Stromal Fraction Research Articles

Oxidant therapy improves adipogenic differentiation of adipose-derived stem cells in human wound healing

BackgroundAdipose-derived stem cells (ASC) and adipocytes are involved in numerous physiological and pathophysiological conditions, which have been extensively described in subcutaneous and visceral fat depots over the past two decades. However, much less is known about ASC and adipocytes outside classical fat tissue depots and their necessity in tissue remodeling after injury. Therefore, we investigated the etiology of adipocytes in human granulation tissue and define their possible role wound healing.MethodsIdentification of human wound tissue adipocytes was determined by immunohistochemical staining of granulation tissue sections from patients undergoing surgical debridement. Stromal cell fractions from granulation tissue and subcutaneous fat tissue were generated by collagenase type II-based protocols. Pro- and anti-inflammatory wound bed conditions were mimicked by THP1- and CD14+ monocyte-derived macrophage models in vitro. Effects of macrophage secretome on ASC differentiation and metabolism were determined by immunoblotting, flow cytometry, and microscopy assessing early and late adipocyte differentiation states. Functional rescuing experiments were conducted by lentiviral transduction of wildtype PPARG, IL1RA, and N-chlorotaurine (NCT) treatment.ResultsSingle and clustered adipocyte populations were detected in 11 out of 13 granulation tissue specimens and single-cell suspensions from granulation tissue showed adipogenic differentiation potential. Pro-inflammatory signaling by IFNG/LPS-stimulated macrophages (M (IFNG/LPS)) inhibited the maturation of lipid droplets in differentiated ASC. In contrast, anti-inflammatory IL4/IL13-activated macrophages (M (IL4/IL13)) revealed minor effects on adipocyte development. The M (IFNG/LPS)-induced phenotype was associated with a switch from endogenous fatty acid synthesis to glycolysis-dominated cell metabolism and increased pro-inflammatory cytokine production. Impaired adipogenesis was associated with increased, but seemingly non-functional, CEBPB levels, which failed to induce downstream PPARG and CEBPA. Neither transgenic PPARG overexpression, nor inhibition of IL1B was sufficient to rescue the anti-adipogenic effects induced by IFNG/LPS-activated macrophages. Instead, macrophage co-treatment during stimulation with NCT, a mild oxidant produced by activated granulocytes present in human wounds in vivo, significantly attenuated the anti-adipogenic effects.ConclusionsIn conclusion, the appearance of adipocytes in wound tissue indicates a prevailing anti-inflammatory environment that could be promoted by NCT treatment and may be associated with improved healing outcomes.Graphical abstract

A Novel Hypothesis and Characterization to Isolate Microvascular Endothelial Cells Simultaneously with Adipose-Derived Stem Cells from the Human Adipose-Derived Stromal Vascular Fraction.

Angiogenesis and vasculogenesis are essential processes for successful tissue regeneration in tissue engineering and regenerative medicine. The adipose-derived stromal vascular fraction (SVF) is not only a source of adipose stem cells (ASC) but also a suitable source of microvascular endothelial cells because it is a rich capillary network. So, we propose a new hypothesis for isolating adipose-derived human microvascular endothelial cells (HMVEC-A) from the SVF and developed a dual isolation system that isolates two cell types from one tissue. To isolate HMVEC-A, we analyzed the supernatant discarded when ASC is isolated from the adipose-derived SVF. Based on this analysis, we assumed that the SVF adherent to the bottom of the culture plate was divided into two fractions: the stromal fraction as the ASC-rich fraction, and the vascular fraction (VF) as the endothelial cells-rich fraction floating in the culture supernatant. VF isolation was optimized and the efficiency was compared, and the endothelial cells characteristics of HMVEC-A were confirmed by flow cytometric analysis, immunocytochemistry (ICC), a DiI-acetylated low-density lipoprotein (DiI-Ac-LDL) uptake, and in vitro tube formation assay. Consistent with the hypothesis, we found a large population of HMVEC-A in the VF and isolated these HMVEC-A by our isolation method. Additionally, this method had higher yields and shorter doubling times than other endothelial cells isolation methods and showed typical morphological and phenotypic characteristics of endothelial cells. Cells obtained by the method according to our hypothesis can be applied as a useful source for studies such as tissue-to-tissue networks, angiogenesis and tissue regeneration, patient-specific cell therapy, and organoid chips.

ALVEOLAR RECONSTRUCTION WITH THE USE OF ESTROMAL FRACTION OF BONE MARROW AUTOLOGOUS: A CASE REPORT

<h3>Background</h3> There is a great challenge in restoring the function and aesthetics of the patient using dental implants, especially when there are alveolar bone defects due to dental losses, often requiring grafting techniques for repair and reconstruction of the affected area. The objective of this study was to analyze the progress of alveolar regeneration and osteogenesis of a clinical case with bone grafting combined with the use of the stromal fraction of bone marrow, identifying the effectiveness of bone repair and exploring possibilities of use in the dental and medical area. <h3>Methods</h3> The 54-year-old patient with alveolar bone loss and atretic ridge was submitted to a graft with synthetic bone and platelet-rich fibrin (PRF), optimized through autologous cell grafting, for osteoinduction and faster alveolar reconstruction for later treatment with dental implants and prosthetic rehabilitation. <h3>Results</h3> The results analyzed through clinical and tomographic examinations, performed periodically, showed the effectiveness of the bone repair process of the patient, with consistent and quick alveolar reconstruction. <h3>Conclusion</h3> The use of stromal fraction of bone marrow combined with PRF proved to be safe and viable for the optimization of bone repair of alveolar defects.

Fresh autologous stromal tissue fraction for the treatment of knee osteoarthritis related pain and disability.

Knee osteoarthritis (KOA) is a very common condition with multifactorial etiology leading to severe pain and disability in the adult population. Although KOA is considered a non-inflammatory arthritis, upregulation of inflammatory and catabolic pathways with increased production of proinflammatory cytokines leading to cartilage degradation and extracellular matrix degeneration has been reported. Intra-articular injection of fresh fat derived stromal vascular fraction (SVF) fraction has been proposed as a valid and alternative treatment for symptomatic KOA that guarantees mechanical support through viscosupplementation, anti-inflammatory, and anabolic action. We retrospectively reviewed a case series of 84 consecutive adult patients with KOA who underwent intra-articular injection of fresh fat derived SVF. Significant improvement in pain levels (NRS score decrease 3.5±1.1, p<0.001), WOMAC pain (-7.02±3.45 score change, p<0.001), WOMAC stiffness (-1.97±1.02, p<0.001), and ROM improvement (+17.13±5.22°, p<0.001). The only complication noted was knee joint swelling lasting for less than 7 days after the injection in 7% of the patients.

A tumor microenvironment-specific gene expression signature predicts chemotherapy resistance in colorectal cancer patients

Studies have shown that tumor microenvironment (TME) might affect drug sensitivity and the classification of colorectal cancer (CRC). Using TME-specific gene signature to identify CRC subtypes with distinctive clinical relevance has not yet been tested. A total of 18 “bulk” RNA-seq datasets (total n = 2269) and four single-cell RNA-seq datasets were included in this study. We constructed a “Signature associated with FOLFIRI resistant and Microenvironment” (SFM) that could discriminate both TME and drug sensitivity. Further, SFM subtypes were identified using K-means clustering and verified in three independent cohorts. Nearest template prediction algorithm was used to predict drug response. TME estimation was performed by CIBERSORT and microenvironment cell populations-counter (MCP-counter) methods. We identified six SFM subtypes based on SFM signature that discriminated both TME and drug sensitivity. The SFM subtypes were associated with distinct clinicopathological, molecular and phenotypic characteristics, specific enrichments of gene signatures, signaling pathways, prognosis, gut microbiome patterns, and tumor lymphocytes infiltration. Among them, SFM-C and -F were immune suppressive. SFM-F had higher stromal fraction with epithelial-to-mesenchymal transition phenotype, while SFM-C was characterized as microsatellite instability phenotype which was responsive to immunotherapy. SFM-D, -E, and -F were sensitive to FOLFIRI and FOLFOX, while SFM-A, -B, and -C were responsive to EGFR inhibitors. Finally, SFM subtypes had strong prognostic value in which SFM-E and -F had worse survival than other subtypes. SFM subtypes enable the stratification of CRC with potential chemotherapy response thereby providing more precise therapeutic options for these patients.

Tumor Hypoxia Regulates Immune Escape/Invasion: Influence on Angiogenesis and Potential Impact of Hypoxic Biomarkers on Cancer Therapies.

The environmental and metabolic pressures in the tumor microenvironment (TME) play a key role in molding tumor development by impacting the stromal and immune cell fractions, TME composition and activation. Hypoxia triggers a cascade of events that promote tumor growth, enhance resistance to the anti-tumor immune response and instigate tumor angiogenesis. During growth, the developing angiogenesis is pathological and gives rise to a haphazardly shaped and leaky tumor vasculature with abnormal properties. Accordingly, aberrantly vascularized TME induces immunosuppression and maintains a continuous hypoxic state. Normalizing the tumor vasculature to restore its vascular integrity, should hence enhance tumor perfusion, relieving hypoxia, and reshaping anti-tumor immunity. Emerging vascular normalization strategies have a great potential in achieving a stable normalization, resulting in mature and functional blood vessels that alleviate tumor hypoxia. Biomarkers enabling the detection and monitoring of tumor hypoxia could be highly advantageous in aiding the translation of novel normalization strategies to clinical application, alone, or in combination with other treatment modalities, such as immunotherapy.

RECONSTRUCTION OF FUNCTIONAL EQUIVALENTS OF THE SKIN USING CELL AND TISSUE TECHNOLOGIES

The work is based on obtaining a living skin equivalent based on a three-dimensional scaffold consisting of type I collagen and other components of the extracellular matrix. The composition of the equivalent includes dermal and epidermal cells, it significantly accelerates and normalizes the healing process of chronic wounds. The living equivalent of the skin is a start for the further development of tissue-engineered constructs-analogs of skin and equivalents of other human tissues: at present, in particular, work is underway to study the stromal fractions of the dermis to control epithelial-mesenchymal interactions in vitro.

Bone marrow-derived NGFR+ cells regulate arterial remodeling and those poor mobilizations in peripheral blood in acute coronary syndrome predicts plaque progression at the non-targeted lesion

Abstract Background In our previous 5-year cohort study, we demonstrated that low gene expression of nerve growth factor receptor (NGFR) in peripheral leucocytes in acute coronary syndrome (ACS) predicted repetitive coronary interventions at the de novo lesions. An NGFR-positive cell has been demonstrated to reside in bone marrow (BM) stromal fraction and to be increased in peripheral blood mononuclear cell (MNCs) fraction in patients with ischemic heart disease. Purpose To investigate whether the BM-NGFR+ cell is associated with arterial remodeling and the relationship between the levels of peripheral NGFR+ cells after ACS and coronary plaque progression in an experimental and prospective clinical study. Methods and results In an experimental study, 8-week-old C57B6/J wild type male mice were subjected to irradiation with 9.6 Gy and transplantation with BM (BMT) isolated from GFP-transgenic NGFR wild type (WT) or knock-out (KO) mice at day 1. Four weeks after BMT, the right carotid artery was ligated for 4 weeks. Induced neointimal area was increased (p&amp;lt;0.05), where cells under apoptosis were decreased (p&amp;lt;0.05) in NGFR-KO-BMT group compared to WT-BMT group (n=4). NGFR+ cells were not detected in wild type sham-operated artery, whereas in the ligated artery in WT-BMT group NGFR+ cells assembled in the developed neointima and exclusively presented double positive with GFP, but absent in NGFR-KO-BMT group (p&amp;lt;0.05, n=4). In a clinical study, thirty patients with ACS who underwent primary percutaneous coronary intervention (PCI) were enrolled. The peripheral blood sample was collected on days 0, 3 and 7, and 9 months follow-up and the number of NGFR+MNCs were measured by flowcytometric analysis. The plaque volume at non-targeted coronary lesion (non-TL:&amp;gt;5 mm proximal or distal to the implanted stents) were quantitatively analysed using gray-scale intravascular ultrasound (IVUS) and Q-IVUS™ software at the acute phase and 9 months follow-up. The number of NGFR+MNCs in peripheral blood was 1.5-fold increased at day 3 (0.064±0.056%) compared to day 0 (0.042±0.030%) (p&amp;lt;0.05). The change in normalized total plaque volume (TAVN) at non-TL at 9 months was negatively correlated with the number of NGFR+MNCs at day 0 (r=−0.51), day 3 (r=−0.51) and 9 months (r=−0.59) after ACS (p&amp;lt;0.05). Multiple regression analysis showed that NGFR+MNCs at day 0 (β=−0.48, p=0.01) and CRP (β=−0.53, P&amp;lt;0.01) are independent factors associating with TAVN change at non-TL at 9 months, regardless of LDL-cholesterol control level. ROC analysis revealed that NGFR+MNCs &amp;lt;0.049 at day 0 predicted the increase of TAVN with AUC 0.78; sensitivity 0.82 and specificity 0.67. Conclusions Bone marrow-derived peripheral NGFR+ cells negatively regulate arterial remodeling through appropriate apoptosis of neointimal cells and the peripheral level of NGFR+ cells in ACS predicts plaque progression at the non-targeted lesion. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): KAKENHI

COMPARISON OF HUMAN ANTRAL FOLLICLES OF XENOGRAFT VERSUS OVARIAN ORIGIN REVEALS DISPARATE MOLECULAR SIGNATURES

The activation, growth and maturation of oocytes to an ovulatory phase, termed folliculogenesis, is governed by the orchestrated activity of multiple specialized cells types within the ovary, yet mechanisms governing diversification and behavior of discrete cellular sub-populations within follicles are poorly understood. We set out to identify and characterize unique subsets among the granulosa (GC), theca (TC) and stromal cell fractions, and to compare the molecular profiles of these sub-populations when isolated from native ovarian versus xenograft-derived antral follicles. Small antral follicles (< 5mm) were isolated from ovaries of organ donors or patients undergoing whole ovarian resection for fertility preservation. These were compared to antral follicles that developed in long term (> 14 weeks) xenografts of cryopreserved/thawed human ovarian cortex. Immunostaining, flow cytometry, bulk and single-cell RNA sequencing were used to segregate and interrogate discrete follicle-derived cell fractions. The GC fraction was first purified from 3 small (2-3 mm) ovary-derived antral follicles and subjected to bulk RNA sequencing to identify candidate surface molecules that may specifically identify follicle cell sub-types. Candidate factors were screened using immune-staining and flow cytometry of xenograft and ovary-derived follicles. 22 unique single-cell library preparations (∼100,000 cells) representing fractions from 13 individual follicles (8 ovary origin, 5 xenograft origin) and 6 women were prepared, sequenced and analyzed. Analysis of GC, TC and stromal subpopulations deconstructed phenotypic diversification during early antral follicle development, identifying secreted factors that are differentially enriched between mural and oophorus GCs, and segregating stromal/support and steroidogenic activity between theca externa and interna, respectively. Numerous factors were differentially expressed in follicles of xenograft versus ovarian origin, highlighting the potential contribution of humoral factors to follicular development that have not been considered, to date. These data shed light on previously undescribed subpopulations of both GC and TC compartments and provide a systems level portrait of cellular diversification in early antral human follicles.

Clinical and genomic landscape of hepatocellular carcinoma subtypes with various proportions of nonleukocyte stromal cells.

Hepatocellular carcinoma (HCC) is one of the most common malignancies and inflicts high mortality worldwide. The effect of tumor microenvironment components on HCC oncogenesis remains unknown. In particular, the nonleukocyte portion of the stromal fraction (SF) is poorly understood. We comprehensively evaluated the proportional cell counts and gene expression data from The Cancer Genome Atlas (TCGA) to examine the contributions of cell components to the tumor microenvironment. Single-cell sequencing data from the Gene Expression Omnibus (GEO) were also analyzed to verify the association between the nonleukocyte SF and genes. We classified HCC using a hierarchical clustering method based on diversity of nonleukocyte SF-related gene expression among different components, and we used an appropriate GEO dataset to verify the clusters with a support vector machine (SVM) model. The prognosis of subtypes and their relationship with tumor microenvironmental cell proportions, clinicopathogenesis factors, and other indicators were evaluated. Based on linear regression, 711 genes related to nonleukocyte SF were selected from the TCGA dataset. We classified HCC into three subtypes using genes related to the nonleukocyte SF. Additionally, the GEO single-cell sequencing data confirmed the relationship between genes and the nonleukocyte SF. The tumor microenvironment of Type 2 contained the most significant mutually reinforcing interaction between the nonleukocyte SF and tumor cells. Meanwhile, Type 2 patients had the poorest prognosis and the most severe tumor-node-metastasis (TNM) stages, histological grades, etc. The analysis based on the GEO dataset verified the classification results with an SVM model. Type 2 was associated with worse clinicopathological characteristics, including tumor grading and staging, than the other types. In addition, the pathway analysis revealed that signals related to the SF and cell proliferation were significantly enhanced in Type 2 compared to the other group, which consisted of Types 1 and 3. The nonleukocyte SF in the tumor microenvironment contributed greatly to HCC oncogenesis. We can use these HCC classification criteria to stratify patients into subtypes for personalized treatment.

Recapitulation of prostate tissue cell type-specific transcriptomes by an in vivo primary prostate tissue xenograft model.

Studies of the normal functions and diseases of the prostate request in vivo models that maintain the tissue architecture and the multiple-cell type compartments of human origin in order to recapitulate reliably the interactions of different cell types. Cell type-specific transcriptomes are critical to reveal the roles of each cell type in the functions and diseases of the prostate. A primary prostate tissue xenograft model was developed using fresh human prostate tissue specimens transplanted onto male mice that were castrated surgically and implanted with a device to maintain circulating testosterone levels comparable to adult human males. Endothelial cells and epithelial cells were isolated from 7 fresh human prostate tissue specimens and from primary tissue xenografts established from 9 fresh human prostate tissue specimens, using antibody-conjugated magnetic beads specific to human CD31 and human EpCAM, respectively. Transcriptomes of endothelial, epithelial and stromal cell fractions were obtained using RNA-Seq. Global and function-specific gene expression profiles were compared in inter-cell type and inter-tissue type manners. Gene expression profiles in the individual cell types isolated from xenografts were similar to those of cells isolated from fresh tissue, demonstrating the value of the primary tissue xenograft model for studies of the inter-relationships between prostatic cell types and the role of such inter-relationships in organ development, disease progression, and response to drug treatments.

Prostate Microstructure in Prostate Cancer Using 3-T MRI with Diffusion-Relaxation Correlation Spectrum Imaging: Validation with Whole-Mount Digital Histopathology.

Background Microstructural MRI has the potential to improve diagnosis and characterization of prostate cancer (PCa), but validation with histopathology is lacking. Purpose To validate ex vivo diffusion-relaxation correlation spectrum imaging (DR-CSI) in the characterization of microstructural tissue compartments in prostate specimens from men with PCa by using registered whole-mount digital histopathology (WMHP) as the reference standard. Materials and Methods Men with PCa who underwent 3-T MRI and robotic-assisted radical prostatectomy between June 2018 and January 2019 were prospectively studied. After prostatectomy, the fresh whole prostate specimens were imaged in patient-specific three-dimensionally printed molds by using 3-T MRI with DR-CSI and were then sliced to create coregistered WMHP slides. The DR-CSI spectral signal component fractions (fA, fB, fC) were compared with epithelial, stromal, and luminal area fractions (fepithelium, fstroma, flumen) quantified in PCa and benign tissue regions. A linear mixed-effects model assessed the correlations between (fA, fB, fC) and (fepithelium, fstroma, flumen), and the strength of correlations was evaluated by using Spearman correlation coefficients. Differences between PCa and benign tissues in terms of DR-CSI signal components and microscopic tissue compartments were assessed using two-sided t tests. Results Prostate specimens from nine men (mean age, 65 years ± 7 [standard deviation]) were evaluated; 20 regions from 17 PCas, along with 20 benign tissue regions of interest, were analyzed. Three DR-CSI spectral signal components (spectral peaks) were consistently identified. The fA, fB, and fC were correlated with fepithelium, fstroma, and flumen (all P < .001), with Spearman correlation coefficients of 0.74 (95% confidence interval [CI]: 0.62, 0.83), 0.80 (95% CI: 0.66, 0.89), and 0.67 (95% CI: 0.51, 0.81), respectively. PCa exhibited differences compared with benign tissues in terms of increased fA (PCa vs benign, 0.37 ± 0.05 vs 0.27 ± 0.06; P < .001), decreased fC (PCa vs benign, 0.18 ± 0.06 vs 0.31 ± 0.13; P = .01), increased fepithelium (PCa vs benign, 0.44 ± 0.13 vs 0.26 ± 0.16; P < .001), and decreased flumen (PCa vs benign, 0.14 ± 0.08 vs 0.27 ± 0.18; P = .004). Conclusion Diffusion-relaxation correlation spectrum imaging signal components correlate with microscopic tissue compartments in the prostate and differ between cancer and benign tissue. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Lee and Hectors in this issue.

1962-P: Role of Transcription Factor TCF21 in Adipocyte Progenitor Cells of Visceral Fat

Accumulation of visceral fat leads to insulin resistance and diabetes. Therefore, it is important to elucidate the mechanism by which adipose progenitor cells (APCs) are maintained and how they differentiate. The bHLH transcription factor 21 (Tcf21) is expressed in visceral, but not in subcutaneous fat, and used as a marker for white adipose tissues. Although Tcf21 promotes IL6 expression in APCs in vitro, little is known about its role in vivo. Therefore, we aimed to clarify the role of Tcf21 in visceral fat using mouse models. Tcf21 was deleted after birth using inducible Cre-LoxP system to circumvent the lethality due to cardiac malformation (iTcf21 KO). The mice were fed high fat diet (HFD) from 6 to 24 weeks of age. Weight gain, blood glucose level, epididymal fat weight, and APC count (0.8x104/g fat for iKO and 2.9x104/g fat for wild type (WT)) of epididymal fat were all low in iTcf21KO compared to WT mice. Insulin sensitivity was increased in iTcf21KO mice. Staining of epididymal fat from Tcf21lacZ/+ mice using X-Gal revealed that Tcf21 was localized in the vascular mural cells. Isolation of Sca1+ Lin- APCs using magnetic activated cell sorting revealed that Tcf21 mRNA was expressed in the APCs, along with Pdgfrb and Periostin. In contrast, Tcf21 expression was attenuated in the adipose tissue of WT mice after HFD, and in adipocyte culture that was differentiated from the vascular stromal fraction (SVF). In addition, Tcf21 expression suppressed by HFD was upregulated in adipocytes undergoing de-differentiation by ceiling culture. Adipocyte induction from SVF resulted in no differences between WT and iTcf21KO mice assessed by oil red O staining. However, adipocyte differentiation and expression of Pparg and Cebpb were suppressed in 3T3-L1 cells in which Tcf21 was overexpressed. These findings suggest that Tcf21 is expressed in the APCs of vascular mural cells, controls the number of APCs, and potentially contributes to the maintenance of their progenitor state through suppression of adipocyte differentiation. Disclosure T. Minamizuka: None. Y. Maezawa: None. H. Udagawa: None. Y. Baba: None. M. Koshizaka: None. Y. Endo: None. K. Yasuda: None. K. Yokote: Research Support; Self; Astellas Pharma Inc., Daiichi Sankyo, Eli Lilly Japan K.K., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Mochida Pharmaceutical Co., Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Pfizer Japan Inc., Sanofi K.K., Shionogi &amp; Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited. Speaker’s Bureau; Self; Abbott, Astellas Amgen, AstraZeneca K.K., Bayer Inc., FUJIFILM Pharmaceuticals U.S.A., Inc., Kaken Pharmaceutical Co., Ltd., Sanwa Kagaku Kenkyusho.

SUN-746 Thyroid Hormone Receptor Alpha Sumoylation Is Important for the Development of Adipose Tissue

Thyroid hormone (TH) plays an essential role in normal development. TH action requires binding to its receptor. There are two types of thyroid hormone receptor, alpha (THRA) and beta (THRB). THRB is important for regulation of the Hypothalamic-Pituitary-Thyroid axis and regulating cholesterol metabolism. THRA is involved in the development and the function of brain, adipose tissue, small intestine, bone and heart. Both THRA and THRB are post-translationally modified by small ubiquitin-like modifier (SUMO). Previously, we showed in an in vitro model that mutation of a THRA sumoylation motif impairs proliferation and differentiation of human primary white adipocytes. This is due to interference of the desumolyated THRA in the cell cycle at G1/S phase and disruption of PPAR signaling. To determine the in vivo effects of a desumoylated THRA, we generated a mouse model carrying a mutation of the THRA gene, which eliminates SUMO conjugation at lysine (K) 283. At 12 weeks of age, there was no difference in body composition (body weight, fat mass, and lean mass) in THRA mutant mice compared to wild-type (WT). From 12 weeks onward mutant mice and WT did not have significant change body weight compared to Wt mice. However, THRA sumoylation mutant mice had much lower percent body fat. Dissected WAT fat pads from mutant mice were significantly smaller compared to WT mice. Histological analysis showed that the cell size of white adipose tissue in mutant mice was significantly smaller than that in Wt mice as well. Serum leptin levels in the mutant mice were significantly lower than that in mice, consistent with reduced fat mass. We isolated fat stem cells from stromal vesicular fraction of 6 week old mice and found that the number of fat stem cells was significant less in mutant mice compared to those in WT mice. This data suggest that de-sumoylated THRA is associated with reduced fat stem cells at a young age, resulting in reduced adipose tissue mass in adult. THRA sumoylation is important for thyroid hormone modulation of fat mass. Reference: (1) Yan-Yun Liu et al, JBC 2012 (2) Yan-Yun Liu et al., JBC 2015

3-Iodothyronamine Affects Thermogenic Substrates' Mobilization in Brown Adipocytes.

We investigated the effect of 3-iodothyronamine (T1AM) on thermogenic substrates in brown adipocytes (BAs). BAs isolated from the stromal fraction of rat brown adipose tissue were exposed to an adipogenic medium containing insulin in the absence (M) or in the presence of 20 nM T1AM (M+T1AM) for 6 days. At the end of the treatment, the expression of p-PKA/PKA, p-AKT/AKT, p-AMPK/AMPK, p-CREB/CREB, p-P38/P38, type 1 and 3 beta adrenergic receptors (β1–β3AR), GLUT4, type 2 deiodinase (DIO2), and uncoupling protein 1 (UCP-1) were evaluated. The effects of cell conditioning with T1AM on fatty acid mobilization (basal and adrenergic-mediated), glucose uptake (basal and insulin-mediated), and ATP cell content were also analyzed in both cell populations. When compared to cells not exposed, M+T1AM cells showed increased p-PKA/PKA, p-AKT/AKT, p-CREB/CREB, p-P38/P38, and p-AMPK/AMPK, downregulation of DIO2 and β1AR, and upregulation of glycosylated β3AR, GLUT4, and adiponectin. At basal conditions, glycerol release was higher for M+T1AM cells than M cells, without any significant differences in basal glucose uptake. Notably, in M+T1AM cells, adrenergic agonists failed to activate PKA and lipolysis and to increase ATP level, but the glucose uptake in response to insulin exposure was more pronounced than in M cells. In conclusion, our results suggest that BAs conditioning with T1AM promote a catabolic condition promising to fight obesity and insulin resistance.

Pancreatic adenocarcinoma: quantitative CT features are correlated with fibrous stromal fraction and help predict outcome after resection.

To identify quantitative imaging features of contrast-enhanced computed tomography (CE-CT) that may be prognostically favorable after resection of smaller (≤ 30mm) pancreatic ductal adenocarcinomas (PDACs) located at head. This retrospective study included two independent cohorts (discovery cohort, n = 212; test cohort, n = 100) of patients who underwent resection of head PDACs ≤ 30mm and preoperative CE-CT. We examined tumor and surrounding parenchymal attenuation differences (deltas), and tumor attenuation changes across phases (ratios). Semantic features of PDACs were evaluated by two radiologists. Clinicopathologic and imaging features for predicting disease-free survival (DFS) and overall survival (OS) were analyzed via multivariate Lasso-penalized Cox proportional-hazards models. Survival rates were derived by Kaplan-Meier method. Imaging features achieved C-indices of 0.766 (discovery cohort) and 0.739 (test cohort) for DFS, and 0.790 (discovery cohort) and 0.772 (test cohort) for OS estimates through incorporation of clinicopathologic features. The most decisive imaging feature was delta 3, denoting attenuation differences between tumor and surrounding pancreas at pancreatic phase (DFS: HR = 2.122; OS: HR = 2.375; both p < 0.001). Compared with inconspicuous (low-delta-3, < 28 HU) tumors, conspicuous (high-delta-3) tumors correlated significantly with more aggressive histologic grades (p = 0.014) and less extensive tumor fibrous stromal fractions (p < 0.001). Patients with low-delta-3 tumors ≤ 20mm experienced the most favorable outcomes (DFS, 36months; OS, 42months), whereas those with high-delta-3 tumors fared poorly, regardless of tumor size (DFS, 12months; OS, 19months). Quantifiable CT imaging features reflect heterogeneous fibrous stromal fractions and histologic grades of PDAC at head locations that help stratify patients with disparate clinical outcomes. • Quantitative and semantic imaging features achieved promising results for the prognosis of resected PDAC (≤ 30mm) at head location, through incorporation of clinicopathologic features. • Attenuation difference at tumor-parenchyma interface (delta 3) emerged as the most decisive imaging feature, enabling further stratification of patients into distinct prognostic subtypes by tumor size. • High delta 3 signifies sharper contrast between tumor and surrounding pancreas, correlating with more aggressive histologic grades and less extensive tumor fibrous stromal fractions.

Single-cell analysis of human adipose tissue identifies depot and disease specific cell types.

The complex relationship between metabolic disease risk and body fat distribution in humans involves cellular characteristics which are specific to body fat compartments. Here we show depot-specific differences in the stromal vascual fraction of visceral and subcutaneous adipose tissue by performing single-cell RNA sequencing of tissue specimen from obese individuals. We characterize multiple immune cells, endothelial cells, fibroblasts, adipose and hematopoietic stem cell progenitors. Subpopulations of adipose-resident immune cells are metabolically active and associated with metabolic disease status and those include a population of potential dysfunctional CD8+ T cells expressing metallothioneins. We identify multiple types of adipocyte progenitors that are common across depots, including a subtype enriched in individuals with type 2 diabetes. Depot-specific analysis reveals a class of adipocyte progenitors unique to visceral adipose tissue, which shares common features with beige preadipocytes. Our human single-cell transcriptome atlas across fat depots provides a resource to dissect functional genomics of metabolic disease.

Effect of Mural or Intra-Arterial Injection Adipose Derived Stromal Fraction on TNBS-Induced Colitis: An Experimental Study

Effect of Mural or Intra-Arterial Injection Adipose Derived Stromal Fraction on TNBS-Induced Colitis: An Experimental Study

Serum Amyloid P and a Dendritic Cell–Specific Intercellular Adhesion Molecule-3–Grabbing Nonintegrin Ligand Inhibit High-Fat Diet–Induced Adipose Tissue and Liver Inflammation and Steatosis in Mice

High-fat diet (HFD)-induced inflammation is associated with a variety of health risks. The systemic pentraxin serum amyloid P (SAP) inhibits inflammation. SAP activates the high-affinity IgG receptor Fcγ receptor I (FcγRI; CD64) and the lectin receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN; CD209). Herein, we show that for mice on an HFD, injections of SAP and a synthetic CD209 ligand (1866) reduced HFD-increased adipose and liver tissue inflammation, adipocyte differentiation, and lipid accumulation in adipose tissue. HFD worsened glucose tolerance test results and caused increased adipocyte size; for mice on an HFD, SAP improved glucose tolerance test results and reduced adipocyte size. Mice on an HFD had elevated serum levels of IL-1β, IL-23, interferon (IFN)-β, IFN-γ, monocyte chemoattractant protein 1 [MCP-1; chemokine (C-C motif) ligand 2 (CCL2)], and tumor necrosis factor-α. SAP reduced serum levels of IL-23, IFN-β, MCP-1, and tumor necrosis factor-α, whereas 1866 reduced IFN-γ. Invitro, SAP, but not 1866, treated cells isolated from white fat tissue (stromal vesicular fraction) produced the anti-inflammatory cytokine IL-10. HFD causes steatosis, and both SAP and 1866 reduced it. Conversely, compared with control mice, SAP knockout mice fed on a normal diet had increased white adipocyte cell sizes, increased numbers of inflammatory cells in adipose and liver tissue, and steatosis; and these effects were exacerbated on an HFD. SAPand1866 may inhibit some, but not all, of the effects of a high-fat diet.

Whole-tumor histogram analysis of monoexponential and advanced diffusion-weighted imaging for sinonasal malignant tumors: Correlations with histopathologic features.

The histopathological basis of monoexponential diffusion-weighted imaging (DWI), intravoxel incoherent motion (IVIM), and diffusion kurtosis imaging (DKI) in the characterization of sinonasal malignant tumors is still unclear. To explore the correlations of histogram metrics from monoexponential DWI, IVIM, and DKI with histopathologic features in sinonasal malignant tumors. Retrospective. In all, 76 patients with sinonasal malignant tumors. Fourteen different b values (b = 0, 50, 100, 150, 200, 250, 300, 350, 400, 800, 1000, 1500, 2000, and 2500 sec/mm2 ) were used to perform different DWI models at 3.0T. The whole-tumor histogram metrics were calculated on the apparent diffusion coefficient (ADC), pure diffusion coefficient (D), pseudodiffusion coefficient (D*), perfusion fraction (f), diffusion kurtosis (K), and diffusion coefficient (Dk) maps. Histopathologic features, including nuclear, cytoplasmic, cellular, stromal fractions, and the nuclear-to-cytoplasmic (N/C) ratio, were measured. Spearman correlations and stepwise multiple linear regression analyses were performed to determine the correlations between histogram metrics and histopathologic features. ADC, Dk, and f histogram metrics showed significant correlations with investigated histopathologic features; D and K histogram metrics were significantly correlated with cellular, stromal, and nuclear fractions (all P < 0.05). Significant correlations between the 75th percentile of D and cytoplasmic fraction and between the kurtosis of K and the N/C ratio were observed (P < 0.05). The skewness of Dk, K, and the 75th percentile of D were independently associated with cellular and nuclear fractions; the skewness of Dk and K were independently associated with stromal fraction (P < 0.05). Monoexponential and advanced DWI histogram parameters were significantly correlated with histopathologic features in sinonasal malignancies. 3 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2020;51:273-285.