrected for weight. A p value of <0.05 was considered significant. Paraffin sections were H&E stained for histologic comparison. Results: PV loop of mutant animals was shifted left and upward compared to controls (figure). Cst was increased in mutants v. control (2.91 60.65 ml/cmH20/kg v. 2.37 60.31 ml/cmH20/kg, p<0.05). Both FEV0.1 and FVC were reduced in mutants v. controls (0.58 60.10ml v. 0.94 60.14 ml and 0.59ml 60.10ml v. 0.97 60.16ml, p<0.05 respectively). There was no difference in FEV0.1/FVC between the two groups (99.2% 6 0.9% v. 96.8% 6 2.8%, p ¼ NS). PEF was decreased in mutants (11.93 6 1.86ml/s v. 16.50 6 2.31ml/s, p<0.05). IC was greatly increased in the mutant group (42.7 6 2.68 ml/kg v. 29.0 6 1.5 ml/kg, p<0.001). Mutant airway spaces appear enlarged on H&E. Conclusions: Mesenchymal specific inhibition of VEGF in mice results in OLD. This transgenic mouse represents an inducible, novel model of OLD and supports a role for VEGF in the pathogenesis of OLD.
FIG. Pressure-volume curves comparing Dermo-1(Cre)-rtTA(flox/flox)-tet(0)-sfit-1 transgenic mice with littermate controls for (A) male and females averaged together and (B) males (N¼2) and females (N¼2) plotted individually. 30.4. Fgfr-Akt-Beta-Catenin and Tgfbeta-Smad3 Signaling Pathways are Associated With Expansion and Epithelial-Mesenchyme Trans-Differentiation of Cd133pos Cd49fpos Cells in Murine Model of Biliary Atresia. N. Mavila,1 D. James,1 P. Shivakumar,2 S. Utley,1 A. Wu,1 K. Mak,1 C. Vendyres,1 J. Bezerra,2 K. Wang1; 1Children’s Hospital Los Angeles, Los Angeles, CA; 2Cincinnati Children’s Hospital Medical Center, Cincinnati, OH Introduction: Biliary atresia (BA) is a progressive obliterative extrahepatic cholangiopathy of infants. Despite successful surgical