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Organ-level Tissue Engineering Using Native Microvascular Beds From Adipose Tissue
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS intravascular access, upper arm fistulas are not only a viable option for primary vascular access, but are likely to be a superior option to classic forearm fistulas.
251
Introduction: One of the main challenges in tissue engineering is the ability to maintain large masses of cells alive following their transfer from in vitro culture conditions into host. To overcome initial tissue mass loss, improved vascularisation of the regenerated tissue is essential. Angiopoietin 1(Ang1) is an angiogenic ligand essential for formation of functional blood vessels and with great potential for promoting tissue vascularisation.However, recombinant Ang1 is a large glycoprotein with variable solubility and biological activity and is difficult to produce. This study aims to develop small, stable, nonantagonizable Ang1 mimetic proteins for use as potential therapeutic lead molecules. Methods: Based on the mechanism by which the native ligand activates its receptor, a small synthetic ligand was designed. DNA sequences encoding the synthetic ligand were constructed and expressed in E.Coli. The synthetic ligand was isolated and purified and its ability to bind the angiopoietin receptor analysed by in vitro ELISA. Cell surface binding was examined by immunoflouresence staining and the ability of the ligand to activate cellular signalling was tested by phospho-specific immunoblotting. Functionally, the influence of the ligand on endothelial cells migration was studied using Boyden chamber chemo tactic assay. Results: A synthetic ligand was produced of molecular mass 12kDa compared with the 70kDa native ligand. This ligand binds angiopoietin receptor in vitro. In cellular assays the ligand interacts with the endothelial cell surface and activates the angiopoietin receptor. In addition it stimulates downstream signalling pathways including the phosphatidylinositol 3-kinase/Akt and Erk1/2 pathways. The ligand activates endothelial cell migration. Conclusions: The novel synthetic ligands are easy to produce, can be expressed in E Coli, are highly soluble and stable, activate the angiopoietin receptor. The properties of these synthetic ligands suggest they may be lead molecules for generating potential therapeutic Ang1 mimetics. In addition the synthetic ligands can be immobilized on a tissue engineering scaffold to improve vascularisation of the engineered tissue.
Cox Proportional Hazards Primary Patency Analysis Covariate Age Comorbidities Hypertension Diabetes Mellitus Current Smoking Current Dialysis Heart disease Pulmonary Disease Cancer Preoperative Labs Albumin Creatinine Preopeartive Measures BMI LVEF (%) Medications ASA Antiplatelet Anticoagulated Statin Surgical Variables Site-Upper arm Side
Hazard Ratio
95% ConfLower
95% ConfUpper
P Value
1
0.975
1.027
0.921
1.086 1.118 0.897 0.897 1.074 0.906 1.005
0.215 0.652 0.504 0.48 0.587 0.501 0.491
5.469 1.919 1.595 1.675 1.962 1.637 2.057
0.984 0.685 0.71 0.733 0.818 0.744 0.988
0.916 0.956
0.731 0.83
1.148 1.101
0.445 0.528
0.997 0.998
0.961 0.988
1.033 1.009
0.849 0.751
0.901 0.524 0.952 1.204
0.515 0.12 0.499 0.667
1.577 2.278 1.815 2.173
0.715 0.389 0.881 0.537
0.578 0.915
0.314 0.437
1.065 1.917
0.079 0.814
29.2. Engineering A Novel Ang1 Mimetic For Regenerative Medicine Applications. E. W. Issa, N. J. London, N. P. Brindle; University of Leicester, Leicester, Leicestershire
29.3. Organ-level Tissue Engineering Using Native Microvascular Beds From Adipose Tissue. M. Sorkin, W. W. Victor, R. Kosaraju, J. P. Glotzbach, K. C. Rustad, M. T. Longaker, G. G. Gurtner; Stanford University, Stanford, CA Introduction: The fabrication of organ-level constructs currently relies on de novo neovascularization to supply exceedingly complex cell/ scaffold units. However, pre-patterned functional vascular beds exist throughout the body and are autologous, explantable, and readily reintegrated with the systemic circulation. We recently reported that explantable microvascular beds (EMBs) in rats could be sustained and manipulated ex vivo on a bioreactor. Thus, we hypothesized that isolation of these vascular networks and subsequent
252
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
decellularization could provide a pre-vascularized scaffold for progenitor cell seeding and organ-level tissue engineering. Methods: Superficial inferior epigastric (SIE) flaps were harvested from Sprague-Dawley rats and maintained on a perfusion bioreactor. EMBs were decellularized with combinations of enzymatic/solvent treatments. Vascular patency was assessed with histological staining and SEM analysis of acrylic-based vascular casts. Efficiency of decellularization and preservation of extracellular matrix was assessed with histologic analysis. Decellularized EMBs were seeded with human adipose-derived stem cells (hASCs) and maintained in culture. A novel oxygen-delivery protein was tested to augment flap oxygenation. Results: SIE/EMB tissue flaps could be effectively decellularized with combinations of peracetic acid and alcohol-based perfusates. Collagenase, sodium dodecyl sulfate, and triton-X-based treatments significantly disrupted vascular and matrix architecture. Vascular patency varied with the type of decellularization treatment and was confirmed with histologic analysis. Microcirculatory networks were maintained during bioreactor perfusion periods of over 24 hours. Stem cells seeded onto decellularized EMBs demonstrated excellent viability. Perfusion of oxygen-delivery proteins significantly improved EMB oxygenation up to normoxic levels fold. Conclusions: Autologous vascular beds can be effectively isolated, decellularized, and seeded with hASCs. Enzymatic/solvent treatments demonstrated variable success and future studies to optimize decellularization while maintaining vascular patency are needed. Strategies to augment ex vivo flap survival through oxygen-delivery proteins appear promising. Together, these preliminary studies suggest that progenitor cell-seeded vascularized scaffolds are a promising approach to organ-level tissue engineering for wounded warriors.
29.4. MARCKS and p27kip1 In Smooth Muscle Cell Signaling: Implications In Intimal Hyperplasia. L. M. Korepta, L. Pradhan, A. Landis, S. Essayagh, C. J. Ferran, F. W. LoGerfo; Beth Israel Deaconess Medical Center, Boston, MA Introduction: Previous experiments conducted in our lab have demonstrated that knocking-down myrisolated, alanine-rich C-kinase substrate (MARCKS) using small interfering RNA (siRNA) significantly reduces smooth muscle cell (SMC) proliferation and migration, the hallmark of intimal hyperplasia whereas there is minimal effect on endothelial cell (EC) function. The present study is aimed at investigating the signaling pathway of MARCKS in human coronary artery SMC (HCASMC) and human aortic EC (HAEC). Methods: HCASMCs and HAECs were transfected with 11nM control or MARCKS siRNA using HiPerfect transfection reagent for 24 hours. 24 hours post-transfection, cells were serum starved for another 24 hours with 0.1% fetal bovine serum (FBS) containing growth media, in order to synchronize all of the cells in the G0 phase of cell cycle. Post starvation, cells were stimulated with a 10% FBS + 50ng/mL PDGF-BB growth media and harvested for total protein extraction at different time points (0, 5, 15 mins and 4, 6, and 22 hours). Western blot analysis was performed to monitor the protein expression of MARCKS, p27kip1,and Meso Scale Discovery MAP kinase Total Protein and Phosphoprotein Triplex Assays for p38 and ERK1/2. Q-PCR was performed to monitor the mRNA expression. Results: MARCKS mRNA and protein expression is significantly reduced in MARCKS siRNA treated HCASMCs and HAECs, with more profound knock-down in HAECs. There is a statistically significant increase in the mRNA expression of p27kip1, an important cell cylce inhibitor, at 1hr and 6hr post stimulation only in HCASMCs but not in HAECs. However, the protein expression of p27kip1 was not found to be changed at any time point in HCASMCs, suggesting a need to investigate the protein expression of p27kip1 at a later time point post MARCKS knock-down. Interestingly, total ERK1/2 protein expression was increased whereas phospho-ERK1/2 expression was unchanged in MARCKS siRNA treated HCASMCs (20%) with no changes observed in HAECs. Both phosphorylated and total p38 MAPK was not changed by MARCKS knock-down. Conclusions: MARCKS may affect smooth
muscle cell proliferation and migration by affecting ERK1/2 and p27kip1 expression, with ERK1/2 possibly being the link between MARCKS and p27kip1. Although MARCKS siRNA knock-down was more profound in HAECs compared to HCASMCs, the fact that this knock-down selectively affects HCASMC signaling with minimal effect on HAEC signaling sets it apart as an important and amenable target for gene silencing in the treatment of intimal hyperplasia. 29.5. Early Modulation Of The Injury Response By The Shortacting NO Donor PROLI/NO Leads To Durable Inhibition Of Neointimal Hyperplasia. A. K. Vavra, J. Martinez, V. R. Lee, Q. Jiang, B. Fu, M. R. Kibbe; Northwestern University, Chicago, IL Introduction: We have previously demonstrated that periadventitial delivery of the NO donor PROLI/NO provides durable inhibition of neointimal hyperplasia for up to 8 weeks in spite of its very short half-life of 2 seconds under physiologic conditions in vitro. The goal of this current study was to examine the effect of PROLI/NO on the arterial wall in the first 7 days following injury. Our hypothesis was that early modulation of cell populations in the arterial wall accounts for the durable efficacy of NO at inhibiting neointimal hyperplasia. Methods: The rat carotid injury model was performed in 10wk old male Sprague Dawley rats. Treatment groups included control, injury, or injury +PROLI/NO (10mg). Arteries were harvested at 2, 24, 72 hours and 7 days for immunohistochemistry as well as protein assessment via Western blot analysis. Total cell counts were performed on H&E stained sections. Proliferation was assessed with BrdU incorporation. Phenotypic differentiation was assessed by desmin and aSMC-actin expression. Results: Compared to uninjured arteries, overall cellularity was increased by 60% 7 days after injury (p<0.001). Increased cellularity was due to an 80% increase in cell number in the intima and adventitia, respectively (p<0.05). However, injury decreased cell number in the media at all time points by up to 85% (p<0.001). Treatment with NO prevented the increased cellularity produced with injury primarily by preventing neointima formation and repopulation of the media. The effects of NO were seen as early as 2 hours. At 7 days NO-treated arteries had cellularity similar to the uninjured artery and 69% fewer cells compared to injury alone (p0.009), with an 87% reduction in the intima and media respectively (p<0.05). NO treated arteries also had fewer cells in the adventitia compared to injury but to a lesser extent with a 54% reduction in cell number at 7 days. NO also inhibited proliferation up to 78%, 95% and 80% in the intima, media and adventitia, respectively (p<0.04). Compared to injury alone, NO-treated arteries inhibited the ubiquitous VSMC marker aSMC-actin at all time points with a 10-fold reduction seen at 7 days compared to injury. In contrast, there was a dramatic decrease in the VSMC specific marker desmin at 2 and 24 hours with NO compared to injury and a subsequent 2-fold increase in expression at the later time points. Conclusions: The short-acting NO-donor PROLI/NO inhibits neointimal hyperplasia via modulation of proliferation and phenotypic differentiation in the arterial wall as early as 2 hours following injury. These data suggest that the early effects of NO on the arterial injury response are ultimately responsible for the durability of this shortacting donor. 29.6. Sodium Nitrite Reverses Vascular Intimal Hyperplasia Via An Autophagic Mechanism. M. J. Alef,1 E. Carchman,1 E. Tzeng,1,2 B. S. Zuckerbraun1,2; 1University of Pittsburgh Medical Center, Pittsburgh, PA; 2VA Pittsburgh Healthcare System, Pittsburgh, PA Introduction: Intimal hyperplasia (IH) limits the patency of revascularization procedures. Nitrite (NO2-), a product of nitric oxide (NO) metabolism, can be enzymatically converted back to vasoregulatory NO within the injured vessel. We hypothesize that nitrite
251
Introduction: One of the main challenges in tissue engineering is the ability to maintain large masses of cells alive following their transfer from in vitro culture conditions into host. To overcome initial tissue mass loss, improved vascularisation of the regenerated tissue is essential. Angiopoietin 1(Ang1) is an angiogenic ligand essential for formation of functional blood vessels and with great potential for promoting tissue vascularisation.However, recombinant Ang1 is a large glycoprotein with variable solubility and biological activity and is difficult to produce. This study aims to develop small, stable, nonantagonizable Ang1 mimetic proteins for use as potential therapeutic lead molecules. Methods: Based on the mechanism by which the native ligand activates its receptor, a small synthetic ligand was designed. DNA sequences encoding the synthetic ligand were constructed and expressed in E.Coli. The synthetic ligand was isolated and purified and its ability to bind the angiopoietin receptor analysed by in vitro ELISA. Cell surface binding was examined by immunoflouresence staining and the ability of the ligand to activate cellular signalling was tested by phospho-specific immunoblotting. Functionally, the influence of the ligand on endothelial cells migration was studied using Boyden chamber chemo tactic assay. Results: A synthetic ligand was produced of molecular mass 12kDa compared with the 70kDa native ligand. This ligand binds angiopoietin receptor in vitro. In cellular assays the ligand interacts with the endothelial cell surface and activates the angiopoietin receptor. In addition it stimulates downstream signalling pathways including the phosphatidylinositol 3-kinase/Akt and Erk1/2 pathways. The ligand activates endothelial cell migration. Conclusions: The novel synthetic ligands are easy to produce, can be expressed in E Coli, are highly soluble and stable, activate the angiopoietin receptor. The properties of these synthetic ligands suggest they may be lead molecules for generating potential therapeutic Ang1 mimetics. In addition the synthetic ligands can be immobilized on a tissue engineering scaffold to improve vascularisation of the engineered tissue.
Cox Proportional Hazards Primary Patency Analysis Covariate Age Comorbidities Hypertension Diabetes Mellitus Current Smoking Current Dialysis Heart disease Pulmonary Disease Cancer Preoperative Labs Albumin Creatinine Preopeartive Measures BMI LVEF (%) Medications ASA Antiplatelet Anticoagulated Statin Surgical Variables Site-Upper arm Side
Hazard Ratio
95% ConfLower
95% ConfUpper
P Value
1
0.975
1.027
0.921
1.086 1.118 0.897 0.897 1.074 0.906 1.005
0.215 0.652 0.504 0.48 0.587 0.501 0.491
5.469 1.919 1.595 1.675 1.962 1.637 2.057
0.984 0.685 0.71 0.733 0.818 0.744 0.988
0.916 0.956
0.731 0.83
1.148 1.101
0.445 0.528
0.997 0.998
0.961 0.988
1.033 1.009
0.849 0.751
0.901 0.524 0.952 1.204
0.515 0.12 0.499 0.667
1.577 2.278 1.815 2.173
0.715 0.389 0.881 0.537
0.578 0.915
0.314 0.437
1.065 1.917
0.079 0.814
29.2. Engineering A Novel Ang1 Mimetic For Regenerative Medicine Applications. E. W. Issa, N. J. London, N. P. Brindle; University of Leicester, Leicester, Leicestershire
29.3. Organ-level Tissue Engineering Using Native Microvascular Beds From Adipose Tissue. M. Sorkin, W. W. Victor, R. Kosaraju, J. P. Glotzbach, K. C. Rustad, M. T. Longaker, G. G. Gurtner; Stanford University, Stanford, CA Introduction: The fabrication of organ-level constructs currently relies on de novo neovascularization to supply exceedingly complex cell/ scaffold units. However, pre-patterned functional vascular beds exist throughout the body and are autologous, explantable, and readily reintegrated with the systemic circulation. We recently reported that explantable microvascular beds (EMBs) in rats could be sustained and manipulated ex vivo on a bioreactor. Thus, we hypothesized that isolation of these vascular networks and subsequent
252
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
decellularization could provide a pre-vascularized scaffold for progenitor cell seeding and organ-level tissue engineering. Methods: Superficial inferior epigastric (SIE) flaps were harvested from Sprague-Dawley rats and maintained on a perfusion bioreactor. EMBs were decellularized with combinations of enzymatic/solvent treatments. Vascular patency was assessed with histological staining and SEM analysis of acrylic-based vascular casts. Efficiency of decellularization and preservation of extracellular matrix was assessed with histologic analysis. Decellularized EMBs were seeded with human adipose-derived stem cells (hASCs) and maintained in culture. A novel oxygen-delivery protein was tested to augment flap oxygenation. Results: SIE/EMB tissue flaps could be effectively decellularized with combinations of peracetic acid and alcohol-based perfusates. Collagenase, sodium dodecyl sulfate, and triton-X-based treatments significantly disrupted vascular and matrix architecture. Vascular patency varied with the type of decellularization treatment and was confirmed with histologic analysis. Microcirculatory networks were maintained during bioreactor perfusion periods of over 24 hours. Stem cells seeded onto decellularized EMBs demonstrated excellent viability. Perfusion of oxygen-delivery proteins significantly improved EMB oxygenation up to normoxic levels fold. Conclusions: Autologous vascular beds can be effectively isolated, decellularized, and seeded with hASCs. Enzymatic/solvent treatments demonstrated variable success and future studies to optimize decellularization while maintaining vascular patency are needed. Strategies to augment ex vivo flap survival through oxygen-delivery proteins appear promising. Together, these preliminary studies suggest that progenitor cell-seeded vascularized scaffolds are a promising approach to organ-level tissue engineering for wounded warriors.
29.4. MARCKS and p27kip1 In Smooth Muscle Cell Signaling: Implications In Intimal Hyperplasia. L. M. Korepta, L. Pradhan, A. Landis, S. Essayagh, C. J. Ferran, F. W. LoGerfo; Beth Israel Deaconess Medical Center, Boston, MA Introduction: Previous experiments conducted in our lab have demonstrated that knocking-down myrisolated, alanine-rich C-kinase substrate (MARCKS) using small interfering RNA (siRNA) significantly reduces smooth muscle cell (SMC) proliferation and migration, the hallmark of intimal hyperplasia whereas there is minimal effect on endothelial cell (EC) function. The present study is aimed at investigating the signaling pathway of MARCKS in human coronary artery SMC (HCASMC) and human aortic EC (HAEC). Methods: HCASMCs and HAECs were transfected with 11nM control or MARCKS siRNA using HiPerfect transfection reagent for 24 hours. 24 hours post-transfection, cells were serum starved for another 24 hours with 0.1% fetal bovine serum (FBS) containing growth media, in order to synchronize all of the cells in the G0 phase of cell cycle. Post starvation, cells were stimulated with a 10% FBS + 50ng/mL PDGF-BB growth media and harvested for total protein extraction at different time points (0, 5, 15 mins and 4, 6, and 22 hours). Western blot analysis was performed to monitor the protein expression of MARCKS, p27kip1,and Meso Scale Discovery MAP kinase Total Protein and Phosphoprotein Triplex Assays for p38 and ERK1/2. Q-PCR was performed to monitor the mRNA expression. Results: MARCKS mRNA and protein expression is significantly reduced in MARCKS siRNA treated HCASMCs and HAECs, with more profound knock-down in HAECs. There is a statistically significant increase in the mRNA expression of p27kip1, an important cell cylce inhibitor, at 1hr and 6hr post stimulation only in HCASMCs but not in HAECs. However, the protein expression of p27kip1 was not found to be changed at any time point in HCASMCs, suggesting a need to investigate the protein expression of p27kip1 at a later time point post MARCKS knock-down. Interestingly, total ERK1/2 protein expression was increased whereas phospho-ERK1/2 expression was unchanged in MARCKS siRNA treated HCASMCs (20%) with no changes observed in HAECs. Both phosphorylated and total p38 MAPK was not changed by MARCKS knock-down. Conclusions: MARCKS may affect smooth
muscle cell proliferation and migration by affecting ERK1/2 and p27kip1 expression, with ERK1/2 possibly being the link between MARCKS and p27kip1. Although MARCKS siRNA knock-down was more profound in HAECs compared to HCASMCs, the fact that this knock-down selectively affects HCASMC signaling with minimal effect on HAEC signaling sets it apart as an important and amenable target for gene silencing in the treatment of intimal hyperplasia. 29.5. Early Modulation Of The Injury Response By The Shortacting NO Donor PROLI/NO Leads To Durable Inhibition Of Neointimal Hyperplasia. A. K. Vavra, J. Martinez, V. R. Lee, Q. Jiang, B. Fu, M. R. Kibbe; Northwestern University, Chicago, IL Introduction: We have previously demonstrated that periadventitial delivery of the NO donor PROLI/NO provides durable inhibition of neointimal hyperplasia for up to 8 weeks in spite of its very short half-life of 2 seconds under physiologic conditions in vitro. The goal of this current study was to examine the effect of PROLI/NO on the arterial wall in the first 7 days following injury. Our hypothesis was that early modulation of cell populations in the arterial wall accounts for the durable efficacy of NO at inhibiting neointimal hyperplasia. Methods: The rat carotid injury model was performed in 10wk old male Sprague Dawley rats. Treatment groups included control, injury, or injury +PROLI/NO (10mg). Arteries were harvested at 2, 24, 72 hours and 7 days for immunohistochemistry as well as protein assessment via Western blot analysis. Total cell counts were performed on H&E stained sections. Proliferation was assessed with BrdU incorporation. Phenotypic differentiation was assessed by desmin and aSMC-actin expression. Results: Compared to uninjured arteries, overall cellularity was increased by 60% 7 days after injury (p<0.001). Increased cellularity was due to an 80% increase in cell number in the intima and adventitia, respectively (p<0.05). However, injury decreased cell number in the media at all time points by up to 85% (p<0.001). Treatment with NO prevented the increased cellularity produced with injury primarily by preventing neointima formation and repopulation of the media. The effects of NO were seen as early as 2 hours. At 7 days NO-treated arteries had cellularity similar to the uninjured artery and 69% fewer cells compared to injury alone (p0.009), with an 87% reduction in the intima and media respectively (p<0.05). NO treated arteries also had fewer cells in the adventitia compared to injury but to a lesser extent with a 54% reduction in cell number at 7 days. NO also inhibited proliferation up to 78%, 95% and 80% in the intima, media and adventitia, respectively (p<0.04). Compared to injury alone, NO-treated arteries inhibited the ubiquitous VSMC marker aSMC-actin at all time points with a 10-fold reduction seen at 7 days compared to injury. In contrast, there was a dramatic decrease in the VSMC specific marker desmin at 2 and 24 hours with NO compared to injury and a subsequent 2-fold increase in expression at the later time points. Conclusions: The short-acting NO-donor PROLI/NO inhibits neointimal hyperplasia via modulation of proliferation and phenotypic differentiation in the arterial wall as early as 2 hours following injury. These data suggest that the early effects of NO on the arterial injury response are ultimately responsible for the durability of this shortacting donor. 29.6. Sodium Nitrite Reverses Vascular Intimal Hyperplasia Via An Autophagic Mechanism. M. J. Alef,1 E. Carchman,1 E. Tzeng,1,2 B. S. Zuckerbraun1,2; 1University of Pittsburgh Medical Center, Pittsburgh, PA; 2VA Pittsburgh Healthcare System, Pittsburgh, PA Introduction: Intimal hyperplasia (IH) limits the patency of revascularization procedures. Nitrite (NO2-), a product of nitric oxide (NO) metabolism, can be enzymatically converted back to vasoregulatory NO within the injured vessel. We hypothesize that nitrite