Inguinal Fat Pads Research Articles

Comparison of Soluble and Liposome Encapsulated, Sustained Release Latanoprost for Focal Adipose Reduction.

Background: To address the lack of non-cytotoxic, non-surgical options to treat undesirable focal adiposity of the face, we propose use of the anti-glaucoma medication and prostaglandin F2α analogue latanoprost, which has a well-described side effect of periorbital adipose shrinkage. Objective: To evaluate the safety and efficacy of soluble and liposomal latanoprost for focal fat reduction. Approach: To compare efficacy, single administrations of either the FDA-approved cytolytic drug deoxycholic acid (DOCA), latanoprost, or liposomal latanoprost were injected into ob/ob mouse inguinal fat pads. Study outcomes included mouse weight, inguinal fat pad volume, architecture, and cytotoxicity. Results: Both DOCA and soluble latanoprost significantly reduced inguinal fat pad volume whereas liposome encapsulation reduced inguinal fat pad volume insignificantly over the 14-day study period. Hematoxylin and eosin demonstrated effective reduction in adipocyte volume without histologic evidence of cytolysis or inflammation whereas DOCA caused dermal ulcerations, adipocyte lysis, and increased tissue inflammation. Conclusion: Latanoprost reduced fat volume without inducing cell lysis or inflammation.

RF10 | PMON205 LH/CG Receptor Activation Protects Mice from Diet-Induced Obesity and Modifies Adipose Tissue Immune Response

Abstract Menopause is associated with the loss of LH ovulatory surges and enhanced visceral adiposity. Visceral fat depots increase from 5-8% at premenopause to 15-20% of total body fat at postmenopause. Here, we report that high-dose LH, hCG, or small molecule LH/CGR agonist ORG43553 injected twice-a-week into 14-weeks-old C57BL/6 male mice protects them from diet-induced obesity. Testosterone levels were elevated in mice treated with LH or hCG, but not with ORG43553. Notably, the anti-obesity action of LH/hCG is independent of testosterone, as blocking the androgen receptor using flutamide yielded similar results. Importantly, male Lhcgr knockout mice on a high-fat diet treated with LH failed to display a reduction in adiposity, confirming the in vivo specificity of action. Furthermore, our data phenocopied Lhcgr haploinsufficiency in mice. We confirmed the presence of Lhcgr in mouse genital and inguinal fat pads, adipose-derived stromal vascular cells, as well as in differentiated and undifferentiated 3T3-L1 murine adipocytes by qPCR, RNAscope in situ hybridization, and immunohistochemistry. Sanger sequencing showed that the extracellular domain of Lhcgr in genital fat depot was identical to the ovarian receptor. Similarly, we identified LHCGR in human subcutaneous and visceral fat depots. Binding of intraperitoneally injected AlexaFluor-488-labeled hCG was found not only in mouse ovary, but also in genital and subcutaneous fat pad, further confirming the presence of LHCGR in adipose tissue. This binding could be competitively displaced in 3T3-L1 cells using unlabeled hCG. LH, hCG and ORG43553 activated ERK1/2 in a dose-dependent manner in undifferentiated and differentiated 3T3-L1 cells, suggesting that the adipose LHCGR is fully functional. LH, hCG, and ORG43553 reduced adipogenic differentiation in 3T3-L1 cells, which is further confirmed by RNA sequencing. Moreover, we observed, that LH and hCG also alters several aspects of immune response in adipose tissue, including inflammatory response and adaptive immunity. In conclusion, we demonstrated that LH/CG receptors are present and fully functional in adipose tissue, and that high-dose intermittent activation of LHCGR in mouse fat depots protects mice from diet-induced obesity and modifies adipose tissue immune response. Presentation: Saturday, June 11, 2022 1:42 p.m. - 1:47 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Mechanical Stress Improves Fat Graft Survival by Promoting Adipose-Derived Stem Cells Proliferation.

Cell-assisted lipotransfer (CAL), defined as co-transplantation of aspirated fat with enrichment of adipose-derived stem cells (ASCs), is a novel technique for cosmetic and reconstructive surgery to overcome the low survival rate of traditional fat grafting. However, clinically approved techniques for increasing the potency of ASCs in CAL have not been developed yet. As a more clinically applicable method, we used mechanical stress to reinforce the potency of ASCs. Mechanical stress was applied to the inguinal fat pad by needling. Morphological and cellular changes in adipose tissues were examined by flow cytometric analysis 1, 3, 5, and 7 days after the procedure. The proliferation and adipogenesis potencies of ASCs were evaluated. CAL with ASCs treated with mechanical stress or sham control were performed, and engraftment was determined at 4 weeks post-operation. Flow cytometry analysis revealed that mechanical stress significantly increased the number as well as the frequency of ASC proliferation in fat. Proliferation assays and adipocyte-specific marker gene analysis revealed that mechanical stress promoted proliferation potential but did not affect the differentiation capacity of ASCs. Moreover, CAL with cells derived from mechanical stress-treated fat increased the engraftment. Our results indicate that mechanical stress may be a simple method for improving the efficacy of CAL by enhancing the proliferation potency of ASCs.

Latent transforming growth factor β binding protein 3 controls adipogenesis

Transforming growth factor-beta (TGFβ) is released from cells as part of a trimeric latent complex consisting of TGFβ, the TGFβ propeptides, and either a latent TGFβ binding protein (LTBP) or glycoprotein-A repetitions predominant (GARP) protein. LTBP1 and 3 modulate latent TGFβ function with respect to secretion, matrix localization, and activation and, therefore, are vital for the proper function of the cytokine in a number of tissues. TGFβ modulates stem cell differentiation into adipocytes (adipogenesis), but the potential role of LTBPs in this process has not been studied. We observed that 72 h post adipogenesis initiation Ltbp1, 2, and 4 expression levels decrease by 74–84%, whereas Ltbp3 expression levels remain constant during adipogenesis. We found that LTBP3 silencing in C3H/10T1/2 cells reduced adipogenesis, as measured by the percentage of cells with lipid vesicles and the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ). Lentiviral mediated expression of an Ltbp3 mRNA resistant to siRNA targeting rescued the phenotype, validating siRNA specificity. Knockdown (KD) of Ltbp3 expression in 3T3-L1, M2, and primary bone marrow stromal cells (BMSC) indicated a similar requirement for Ltbp3. Epididymal and inguinal white adipose tissue fat pad weights of Ltbp3−/- mice were reduced by 62% and 57%, respectively, compared to wild-type mice. Inhibition of adipogenic differentiation upon LTBP3 loss is mediated by TGFβ, as TGFβ neutralizing antibody and TGFβ receptor I kinase blockade rescue the LTBP3 KD phenotype. These results indicate that LTBP3 has a TGFβ-dependent function in adipogenesis both in vitro and possibly in vivo. SignificanceUnderstanding the control of mesenchymal stem cell fate is crucial for the potential use of these cells for regenerative medicine.

Adipose-derived mesenchymal stem cells reduced transient cerebral ischemia injury by modulation of inflammatory factors and AMPK signaling

Stem cell-based treatments have been recommended as a feasible therapy for stroke victims due to their potential for angiogenesis, neurogenesis, and synaptic plasticity. The intracellular mechanisms of stem cells against cerebral hypoperfusion are not well recognized. In this study, by releasing the clips, the reperfusion period was extended to 96 h, and two hours after cerebral ischemia, animals received adipose-derived MSCs. MSCs were isolated from the inguinal fat pads of rats and injected into two-vessel occlusion (2VO) rats 1 h after ischemia induction. Ninety-six hours after 2VO induction, behavioral and molecular tests were assessed. Adipose-derived MSCs treatment improves neurological scores, passive avoidance memory, and novel object recognition tests in the 2VO model compared to 2VO rats (P < 0.001). MSCs treatment decreased TNF-α (P < 0.01) and IL-6 (P < 0.01) and apoptotic factors (Bax/Bcl-2 ratio and caspase-3 level (P < 0.01)) compared with ischemic rats. MSCs treatment of ischemic rats could enhance Klotho-α and AMPK-α compared with ischemic rats (P < 0.001). The study disclosed that adipose-derived MSCs could improve neurological damage and memory deficits by reducing neuronal death in cerebral ischemia. Data proposed that adipose-derived MSCs inhibit pro-inflammatory factors such as IL-6 and TNF-α, consequently decreasing apoptosis in the hippocampus of CCAO rats. Besides, the Klotho-α and AMPK-α measurements found that MSCs might induce intracellular neuroprotective pathways via activation of Klotho-α/AMPK-α signaling.

QS42. Closing The Gap on Clinical Translation of Cell-Based Therapies: A Novel Technique For Intraoperative Isolation of the Stromal Vascular Fraction for Bone Tissue Engineering

Purpose: Adipose-derived stems cells (ASC) have demonstrated promise across many areas of regenerative medicine, including bone tissue engineering. One major barrier to clinical translation and broad application of these therapeutics is current protocols which require laboratory cell culture and enzymatic digestion to produce stem cell rich product. More optimal methodologies would circumvent the current protocols, and instead, employ mechanically-based techniques to produce ASC rich stromal vascular fraction (SVF) intraoperatively for immediate clinical application. Therefore, the purpose of this study was to develop a clinically translatable technique for intra-operative harvest, isolation, and implantation of SVF with the primary aim of enhancing bone healing at irradiated fracture sites. Methods: Male Lewis rats (n=29) were divided into groups: Fracture (Fx), Radiation with Fracture (XRT), and Radiation with Fracture and SVF implantation (SVF). Experimental groups received 35Gy of radiation. All groups underwent mandibular osteotomy and external fixation. Inguinal fat pads were minced and serially processed using Tulip Sizing Transfers (2.4mm, 1.4mm, 1.2mm). Serial filtration (800 micron, 400 micron) and centrifugation was performed. The resultant oil and aqueous layers were discarded and the cell pellet was collected for immediate implantation at the osteotomy site. Animals were sacrificed on post-operative day 40. Gross pathology and MicroCT analysis were utilized to determine union rates and the quality of the bone formed at the osteotomy site. Biomechanical strength testing was performed until failure to evaluate yield and ultimate load of new bone at the osteotomy site. Results: Immediate implantation of SVF increased union rates compared to XRT alone (79% vs. 20%). Additionally, MicroCT analysis demonstrated high quality new bone formation in irradiated fractures treated with SVF compared to the control based on bone mineral density (666.2 ± 32.0 vs. 312.2 ± 51.7; p=0.000) and bone volume fraction (0.744 ± 0.072 vs. 0.350 ± 0.041; p=0.000). In fact, implantation of SVF into irradiated fracture sites resulted in bone quality similar to the bone formed at non-irradiated fracture sites, as there was no significant difference found between groups (BMD: 666.2 ± 32.0 vs. 710.3 ± 38.0; p=0.390, BVF: 0.744 ± 0.072 vs. 0.803 ± 0.04; p=0.300). Radiation significantly diminished the biomechanical properties of bone, including yield (23.6 ± 28.2 vs. 81.9 ± 31.3; p=0.002) and ultimate load (33.7 ± 30.9 vs. 87.3 ± 26.7; p=0.005). SVF implantation improved yield (52.0 ± 28.6 vs. 81.9 ± 31.3; p=0.161) and ultimate load (33.7 ± 30.9 vs. 87.3 ± 26.7; p=0.145) of the irradiated bone to the level of the non-irradiated control, as there was no significant difference between groups. Conclusions: Use of the stromal vascular fraction for bone tissue engineering demonstrates great potential, including applications in irradiated fracture healing. In this study, we developed a novel approach that eliminates laboratory dependent techniques and instead, utilizes mechanical methods that would enable intraoperative SVF harvest, isolation, and immediate implantation. While further studies are required to optimize this approach, the results of this study are incredibly promising for the long-awaited translation of cell-based therapeutics into the clinical arena.

QS34. Prostaglandin F2a Analog Focally Depletes Adipose Volume Without Concurrent Cytolysis

Purpose: Adipose is one of the most mutable and commonly treated tissues in plastic surgery. Adipose tissue is often the thickest and most variable of the cutaneous layers and is a key contributor to smooth contour, feature, and expression. Unwanted focal adiposity is a common complaint and can arise physiologically, pathologically as lipomas, or iatrogenically after fat grafting. While some indications are amenable to liposuction or resection, not all patients want invasive procedures. Non-invasive ablative procedures have arisen to fill this need including cryolipolysis and the injectable cytolytic deoxycholic acid (Kybella). This agent, however, caries significant comorbid risk. In this study, we investigated repurposing a potent FDA approved prostaglandin F2α (PGF2a) analogue, Latanoprost, currently used as first-line treatment for glaucoma, as an alternative adipolytic agent for small volume focal adipose reduction. Methods: Human adipose obtained under IRB exemption was incubated for 14-days and treated with Kybella and either low- or high-dose soluble Latanoprost every 48-hours. Mature adipocytes and the stromal vascular fractions were collected via enzymatic dissolution and size, count, morphology, and viability were assessed using Calcein-AM/PI imaging. Additionally, single administration of Kybella (500µl) or Latanoprost (1.5 or 150mcg) was performed in vitro to murine fat pads and maintained 2-weeks. These samples were harvested and evaluated via H&E, Pentachrome to assess inguinal fat volume, and fibrotic tissue architecture. Samples were further interrogated via immunofluorescence against Perilipin, Hypoxia Inducible Factor 1-alpha (Hif1a), Uncoupling Protein 1 (UCP1), and hexokinase 2 to assess the variable contributions of Latanoprost therapy to white/brown adipose ratio, focal hypoxia, and metabolic flux. Results: Incubation of lipoaspirate with Kybella resulted in total tissue dissolution and loss of viability. In contrast, Latanoprost did not negatively affect viability of stromal cells or adipocytes. In vivo, both 150 mcg Latanoprost and Kybella significantly decreased inguinal fat pad volume at 2-weeks. Kybella treatment resulted in consistent dermal lesions and histologic evidence of inflammation that were not present in Latanoprost treatment. Furthermore, Latanoprost and vehicle-control treated samples did not significantly vary in presence of fibrotic collagen, Hif1a activation, or presence of UCP1-positive brown fat. Conclusion: Single administration of soluble Latanoprost safely reduced fat pad volume at two weeks post injection in vivo with no ex vivo loss of viability in human tissues. This supports a potential application for reduction in focal adiposity without undesirable cytolysis and future studies will be conducted to optimize dose and determine duration of the effect.

Correlation between body condition score and body composition in a rat model for obesity research

The incidences of obesity-associated chronic diseases are increasing worldwide. Research into the causes of obesity as well as potential treatments has highlighted the crucial role of preclinical studies using animal models. Rats are one of the most widely used species in obesity research. However, even with decades of research in both genetically obese rats and diet-induced obese rat models, definitive criteria to practically classify levels of obesity in the rat are not well established. The current study proposes new criteria modified from a 5-point body condition score (BCS) using in an animal health monitoring system and added a half-point scale to extend the range of body weight associated with subcutaneous fat deposition. The modified criteria were tested and compared with body composition from dual energy X-ray absorptiometry scans and selected adipose tissue weights. The results showed that the modified body condition scale was highly correlated with fat deposition in the rat body, particularly the visceral and inguinal fat pads. Both pads were closely related to changes in some specific landmarks used for the scale determination. These finding should extrapolate to obese rats in other models, with the advantage that data classified in BCS can pair the animal data with human body mass index. This will enhance the value of information from preclinical studies to design and predict outcomes of subsequent human clinical trials.

Abstract 1350: The role of phosphatidylserine scramblases in tumor progression

Abstract Introduction: Phosphatidylserine (PS), an anionic phospholipid, is asymmetrically distributed in the inner leaflet of the phospholipid bilayer in normal cells under homeostasis. However, PS is constitutively externalized in the tumor microenvironment (TME); on viable cells that undergo stress, such as hypoxic tumor cells, stressed tumor vascular endothelial cells, as well as on cells undergoing apoptosis. This high PS harbors a pro-tumorigenic micro-environment, as myeloid cells expressing PS receptors (Mertk, Tyro3 and Axl) engage with externalized PS and secrete immunosuppressive cytokines. PS is externalized on viable cells by a calcium activated scramblase TMEM16F and on apoptotic cells by a caspase activated scramblase called Xkr8. Here, to study the mechanisms by which PS is externalized in the TME, we hypothesize that TMEM16F and Xkr8 are the major scramblases responsible for creating an immune suppressive milieu in the tumor microenvironment. Methods: To study the mechanisms of PS dysregulation in the tumor microenvironment, TMEM16F and Xkr8 were knocked out in E0771 murine breast cancer cell line using CRISPR/Cas9. The knockouts were validated by western blotting, q-RT-PCR and surveyor assays. Results: TMEM16F KO E0771 cells express lower basal levels of PS compared to WT E0771 cells when detected by Annexin V staining by flow cytometry. TMEM16F KO cells are also less responsive to activation by calcium ionophore. TMEM16F KO, Xkr8 KO and WT E0771 cells have similar viability and migration patterns in response to Gas6, indicating that the cells themselves do not have altered oncogenic potential. TMEM16F KO E0771 cells injected into the inguinal mammary gland fat pad of C57BL6 syngeneic mice showed modest but significantly reduced tumor growth compared to the E0771 WT tumors, suggesting that TMEM16F mediated PS externalization could play a role in exacerbating tumor progression. Conclusion: TMEM16F driven PS externalization contributes to tumor growth. The effect of TMEM16F KO on immune regulation of the tumor milieu will be discussed. These data corroborate previous studies targeting PS in tumors therapeutically using anti-PS antibodies and suggests a role for scramblases in pleiotropic immune-suppression in the tumor. Citation Format: Varsha Gadiyar, Viralkumar Davra, David Calianese, Kevin Lahey, Raymond B. Birge. The role of phosphatidylserine scramblases in tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1350.

Abstract 2373: The RNA-binding protein HuR mediates impaired adipogenesis in pancreatic cancer cachexia

Abstract Objective: Cachexia, a wasting condition defined by chronic loss of fat and muscle, is a frequent complication of pancreatic ductal adenocarcinoma (PDAC) that negatively impacts quality of life and confers poor patient survival. Cachectic wasting cannot be abrogated by nutritional supplementation, and the mechanisms underlying this phenotype are unclear. Recent work demonstrates that human antigen R (HuR), an RNA-binding protein that regulates expression of genes involved in key cellular processes such as inflammation, stress response, and apoptosis, is also a major repressor of adipogenesis. Thus, we sought to elucidate the relative contributions of enhanced catabolism and impaired anabolism on fat wasting by investigating adipose tissue response to different nutritional contexts and HuR inhibition in cachectic mice. Methods: Adult C57BL/6J mice received orthotopic PDAC tumor injections (KrasG12D; p53R172H/+; Pdx1-cre) or sham injections. Mice were fed ad libitum, fasted 24h or fasted and refed 24h at mid-cachexia (9 days after injection). A separate cohort of PDAC mice were treated with the HuR inhibitor KH3 (100 mg/kg) at 6-, 8-, and 10-days post-injection and subjected to the same fast/refeed paradigm. Gross fat pads were weighed to assess anabolism. Adipose tissue mRNA levels were measured using RNAseq and validated with qPCR. Lipolytic rate was quantified as normalized quantity of glycerol released from inguinal (iWAT) and gonadal (gWAT) fat pads ex vivo. Results: PDAC and control mice exhibited equivalent loss of adipose tissue as a result of food restriction. Fat pads from PDAC mice displayed decreased lipolysis ex vivo and decreased lipase (Atgl &amp; Lipe) mRNA expression. In the fast/refeed model, PDAC mice were unable to restore iWAT or gWAT mass after refeed compared to controls. RNAseq revealed 614 differentially expressed genes between gWAT from PDAC and control mice. Downregulated (n=111) genes were most closely associated with adipogenesis (adj p=5x10-3), and expression of adipogenesis master regulators Pparg and Cebpa were reduced in both iWAT and gWAT from PDAC mice. Inhibition of HuR by KH3 restored lipogenesis in refed animals with a concomitant increase in fat pad mass and associated genes regulating adipogenesis (Pparg, Cebpa, Retn, Adipoq, Fasn). Conclusion: Adipose tissue wasting during PDAC cachexia can result from impaired anabolism in the absence of excess lipolysis or malabsorption. Adipose anabolism in PDAC mice is mediated by increased HuR activity in adipocytes. Restoring anabolic potential in adipose tissue using the HuR inhibitor KH3 may provide a novel approach to alleviating PDAC cachexia. Citation Format: Beth L. Worley, Katherine Pelz, Grace McCarthy, Heike Mendez, Roberto Di Niro, Alex Chitsazan, Jonathan Brody, Aaron Grossberg. The RNA-binding protein HuR mediates impaired adipogenesis in pancreatic cancer cachexia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2373.

Metformin Inhibits Lipid Droplets Fusion and Growth via Reduction in Cidec and Its Regulatory Factors in Rat Adipose-Derived Stem Cells.

Metformin is still being investigated due to its potential use as a therapeutic agent for managing overweight or obesity. However, the underlying mechanisms are not fully understood. Inhibiting the adipogenesis of adipocyte precursors may be a new therapeutic opportunity for obesity treatments. It is still not fully elucidated whether adipogenesis is also involved in the weight loss mechanisms by metformin. We therefore used adipose-derived stem cells (ADSCs) from inguinal and epididymal fat pads to investigate the effects and mechanisms of metformin on adipogenesis in vitro. Our results demonstrate the similar effect of metformin inhibition on lipid accumulation, lipid droplets fusion, and growth in adipose-derived stem cells from epididymal fat pads (Epi-ADSCs) and adipose-derived stem cells from inguinal fat pads (Ing-ADSCs) cultures. We identified that cell death-inducing DFFA-like effector c (Cidec), Perilipin1, and ras-related protein 8a (Rab8a) expression increased ADSCs differentiation. In addition, we found that metformin inhibits lipid droplets fusion and growth by decreasing the expression of Cidec, Perilipin1, and Rab8a. Activation of AMPK pathway signaling in part involves metformin inhibition on Cidec, Perilipin1, and Rab8a expression. Collectively, our study reveals that metformin inhibits lipid storage, fusion, and growth of lipid droplets via reduction in Cidec and its regulatory factors in ADSCs cultures. Our study supports the development of clinical trials on metformin-based therapy for patients with overweight and obesity.

Preoperative Short-term High-Carbohydrate Diet Provides More High-Quality Transplantable Fat and Improves the Outcome of Fat Grafts in Mice.

Patients with a low BMI may have inadequate high-quality adipose tissue for transplantation. The influence of high-energy diets on adipose tissue and graft retention remains unknown. The authors explored inguinal fat pad alternation in mice fed on a short-time high-fat diet (HFD) or a high-carbohydrate diet (HCD) preoperatively and the morphological and histological differences after transplantation. Mice were fed HFD (60% kcal from fat, 20% from carbohydrate), HCD (9.3% kcal from fat, 80.1% from carbohydrate), or normal (12% kcal from fat, 67% kcal from carbohydrate) diets for 2 or 4 weeks. Histological analyses were carried out following hematoxylin and eosin staining as well as CD34 and proliferating cell nuclear antigen immunostaining. The uncoupling protein-1 expression was determined by western blotting. Fat pads from each group were grafted into the dorsal region of the recipient mice, and morphological and histological changes were determined 4, 8, and 12 weeks posttransplantation. Vascular endothelial growth factor-α and platelet-derived growth factor-α expression were determined using quantitative polymerase chain reaction. The inguinal fat pad volume increased in the HFD and HCD groups. The presence of multilocular adipocytes in inguinal fat of HCD-fed mice, combined with the increased uncoupling protein-1 content, suggested adipocyte browning. HCD grafts showed higher volume retention and reduced oil cyst formation, possibly attributed to better angiogenesis and adipogenesis. HCD enlarged adipose tissue and improved graft survival rates, which may be due to the browning of fat before grafting and enhanced angiogenesis after grafting.

A Short-Term High-Fat Diet Improved the Survival of Fat Grafts in Mice by Promoting Macrophage Infiltration and Angiogenesis.

Background: Adipose tissue is an ideal filler material that is widely used for soft tissue defects. But the low survival rate and complications associated with such grafts pose a serious challenge, which limits their clinical application. Adipose tissue is a metabolic diet-responsive tissue; however, the influence of diets on fat grafting remains ambiguous. Methods: We extracted inguinal fat pads from C57/BL6 male mice, and transplanted them into the dorsal region of recipient mice (0.3 ml). Post-fat-grafting, mice (n = 54) were randomized into three groups, namely normal diet (ND), high carbohydrate diet (HC), and high-fat diet (HF). Structural changes were assessed by histological staining. Lipolysis activity and vascular regeneration of grafts on day 30 were analyzed using real-time polymerase chain reaction, immunofluorescence, and western blotting. Results: The grafts of mice on HC and HF diets exhibited significantly fewer oil cysts and larger volume retention (0.18 ± 0.01, 0.21 ± 0.01, and 0.25 ± 0.01 ml, for ND, HC, and HF group, respectively, p < 0.05) on day 90. In comparison, grafts for the mice belonging to the HF groups exhibited higher expression of lipolysis-related genes, including adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and carnitine palmitoyltransferase 1 (CPT1), on day 30. Furthermore, increased infiltration of macrophages (F4/80+) and the higher expression of angiogenesis genes were reported in the HF groups. Conclusion: Altogether, the administration of short-term HF diet remarkably enhanced angiogenesis and improved the quality of fat grafts, which was characterized by fewer oil cysts and higher long-term volume retention. The possible mechanisms may be due to the increased macrophage infiltration, and the promoted angiogenesis in HF grafts.

Intramuscular Fat Graft Results in Lower Retention but Similar Quality Compared with Subcutaneous Counterpart.

The adipose tissue has been injected into both subcutaneous and intramuscular planes for volume augmentation. However, the differences in their outcomes have yet to be fully elucidated. To investigate the differences of intramuscular and subcutaneous graft outcome, adipose tissue was harvested from the inguinal fat pad of mice and then placed into the quadriceps femoris or the subcutaneous plane, respectively. At 8 weeks, the graft outcome was evaluated by gross weight assessment, hema-toxylin and eosin staining, and CD31 staining. The authors found out that though the intramuscular graft had lower weight retention than the subcutaneous graft, the histologic quality and vascularity were similar between the intramuscular and subcutaneous graft. To summarize, the muscle is a feasible plane for fat grafting clinically. While performing intramuscular fat grafting, moderate overcorrec-tion may be necessary to achieve satisfactory results.

The effect of stromal vascular fraction in an experimental frostbite injury model

BackgroundDespite current treatment modalities, frostbite remains an injury with a poor prognosis which may cause functional morbidities. Several experimental and clinical studies have demonstrated that stromal vascular fraction is an autologous mixture, which can improve wound healing and vasculogenesis. The aim of this study was to show the beneficial effects of stromal vascular fraction on experimental frostbite healing. Material and methodsStromal vascular fraction (SVF) was harvested from 5 rats after excision of the inguinal fat pads. Another 20 rats were separated into 2 groups of 10 as the SVF group and the control group. A frostbite injury was created on each rat using a cryoprobe frozen with liquid nitrogen (−196 °C). SVF was applied to the SVF group and phosphate-buffered saline to the control group. All injections were performed subcutaneously within the frostbite injury area. Biopsies were performed on days 5 and 14 for histopathological and immunochemical evaluations. The tissue perfusion rates of both groups were assessed on day 14 using indocyanine green angiography (SPY system). ResultsThe increase in mean tissue perfusion was 373.3% ( ± 32.1) in the SVF group and 123.8% ( ± 16.3) in the control group (p < 0.001). The macroscopic wound reduction rates of the SVF and control groups were 25.5% ( ± 19.1) and 18.0% ( ± 5.9), respectively on day 5%, and 78.2% ( ± 9.2) and 57.3% ( ± 16.7) on day 14 (p = 0.007; p = 0.003). Acute inflammation and the fibrosis gradient were significantly decreased in the SVF group compared to the control group (p = 0.004, p = 0.054 respectively on day 14). Granulation tissue amount, re-epithelialization score and neovascularization were significantly increased in the SVF group (p = 0.006, p = 0.010 and p = 0.021, respectively on day 14). ConclusionsThe study results demonstrated that SVF increases frostbite wound healing by increasing tissue perfusion rate, neovascularization and re-epithelialization, and modulating acute inflammation and fibrosis.

Abstract PD3-03: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

Abstract Introduction: Triple negative breast cancers (TNBC) are frequently poorly differentiated with higher propensity for metastasis than all other subtypes. Enhancer of Zeste Homolog 2 (EZH2) is a lysine methyltransferase that mediates transcriptional repression of pro-differentiation genes in normal and neoplastic cells. The oncogenic role of EZH2 through H3K27me3 is well established. However, non-canonical H3K27me3-independent functions are unclear. We have reported that p38 phosphorylates T367 of EZH2, increasing TNBC metastasis. Recent reports show that EZH2 can regulate signaling pathways through direct methylation of proteins suggesting the hypothesis that EZH2 may methylate p38 in TNBC. Methods: Human tissue samples from 16 primary invasive breast carcinomas and matched distant metastasis arrayed in tissue microarrays were interrogated for pEZH2 T367 and p-p38 by immunohistochemistry. To study p38 methylation, we performed LC-MS/MS analyses GST-p38α, and GST-EZH2 were each incubated with or without recombinant PRC2 complex (EZH2/E ED/SUZ12/RbAp48/AEBP2) and S-adenosyl methionine methyl donor. MDA-MB-231, mouse and human primary TNBC cells and murine 4T1 cells were used in funcional assays in vitro and in vivo. EZH2 knockdown and rescue was carried out using lentiviral transduction of EZH2 with pBabe-myc-EZH2 (wild-type) or pBabe-myc-EZH2 (T367A). We developed p38 methylation mutants: HA-p38a K139A, HA-p38a K165A, and HA-p38a K130A/K165K. To investigate the importance of EZH2 and p38 enzymatic activities we used the EZH2 methyltransferase inhibitors GSK-343 and EPZ-6438 and the p38 inhibitor SB202190 in vitro and in vivo. For animal studies, MDA-MB-231 and 4T1 cells were orthotopically injected into the right inguinal mammary fat pad of eight-week old NOD/SCID or BALBc mice, respectively. When tumors reached 100 mm3 mice were treated 5 days/week by intraperitoneal injection with SB202190 (1mg/kg/day), EPZ-6438 (10mg/kg/day), combination of SB202190 and EPZ-6438, or control. We monitored tumor growth for 50 days, and harvested primary tumors, lungs, and sites of metastasis for histopathology, Western blot, and immunohistochemistry. Primary xenografts from the control and treatment groups were subjected to RNA sequencing. Results: EZH2 methylates p38 at lysines 139 and 165 leading to enhanced p38 stability and increased invasion, and p38 activation requires T367 phosphorylation of EZH2. Dual inhibition of EZH2 methyltransferase and p38 kinase activities downregulates pEZH2 T367, H3K27me3, and p-p38 in vivo, reduces TNBC growth and metastasis, and results in gene expression changes towards a better differentiated phenotype. pEZH2 T367 and p-p38 proteins are significantly coexpressed in human primary and metastatic breast cancer. Conclusions: We provide direct evidence that EZH2 methylates p38 with resultant p38 activation, and that EZH2 phosphorylation at T367 is important for this function. Dual targeting of EZH2 methyltransferase and p38 kinase activities with specific inhibitors reduces breast cancer growth and metastasis indicating a cooperation between EZH2 canonical and non-canonical mechanisms and suggesting therapeutic strategies. Citation Format: Maria E Gonzalez, Shilpa R. Tekula, Talha Anwar, Shoshana A. Leflein, Celina G. Kleer. EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-03.

Oral administration of megestrol acetate can increase fat graft survival in a rat model

Insufficient and inconsistent survival is a significant shortcoming of fat grafts. Reportedly, megestrol acetate (MA) could induce proliferation, migration, and adipogenic differentiation of adipose-derived stem cells in vitro. Thus, we tested whether MA could promote fat graft survival in a rat model. Twenty-eight Sprague-Dawley rats (8 weeks old, male) were divided into two groups: experimental (MA group, n=14) and control (n=14). The inguinal fat pad (1g) was extracted en bloc and re-implanted under the scalp in both groups. MA (100mg/kg/day) was administered orally for 14 postoperative days in the experimental group. After 6 weeks, the volume and weight of the grafted fat were measured. Histologic examination with hematoxylin and eosin (HE) and real-time polymerase chain reaction (PCR) for vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and CCAAT/enhancer-binding protein alpha (C/EBP-α) were performed. Perilipin staining was performed to check the viability of grafted fat. Graft fat volume was greater in the MA group, compared with that in the control (P=0.023). The MA group also had more viable cells, including more adipocytes, and less fibrosis or vacuoles than the control on HE and perilipin staining. MA upregulated the expression of FGF2 (P<0.001), VEGF (P=0.008), and C/EBP-α (P=0.002) at the second postoperative week. MA increased survival of grafted fat in an animal model. Increased vascularization and adipogenesis were related to these results. Further human clinical trials are necessary to evaluate adjunctive oral administration of MA after fat grafting to promote graft survival.

Abstract PO-023: Impaired adipose anabolism drives fat wasting in pancreatic cancer cachexia

Abstract Purpose/Objectives: The disease associated wasting condition, cachexia, is a common complication of pancreatic ductal adenocarcinoma (PDAC) that impacts quality of life and portends poor survival. Cachexia remains refractory to nutritional supplementation, but the mechanisms underlying this phenotype are unclear. By examining the adipose response to different nutritional contexts in cachectic mice, we sought to understand the relative contributions of enhanced catabolism and impaired anabolism on adipose tissue wasting. Materials/Methods: Adult C57BL/6J mice received orthotopic PDAC tumor injections (KrasG12D; p53R172H/+; Pdx1-cre) or sham injections. Mice were fasted 24 h or fasted and refed 24 h at mid-cachexia time point, and gross fat pads were weighed to assess anabolic potential. 3T3-L1 cells were treated with PDAC cell conditioned media during or after differentiation to model cachexia in vitro. Lipolytic rate was quantified as normalized quantity of glycerol released from inguinal (iWAT) and gonadal (gWAT) fat pads ex vivo or differentiated 3T3-L1 cells in vitro. Lipogenesis was measured in vitro using Oil-red-O staining. Adipose tissue mRNA levels were measured using bulk RNAseq and confirmed with qPCR. Malabsorption was assessed via fecal protease activity and total fecal protein and lipid content. Results: Adipose tissue in PDAC and control mice were equally sensitive to fasting/food restriction. Both iWAT and gWAT fat pads from PDAC mice showed decreased lipolysis ex vivo, associated with decreased lipase (Atgl &amp; Lipe) mRNA expression. In the fast/refeed paradigm, PDAC mice were unable to restore both iWAT and gWAT mass after refeed, in contrast to control mice. We found no evidence of malabsorption at this timepoint by any measure. RNAseq on gWAT revealed 614 differentially expressed genes between PDAC and control mice. Downregulated (n=111) genes were most closely associated with adipogenesis (adj p&amp;lt;.05), and expression of adipogenesis master regulators Pparg and Cebpa reduced in WAT from PDAC mice. Upregulated genes were associated with increased RELA and NFkB activity (adj p&amp;lt;5 × 10−22) and inflammatory response hallmarks. Treatment of 3T3-L1 cells with PDAC conditioned media phenocopied in vivo model, showing impaired lipolysis and lipogenesis, associated with decreased expression of lipolytic, adipogenic, and lipogenic genes. Conclusions: Adipose tissue wasting in PDAC cachexia can result from impaired anabolism in the absence of enhanced lipolysis or malabsorption. This deficit in adipogenesis is mediated by a cancer cell secreted product and is associated with increased inflammatory signaling in adipocytes, most likely via RELA and NFkB activity. Restoring adipose anabolic potential may provide a novel approach to cachexia treatment in PDAC. Citation Format: Katherine Pelz, Grace McCarthy, Heike Mendez, Samantha Z. Brown, Jonathan R. Brody, Aaron J. Grossberg. Impaired adipose anabolism drives fat wasting in pancreatic cancer cachexia [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-023.

Peri-prosthetic Fat Grafting Decreases Collagen Content, Density, and Fiber Alignment of Implant Capsules.

Background:Lower capsular contracture rates have been observed with peri-prosthetic fat grafting. We investigated the effect of fat grafting on capsular characteristics and peri-prosthetic collagen density, content, and fiber alignment.Methods:Forty miniature tissue expanders were placed on the backs of 20 rats. After four weeks, both inguinal fat pads were harvested, homogenized, and injected into peri-prosthetic tissue of the right tissue expander (fat graft) while the left served as control. The animals were killed at three (10 rats) and 12 weeks (10 rats), and full thickness peri-prosthetic samples were histologically processed for morphology (H&E) and collagen type and content (picrosirius red).Results:An 8.1% increase in adipose peri-prosthetic thickness was associated with a 10% decrease in collagen content at any time point (P = 0.004). Fat-grafted capsules displayed a 59% reduction in % total collagen when compared with controls (P < 0.001). There were no differences in capsular thickness. Fat-grafted samples were 54 times more likely to have a higher inflammation score and 69 times more likely to have a lower capsular density score than their nongrafted counterparts (P < 0.001 and P = 0.001, respectively). The extent of inflammation decreased over time in all samples (P = 0.002). Additionally, fat-grafted samples were 67 times more likely to have a lower fiber alignment score than the controls (P < 0.001).Conclusions:Enhancement of peri-prosthetic tissue with fat grafting decreases collagen content, density, and fiber alignment of implant capsules. These findings support clinical application of fat grafting in prosthetic breast surgery to potentially decrease capsular contracture.

Selective Microvascular Tissue Transfection Using Minicircle DNA for Systemic Delivery of Human Coagulation Factor IX in a Rat Model Using a Therapeutic Flap.

Gene therapy is a promising treatment for protein deficiency disorders such as hemophilia B. However, low tissue selectivity and efficacy are limitations of systemic vector delivery. The authors hypothesized that selective transfection of rat superficial inferior epigastric artery flaps could provide systemic delivery of coagulation factor IX, preventing the need for systemic vector administration. Minicircle DNA containing green fluorescent protein, firefly luciferase, and human coagulation factor IX was created. Vector constructs were validated by transfecting adipose-derived stromal cells isolated from Wistar rat superficial inferior epigastric artery flaps and evaluating transgene expression by fluorescence microscopy, bioluminescence, and enzyme-linked immunosorbent assay. Minicircle DNA luciferase (10 and 30 μg) was injected into murine (wild-type, C57/BL/6) inguinal fat pads (n = 3) and followed by in vivo bioluminescence imaging for 60 days. Wistar rat superficial inferior epigastric artery flaps were transfected with minicircle DNA human coagulation factor IX (n = 9) with plasma and tissue transgene expression measured by enzyme-linked immunosorbent assay at 2 and 4 weeks. Transfected adipose-derived stromal cells expressed green fluorescent protein for 30 days, luciferase for 43 days, and human coagulation factor IX (21.9 ± 1.2 ng/ml) for 28 days in vitro. In vivo murine studies demonstrated dose-dependence between minicircle DNA delivery and protein expression. Ex vivo rat superficial inferior epigastric artery flap transfection with minicircle DNA human coagulation factor IX showed systemic transgene expression at 2 (266.6 ± 23.4 ng/ml) and 4 weeks (290.1 ± 17.1 ng/ml) compared to control tissue (p < 0.0001). Rat superficial inferior epigastric artery flap transfection using minicircle DNA human coagulation factor IX resulted in systemic transgene detection, suggesting that selective flap or angiosome-based tissue transfection may be explored as a treatment for systemic protein deficiency disorders such as hemophilia B.