67.07 Developing Collateral Arteries Unravel Their Internal Elastic Laminae for Diameter Growth

Posted on January 31, 2019

R. M. McEnaney1,2, D. D. McCreary1, E. Tzeng1,2  1VA Pittsburgh Healthcare System,Vascular Surgery,Pittsburgh, PA, USA 2University Of Pittsburgh School of Medicine,Vascular Surgery,Pittsburgh, PA, USA

Introduction:   Collateral artery growth is a natural and sometimes life-preserving response to arterial occlusive disease.  This is because collateral arteries maintain end organ perfusion once a conductance artery becomes occluded.  “Outward remodeling” describes a process of active cellular activity and matrix turnover that expands the vessel diameter.   For outward remodeling to occur, matrix constraints on arterial lumen diameter must be released. These constraints exist due to the matrix structure of an artery. Elastic fibers coalesce to form lamellae and are the major structural elements within the intima and media.  These matrix elements create the elasticity and the luminal topography characteristic of arteries.  However, it is likely that the elastic lamellar structure also creates diameter restraint and must be degraded to achieve outward remodeling.

Methods:   A modified procedure of hind limb ischemia was performed in rats, as per our previously published report.  Animals were euthanized, and collateral artery tissues were harvested at up to 12 weeks and preserved in paraformaldehyde.  Microscopy was performed with an Olympus FluoView MPE Multiphoton microscope with second-harmonic generation to view elastin and collagen structure.

Results:  Striking structural alterations occur along with arterial diameter expansion that are persistent at 12 weeks. (Figure) We observed in collateral vessels which increased diameter up to threefold, a change in the internal elastic lamina from a nearly continuous, wrinkled and fenestrated sheet to a web-like appearance.  Collagen orientation in collateral arteries appears perturbed, with straightening of groups rather than the typical wavy, ribbon-like appearance.

Conclusion:  Outward remodeling is an important vascular adaptation capable of producing functional collateral arteries which are relevant to patients with cardiovascular diseases.  This study shows that during outward remodeling, the normal elastic lamellar structure of an artery becomes irrevocably altered. This also results in a topographical change in the luminal surface of the collateral artery.  Elastic fibers, structural ECM components unique to vertebrates, are integral to the function of the cardiovascular system and require a complex assembly known to occur only in development.  Perhaps more clinically relevant is understanding the mechanisms underlying this elastic remodeling to develop enhanced collateral artery growth for patients with arterial occlusive disease.

 

67.06 Lipidomic Analysis of Exosomes in Mesenteric Lymph after Intestinal Ischemia-Reperfusion Injury.

Posted on January 31, 2019

A. Senda1, K. Morishita1, M. Kojima1, S. Doki2, M. Yagi1, T. Kobayashi2, J. Aiboshi1, Y. Otomo1  1Tokyo Medical And Dental University,Departments Of Acute Critical Care And Disaster Medicine,Bunkyo-ku, Tokyo, Japan 2Ochanomizu University,Faculty Of Science,Bunkyo-Ku, TOKYO, Japan

Introduction: Intestinal ischemia-reperfusion (IR) leads to gut barrier failure that initiates a systemic inflammatory response resulting in acute lung injury/multiple organ dysfunction syndrome. Previous studies have shown that mesenteric lymph (ML) has a crucial role in driving the gut-mediated inflammation after IR. Our group have reported that lipid mediators, such as lysophosphatidylcholine (LPC) and arachidonic acid, contained in ML mediate organ injury following trauma/hemorrhagic shock (T/HS) via activation of immune cells. Concurrently, there is a research proving exosome as the major component of inflammatory response. Therefore, we investigated the bioactivity of exosomes released to the ML during intestinal IR injury. We also analyzed the profiles of exosomal lipids of ML to identify the specific mediators carried by ML exosomes.

Methods: Male Sprague-Dawley rats underwent laparotomy followed by ML duct cannulation. Animals were then subjected to 60 minutes of superior mesenteric artery clumping and de-clamping to induce gut IR injury. ML was obtained before (Pre) and after IR (Post-I/R). Exosomes were isolated from ML by ultracentrifugation. The biological activity of ML exosomes in each period was measured by monocyte NF-κB activation. The exosomal lipids in each group were extracted using of Bligh and Dyer method and performed liquid chromatography electrospray ionization mass spectrometry.

Results:The pro-inflammatory activities of exosomes harvested after IR were confirmed by an increase in NF-κB activation (5.6-fold increase compared to control). Lipid analysis of exosomes fraction of ML revealed significant increase in the concentration of polyunsaturated fatty acid (PUFA) (C18:2, C20:4) containing LPCs after IR when compared to that of control ML exosomes?(see Figure).

Conclusion:In response to IR injury, exosome fraction of ML shows significant increase in lipid mediators and triggers inflammation.

67.05 Evaluation of Prolonged Permissive Hypotension: Results from a Six Hour Hemorrhage Protocol in Swine

Posted on January 31, 2019

C. G. Morgan1, E. N. Hathaway2, L. E. Neidert1, L. Schaub1, J. J. Glaser1  1Naval Medical Research Unit San Antonio,JBSA-Ft. Sam Houston, TX, USA 2Brooke Army Medical Center,JBSA-Ft. Sam Houston, TX, USA

Introduction: Tactical Combat Casualty Care (TCCC) guidelines for hemorrhage recommend resuscitation to systolic blood pressure (SBP) 80-90mmHg, only fully resuscitating after surgical hemostasis. Success depends on rapid transport to definitive care, within the ‘golden hour’. Future conflicts may demand longer prehospital/transport times. We sought to determine safety of prolonged permissive hypotension (PH) over 6 hours.

Methods:  Adult male swine were randomized to 1) hemorrhage + hypotension 2) hypotensive sham 3) anesthesia sham. Group 1 (G1) underwent hemorrhage to mean arterial blood pressure (MAP) 30mmHg for 30 min followed by 6 hr prolonged field care (PFC) with goal SBP 85±5mmHg maintained with crystalloid. Animals were then resuscitated with whole blood simulating hospital care, and observed for 24 hrs. Group 2 (G2) was bled to SBP 85±5mmHg during PFC then recovered. Group 3 (G3) underwent anesthesia only. Physiologic variables, blood, tissue samples, and neurologic (Tarlov) scores were collected. Significance = p<0.05.

Results: Survival of all groups was 100% at 24 hrs, with no significant differences in Tarlov score (G1: 6.2±6.8, G2: 2.5±4.5, G3: 0.0±0.0). Mean blood loss after hemorrhage was 37.2±7.3%. At PFC Hr 1 G1 lactate level (mmol/L) was significantly higher than G2 and G3 (5.1±2.2 vs 2.6±1.8 vs 2.1±1.1) and at PFC Hr 2 and PFC Hr 4 compared to G3 (PFC Hr 2: 5.7±2.9 vs 1.6±0.5; PFC Hr 4: 4.0±2.4 vs 1.3±0.1). Crystalloid requirement (mL) to maintain blood pressure in PFC was significantly higher in G1 than G2 and G3 (G1: 4728±1410 vs G2: 1214±608 vs G3: 155±347). PaO2/FiO2 ratio remained above 300 in all groups throughout.

Conclusion: After 6 hrs, a prolonged PH strategy showed no detrimental effect on survival or neurologic outcome despite the increased ischemic burden of hemorrhage. Significant fluid volume was required to maintain SBP- a potential logistic burden for prehospital care. Further work to define maximum allowable time of PH is needed.

 

67.04 ER Stress Increases Susceptibility to Sporadic Aortic Aneurysm and Dissection in Mice

Posted on January 31, 2019

W. Luo1,2, Y. Wang1,2, L. Zhang1,2, P. Ren1,2, C. Zhang1,2, J. Guo1,2, C. Joseph S1,2,3, Y. Shen1,2,3, S. LeMaire1,2,3  1Baylor College Of Medicine,Division Of Cardiothoracic Surgery,Houston, TX, USA 2Texas Heart Institute,Department Of Cardiovascular Surgery,Houston, TX, USA 3Baylor College Of Medicine,Cardiovascular Research Institute,Houston, TX, USA

Introduction: Aortic aneurysm and dissection (AAD) carries significant morbidity and mortality. One of the significant features of AAD is progressive smooth muscle cell (SMC) dysfunction and depletion, which leads to aortic dilatation, biomechanical failure, and ultimately rupture. However, the molecular mechanisms underlying the progressive SMC dysfunction and depletion are poorly understood. Increasing evidence has suggested a critical role of endoplasmic reticulum (ER) stress in cell dysfunction. However, whether ER stress can promote aortic destruction, dysfunction and AAD development is unknown. In this study, we examined the effect of ER stress on aortic destruction and AAD formation in mice.

Methods:  The activation of ER stress was compared in aortic tissues from patients with sporadic ascending thoracic AAD with that in tissues from age-matched organ donor controls. The contribution of ER stress to AAD development was assessed by comparing AAD development in C57BL/6J mice that were given thapsigargin (Tg) (n=39) or vehicle (n=40) before challenged with low-dose angiotensin infusion (AngII, 1000 ng/min/kg) for four weeks. 

Results: We observed significant upregulation of ER stress markers in patient aortic tissues, especially in smooth muscle cell (SMC). Thapsigargin itself did not cause AAD or dilatation. However, thapsigargin significantly increased the challenge-induced incidence of AAD (15% in challenged mice vs 44% in Tg treated challenged mice, p=0.005), severe AAD (8% in challenged mice vs 23% in Tg treated challenged mice, p=0.05). Thapsigargin showed similar effect in male and female mice. Furthermore, aortic tissues from challenged mice treated with Tg showed markedly increased aortic destruction and elastic fiber fragmentation.

Conclusion: Our findings demonstrate that inducing ER stress with thapsigargin increases susceptibility to sporadic aortic aneurysm and dissection in mice. 

 

67.03 Histone Methylation Directs Macrophage Toll-like Receptor 4 Expression and Regulates Wound Healing

Posted on January 31, 2019

F. M. Davis1, A. Kimball1, A. Joshi1, A. DenDekker1, A. Obi1, K. Singer2, B. Moore3, S. Kunkel4, K. Gallagher1  2University Of Michigan,Pediatric Endocrinology,Ann Arbor, MI, USA 3University Of Michigan,Microbiology And Immunology,Ann Arbor, MI, USA 4University of Michigan,Pathology,Ann Arbor, MI, USA 1University of Michigan,Surgery,Ann Arbor, MI, USA

Introduction:  Macrophages (MΦ) are critical for both the initiation and resolution of the inflammatory phase of wound repair.  Toll-like receptors (TLRs), particularly TLR4, play a critical role in regulating macrophage-mediated inflammation and tissue regeneration. Since an initial inflammatory phase is vital for tissue repair, we hypothesized that TLR4 regulates macrophage-mediated inflammation in wound repair.

Methods:  Using a murine wound healing model, percent wound healing was analyzed between control and TLR4 deficient (TLR4-/-) mice (n= 10/group).  Bone marrow-derived macrophages (BMDMs) were isolated and cultured in standard fashion. Expression of inflammatory genes (IL1β, IL12, TNFα) were determined by qPCR. Chromatin immunoprecipitation was used to analyze H3K4 methylation.  Adoptive transfer was performed with intravenous injection of CD11b+ cells isolated from spleens of control and TLR4-/- mice. Statistical significance was determined using Student t-tests or ANOVA.

Results: TLR4 expression in wound MΦs was significantly increased throughout the wound healing course (p<0.05).  We identified that changes in MΦ TLR4 during the course of wound healing correlated with increased expression of the histone methyltransferase Mix-lineage leukemia-1 (MLL1) and its activating epigenetic marker, histone 3 lysine 4 tri-methylation (H3K4me3), on the TLR4 promoter. Mice deficient in TLR4 demonstrated delayed wound healing at all time points post-injury. Furthermore, in vitro BMDMs and in vivo wound macrophages isolated from TLR4-/- wounds had decreased inflammatory cytokine production (p<0.05). Importantly, adoptive transfer of monocyte/macrophages from wild-type mice restored normal healing in the TLR4-/- mice.  

Conclusion: These results define a role for macrophage specific TLR4 in the inflammatory response following cutaneous tissue injury and suggest that MLL1 regulates TLR4 expression in wound MΦs.

 

67.02 PAD2 Knockout Improves Survival and Attenuates Organ Injuries in a Mouse Model of Hemorrhagic Shock

Posted on January 31, 2019

J. ZHOU1,2, Y. Tian1,3, Q. Deng1,3, P. Chang1,2, A. M. Williams1, U. F. Bhatti1, B. E. Biesterveld1, B. Liu1, J. Zhang1, T. Wang2, Y. Li1, H. B. Alam1  1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA 2Peking University People’s Hospital,Trauma Center, Department Of Orthopedic And Traumatology,Beijing, BEIJING, China 3Central South University,Xiangya Hospital,Changsha, HUNAN, China

Introduction: The peptidylarginine deiminase (PAD) family consists of five isozymes (PADs 1, 2, 3, 4, and 6), which convert arginine into citrulline through protein citrullination. Our recent studies demonstrate that a pan-PAD inhibitor can improve survival in a rat model of hemorrhagic shock (HS). However, the impact of specific PAD isozyme inhibition in improving survival has not been well studied. In this study, we sought to determine whether selective knockout (KO) of PAD2 could improve outcomes in models of HS.

Methods: I) Survival experiment: PAD2 KO and wild type (WT) mice (n=5/cohort) were subjected to lethal HS (55% volume hemorrhage), and survival was monitored for 10 days. II) Mechanistic studies: PAD2 KO and WT mice (n=3/group) were subjected to sub-lethal HS (30% volume hemorrhage), and the animals were euthanized 12 hours (h) later. Serum and tissues were harvested to analyze the inflammatory response and organ damage. Sham (no HS) was used as a control. Survival was assessed using Kaplan-Meier with log-rank testing, while analysis of variance was used for comparisons among groups.

Results:PAD2 KO mice demonstrated significantly better survival (Figure) compared to WT mice (100 vs 0%; P=0.0017). All the WT mice died within the first hour following HS. Serum IL-1β level was significantly decreased in PAD2 KO mice compared to WT mice 12 hours following sub-lethal HS. Furthermore, the degree of acute lung injury (pulmonary neutrophil infiltration and alveolar edema) and intestinal injury (breakdown of the microvilli) was significantly attenuated in the PAD2 KO mice compared to the WT mice (Figure).

Conclusion:This is the first study to demonstrate that knocking out the PAD2 enzyme improves survival in a rodent model of HS, and attenuates the markers of systemic inflammatory response, acute lung injury, and intestinal damage.

 

67.01 Encapsulation Of Arteriogenic Macrophages Enhances Cell Retention And Ischaemic Limb Perfusion

Posted on January 31, 2019

F. E. Ludwinski1, G. Damodaran1, A. S. Patel1, J. Cho1, S. Jayasinghe2, A. Smith1, B. Modarai1  1King’s College London School Of Medicine,School Of Cardiovascular And Medical Sciences,London, London, United Kingdom 2University College London,Department Of Mechanical Engineering,London, LONDON, United Kingdom

Introduction:
Cell therapy has been proposed as a means of improving perfusion of ischaemic limbs. Direct injection of pro-angiogenic cells has, however, shown only modest outcomes in clinical trials, perhaps because of their loss from the site of injection. Here, we investigate alginate microsphere encapsulation as a means of enhancing retention and improving reperfusion of the ischaemic hindlimb.

Methods:
150μm sodium alginate microspheres, containing 108cells/ml of Tie2-expressing murine bone marrow-derived macrophages (eTie2-iBMMs), were generated using a clinical-grade cell encapsulator. Comparisons were made between eTie2-iBMMs and non-encapsulated cells (nTie2-iBMMs), with respect to: cell viability and phenotype; in vitro pro-angiogenic function after angiopoietin-mediated stimulation of Tie2 (VEGF expression and endothelial tubule formation); cell retention (IVIS biofluorescent imaging); and ability to promote revascularisation in the ischaemic murine hindlimb.

Results:
Encapsulation did not affect Tie2-iBMM viability or phenotype up to 7days in vitro, the expression of VEGF, or formation of endothelial tubules (p=>0.05), although stimulated eTie2-iBMMs secreted significantly greater levels of MCP-1 (p=0.0022). Cell retention was significantly enhanced in ischaemic murine hindlimbs treated with eTie2-iBMMs (p=0.0083) and this was associated with significantly greater limb perfusion over 21 days (p=0.0005).

Conclusion:
Alginate encapsulation of pro-arteriogenic macrophages is not detrimental to their viability or phenotype, and increases their retention and regenerative capacity in the ischaemic hindlimb. Translation of this methodology to a clinical setting may enhance the efficacy of novel cell-based therapies for the treatment of CLI.
 

66.10 The Effects of Immunosuppressant Fingolimod (FTY720) for Hypertrophic Scars

Posted on January 31, 2019

A. KONDO1, M. Aoki1, N. Matsunaga1, A. Honda1, Y. Okubo1, R. Ogawa1  1Nippon Medical School,Plastic, Reconstructive, And Aesthetic Surgery,Bunkyo, TOKYO, Japan

Background:Keloids and hypertrophic scars (HSs) are excessive scar formations with chronic inflammation and capillary vasculogenesis. Keloids, in particular, are refractory as they are resistant to treatments including steroids. Fingolimod (FTY 720) is an analog of sphingosine-1-phosphate (S1P). S1P is a lipid mediator, which is involved in inflammatory cell recruitment and angiogenesis. FTY720 is a functional antagonist of S1P receptor 1 (S1PR1), and inhibits sphingosine kinase 1 (SphK1), which produces S1P. We hypothesized that topical application of FTY720 would decrease inflammatory cells in the local region, and inhibit angiogenesis. We examined the effects of topical FTY720 injection on mechanical force-induced scarring.

Methods: C57BL6/J mice (8 weeks of age, male) were used. VECTOR 620 expansion screws (SCHEU, Iserlohn, Germany) were used for biomechanical loading. Two-cm linear full-thickness incisions were made on the dorsum of the mouse and then sutured with 4–0 vicryl. On day 6, the vicryl sutures were removed and loading devices were secured with 5–0 nylon sutures, and mechanical force was applied. On days 8, 10, and 12, intra-cutaneous FTY720 (10μM) or control injections were performed, and the tension on the scar was reapplied. On day 14, the tissues were harvested for analysis.

Results:Compared to the control group, reduction of the scar was observed in the FTY720 group. Histological analysis showed that the size of scar area was significantly decreased in the FTY720 group compared to the control group. Further, CD34 immunostaining was performed and the number of capillary vessels was counted. Significant increase in the number of blood vessels was observed in the FTY720 group compared to the control group. No significant difference was observed in the blood lymphocyte fraction.

Conclusions:FTY720 acts with a unique mechanism that differs from conventional immunosuppressive agents. Topical application of FTY720 is expected to be effective for various inflammatory and allergic diseases and may be a novel therapeutic agent for treating keloids/HSs.

 

66.09 A chemotactic functional scaffold enhances BMP2-induced bone regeneration in a rodent model

Posted on January 31, 2019

D. Khalil1, R. Bakshi1, L. Wang1, S. Zhou1, K. Rezzadeh1, A. Hokugo1, R. Jarrahy1  1David Geffen School of Medicine at UCLA,Department Of Surgery,Los Angeles, CA, USA

Introduction:  Current commontechniques for repairing full thickness calvarial defects include autologous bone grafting and the use of alloplastic implants, both of which have significant limitations. In this study, we investigate a novel alternative approach to bone repair based on tissue engineering principles. Specifically, we examine the ability of nanoscale peptide amphiphile gels (PAs) engineered tocontrolrelease of VEGF to recruit circulating stem cells to a site of bone regeneration and to help facilitate large-scale bone healing by BMP-2. 

Methods:  Chemotactic functional scaffolds (CFS) were fabricated by combining collagen sponges with PAs to which VEGF was bound. The in vitro chemotactic activity of these constructs was evaluated by measuring human mesenchymal stem cell (hMSC) movement across a semipermeable membrane when exposed to the CFS. In vivo,CFS function was assessed by implantation of scaffolds into dorsal subcutaneous pockets in rodentsand analysis of migration of peripherally injected hMSCs to the CFS. Large-scale rodent cranial bone defectswas created. CFS and other control materials were implanted and bone regeneration was evaluated.

Results: Migration of hMSCs through semipermeable membranes was significantly greater in scaffolds exposed to CFS compared to control scaffolds (P<0.05). In vivo chemotaxis was evidenced by migration of circulating DiR-tagged hMSCs to the CFS. Successful bone regeneration was noted in the defects treated with CFS. 

Conclusion: Our observations suggest that this bioengineered construct successfully acts as a chemo-attractantfor circulating hMSCs, likely due to controlled release of VEGF from the CFS. The CFS may play a role in the future design of clinically relevant bone graft substitutes.?

 

66.08 Application of Purified Exosome Product Improved Wound Healing In Ischemic Wounds: A Rabbit Model

Posted on January 31, 2019

M. F. Bowers1,2, T. C. Huang1, M. D. Sabbagh1, S. L. Moran1  1Mayo Clinic,Rochester, MN, USA 2Meharry Medical College,Nashville, TN, USA

Introduction: Exsomes are extracellular vesicles that are secreted by cells and execute intracellularcommunication1. Exosomes convey cell communication through the export of their contents. Exosomes are known to contain tissue-specific mRNA, miRNA and proteins. Their ability to transfer these contents has been implicated in physiological processes including stem cell renewal, tumor formation, and inflammation reduction2. One of the standard therapies for advanced and tunneled wounds is application of Integra flowable wound matrix [Integra LifeSciences Corporation, NJ, USA] and a subsequent wound vacuum. Previous literature has shown that PEPs can accelerate cutaneous wound healing by upregulating blood vessel and collagen synthesis. However, the effect of PEPs in ischemic or tunneled cutaneous wounds has yet to be examinated in depth. The aim of this study is determine the effectivenss of purified exosome product (PEP) in healing full-thickness and ischemic wounds. 

Methods: 10 New Zealand White rabbits were used for this study and randomly distributed into thee groups:  Control (Saline + Integra, n=3), 5% PEP +Integra (n=4), and 20% PEP + Integra (n=3). Each underwent a surgical creation of an ischemic wound in the right ear while under general anesthesia. Two of the three ear neurovascular bundles were dissected and ligated within the ear. A circumferential skin tunnel was created and a 2cm full thicknesss wound was created by punch biopsy. Integra with either saline or PEP was applied on the wound for complete coverage and sealed with a Tegaderm film (3M). The incisions were closed with sutures and the ear was wrapped in a light dressing. Wound tissue samples were collected after three weeks and wound healing was analyzed via analyzed histologically and through 3D scanning.  Gene Expression profiles for each wound were quantified via reverse transcriptase real-time PCR. 

Results: Control wounds were indurated while all of treated wounds (n=7) showed less induration and inflammation on observation compared to the control group. Analysis from RT-qPCR data showed that the 5% PEP group had up-regulation of TGF-B, TNF-A, CTGF, and PDGF and down-regulation of COL-1 and FGF-1 compared to the control group. The 20% PEP group had down-regulation of BMP-2, COL-1, TNF-A, CD3, CD31, and TGF-B among others. There was no up-regulation of any genes studied in the 20% compared to control. None of the Control wounds showed evidence of epidermal regeneration of histological analysis while half of the 5% wounds, and all of the 20% wounds showed epidermal regeneration. 

Conclusion: PEP use in conjunction with Integra matrix correlated with morphological improvement in ischemic wound healing vs Integra alone. Along with that, wounds treated with PEP showed gene expression profiles indicative of more mature wound healing. Also, data shows that PEP may enhance epidermal regeneration of ischemic wounds and increase the capillary density of these wounds through an unknown mechanism.    
 

66.07 A Mouse Model of Radiation-Induced Limb Contracture which is Rescued by Fat Grafting

Posted on January 31, 2019

M. R. Borrelli1, D. Irizarry1, R. E. Brewer1, R. Patel1, C. Blackshear1, J. Sokol1, A. Momeni1, M. T. Longaker1, D. C. Wan1  1Stanford University,Plastic Surgery,Palo Alto, CA, USA

Introduction Radiotherapy (RT) is extremely effective at shrinking tumor size and reducing local cancer recurrence but causes significant damage to the healthy soft tissue in the radiation field. This collateral soft tissue fibrosis can cause, in addition to cosmetic disturbances, significant functional alterations such as contracture of extremities. Fat grafting is increasingly recognized as a technique able to prevent or reverse the radiation-induced fibrosis in the skin, and clinical reports have demonstrated functional improvements for patients following RT for both breast and head and neck cancer. Animal models to study this effect, however, are limited.

Methods We irradiated the right hind limbs of Prrx1Cre/mTmG mice, a transgenic mouse where the fibrogenic subpopulation of fibroblasts in ventral skin (Prrx1+) are labelled by embryonic expression Cre. Mice received 30 Gy fractionated in 5 Gy doses every two days for a total of 12 days. After a one-month recovery period followed to allow contracture to develop, the mice were sacrificed, and hind limbs were processed for histology. To explore the therapeutic effects of fat graft grafting, CD-1 nude mice were irradiated using the same dosing regimen. At one-month post recovery, the irradiated hind limbs were injected with 200ul of human lipoaspirate fat grafts or lipoaspirate with enriched SVFs (stromal vascular fraction cells, 10,000 cells/ml) into the subcutaneous space. Lipoaspirate was obtained from healthy female donors (n=3).  Control mice were injected with saline or received sham surgery with no injection. Limb extensibility was measured every two weeks for a total of 12-weeks. Mice were then sacrificed, and hind limbs were processed for histology. Finally, hind limb skin underwent mechanical strength testing (MST).

Results: Hind limb irradiation significantly reduced limb extensibility (Fig1A). These functional differences were associated with a significant increase in the fibrogenic fibroblast subpopulation (Fig1B). Fat grafting progressively improved limb extension (Fig1C) and reduced skin stiffness; the greatest benefit was observed in the mice who received fat supplemented with SVFs, with little or no benefit seen in mice who received saline or sham treatment.

Conclusion: We have created a mouse model of radiation-induced hind limb contracture which allows for detailed dissection of the interaction between the transfer of autologous transferred fat and radiation-induced soft tissue contracture. Using this model, we have demonstrated radiation-induced functional hindlimb contractures to be associated with increased fibrogenic subpopulations within the dermis. Transfer of fat also resulted in improved limb extension and reduction in stiffness.

 

66.06 An Analysis of the Growth Factors Involved in Accelerated Wound Healing Due to Platelet-Rich Plasma

Posted on January 31, 2019

G. Garg1,2, B. C. Carney1,3, B. J. Browne1, L. T. Moffatt1,3, J. W. Shupp1,2,3,4  1MedStar Health Research Institute,Firefighters’ Burn & Surgical Research Laboratory,Washington, DC, USA 2MedStar Washington Hospital Center,The Burn Center,Washington, DC, USA 3Georgetown University School of Medicine,Department Of Biochemistry,Washington, DC, USA 4Georgetown University School of Medicine,Department Of Surgery,Washington, DC, USA

Introduction: Rapid wound closure to maintain a barrier against environmental insult is critical, and is especially relevant to the surgical field. Platelet-Rich Plasma (PRP) is an autologous suspension that acts as a “biobandage” to contribute to positive healing. The mechanism is thought to be the release of concentrated growth factors, including VEGF, TGFβ1, and FGF2, upon platelet activation. Platelets also contain polyP, a phosphate polymer which has been shown to have effects on wound healing, coagulation, and bone remodeling. In this experiment, PRP was studied as a means for accelerating wound healing in acute excisional wounds. Furthermore, the effects of individual growth factors on wounds treated with PRP were measured.

Methods: PRP was created by multiple rounds of centrifugation of citrated whole blood, and platelet concentration was confirmed via Wright-Giemsa staining. 6 mm punch biopsies were used to create two wounds on the dorsum of C57BL/6 mice. Splints were placed on the wounds to allow healing by re-epithelialization instead of contraction. Mice were divided into PRP-treated and untreated groups. PRP was activated by CaCl2 and recombinant thrombin to form a gel for application. Photos of wounds were taken prior to necropsy on post-injury days 3, 5, and 7, and whole wounds were fixed in formalin. Open wound areas were quantified using Image J to assess healing rate. Fixed wounds were stained with H&E and Cytokeratin 16 and were examined histomorphometrically for re-epithelialization. Immunofluorescent staining was used to quantify the expression of VEGF, TGFβ1, and FGF2 in the wounds collected on post-injury day 3. 5 images were obtained from tissues adjacent to the edge of the healing epithelium from each wound and the Cy3 fluorescence intensity was quantified using Zen Imaging Software.

Results: PRP-treated wounds re-epithelialized faster compared to untreated wounds when wound photos were analyzed at days 3 and 5 (n = 6 wounds, p < 0.05). Open wound areas were smaller in treated wounds when H&E and Cytokeratin 16 sections were analyzed. None of the growth factors investigated showed significantly different fluorescence intensity between treated and untreated groups (VEGF: 867.0 ± 292.1 vs. 518.9 ± 5.48, p = 0.1750; TGFβ1: 1453 ± 222.3 vs. 1491 ± 192.7, p = 0.8534; FGF2: 663.5 ± 182.7 vs. 503.6 ± 218.0, p = 0.4484).

Conclusion: Due to its autologous nature, PRP is a safe and efficacious option for accelerating wound healing. Previous work has demonstrated that polyP can accelerate healing in an in vitro keratinocyte scratch model. Our data shows that concentrated growth factors found in PRP do not exert a significant effect early in wound healing, suggesting that polyP may be primarily responsible for the accelerated healing seen here. Future work will analyze the effect that spiking PRP with an excess amount of polyP has on wound healing.

66.05 Human Abdominal Adhesion Fibroblasts Upregulate PDGFRa Surface Expression and EMT Pathway Genes

Posted on January 31, 2019

D. S. Foster1,2, C. D. Marshall1,2, G. Gulati1, R. E. Jones1, A. Titan1,2, A. Nguyen1, A. Salhotra1, R. C. Ransom1, S. Mascharak1, R. E. Brewer1, J. A. Norton1,2, C. J. Kin2, A. A. Shelton2, M. T. Longaker1  1Stanford University,Hagey Laboratory For Pediatric Regenerative Medicine,Stanford, CA, USA 2Stanford University,General Surgery,Palo Alto, CA, USA

Introduction: Intra-abdominal adhesions are a common cause of small bowel obstruction after surgery and cost the U.S. health care system $5 billion annually. Fibroblasts are activated in the context of abdominal surgery and deposit fibrotic scar tissue. Despite the prevalence of adhesions, no effective therapeutics exist for prevention or treatment.

Methods: Human adhesion tissues, along with control peritoneum from patients without surgical history, were harvested from patients undergoing elective operations at Stanford Hospital (Fig. 1A). Adhesion-forming and quiescent fibroblasts were fluorescence-activated cell sorting (FACS)-isolated using an unbiased, lineage-negative approach (Fig. 1B-C). These cells were screened for expression of known fibroblast surface markers. Bulk RNA-seq was conducted; gene expression was compared between adhesion and control cohorts, as well as with bulk RNA-seq data acquired from mouse abdominal adhesions and control specimens. Adhesion specimens were fixed, sectioned and stained. Histological data was compared with control and mouse tissue specimens. All experiments were approved by Stanford University’s IRB and APLAC, as appropriate. 

Results: Human adhesion-forming fibroblasts show significantly increased cell surface expression of PDGFRa compared with non-adhesion fibroblasts (Fig. 1D). PDGFRa is also highly-expressed on the surface of adhesion-forming fibroblasts in mice. Histologically, PDGFRa+ fibroblasts appear to closely associate with collagen fibers (Fig. 1E). Adhesion fibroblasts also express the cell surface marker CD26 in both human (Fig. 1D) and mice. These markers are also associated with scar-forming fibroblasts in skin. Bulk RNA-seq of human adhesion fibroblasts shows significant upregulation of Col24a1 expression compared to control, which is a member of the collagen gene family involved in type 1 collagen regulation and is also highly expressed by mouse adhesion fibroblasts. In addition, multiple genes involved in the epithelial-mesenchymal transition (EMT) pathway are upregulated by both human and mouse adhesion fibroblasts (eg. SPP1, SDC1, Comp, CDH2) (data not shown). These present potential therapeutic targets.

Conclusion: This research identifies PDGFRa-expressing fibroblasts as major contributors in abdominal adhesion formation and provides in depth tissue level and gene expression characterization of adhesion fibroblasts in humans. These data provide insight into key signaling processes responsible for adhesion formation as well as identifying a set of potential therapeutic targets common to human and mice. These targets can be validated preclinically and suggest promise for the development of therapeutics that could change the course of this challenging disease.
 

66.04 DEL-1 Interacts with Integrins of BCSPs to Promote Fracture Healing

Posted on January 31, 2019

D. M. Struck1, T. V. Boyko1, R. K. Mann4, M. T. Longaker4, C. K. Chan4, G. Yang1,2,3  1Stanford University,Department Of Surgery,Palo Alto, CA, USA 2University Of Alabama at Birmingham,Department Of Surgery,Birmingham, Alabama, USA 3Birmingham VA Medical Center,Birmingham, ALABAMA, USA 4Stanford University,Stanford University School Of Medicine,Palo Alto, CA, USA

Introduction:  Of the millions of people who suffer a bone fracture every year, 5-10% will have delayed or impaired healing leading to major economic and quality of life burdens. We have shown the developmentally-regulated endothelial cell locus 1 protein (DEL-1) acts as a growth factor for mouse Bone Cartilage Stroma Progenitors (BCSPs), the skeletal progenitor population most important in fracture healing. DEL-1 is a modular protein with three EGF like repeats, two discoidin domains and an RGD motif. Deletion of Del-1 leads to mice that heal fractures with decreased bone. Based on this phenotype and the presence of an RGD motif, we hypothesized that DEL-1 interacts with integrins at this binding site to promote BCSP proliferation to the fracture callus during bone healing.

Methods:  Polymerase chain reaction mutagenesis was used to create a mutant Del-1 DNA construct where the RGD (Arg-Gly-Asp) sequence was substituted with RAD (Arg-Ala-Asp). Both DEL-1 wildtype (WT) and DEL-1 RAD mutant (Mut) was produced using a baculovirus expression system, and purified with immobilized metal affinity and size-exclusion chromatography. Del-1 knockout mice underwent mid-femoral fractures with internal fixation followed by local application of hydrogels loaded with DEL-1 WT,  DEL-1 Mut or blank controls. Additional controls were wild type mice treated with blank hydrogel. BCSPs were isolated from the fracture callus 7 days after fracture using FACS. Untreated Del-1 knockout BCSPS were also plated on 24 well plates coated with DEL-1 WT, DEL-1 RAD Mutant or BSA control. Proliferation was measured every day for a week.

Results: The addition of DEL-1 WT to Del-1 knockout mice resulted in increased callus formation compared to control (p<0.05). This was correlated with increased BCSPs at the fracture callus (p< 0.05). In vitro, the addition of DEL-1 WT led to increased proliferation of BCSPs (p< 0.05). In contrast, the addition of DEL-1 Mut had no effect on either the size of the fracture callus or the number of BCSPs at the fracture site. In vitro, there was no impact on proliferation of DEL-1 Mut on BCSPs.

Conclusion: DEL-1 WT rescued the phenotype of poor fracture healing in the Del-1 knockout mice leading to increased fracture callus size with increased numbers of BCSPs. This was correlated with stimulation of proliferation in vitro. In contrast, the DEL-1 Mut had no impact on fracture callus size, mobilization of BCSPs or proliferation of BSCPs. Based on the known function of the RGD motif, we conclude that DEL-1 affects BCSPs through binding of integrins using the RGD motif.

 

66.03 Nerve-dependent progenitor expansion is critical in post-injury mandibular repair

Posted on January 31, 2019

R. Jones1, A. Salhotra1, C. Ransom1, D. Foster1, T. Chari1, K. Robertson1, D. Wan1, M. T. Longaker1  1Hagey Laboratory for Pediatric Regenerative Medicine,Division Of Plastic And Reconstructive Surgery, Department Of Surgery,Palo Alto, CA, USA

Introduction:
We have previously established a mouse model of inferior alveolar nerve (IAN) disruption, and demonstrated via fluorescence active cell sorting that IAN denervation impairs skeletal stem cell (SSC) recruitment function after fracture injury.However, the impact of denervation on healing morphology and tissue-specific progenitor activity has yet to be examined.

Methods:
Wild-typemice were subjected to surgical disruption of the right IAN, and two weeks were allowed for complete nerve degeneration. This model of denervation has been validated in our previous studies (data not shown). Using innervated mandibles as a control, mice underwent unicortical osteotomy on the buccal surface of the right mandible (Fig 1a). Hemimandibles were harvested 2 weeks after fracture, then sectioned and stained for analysis. The same surgical protocol was enacted on Actin-CreERT2::Rosa26-VT2/GK3 mice, or actin rainbow mice, which house a multicolor Cre-dependent reporter. Upon Cre recombinase activation by tamoxifen, individual cells randomly and permanently express one of four fluorochromes: eGFP (cytoplasmic green), mCerulean (membrane blue), mCherry (membrane red), and mOrange (membrane yellow). Selection of the ubiquitous actin Cre driver permits visualization of clonal expansion of cells, with clones defined as continuous regions of a single color. Denervated and innervated mandibles from actin rainbow mice were harvested two weeks postoperatively after unicortical fracture and mounted whole. They were examined with stereoscopic microscopy and corrected fluorescence intensity of fracture area was computed in ImageJ.

Results:
Denervated fractures healed with gross morphological abnormalities (Fig 1b). Compared to the compact callus formed in innervated bone healing, denervated tissue exhibits large gaps in bone tissue repair. Fluorochrome intensity was measured and compared between innervated and denervated fracture sites in actin rainbow mandibles. The denervated tissue exhibited significantly less fluorescent signal than the innervated samples in green, red, and merge channels (p= 0.0349*, 0.0079**, and 0.0097**, respectively) (Fig 1c). Images of fluorochrome signals are exhibited (Fig 1d), consistent with a striking fault in clonal expansion of denervated fracture healing tissue.

Conclusion:
With our novel mouse model of mandibular denervation, we show that bony healing of denervated mandibular fracture is morphologically aberrant. This defect is paired with lack of clonal expansion into the zone of healing, indicating that impaired stem/progenitor activity is responsible for deficient injury repair in denervated healing. Our data suggest that mandibular fracture healing is enacted via stem/progenitor cells which exhibit a high degree of nerve dependency.
 

66.02 Sex Differences in Murine Myocutaneous Flap Revascularization

Posted on January 31, 2019

J. S. Brandenburg1, R. M. Clark1, B. B. Coffman4, G. Sharma2, H. J. Hathaway3, E. R. Prossnitz2, T. R. Howdieshell1,3  1University Of New Mexico HSC,Surgery,Albuquerque, NM, USA 2University Of New Mexico HSC,Molecular Medicine,Albuquerque, NM, USA 3University Of New Mexico HSC,Cell Biology,Albuquerque, NM, USA 4University Of New Mexico HSC,Pathology,Albuquerque, NM, USA

 

Introduction:   Sex differences in susceptibility to ischemia/reperfusion injury have been documented in humans.  Premenopausal women have a lower risk of ischemic heart disease than age-matched men, whereas after menopause, the risk is similar or even higher in women.  However, little is known about the effects of sex on cutaneous wound revascularization.

Methods:   A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of 10-12 week old male (n=10) and female (n=10) C57BL6 mice.  Planimetric analysis of digital photographic images was utilized to determine flap viability.  Real-time flap perfusion, ischemia, and functional revascularization was determined by laser speckle contrast imaging (LSCI).  Image analysis of CD31-immunostained sections confirmed flap microvascular anatomy, density, and surface area.  Values are expressed as means ± SEM.  Student’s t test was performed when comparing two groups, and two-way ANOVA used when comparing multiple groups.  Statistical significance was accepted at a p value < 0.05.

Results:  Flaps created on female mice were engrafted to the recipient site resulting in nearly complete viability at post-operative day 10.  In contrast, distal full thickness myocutaneous necrosis was evident at 10 days post-surgery in male mice (percent flap necrosis: female 5.8 ± 0.9% versus male 30.8 ± 4.7%, p < 0.05).  Over the 10 day study interval, LSCI documented functional flap revascularization in all regions of interest (ROI) in female mice, but minimal distal flap reperfusion in male mice (day 10 distal ROI perfusion: female 191.5 ± 9.2 PU versus male 138.9 ± 11.7 PU, p < 0.05).  Day 10 histologic sections immunostained to detect CD31 confirmed significant increases in distal flap vessel count and vascular surface area in female compared to male mice (vessel count: female 127 ± 12 vessels/mm2 versus male 38 ± 6 vessels/mm2, p < 0.05; vascular surface area: female 7,899 ± 85 µ2/mm2 versus male 2,335 ± 40 µ2/mm2, p < 0.05).

Conclusions:  In a graded-ischemia wound healing model, flap revascularization was more effective in female mice.  The recognition and identification of sex-specific wound healing differences may lead to a better understanding of the underlying mechanisms of myocutaneous revascularization and drive novel discovery to improve soft tissue wound healing.

 

66.01 Neural Input Modulates Aberrant Cell Fate After Extremity Trauma

Posted on January 31, 2019

C. Hwang1, C. A. Kubiak1, M. Sorkin1, C. A. Pagani1, T. Rehse1, M. A. Garada1, Z. N. Khatib1, P. Kotha1, J. Lisiecki1, D. M. Stepien1, N. D. Visser1, K. Vasquez1, A. W. James1, Y. S. Niknafs1, P. S. Cederna1, S. W. Kemp1, B. Levi1  1University Of Michigan,Surgery,Ann Arbor, MI, USA

Introduction: Heterotopic ossification (HO) is a painful, debilitating formation of ectopic bone, often found after severe polytrauma, burn and neural injury. Literature has implicated neurotrophic signals such as nerve growth factor (NGF) as crucial signals for normal bone development. Additionally,  secreted neural peptides including substance P (SP) have been demonstrated as capable of regulating osteoblast behavior, modulating osteogenic cues, and producing ectopic bone when exogenously introduced. Thus, we hypothesized that innervation is crucial to pathologic stem cell differentiation and resultant HO.

Methods: C57BL/6J male mice were stratified to burn/tenotomy (BT) or BT+neurectomy. The Achilles tendon was bisected and accompanied by 30% TBSA dorsal burn. Sciatic/sural nerves were transected at midthigh (proximal/distal to bifurcation, respectively). Hindlimbs were analyzed via µCT at 9 weeks (n=3-5/group). Sections (n=1) from the ankle were immunolabeled (IF) at 1 and 3 weeks. Myeloperoxidase (MPO) activity was measured via in vivo imaging system at the ankle at 4 days (n=4-5/group). From BT mice, mRNA was harvested from injured and uninjured tendon (n=3/group) and hybridized to Affymetrix microarray (1 week) or processed for whole transcriptome via RNAseq (3 weeks). Microarray data of human ligament cells from GEO dataset GSE5464 were analyzed using linear modeling with empirical Bayes method for differential expression.

Results: Sciatic neurectomy upon BT significantly reduced total HO (Fig A, 4.7 v. 1.57mm3, p=0.036). IF imaging of NGF and SP showed robust protein expression at 1 week with limited colocalization of F4/80 or Ly6G (Fig. B,C). Neurectomies did not cause changes in MPO levels (Fig. D). BT (Fig. E, right) induced upregulation of characteristic genes of inflammation (Il6, Ptgs2, Ptger1, Il1a, Tacr1) vs. ininjured tendon at 1 week (left). Similar upregulation was observed at 3 weeks (Fig. F). Notable neurotrophins, Ngf, Gdnf, and Brdf, were also upregulated.  GEO data exhibited parallel trends of Ngf and Brdf in human spinal ligament cells subjected to cyclic strain (Fig. G); a loading shown to be associated with ossification of connective tissues.

Conclusion: Interruption of innervation to an injury site inhibits post-traumatic ossification independent of myeloid cell infiltration (MPO) during the acute response. In mice, BT induces upregulation of neurotrophin genes in both acute and sub-acute timepoints, concordant with upregulation seen in strained human cells. BT exhibited robust labeling of NGF, spatially distinct from macrophages and PMNs, along with SP, a well characterized regulator of osteogenesis. This data suggests neural signals modulate aberrant wound healing as demonstrated by HO formation.

65.10 Transcription Factor Profiling Identifies Mediators of Invasion in Follicular Thyroid Cancer

Posted on January 31, 2019

N. G. Nicolson1, J. Paulsson2, C. Juhlin2, R. Korah1, T. Carling1  1Yale University School Of Medicine,Department Of Surgery,New Haven, CT, USA 2Karolinska Institute,Solna, STOCKHOLM COUNTY, Sweden

Introduction:  Follicular thyroid cancer (FTC) is generally indolent with low risk of recurrence or death, but some cases are of encapsulated angio-invasive (eaFTC) or widely invasive (wiFTC) histological subtypes, portending a significantly worse prognosis. The mechanism and associated biomarkers of this invasive behavior have not been fully elucidated.

Methods:  Tissue samples including minimally invasive FTC (miFTC), eaFTC, and wiFTC tumors, as well as histologically normal thyroid adjacent to benign follicular adenomas, were selected from a cohort (n=21) of thyroid tumor patients. Histology was confirmed by experienced endocrine pathologists to designate the invasiveness of each sample as described in the 2017 WHO Guidelines. Total RNA was isolated, reverse transcribed, and subjected to a quantitative PCR array containing 84 transcription factor probes, with relative expression levels determined by a modified Livak method. Genes differentially expressed in invasive FTC were determined. In silico network analysis was performed to highlight involved signaling networks. Immunohistochemistry was performed to assess spatial expression patterns of selected transcription factors.

Results: Of the 84 transcription factors studied, 30 were differentially expressed between FTC and normal, or between invasive subtypes of FTC. E2F transcription factor 1 (E2F1), a ubiquitous transcription factor, was over-expressed in all 3 FTC subtypes (p<0.01, Figure 1). Specificity factor 1 (SP1), previously shown to modulate invasion in breast, prostate, and gastric cancers, was differentially expressed in eaFTC and wiFTC (p<0.05, Figure 1). Transcription factor 7-like 2 (TCF7L2), an established inducer of epithelial-to-mesenchymal transition and associated cancer invasion, was significantly upregulated in widely invasive tumors specifically (p<0.05, Figure 1). Thirteen transcription factors were differentially expressed in eaFTC and wiFTC compared to miFTC, and network interaction analysis suggested a role for Wnt signaling and associated networks in the invasive phenotype. Immunohistochemistry revealed differential expression of Yin and Yang 1 protein (YY1) and Myc-Associated Factor X (MAX) along the tumor invasive front relative to the central tumor, suggesting that invasiveness may be a local phenomenon rather than a property of the tumor at large.

Conclusion: Invasive FTC is rare but has a high risk of recurrence and death relative to minimally invasive FTC. This study identifies differential transcription factor expression associated with invasive subtypes of FTC and identifies dysregulated signaling pathways by investigating transcriptional regulation. Our study lays the groundwork for novel therapies targeting invasion pathways in FTC.

65.09 Histotripsy Tumor Ablation Stimulates Systemic Melanoma-Specific Immune Responses in Mice

Posted on January 31, 2019

S. Qu1,2, A. Kevelin1, M. Toma1, A. Pepple1, A. Felsted1, T. Worlikar3, R. Hubbard3, T. K. Luther1, Z. Xu3, C. S. Cho1,4  1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA 2Xijing Hospital, Fourth Military Medical University,Department Of Hepatobiliary Surgery,Xi’an, SHAANXI, China 3University Of Michigan,Department Of Biomedical Engineering,Ann Arbor, MI, USA 4Ann Arbor VA Healthcare System,Ann Arbor, MI, USA

Introduction: Despite its revolutionary successes, many patients with poorly immunogenic cancers have not been able to reap the benefits of immunotherapy. It has been speculated that tumor-directed therapies like radiation and thermal ablation may be able to enhance the immunogenicity of these refractory cancers to stimulate tumor-specific immune responses. Unfortunately, these therapies disrupt subcellular integrity and molecular activity while having limited effects on antitumor immune responses. A novel non-invasive, non-thermal ablation therapy termed histotripsy (HT) uses acoustic cavitation to disrupt cell membrane integrity while preserving subcellular integrity. Recent studies have shown that immunogenic cell death (ICD), characterized by the release of damage-associated molecular patterns (DAMPs) such as HMGB1, can provoke immune responses against cancer.  Thus, we hypothesize that HT may be uniquely capable of stimulating a significant anti-tumor immune response.

Methods: We inoculated C57BL/6 mice with B16 melanoma cells unilaterally or bilaterally. Radiation therapy (XRT), radiofrequency ablation (RFA), or HT ablation was performed on day 10 after tumor inoculation, and tumor growth was monitored every 3 days. Tumor, spleen, inguinal tumor-draining lymph nodes (TDLN), and axillary non-tumor-draining lymph nodes (NTDLN) were harvested and processed for flow cytometry to assess the immune response on days 3 and 10 after HT treatment. Similarly, tumor and serum were harvested and processed for ELISA to measure HMGB1 on days 1 and 8 after HT treatment.

Results: HT suppressed the growth of treated tumors and resulted in significant increases in CD8+ T cells locally within tumors, but also increased the numbers of tumor-specific CD8+ T cells regionally within TDLN (but not NTDLN), and systemically within the spleen. HT also increased the ratio of CD8+GP33+ lymphocytes to Treg cells in the systemic circulation. The ability of HT to promote intratumoral CD8+ TIL was stronger than irradiation or radiofrequency ablation. Surprisingly, histotripsy also suppressed the growth of untreated, contralateral tumors. In untreated tumors, HT significantly increased CD8+ tumor infiltrating lymphocytes. HT also upregulated circulating NK cells and neutrophils. Moreover, HT resulted in significant increases of extracellular HMGB1 in tumors and serum.

Conclusion: These data suggest, for the first time, that HT is capable of stimulating potent anti-tumor immune responses.  The immunostimulatory effect of HT appears to be much stronger than that of other tumor-directed therapies, and may be mediated by its ability to promote the release of tumor-associated antigen DAMPs like HMGB1.

 

65.08 Human Preadipocytes Promote Pancreatic Cancer Cellular Proliferation in a Diabetes-Specific Manner

Posted on January 31, 2019

L. Delrosario1, C. G. Flesher1, N. A. Baker1, R. W. O’Rourke1, T. Frankel1  1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA

Introduction: Type 2 diabetes mellitus (DM) is a risk factor for pancreatic ductal adenocarcinoma (PDAC) but mechanisms underlying this association remain poorly understood. Multiple reports implicate adipocytes and preadipocytes as putative promoters of PDAC in humans and mice. To test whether preadipocytes or adipocytes regulate PDAC tumorigenic functions in the context of DM, we explored the effects of conditioned media prepared from preadipocytes from nondiabetic (NDM) and diabetic patients on PDAC cells.

Methods:  Preadipocytes were isolated from omental adipose tissue obtained from obese NDM and DM patients undergoing bariatric surgery. Serum-free conditioned media was prepared from these preadipocytes cultured in vitro for 72 hours. Sphere assays using T3M4 and Capan1 PDAC lines were performed in the presence or absence of conditioned media, and sphere surface area was measured over 2-3 weeks. Modifications of the sphere assays, including boiling of conditioned media and addition of neutralizing antibodies, was performed. A proteome profiler array was used to identify differences in chemokine expression between DM and NDM preadipocytes.

Results: Conditioned media from human DM preadipocytes, compared to media from NDM preadipocytes, increased PDAC cell sphere surface area in both T3M4 and Capan1 cell lines. Conditioned media from mature differentiated human adipocytes did not regulate PDAC cell sphere area. Sphere surface area correlated directly with serum hemoglobin A1c (HbA1c) but not gender or body mass index of the human subjects from which preadipocytes were derived. Boiling of preadipocyte supernatants abrogated their pro-proliferative effects, suggesting a peptide or protein mediator, and ultracentrifugation of preadipocyte supernatants localized activity to the non-exosome fraction. Proteome profiler array analysis identified different proteins that were differentially expressed by DM and NDM preadipocytes, among which chitinase-3-like protein 1 (CHI3L1) was increased over 2-fold in DM relative to NDM preadipocyte supernatants. Neutralizing antibody to human CHI3L1 attenuated the increase in PDAC sphere surface area in response to DM preadipocyte conditioned media in a dose-dependent manner.

Conclusion: Preadipocyte-PDAC cell crosstalk regulates PDAC cell proliferation in a DM-specific and CHI3L1-dependent manner. These results suggest that the positive clinical correlation between DM and PDAC may be mediated by preadipocytes expressing higher levels of CHI3L1. Preadipocytes and CHI3L1 represent targets for further research directed towards developing novel therapies for PDAC based on manipulation of the tumor microenvironment.

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