Stromal Vascular Cells Research Articles

Endogenous Omega-3 Polyunsaturated Fatty Acids Reduce the Number and Differentiation of White Adipocyte Progenitors in Mice.

Reducing the increased number of white adipocyte progenitors (WAP) is considered a novel approach to controlling obesity. The role of omega-3 polyunsaturated fatty acids (PUFA) in regulating the WAP resident population is unclear. The objective of this study was to investigate the effect of omega-3 PUFA on the niche composition of adipose-derived stem cells. Stromal vascular cell fraction (SVF) was collected from subcutaneous fat of wild-type (WT) and transgenic micecarryinga fat-1 gene from Caenorhabditis elegans (Fat-1 mice), which are capable of synthesizing omega-3 PUFA and have much higher tissue levels of omega-3 PUFA relative to WT mice. The isolated SVF cells were cultured and used for theexamination of adipocyte differentiation, adipogenic markers, fatty acid composition, and WAPnumbers. SVF isolated from Fat-1 mice (Fat-1-SVF) exhibited markedly fewer differentiated adipocytes with smaller cell size and less lipid content than that of WT mice (WT-SVF). Accordingly, adipogenesis-related genes and the white adipocyte surface marker ASC-1 were downregulated in Fat-1-SVF relative to WT-SVF. Furthermore, WAP numbers and adipose tissue macrophages were lower in Fat-1-SVF than WT-SVF. Omega-3 PUFA can both limit the WAP resident population and suppress their differentiation to white adipocytes, suggesting a new mechanism for the antiobesity effect of omega-3 PUFA.

Complete Human Penile Scaffold for Composite Tissue Engineering: Organ Decellularization and Characterization

Reconstruction for total penile defects presents unique challenges due to its anatomical and functional complexity. Standard methods suffer from high complication rates and poor functional outcomes. In this work we have developed the first protocol for decellularizing whole-organ human penile specimens for total penile tissue engineering. The use of a hybrid decellularization scheme combining micro-arterial perfusion, urethral catheter perfusion and external diffusion enabled the creation of a full-size scaffold with removal of immunogenic components. Decellularization was complete as assessed by H&E and immunohistochemistry, while quantification of residual DNA showed acceptably low levels (<50 ng/mg). An intact ECM was maintained with histologic architecture preservation on H&E and SEM as well as preservation of key proteins such as collagen-1, laminin and fibronectin and retention of growth factors VEGF (45%), EGF (57%) and TGF-beta1 (42%) on ELISA. Post-decellularization patency of the cavernosal arteries for future use in reseeding was demonstrated. Scaffold biocompatibility was evaluated using human adipose-derived stromal vascular cells. Live/Dead stains showed the scaffold successfully supported cell survival and expansion. Influence on cellular behavior was seen with significantly higher expression of VWF, COL1, SM22 and Desmin as compared to cell monolayer. Preliminary evidence for regional tropism was also seen, with formation of microtubules and increased endothelial marker expression in the cavernosa. This report of successful decellularization of the complete human phallus is an initial step towards developing a tissue engineered human penile scaffold with potential for more successfully restoring cosmetic, urinary and sexual function after complete penile loss.

Impact of the Different Preparation Methods to Obtain Human Adipose-Derived Stromal Vascular Fraction Cells (AD-SVFs) and Human Adipose-Derived Mesenchymal Stem Cells (AD-MSCs): Enzymatic Digestion Versus Mechanical Centrifugation.

Autologous therapies using adipose-derived stromal vascular fraction (AD-SVFs) and adult adipose-derived mesenchymal stem cells (AD-MSCs) warrant careful preparation of the harvested adipose tissue. Currently, no standardized technique for this preparation exists. Processing quantitative standards (PQSs) define manufacturing quantitative variables (such as time, volume, and pressure). Processing qualitative standards (PQLSs) define the quality of the materials and methods in manufacturing. The purpose of the review was to use PQSs and PQLSs to report the in vivo and in vitro results obtained by different processing kits that use different procedures (enzymatic vs. non-enzymatic) to isolate human AD-SVFs/AD-MSCs. PQSs included the volume of fat tissue harvested and reagents used, the time/gravity of centrifugation, and the time, temperature, and tilt level/speed of incubation and/or centrifugation. PQLSs included the use of a collagenase, a processing time of 30 min, kit weight, transparency of the kit components, the maintenance of a closed sterile processing environment, and the use of a small centrifuge and incubating rocker. Using a kit with the PQSs and PQLSs described in this study enables the isolation of AD-MSCs that meet the consensus quality criteria. As the discovery of new critical quality attributes (CQAs) of AD-MSCs evolve with respect to purity and potency, adjustments to these benchmark PQSs and PQLs will hopefully isolate AD-MSCs of various CQAs with greater reproducibility, quality, and safety. Confirmatory studies will no doubt need to be completed.

Immunohistochemical assessment of Eph/ephrin expression in oral squamous cell carcinoma and precursor lesions.

To evaluate erythropoietin-producing hepatocellular carcinoma (Eph)/Eph receptor-interaction protein (ephrin) expression in oral squamous cell carcinoma (OSCC) and oral epithelial precursor lesions (OEPLs), EphA2, EphB4, and ephrinB2 were examined and compared with microvessel density (MVD) and lymphatic vessel density (LVD). Samples from 73 OSCC and 43 OEPLs patients were immunohistochemically analyzed with antibodies against EphA2, EphB4, ephrinB2, CD34, and D2-40. Results were compared with clinicopathological findings. Immunohistochemical reactivity for EphA2, EphB4, and ephrinB2 was detected in epithelial cells and some stromal vascular cells in OEPLs and OSCC, proportionately with the level of malignancy. The number of blood vessel endothelial cells stained with CD34 and lymphatic vessel endothelial cells stained with D2-40 was increased in OEPLs and OSCC. In OSCC, ephrinB2 and EphB4 exhibited significant correlation with recurrence and invasion depth, respectively. MVD was significantly lower in slight lymphocytic reaction than in prominent stromal reaction. Association was found between LVD and T classification, postoperative metastasis, survival, mode of invasion, and invasion depth. Expression of EphA2, EphB4, ephrinB2, MVD, and LVD might be associated with malignant potential of the oral epithelium. Angiogenesis and lymphangiogenesis appear to be related to progression of potentially malignant oral lesions.

Point-of-Care Procedure for Enhancement of Meniscal Healing in a Goat Model Utilizing Infrapatellar Fat Pad–Derived Stromal Vascular Fraction Cells Seeded in Photocrosslinkable Hydrogel

Background: Large radial tears of the meniscus involving the avascular region can compromise meniscal function and result in poor healing and subsequent osteochondral degeneration. Augmentation of surgical repairs with adipose-derived stromal vascular fraction (SVF), which contains mesenchymal stromal cells, may improve meniscal healing and preserve function (ie, chondroprotection). Purposes: (1) To develop a goat model of a radial meniscal tear with resulting osteoarthritis and (2) to explore the efficacy of a 1-step procedure utilizing infrapatellar fat pad–derived SVF cells seeded in a photocrosslinkable hydrogel to enhance meniscal healing and mitigate osteochondral degeneration. Study Design: Controlled laboratory study. Methods: A full-thickness radial tear spanning 90% of the medial meniscal width was made at the junction of the anterior and middle bodies of the goat stifle joint. Tears received 1 of 3 interventions (n = 4 per group): untreated, repair, or repair augmented with photocrosslinkable methacrylated gelatin hydrogel containing 2.0 × 106 SVF cells/mL and 2.0 µg/mL of transforming growth factor β3. The contralateral (left) joint served as a healthy control. At 6 months, meniscal healing and joint health were evaluated by magnetic resonance imaging (MRI) and assessed by histological and macroscopic scoring. The Whole-Organ Magnetic Resonance Imaging Score and the presence of a residual tear, as evaluated with T2 MRI sequences, were determined by a single blinded orthopaedic surgeon. Results: When compared with tears left untreated or repaired with suture alone, augmented repairs demonstrated increased tissue formation in the meniscal tear site, as seen on MRI and macroscopically. Likewise, the neotissue of augmented repairs possessed a histological appearance more similar, although still inferior, to healthy meniscus. Osteochondral degeneration in the medial compartment, as evaluated by the Whole-Organ Magnetic Resonance Imaging Score and Inoue (macroscopic) scale, revealed increased degeneration in the untreated and repair groups, which was mitigated in the augmented repair group. Histological evaluation with a modified Mankin score showed a similar trend. In all measures of osteochondral degeneration, the augmented repair group did not differ significantly from the uninjured control. Conclusion: A radial tear spanning 90% of the medial meniscal width in a goat stifle joint showed poor healing potential and resulted in osteochondral degeneration by 6 months, even if suture repair was performed. Augmentation of the repair with a photocrosslinkable hydrogel containing transforming growth factor β3 and SVF cells, isolated intraoperatively by rapid enzymatic digestion, improved meniscal healing and mitigated osteoarthritic changes. Clinical Relevance: Repair augmentation with an SVF cell–seeded hydrogel may support successful repair of meniscal tears previously considered irreparable.

Risk factors and complications after body-contouring surgery and the amount of stromal vascular fraction cells found in subcutaneous tissue.

Body contouring surgery following massive weight loss is often prone to complications. Subcutaneous adipose tissue is a rich source of stromal vascular fraction (SVF) cells, and moreover it plays an important role in the pathophysiology of obesity, metabolic syndrome, and wound healing. In this retrospective, single-centred appraisal, complications are examined and correlated with individual SVF numbers in abdominal subcutaneous fat tissue. We analysed whether the weight loss method affected complications. Eighty seven massive weight loss patients undergoing body contouring surgery between 2010 and 2017 were included in the study. In total, 57 cases with at least one complication were recorded (65.5%). Maximum lifetime weight was 109.6 kg (range 48-184 kg). Half of the complications (50.8%) were minor complications without the need for surgical revision. The mean number of SVF found in the resected tissue was 714 997.63 cells/g fat tissue. We found no statistical difference in complication rates dependent on cell numbers. Smoking (P = .049) and a high BMI at the time point of surgery (P = .031) led to significantly more complications. Also, a high resection weight (P = .057) showed a tendency for impaired wound healing. However, there was no difference in complication rates following body contouring procedures attributable to the method of weight loss in this study.

5221Knockout of purinergic receptor Y13 (P2Y13) results in an improved outcome in metabolic syndrome in mice

Abstract Background Metabolic syndrome clusters the main risk factors for cardiovascular diseases and endocrine dysfunction. Novel studies show that the important underlying mechanism is a long term inflammation of adipose tissue with a gradual shift from the anti-inflammatory M2 towards the pro-inflammatory M1 macrophages (pathognomonic). Massive hypertrophy induces adipocyte death, which releases DAMPs like nucleotides recognized by purinergic receptors orchestrating ongoing inflammatory process. Interestingly, we found Gi coupled P2Y13R only expressed on M1 macrophages, not in M0 (unstimulated) and M2 macrophages. Nevertheless, the role of P2Y13R in the immune system and especially macrophages is currently unknown. Given its pivotal role in central metabolic processes (P2Y13R has been described in insulin secretory signalling) together with its unique expression in its pathognomonic inflammatory macrophage subtype makes it an interesting candidate to investigate its role in metabolic syndrome. Purpose Due to the unique expression of P2Y13R on M1 macrophages we hypothesise an improved outcome in a high-fat diet induced metabolic syndrome by interfering with the P2Y13 signalling cascade. Methods BMDM differentiation to macrophages using M-CSF and subsequent stimulation with medium (M0), LPS and IFNγ (M1) or IL4 (M2); Expression of P2Rs quantified using Taqman qPCR. Male C57Bl6/J wild-type (WT) and P2Y13-deficient (KO) mice were fed with a high-fat diet (HFD) for 20 weeks; On week 15 we performed the ITT, on week 16 the GTT. Metabolic performance was monitored by metabolic caging. Results We observed a unique expression of P2Y13R on M1 macrophages. Adult P2Y13-deficient mice showed a higher O2 consumption compared to adult C57Bl6/J wild-type mice (AUC of O2 consumption 2nd day= KO: 61620±2261mL/kg vs WT: 53830±916.1mL/kg, p=0.0331). Although both P2Y13−/− mice and WT littermates consumed comparable amount of food (daily food intake per mouse → KO: 3.97±0.25g vs WT: 3.76±0.18g), P2Y13 deficient animals showed significantly decelerated weight gain (e.g. on week 15 → KO: 142±2% (n=10) vs WT: 198±5% (n=10), p&lt;0.0001). Obese P2Y13−/− animals outperformed obese WT littermates in a peritoneal glucose tolerance test (2h after glucose injection → KO: 272.9±21.0 mg/dL (n=10) vs WT: 532.6±21.2 (n=10) mg/dL, p&lt;0.0001). There was no difference in the cell amount of stromal vascular fraction cells. Conclusion Global P2Y13 deficiency leads to an improved outcome in metabolic syndrome with an increased protection against developing an insulin resistance as shown through an improved glucose tolerance and basal glucose levels, a decelerated weight gain despite comparable food consumption and a better metabolic turnover. Observing these beneficial metabolic improvements, we hypothesise that antagonization of P2Y13R could be a promising therapeutic target in the field of metabolic syndrome.

Conditioned medium from 3D culture system of stromal vascular fraction cells accelerates wound healing in diabetic rats.

Aim: We investigated the healing effects of conditioned medium (CM) derived from a physiological 3D culture system engineered to use an extracellular matrix/stromal vascular fraction (SVF) gel enriched for adipose on diabetic wounds in rats. This CM (Gel-CM) was compared with that from a 2D culture system that used SVF cells (SVF-CM). Materials & methods: Keratinocytes, fibroblasts and wounds were treated with Gel-CM and SVF-CM, and cytokine levels in the CM types were quantified. Results: Proliferation and migration of keratinocytes and fibroblasts were significantly higher after treatment with Gel-CM than with SVF-CM. Collagen secretion by fibroblasts and wound closure were highly stimulated by Gel-CM. Proteomic analyses revealed a higher concentration of growth factors in Gel-CM than in SVF-CM. Conclusion: Gel-CM is a promising therapeutic option for treating diabetic wounds.

Wound Healing Effect of Autologous Stromal-Vascular Fraction Cells on Induced Damage to Skin

A course of reparative regeneration after skin damage caused by a jagged wound under the effect of the autologous stroma-vascular fraction cell suspension has been studied. The methods for clinical observation, the studies using photo-optical biopsy instrumentation, and the macro- and microscopic morphometric measurements were used. The cell material was derived from the animal’s withers with the liposuction technique. The autologous adipose tissue was used to prepare the cell suspension containing a cell group (of fibroblasts, pericytes, acrophags, adipocytes, and endothelial cells) within the stromal vascular fraction. The obtained data indicate that the cellular product contributes to a faster wound-healing process and enhanced formation of regenerator morphologically similar to the intact skin surrounding the wound (when compared to the control). In the regenerator structure, the restored areas of the skin structure layers (epidermis, dermis, and dermal adipocyte tissue) were microscopically revealed. A small site of a scar structure tended to remain in the regenerator’s central part, which was significantly smaller than that in the analogue control. It has been concluded that the cellular products have a potential to enhance the processes of reparative regeneration and can be efficient in skin-wound healing.

Role of progranulin in adipose tissue innate immunity

Regulation of progranulin in adipocytes and its role in inflammation is poorly understood. Aim: (i) to investigate regulation of progranulin in adipocyte differentiation and adipose tissue compartments, (ii) to address progranulin expression in two murine (C57BL/6) models of inflammation. Results: Progranulin expression was induced during adipocyte differentiation. Neither estradiol nor testosterone or metabolic stimuli such as glucose and insulin modified progranulin synthesis. Fatty acids, bile acids and incretins GLP-1 and GIP-1 exerted potent and differential effects on progranulin secretion. LPS, TNF and IL6 significantly increased progranulin secretion. TLR9 agonists decreased and TLR1/2, TLR3, TLR5, and TLR2/6 ligands increased progranulin expression. TLR3-mediated progranulin induction was abrogated by inhibitors of NF-κB and PI3K pathways. Progranulin expression between murine epididymal and subcutaneous adipose tissue did not differ in total adipose tissue, in isolated adipocytes or in the stromal-vascular cell fraction (SVC). However, SVC expressed significantly higher levels of progranulin than adipocytes at all sites. In adipocytes, female mice had significantly higher progranulin expression at all sites. An intra-peritoneal LPS challenge in mice did not affect adipose tissue progranulin expression, whereas peritoneal infection by S. aureus increased progranulin expression after 24 h. Conclusions: There are relevant sex-, site- and cell-specific effects on progranulin gene expression that is induced during adipocyte differentiation and modulated by various inflammatory and metabolic factors. Most importantly, ligands for TLR1/2 and TLR2/6 (recognizing S. aureus) in vitro and infection by S. aureus in vivo induce progranulin expression suggesting a role of adipocytes in protection against infection by gram-positive bacteria.

First-in-human study of TK-positive oncolytic vaccinia virus delivered by adipose stromal vascular fraction cells

BackgroundACAM2000, a thymidine kinase (TK)-positive strain of vaccinia virus, is the current smallpox vaccine in the US. Preclinical testing demonstrated potent oncolytic activity of ACAM2000 against several tumor types. This Phase I clinical trial of ACAM2000 delivered by autologous adipose stromal vascular fraction (SVF) cells was conducted to determine the safety and feasibility of such a treatment in patients with advanced solid tumors or acute myeloid leukemia (AML).MethodsTwenty-four patients with solid tumors and two patients with AML participated in this open-label, non-randomized dose-escalation trial. All patients were treated with SVF derived from autologous fat and incubated for 15 min to 1 h with ACAM2000 before application. Six patients received systemic intravenous application only, one patient received intra-tumoral application only, 15 patients received combination intravenous with intra-tumoral deployment, 3 patients received intravenous and intra-peritoneal injection and 1 patient received intravenous, intra-tumoral and intra-peritoneal injections. Safety at each dose level of ACAM2000 (1.4 × 106 plaque-forming units (PFU) to 1.8 × 107 PFU) was evaluated. Blood samples for PK assessments, flow cytometry and cytokine analysis were collected at baseline and 1 min, 1 h, 1 day, 1 week, 1 month, 3 months and 6 months following treatment.ResultsNo serious toxicities (> grade 2) were reported. Seven patients reported an adverse event (AE) in this study: self-limiting skin rashes, lasting 7 to 18 days—an expected adverse reaction to ACAM2000. No AEs leading to study discontinuation were reported. Viral DNA was detected in all patients’ blood samples immediately following treatment. Interestingly, in 8 patients viral DNA disappeared 1 day and re-appeared 1 week post treatment, suggesting active viral replication at tumor sites, and correlating with longer survival of these patients. No major increase in cytokine levels or correlation between cytokine levels and skin rashes was noted. We were able to assess some initial efficacy signals, especially when the ACAM2000/SVF treatment was combined with checkpoint inhibition.ConclusionsTreatment with ACAM2000/SVF in patients with advanced solid tumors or AML is safe and well tolerated, and several patients had signals of an anticancer effect. These promising initial clinical results merit further investigation of therapeutic utility.Trial registration Retrospectively registered (ISRCTN#10201650) on October 22, 2018.

Effect of testosterone on the differentiation control of stromal vascular cells isolated from longissimus muscle of Hanwoo beef cattle

Testosterone, as an influential factor in marbling score, requires strict management for uniform development of adipocytes in-between muscle bundles. Present study investigated effect of castration timing and testosterone levels on adipocyte development using SVCs. Isolated SVCs exhibited classical MSC markers, CD31−, CD34−, CD45−, CD90+, and CD105+. ELISA analysis indicated that serum testosterone concentration was highest in non-castrated calves while no significant difference was observed between female, early and late castrated calves. CCK-8 assay showed that concentration of testosterone had no effect on cell proliferation. However, the real-time PCR demonstrated that 20 ng/ml of testosterone suppressed expression of preadipocyte markers, pref-1 and zfp423, but encouraged expression of myoblast markers, myf5 and myoD, via the AR. Consequently, expression of adipogenic markers C/EBPα and PPARγ, as well as accumulation of triglyceride, were decreased in 20 ng/ml testosterone treatment under adipogenic conditions. These findings suggest that by castrating calves before level of testosterone increases, may improve marbling development in the Hanwoo beef industry.

Model of Radiation-induced Hind Limb Contracture and Skin Fibrosis Rescued by Fat Grafting

INTRODUCTION: Radiotherapy is an effective anti-cancer treatment, able to reduce tumor size and decrease local cancer recurrence. However, the long-term outcome of radiotherapy is significant and pathologic fibrosis of the soft tissue surrounding the malignancy. Radiation-induced soft tissue fibrosis can disrupt tissue esthetic appears and impair function, such as impaired swallowing and limb contracture. Fat grafting is gaining popularity as a surgical technique able to prevent or reverse the radiation-induced soft tissue fibrosis. We developed a mouse model of radiation-induced hind limb contracture and investigated the potential of grafted fat to restore mobility to the irradiated hind limb. METHODS: The hind limbs of Prrx1Cre;R26mTmG mice were irradiated with 30 Gy fractionated in 5 Gy doses every 2 days for a total of 12 days. The Prrx1Cre;R26mTmG mice were used to label a fibrogenic subpopulation of fibroblasts in ventral skin (PRRX-1+) by embryonic expression Cre. A 4-week period followed irradiation to allow limb contracture to develop, and mice were then sacrificed, and hind limbs were processed for histology. To explore the therapeutic effects of fat grafting, CD-1 nude mice were irradiated with the same irradiation protocol, and at 4 weeks, the mice were injected with 200 μl of human lipoaspirate fat or lipoaspirate enriched with stromal vascular cells (10,000 cells/200 μl) directly into the subcutaneous space on the ventral surface of the irradiated hind limbs. We used 2 control mice; mice injected with 200 μl of saline or mice who received sham surgery with no injection. Limb extension was measured every 2 weeks for a total of 12 weeks, and mice were then sacrificed for hind limb skin mechanical strength testing and histologic analysis. RESULTS: Hind limb irradiation significantly reduced limb extensibility compared to the nonirradiated side, and contracture was associated with a significant increase in the fibrogenic Prrx1+ fibroblast subpopulation in mouse ventral skin. Fat grafting progressively increased limb extension, reduced skin stiffness, and reversed the fibrotic histologic changes in the skin. The greatest improvements were found in mice who received fat grafted with stromal vascular cells. CONCLUSION: We present a mouse model of radiation-induced hind limb contracture which we use to show that grafted fat can reverse the fibrotic changes seen in irradiated skin and can improve the extensibility of contracted limbs postirradiation.

Эффективность выделения клеток стромально-васкулярной фракции из липоаспирата без ферментативной обработки

Эффективность выделения клеток стромально-васкулярной фракции из липоаспирата без ферментативной обработки

Synergistic activation of thermogenic adipocytes by a combination of PPARγ activation, SMAD3 inhibition and adrenergic receptor activation ameliorates metabolic abnormalities in rodents.

To treat obesity and related diseases, considerable effort has gone into developing strategies to convert white adipocytes into thermogenic brown-like adipocytes ('browning'). The purpose of this study was to identify the most efficient signal control for browning. To identify the most efficient signal control for browning, we examined rat stromal vascular fraction cells. In addition, physiological changes consequent to signal control were examined in vivo using lean and diet-induced obese (DIO) C57BL/6J mice. Combined treatment with the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone, the SMAD3 inhibitor SIS3 and the adrenergic receptor agonist noradrenaline (norepinephrine) synergistically induced Ucp1, Fgf21 and Cited1 expression, triggering brown adipogenesis. Synergistic induction of Ucp1 by the three agents was negatively regulated by forkhead box O (FOXO)3 via the inhibition of PPARγ-dependent gene transcription. Moreover, the administration of rosiglitazone, SIS3 and the selective β3 adrenergic receptor agonist CL316,243 to DIO mice reduced the amount of body-fat deposits (body weight from day 0 to 14, 12.3% reduction), concomitant with morphological changes in white adipose tissue, an increase in mitochondrial biosynthesis and a marked induction of uncoupling protein 1 (UCP1). Furthermore, administration of the three agents significantly increased serum adiponectin levels (mean 65.56μg/ml with the three agents vs 20.79μg/ml in control mice, p< 0.05) and improved glucose and lipid tolerance. These results suggest that the combined regulation of PPARγ, SMAD and the adrenergic receptor signalling pathway synergistically induces brown adipogenesis and may serve as an effective strategy to treat obesity and related diseases, including type 2 diabetes.

Cryopreservation of Stromal Vascular Fraction Cells Reduces Their Counts but Not Their Stem Cell Potency.

Background:Adipose-derived stem cells are derived from the nonfat component of adipose tissue termed the stromal vascular fraction (SVF). The use of freshly isolated autologous SVF cells as an alternative to adult stem cells is becoming more common. Repeated SVF administration for improved clinical outcomes is complicated by the need for repeated liposuction. This can be overcome by cryopreservation of SVF cells. The current study aimed to assess whether SVF cells retain their stem cell potency during cryopreservation.Methods:SVF cells isolated from lipoaspirates (donor age: 46.1 ± 11.7 y; body mass index: 29.3 ± 4.8 kg/m2) were analyzed either immediately after isolation or following cryopreservation at −196°C. Analyses included assessment of nucleated cell counts by methylene blue staining, colony-forming unit fibroblast counts, surface marker expression using a flow cytometric panel (CD45, CD34, CD31, CD73, CD29, and CD105), expansion in culture, and differentiation to fat and bone.Results:While cryopreservation reduced the number of viable SVF cells, stem cell potency was preserved, as demonstrated by no significant difference in the proliferation, surface marker expression in culture, bone and fat differentiation capacity, and the number of colony-forming unit fibroblasts in culture, in cryopreserved versus fresh SVF cells. Importantly, reduced cell counts of cryopreserved cells were due, mainly, to a reduction in hematopoietic CD45+ cells, which was accompanied by increased proportions of CD45−CD34+CD31− stem cell progenitor cells compared to fresh SVF cells.Conclusions:Cryopreservation of SVF cells did not affect their in vitro stem cell potency and may therefore enable repeated SVF cell administrations, without the need for repeated liposuction.

The Local Regulation of Vascular Function: From an Inside-Outside to an Outside-Inside Model.

Our understanding of the regulation of vascular function, specifically that of vasomotion, has evolved dramatically over the past few decades. The classic conception of a vascular system solely regulated by circulating hormones and sympathetic innervation gave way to a vision of a local regulation. Initially by the so-called, autacoids like prostacyclin, which represented the first endothelium-derived paracrine regulator of smooth muscle. This was the prelude of the EDRF-nitric oxide age that has occupied vascular scientists for nearly 30 years. Endothelial cells revealed to have the ability to generate numerous mediators besides prostacyclin and nitric oxide (NO). The need to classify these substances led to the coining of the terms: endothelium-derived relaxing, hyperpolarizing and contracting factors, which included various prostaglandins, thromboxane A2, endothelin, as well numerous candidates for the hyperpolarizing factor. The opposite layer of the vascular wall, the adventitia, eventually and for a quite short period of time, enjoyed the attention of some vascular physiologists. Adventitial fibroblasts were recognized as paracrine cells to the smooth muscle because of their ability to produce some substances such as superoxide. Remarkably, this took place before our awareness of the functional potential of another adventitial cell, the adipocyte. Possibly, because the perivascular adipose tissue (PVAT) was systematically removed during the experiments as considered a non-vascular artifact tissue, it took quite long to be considered a major source of paracrine substances. These are now being integrated in the vast pool of mediators synthesized by adipocytes, known as adipokines. They include hormones involved in metabolic regulation, like leptin or adiponectin; classic vascular mediators like NO, angiotensin II or catecholamines; and inflammatory mediators or adipocytokines. The first substance studied was an anti-contractile factor named adipose-derived relaxing factor of uncertain chemical nature but possibly, some of the relaxing mediators mentioned above are behind this factor. This manuscript intends to review the vascular regulation from the point of view of the paracrine control exerted by the cells present in the vascular environment, namely, endothelial, adventitial, adipocyte and vascular stromal cells.

Effects of 1,25-dihydroxyvitamin D3 on Inflammatory Responses of Stromal Vascular Cells and Adipocytes from Control and Obese Mice (FS12-04-19)

ObjectivesSeveral in vitro studies showed that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment could inhibit chronic inflammation in mouse or human adipocytes; however there have been a few in vivo studies. We investigated whether vitamin D supplementation affects subpopulation of immune cells in adipose tissue and the effects of in vitro 1,25(OH)2D3 treatment on pro-inflammatory cytokine secretion by stromal vascular cells (SVC) and adipocytes. MethodsFive-wk old C57BL/6 N mice were divided into 4 groups and fed diets differ in fat amount (10 or 45% kcal fat: CON or HFD) and vitamin D content (1000 or 10,000 IU/kg of diet: DC, or DS) for 13 wks. Subpopulation of immune cells (macrophage, NK cell, CD 4 T cell, CD 8 T cell, B cell) from adipose tissue-derived SVC was determined by FACS analysis. Five-wk old C57BL/6 mice were fed control or HFD diets (10 or 60% kcal fat, CON or HFD) for 12 wks. Adipocytes and SVC, isolated from visceral adipose tissue, were cultured with or without 10 nM of 1,25(OH)2D3 for 48 h and stimulated with LPS during the last 24 h. Pro-inflammatory cytokines produced by SVC and adipocytes were measured by ELISA. The mRNA levels of Tlr2, Tlr4, Dusp1, and Dusp10 were determined in SVC by real-time PCR. ResultsThe number of macrophages and NK cells within adipose tissue were higher in the HFD groups than CON groups. Dietary vitamin D did not alter the number of immune cells in adipose tissue. The production of IL-6 and MCP-1 from SVC and adipocytes were higher in the HFD groups compared with CON groups. In vitro 1,25(OH)2D3 treatment decreased IL-6, MCP-1, IL-1β production by SVC from HFD group and decreased IL-6 production by SVC from CON group. SVC Tlr2 mRNA levels, which were higher in the HFD group, decreased significantly by in vitro 1,25(OH)2D3 treatment. mRNA levels of Dusp1, which inhibits MAPK signaling, were increased by in vitro 1,25(OH)2D3 treatment. However, pro-inflammatory cytokine secretion from adipocytes was not affected by in vitro 1,25(OH)2D3 treatment. ConclusionsAlthough vitamin D supplementation did not reduce macrophage and NK cell numbers in adipose tissue, 1,25(OH)2D3 seemed to decrease pro-inflammatory cytokine production from SVCs by regulating Tlr2 and Dusp1. Funding SourcesSupported by the grant from the National Research Foundation (NRF) of Korea (NRF-2018R1D1A1B070491).

PTX3 Regulates miR-21 Expression and Secretion in Brown Adipocytes During Lipopolysaccharide-induced Inflammation (P21-072-19)

ObjectivesMetabolic endotoxemia is known to induce systemic inflammation and adipose tissue dysfunction during obesity. Pentraxin 3 (PTX3), a member of the pentraxin family can be induced by inflammatory stimuli in adipocytes. However, how PTX3 regulates inflammation in adipocytes is not fully understood. MicroRNAs (miRNAs) are key regulators of inflammatory gene expression. Specifically, miR-21 plays a role in the resolution of inflammation through inhibiting Toll-like receptor 4 (TLR4) signaling pathway. This study aimed to investigate how PTX3 regulates inflammatory homeostasis involving miR-21 during lipopolysaccharide (LPS) stimulation in brown adipocytes. MethodsUsing PTX3 knockout (KO) mouse and primary stromal-vascular (SV) cell culture models, we determined the effect of PTX3 deficiency on miR-21 expression, secretion, and inflammatory response in brown adipocytes, as well as the rescue effect of recombinant PTX3 on LPS-induced inflammation. ResultsBrown adipose tissue (BAT) had significantly higher levels of Dicer protein expression than inguinal and epididymal adipose depots; fully differentiated brown adipocytes expressed higher levels of Dicer than SV cells. These results suggest that BAT is the major site of miRNA production, and brown adipocyte is the main cell type that produces miRNAs. Moreover, we found brown adipocytes but not SV cells are the LPS responsive cells in miR-21 expression and secretion. In WT brown adipocytes, LPS stimulation significantly up-regulated cellular levels of miR-21. Interestingly, PTX3 deficiency led to a significant increase in the basal levels of both cellular and secreted miR21 compared to WT cells. Unlike in WT cells, LPS treatment suppressed cellular expression as well as secretion of miR-21 in PTX3 KO brown adipocytes. Treatment of recombinant PTX3 slightly up-regulated cellular expression of miR-21, but significantly increased the secretion of miR-21 in PTX3 KO brown adipocytes. Moreover, Treatment of recombinant PTX3 significantly attenuated LPS-stimulated increase in the expression of TNFα and MCP1 genes in PTX3 KO adipocytes. ConclusionsWe conclude that PTX3 plays an anti-inflammatory role in part via regulating the expression and secretion of miR-21. Funding SourcesAmerican Diabetes Association.

1786-P: ACSL1 Contributes to Thermogenesis and Differentiation of Brown Adipose Tissue

Long chain acyl-CoA synthetases (ACSLs) convert fatty acids (FA) to fatty acyl-CoAs that are utilized for various cellular processes, including triglyceride synthesis, activation of transcription factors, signal transduction, and providing energy sources via oxidation. Among 5 isotypes (ACSL1, 3, 4, 5, and 6), ACSL1 is the predominant isotype in adipocytes, and it is known to play important roles in adipocyte physiology, including FA uptake, triglyceride (TG) synthesis, thermogenesis, and FA oxidation. However, the roles of ACSL1 in brown adipocyte differentiation is incompletely understood. During the in vitro differentiation of brown adipocytes from stromal vascular cells (SVC), mRNA expression of ACSL1 was drastically increased. Pharmacological inhibition of ACSLs by triacsin C suppressed the differentiation of brown adipocytes. Interestingly, triacsin C inhibited the expression of uncoupled protein 1 (UCP1), which plays the major role in thermogenesis in mitochondria. Thus, we hypothesize that ACSL1 regulates thermogenesis and energy utilization by regulation of brown adipocyte differentiation. To examine the role of ACSL1 in brown adipose tissue (BAT), we generated BAT specific ACSL1 knockout mice by breeding UCP1-Cre with ACSL1 flox/flox mice (ACSL1BATKO). ACSL1 deficiency in BAT reduced the weight gain and glucose intolerance in high fat diet (HFD)-fed mice. ACSL1BATKO mice were severely cold intolerant. Furthermore, ACSL1BATKO mice were resistant to beta 3-agonist (CL-316243)-stimulated weight loss. Fat mass in HFD-fed ACSL1BATKO was lower than HFD-fed wild type mice. These data suggest that ACSL1 in BAT contributes to BAT differentiation, which causes the impairment of FA utilization. However, the mechanisms by which deficiency of ACSL1 in BAT reduces HFD-induced weight gain need to be further investigated. Disclosure G. Ren: None. J. Kim: None. Funding National Institutes of Health (R01HL128695R03AG058078)