Human Adipose Tissue Research Articles

Proposal of Simplified Standardization of the Cell-Growth-Promoting Activity of Human Adipose Tissue Mesenchymal Stromal Cell Culture Supernatants

The conditioned medium (CM) obtained from mesenchymal stromal cell (MSC) culture has excellent cell growth-promoting activity and is used for cosmetics and healthcare products. Unlike pharmaceuticals, strict efficacy verification is not legally required for these products. However, their efficacy must be substantiated as commercial products. We attempted to simplify CM production and to standardize the evaluation of the growth-promoting activity of CM. CM was obtained through the culturing of two lines of commercially available human adipose tissue-derived MSCs using MEMα with or without 10% fetal bovine serum (FBS) for 24 h. Non-CM control media were produced by the same protocol without MSCs. Growth-promoting activities of the CM were estimated by [3H]-thymidine pulse. CM were subjected to molecular weight fractionation with ultrafiltration using 10 k-, 30 k-, 50 k-, and 100 k-membranes. The FBS-free CMs showed 1.34- to 1.85-fold increases and FBS-containing CMs showed 1.45- to 1.67-fold increases in proliferation-promoting activity compared with non-CM controls, regardless of the source of the cell. The thymidine incorporation levels were approximately three times higher in FBS-containing CMs. Aged cells also showed 1.67- to 2.48-fold increases in the activity due to FBS-containing CM, but not to FBS-free CM. The CM activities were sustained even after 1 year at 4 °C. Molecular weight fractionation showed that the activity was recovered in the fraction above 100 k. Clear and stable cell-growth-promoting activity was confirmed with CMs of commercially available adipose tissue MSCs. The activity was detected in the fraction over 100 k. We propose here the importance of standardizing the production and evaluation of CMs to indicate their specific action.

The beta-2-adrenoreceptor agonist fenoterol increases resting enery expenditure without activation of brown adipose tissue in humans

The beta-2-adrenoreceptor agonist fenoterol increases resting enery expenditure without activation of brown adipose tissue in humans

Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing

Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing

Abstract PO3-13-08: D17104: Aryl Hydrocarbon Receptor Signaling and Other Biomarkers of Fat Metabolism in Human Mammary Adipose Tissue from Women with Malignant Breast Disease

Abstract Background: Obesity is a recognized risk factor for the development of metabolic disease and breast cancer possibly due to chronic inflammation mediated by cytokines secreted by adipose tissue. Given this, our group examined differential expression of several markers of chronic inflammation in adipose tissue of both benign and malignant breast tissue from patients. The aryl hydrocarbon receptor (AhR) is an important cytosolic, ligand-dependent transcription factor found within hepatocytes and adipocytes that is upregulated in many types of cancer. AhR promotes the initiation, progression, and metastasis of cancer cells allowing certain cancers to evade immune recognition (Xue et al. Front. Immunology 2018). Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme that generates kynurenine (KYN) from tryptophan (TRP), which acts as an AhR agonist. We sought to determine expression of IDO1 via immunohistochemistry (IHC) in mammary adipose tissue associated with benign or malignant breast. We hypothesized that adipose tissue adjacent to malignant breast tissue may have heightened expression of IDO1 in addition to Cyp1b1, and SPP1; both of which are also markers of increased aryl-hydrocarbon transcriptional activity, compared to adipose tissue noncontiguous to malignant lesions. KYN affects gene expression for insulin resistance and levels are elevated in plasma of older obese women compared to lean women; however, the relationship to breast malignancy is not known. Methods: IDO1 (Cell Signaling, 1:200), Cyp1b1 (Thermo Fisher, 1:300), and SPP1 (Thermo Fisher, 1:200) IHC was performed according to the manufacturers' protocols. Adipose tissue immediately adjacent to, and >2 mm away from, tumors (three DCIS, 11 invasive carcinomas) was evaluated in 14 patients. Adipose tissue in separate tissue blocks from benign breast tissue (three ipsilateral, 11 contralateral) in the same patients was also evaluated. Human plasma KYN levels were assayed by HPLC in the Dartmouth Clinical Pharmacology Shared Resource as described previously (Rojas et al. Obesity 2021). Results: In this preliminary pilot study, there was no difference in staining pattern for IDO1 in adipose tissue present in malignant and benign samples. Two patients showed increased Cyp1b1 and SPP1 staining immediately adjacent to the invasive carcinoma (Figure 1). Patients with benign breast disease did not show increased Cyp1b1 or SPP1 staining. Results will be confirmed using qPCR for mRNA. Plasma samples from patients with benign disease showed a lower level of KYN expression on average (0.370 ug/mL, N = 8) compared to plasma from patients with malignant disease (0.424, N = 10). Additionally, the ratio of KYN:TRP was lower in the benign group (0.065) compared to the cohort with malignancy (0.080). Univariate linear regression will be used to further assess relationships between continuous variables in the study population (e.g., patient age, BMI, KYN plasma levels, benign vs malignant breast disease). Regression lines and the associated slope estimates (b-value) and p-values for regression models will be provided on scatter plots. Conclusions: While there was no difference in IDO1 staining patterns in adipose tissue of malignant and benign samples in this group, tissue samples from patients with invasive carcinoma demonstrated increased Cyp1b1 and SPP1 staining adjacent to the malignant tumor; while benign tissue samples did not show a positive stain. This suggests increased aryl-hydrocarbon transcriptional activity in invasive breast carcinoma—further linking AhR signaling with adipose tissue immune dysregulation and invasive malignancy. Additionally, KYN plasma levels were higher in patients with malignant disease indicating a possible relationship between insulin resistance and breast malignancy. Additional research may offer new insights relating to this important pathway and ultimately treatments aimed at blunting its nefarious effects. Citation Format: Adam Ephraim, Jonathan Pirruccello, Scott Palisoul, Jonathan Marotti, Mary Chamberlin. D17104: Aryl Hydrocarbon Receptor Signaling and Other Biomarkers of Fat Metabolism in Human Mammary Adipose Tissue from Women with Malignant Breast Disease [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-08.

Deletion of CD44 promotes adipogenesis by regulating PPARɣ and cell cycle-related pathways.

CD44, a cell surface adhesion receptor and stem cell biomarker, is recently implicated in chronic metabolic diseases. Ablation of CD44 ameliorates adipose tissue inflammation and insulin resistance in obesity. Here, we investigated cell type specific CD44 expression in human and mouse adipose tissue and further studied how CD44 in preadipocytes regulates adipocyte function. Using Crispr Cas9-mdediated gene deletion and lentivirus-mediated gene re-expression, we discovered that deletion of CD44 promotes adipocyte differentiation and adipogenesis, whereas re-expression of CD44 abolishes this effect and decreases insulin responsiveness and adiponectin secretion in 3T3-L1 cells. Mechanistically, CD44 does so via suppressing Pparg expression. Using quantitative proteomics analysis, we further discovered that cell cycle-regulated pathways were mostly decreased by deletion of CD44. Indeed, re-expression of CD44 moderately restored expression of proteins involved in all phases of the cell cycle. These data were further supported by increased preadipocyte proliferation rates in CD44 deficient cells and re-expression of CD44 diminished this effect. Our data suggest that CD44 plays a crucial role in regulating adipogenesis and adipocyte function possibly through regulating PPARɣ and cell cycle-related pathways. This study provides evidence for the first time that CD44 expressed in preadipocytes plays key roles in regulating adipocyte function outside immune cells where CD44 is primarily expressed. Therefore, targeting CD44 in (pre)adipocytes may provide therapeutic potential to treat obesity-associated metabolic complications.

The Vascular Function of Resistance Arteries Depends on NADPH Oxidase 4 and Is Exacerbated by Perivascular Adipose Tissue

The NADPH oxidase NOX4 that releases H2O2 can mediate vasoprotective mechanisms under pathophysiological conditions in conductive arteries. However, the role of NOX4 in resistance arteries and in perivascular adipose tissue is not well understood. We hypothesized that NOX4 is of functional importance in resistance arteries and perivascular adipose tissue under dyslipidemia conditions. We detected elevated NOX4 expression in murine and human vessels under dyslipidemia. Diminishing Nox4 under these conditions led to endothelial dysfunction in resistance arteries. The mesenteric arteries of Nox4−/−/Ldlr−/− mice revealed decreased eNos mRNA expression. Inhibition of eNOS in those vessels did not affect vascular function, while in Ldlr−/− mice endothelial function was significantly altered. Anticontractile properties of perivascular adipose tissue at resistance arteries were diminished in Nox4−/−/Ldlr−/− compared with Ldlr−/− mice. In addition, the presence of perivascular adipose tissue further worsened endothelial dysfunction in mesenteric arteries under dyslipidemia conditions. Perivascular adipose tissue from mesenteric arteries revealed a higher expression of markers of white adipocytes compared to markers of beige/brown adipocytes. Among those white adipocyte markers, leptin was significantly less expressed in perivascular adipose tissue from Nox4−/−/Ldlr−/− mice compared with Ldlr−/− mice. Furthermore, in human perivascular adipose tissue with a profound pattern of white adipocyte marker genes, we detected a correlation of NOX4 and LEP expression. In addition, incubating arterial vessels with leptin induced nitrite release, indicating increased eNOS activity. In humans, a higher expression of leptin in perivascular adipose tissue correlated with eNOS expression in the corresponding left internal mammary artery. In conclusion, vascular function of resistance arteries was dependent on Nox4-derived H2O2, especially under dyslipidemia conditions. Perivascular adipose tissue of the mesenteric arteries with white adipose tissue characteristics further aggravated endothelial function through reduced leptin-eNOS signaling.

MALAT1: A Long Non-Coding RNA with Multiple Functions and Its Role in Processes Associated with Fat Deposition.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) belongs to the lncRNA molecules, which are involved in transcriptional and epigenetic regulation and the control of gene expression, including the mechanism of chromatin remodeling. MALAT1 was first discovered during carcinogenesis in lung adenocarcinoma, hence its name. In humans, 66 of its isoforms have been identified, and in pigs, only 2 are predicted, for which information is available in Ensembl databases (Ensembl Release 111). MALAT1 is expressed in numerous tissues, including adipose, adrenal gland, heart, kidney, liver, ovary, pancreas, sigmoid colon, small intestine, spleen, and testis. MALAT1, as an lncRNA, shows a wide range of functions. It is involved in the regulation of the cell cycle, where it has pro-proliferative effects and high cellular levels during the G1/S and mitotic (M) phases. Moreover, it is involved in invasion, metastasis, and angiogenesis, and it has a crucial function in alternative splicing during carcinogenesis. In addition, MALAT1 plays a significant role in the processes of fat deposition and adipogenesis. The human adipose tissue stem cells, during differentiation into adipocytes, secrete MALAT1 as one the most abundant lncRNAs in the exosomes. MALAT1 expression in fat tissue is positively correlated with adipogenic FABP4 and LPL. This lncRNA is involved in the regulation of PPARγ at the transcription stage, fatty acid metabolism, and insulin signaling. The wide range of MALAT1 functions makes it an interesting target in studies searching for drugs to prevent obesity development in humans. In turn, in farm animals, it can be a source of selection markers to control the fat tissue content.

Glucose regulation of adipose tissue browning by CBP/p300- and HDAC3-mediated reversible acetylation of CREBZF

Glucose is required for generating heat during cold-induced nonshivering thermogenesis in adipose tissue, but the regulatory mechanism is largely unknown. CREBZF has emerged as a critical mechanism for metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as nonalcoholic fatty liver disease (NAFLD). We investigated the roles of CREBZF in the control of thermogenesis and energy metabolism. Glucose induces CREBZF in human white adipose tissue (WAT) and inguinal WAT (iWAT) in mice. Lys208 acetylation modulated by transacetylase CREB-binding protein/p300 and deacetylase HDAC3 is required for glucose-induced reduction of proteasomal degradation and augmentation of protein stability of CREBZF. Glucose induces rectal temperature and thermogenesis in white adipose of control mice, which is further potentiated in adipose-specific CREBZF knockout (CREBZF FKO) mice. During cold exposure, CREBZF FKO mice display enhanced thermogenic gene expression, browning of iWAT, and adaptive thermogenesis. CREBZF associates with PGC-1α to repress thermogenic gene expression. Expression levels of CREBZF are negatively correlated with UCP1 in human adipose tissues and increased in WAT of obese ob/ob mice, which may underscore the potential role of CREBZF in the development of compromised thermogenic capability under hyperglycemic conditions. Our results reveal an important mechanism of glucose sensing and thermogenic inactivation through reversible acetylation.

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue.

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk. hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function. hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

Adipose-derived stem cells derived decellularized extracellular matrix enabled skin regeneration and remodeling.

The tissues or organs derived decellularized extracellular matrix carry immunogenicity and the risk of pathogen transmission, resulting in limited therapeutic effects. The cell derived dECM cultured in vitro can address these potential risks, but its impact on wound remodeling is still unclear. This study aimed to explore the role of decellularized extracellular matrix (dECM) extracted from adipose derived stem cells (ADSCs) in skin regeneration. Methods: ADSCs were extracted from human adipose tissue. Then we cultivated adipose-derived stem cell cells and decellularized ADSC-dECM for freeze-drying. Western blot (WB), enzyme-linked immunosorbent assay (ELISA) and mass spectrometry (MS) were conducted to analyzed the main protein components in ADSC-dECM. The cell counting assay (CCK-8) and scratch assay were used to explore the effects of different concentrations of ADSC-dECM on the proliferation and migration of human keratinocytes cells (HaCaT), human umbilical vein endothelia cells (HUVEC) and human fibroblasts (HFB), respectively. Moreover, we designed a novel ADSC-dECM-CMC patch which used carboxymethylcellulose (CMC) to load with ADSC-dECM; and we further investigated its effect on a mouse full thickness skin wound model. Results: ADSC-dECM was obtained after decellularization of in vitro cultured human ADSCs. Western blot, ELISA and mass spectrometry results showed that ADSC-dECM contained various bioactive molecules, including collagen, elastin, laminin, and various growth factors. CCK-8 and scratch assay showed that ADSC-dECM treatment could significantly promote the proliferation and migration of HaCaT, human umbilical vein endothelia cells, and human fibroblasts, respectively. To evaluate the therapeutic effect on wound healing in vivo, we developed a novel ADSC-dECM-CMC patch and transplanted it into a mouse full-thickness skin wound model. And we found that ADSC-dECM-CMC patch treatment significantly accelerated the wound closure with time. Further histology and immunohistochemistry indicated that ADSC-dECM-CMC patch could promote tissue regeneration, as confirmed via enhanced angiogenesis and high cell proliferative activity. Conclusion: In this study, we developed a novel ADSC-dECM-CMC patch containing multiple bioactive molecules and exhibiting good biocompatibility for skin reconstruction and regeneration. This patch provides a new approach for the use of adipose stem cells in skin tissue engineering.

Adipose-tissue Tregcells restrain differentiation of stromal adipocyte precursors to promote insulin sensitivity and metabolic homeostasis.

Regulatory T (Treg) cells in epidydimal visceral adipose tissue (eVAT) of lean mice and humans regulate metabolic homeostasis. We found that constitutive or punctual depletion of eVAT-Treg cells reined in the differentiation of stromal adipocyte precursors. Co-culture of these precursors with conditional medium from eVAT-Treg cells limited their differentiation invitro, suggesting a direct effect. Transcriptional comparison of adipocyte precursors, matured in the presence or absence of the eVAT-Treg-conditioned medium, identified the oncostatin-M (OSM) signaling pathway as a key distinction. Addition of OSM to invitro cultures blocked the differentiation of adipocyte precursors, while co-addition of anti-OSM antibodies reversed the ability of the eVAT-Treg-conditioned medium to inhibit invitro adipogenesis. Genetic depletion of OSM (specifically in Treg) cells or of the OSM receptor (specifically on stromal cells) strongly impaired insulin sensitivity and related metabolic indices. Thus, Treg-cell-mediated control of local progenitor cells maintains adipose tissue and metabolic homeostasis, a regulatory axis seemingly conserved in humans.

Distinct subpopulations of human subcutaneous adipose tissue precursor cells revealed by single-cell RNA sequencing.

Adipose-derived stem cells (ADSCs) play an important role in the differential capacity for excess energy storage between upper body abdominal (ABD) adipose tissue (AT) and lower body gluteofemoral (GF) AT. We cultured ADSCs from subcutaneous ABD AT and GF AT isolated from eight women with differential body fat distribution and performed single-cell RNA sequencing. Six populations of ADSCs were identified and segregated according to their anatomical origin. The three ADSC subpopulations in GF AT were characterized by strong cholesterol/fatty acid (FA) storage and proliferation signatures. The two ABD subpopulations, differentiated by higher expression of committed preadipocyte marker genes, were set apart by differential expression of extracellular matrix and ribosomal genes. The last population, identified in both depots, was similar to smooth muscle cells and when individually isolated and cultured in vitro they differentiated less than the other subpopulations. This work provides important insight into the use of ADSC as an in vitro model of adipogenesis and suggests that specific subpopulations of GF-ADSCs contribute to the more robust capacity for GF-AT to expand and grow compared with ABD-AT in women.NEW & NOTEWORTHY Identification of distinct subpopulations of adipose-derived stem cells (ADSCs) in upper body abdominal subcutaneous (ABD) and lower body gluteofemoral subcutaneous (GF) adipose tissue depots. In ABD-ADSCs, subpopulations are more committed to adipocyte lineage. GF-ADSC subpopulations are enriched for genes involved in lipids and cholesterol metabolism. Similar depot differences were found in stem cell population identified in freshly isolated stoma vascular fraction. The repertoire of ADSCs subpopulations was different in apple-shaped versus pear-shaped women.

Inhibition of the glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 drives concurrent 11-oxygenated androgen excess.

Aldo-keto reductase 1C3 (AKR1C3) is a key enzyme in the activation of both classic and 11-oxygenated androgens. In adipose tissue, AKR1C3 is co-expressed with 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1), which catalyzes not only the local activation of glucocorticoids but also the inactivation of 11-oxygenated androgens, and thus has the potential to counteract AKR1C3. Using a combination of invitro assays and in silico modeling we show that HSD11B1 attenuates the biosynthesis of the potent 11-oxygenated androgen, 11-ketotestosterone (11KT), by AKR1C3. Employing exvivo incubations of human female adipose tissue samples we show that inhibition of HSD11B1 results in the increased peripheral biosynthesis of 11KT. Moreover, circulating 11KT increased 2-3 fold in individuals with type 2 diabetes after receiving the selective oral HSD11B1 inhibitor AZD4017 for 35 days, thus confirming that HSD11B1 inhibition results in systemic increases in 11KT concentrations. Our findings show that HSD11B1 protects against excess 11KT production by adipose tissue, a finding of particular significance when considering the evidence for adverse metabolic effects of androgens in women. Therefore, when targeting glucocorticoid activation by HSD11B1 inhibitor treatment in women, the consequently increased generation of 11KT may offset beneficial effects of decreased glucocorticoid activation.

Dissection, Histological Processing, and Gene Expression Analysis of Murine Supraclavicular Brown Adipose Tissue.

Brown adipose tissue (BAT)-mediated thermogenesis plays an important role in the regulation of metabolism, and its morphology and function can be greatly impacted by environmental stimuli in mice and humans. Currently, murine interscapular BAT (iBAT), which is located between two scapulae in the upper dorsal flank of mice, is the main BAT depot used by research laboratories to study BAT function. Recently, a few previously unknown BAT depots were identified in mice, including one analogous to human supraclavicular brown adipose tissue. Unlike iBAT, murine supraclavicular brown adipose tissue (scBAT) is situated in the intermediate layer of the neck and thus cannot be accessed as readily. To facilitate the study of newly identified mouse scBAT, presented herein is a protocol detailing the steps to dissect intact scBAT from postnatal and adult mice. Due to scBAT's small size relative to other adipose depots, procedures have been modified and optimized specifically for processing scBAT. Among these modifications is the use of a dissecting microscope during tissue collection to increase the precision and homogenization of frozen scBAT samples to raise the efficiency of subsequent qPCR analysis. With these optimizations, the identification of, morphological appearance of, and molecular characterization of the scBAT can be determined in mice.

Comparison of the therapeutic effects of mesenchymal stem cells derived from human dental pulp (DP), adipose tissue (AD), placental amniotic membrane (PM), and umbilical cord (UC) on postmenopausal osteoporosis.

Background: Osteoporosis is a systemic bone disease characterized by bone loss and microstructural degeneration. Recent preclinical and clinical trials have further demonstrated that the transplantation of mesenchymal stem cells (MSCs) derived from human adipose tissue (AD), dental pulp (DP), placental amniotic membrane (AM), and umbilical cord (UC) tissues can serve as an effective form of cell therapy for osteoporosis. However, MSC-mediated osteoimmunology and the ability of these cells to regulate osteoclast-osteoblast differentiation varies markedly among different types of MSCs. Methods: In this study, we investigated whether transplanted allogeneic MSCs derived from AD, DP, AM, and UC tissues were able to prevent osteoporosis in an ovariectomy (OVX)-induced mouse model of osteoporosis. The homing and immunomodulatory ability of these cells as well as their effects on osteoblastogenesis and the maintenance of bone formation were compared for four types of MSCs to determine the ideal source of MSCs for the cell therapy-based treatment of OVX-induced osteoporosis. The bone formation and bone resorption ability of these four types of MSCs were analyzed using micro-computed tomography analyses and histological staining. In addition, cytokine array-based analyses of serological markers and bioluminescence imaging assays were employed to evaluate cell survival and homing efficiency. Immune regulation was determined by flow cytometer assay to reflect the mechanisms of osteoporosis treatment. Conclusion: These analyses demonstrated that MSCs isolated from different tissues have the capacity to treat osteoporosis when transplanted in vivo. Importantly, DP-MSCs infusion was able to maintain trabecular bone mass more efficiently with corresponding improvements in trabecular bone volume, mineral density, number, and separation. Among the tested MSC types, DP-MSCs were also found to exhibit greater immunoregulatory capabilities, regulating the Th17/Treg and M1/M2 ratios. These data thus suggest that DP-MSCs may represent an effective tool for the treatment of osteoporosis.

CD28-expressing δ T cells are increased in perivascular adipose tissue of hypertensive mice and in subcutaneous adipose tissue of obese humans.

γδ T-lymphocytes play a role in angiotensin II (AngII)-induced hypertension, vascular injury and T-cell infiltration in perivascular adipose tissue (PVAT) in mice. Mesenteric arteries of hypertensive mice and subcutaneous arteries from obese humans present similar remodeling. We hypothesized that γδ T-cell subtypes in mesenteric vessels with PVAT (MV/PVAT) from hypertensive mice and subcutaneous adipose tissue (SAT) from obese humans, who are prone to develop hypertension, would be similar. Mice were infused with AngII for 14 days. MV/PVAT T-cells were used for single-cell RNA-sequencing (scRNA-seq). scRNA-seq data (GSE155960) of SAT CD45+ cells from three lean and three obese women were downloaded from the Gene Expression Omnibus database. δ T-cell subclustering identified six δ T-cell subtypes. AngII increased T-cell receptor δ variable 4 (Trdv4)+ γδ T-effector memory cells and Cd28high δ TEM-cells, changes confirmed by flow cytometry. δ T-cell subclustering identified nine δ T-cell subtypes in human SAT. CD28 expressing δ T-cell subclustering demonstrated similar δ T-cell subpopulations in murine MV/PVAT and human SAT. Cd28+ γδ NKTEM and Cd28high δ TEM-cells increased in MV/PVAT from hypertensive mice and CD28high δ TEM-cells in SAT from obese women compared to the lean women. Similar CD28+ δ T-cells were identified in murine MV/PVAT and human SAT. CD28high δ TEM-cells increased in MV/PVAT in hypertensive mice and in SAT from humans with obesity, a prehypertensive condition. CD28+ δ T-lymphocytes could have a pathogenic role in human hypertension associated with obesity, and could be a potential target for therapy.

IL6/adiponectin/HMGB1 feedback loop mediates adipocyte and macrophage crosstalk and M2 polarization after myocardial infarction.

Differences in border zone contribute to different outcomes post-infarction, such as left ventricular aneurysm (LVA) and myocardial infarction (MI). LVA usually forms within 24h of the onset of MI and may cause heart rupture; however, LVA surgery is best performed 3 months after MI. Few studies have investigated the LVA model, the differences in border zones between LVA and MI, and the mechanism in the border zone. The LVA, MI, and SHAM mouse models were used. Echocardiography, Masson's trichrome staining, and immunofluorescence staining were performed, and RNA sequencing of the border zone was conducted. The adipocyte-conditioned medium-treated hypoxic macrophage cell line and LVA and MI mouse models were employed to determine the effects of the hub gene, adiponectin (ADPN), on macrophages. Quantitative polymerase chain reaction (qPCR), Western blot analysis, transmission electron microscopy, and chromatin immunoprecipitation (ChIP) assays were conducted to elucidate the mechanism in the border zone. Human subepicardial adipose tissue and blood samples were collected to validate the effects of ADPN. A novel, simple, consistent, and low-cost LVA mouse model was constructed. LVA caused a greater reduction in contractile functions than MI owing to reduced wall thickness and edema in the border zone. ADPN impeded cardiac edema and promoted lymphangiogenesis by increasing macrophage infiltration post-infarction. Adipocyte-derived ADPN promoted M2 polarization and sustained mitochondrial quality via the ADPN/AdipoR2/HMGB1 axis. Mechanistically, ADPN impeded macrophage HMGB1 inflammation and decreased interleukin-6 (IL6) and HMGB1 secretion. The secretion of IL6 and HMGB1 increased ADPN expression via STAT3 and the co-transcription factor, YAP, in adipocytes. Based on ChIP and Dual-Glo luciferase experiments, STAT3 promoted ADPN transcription by binding to its promoter in adipocytes. In vivo, ADPN promoted lymphangiogenesis and decreased myocardial injury after MI. These phenotypes were rescued by macrophage depletion or HMGB1 knockdown in macrophages. Supplying adipocytes overexpressing STAT3 decreased collagen disposition, increased lymphangiogenesis, and impaired myocardial injury. However, these effects were rescued after HMGB1 knockdown in macrophages. Overall, the IL6/ADPN/HMGB1 axis was validated using human subepicardial tissue and blood samples. This axis could serve as an independent factor in overweight MI patients who need coronary artery bypass grafting (CABG) treatment. The IL6/ADPN/HMGB1 loop between adipocytes and macrophages in the border zone contributes to different clinical outcomes post-infarction. Thus, targeting the IL6/ADPN/HMGB1 loop may be a novel therapeutic approach for cardiac lymphatic regulation and reduction of cell senescence post-infarction.

Abstract 178: Adipose tissue upregulates expression of RANKL in B-cell acute lymphoblastic leukemia through prostaglandin E2 signaling

Abstract Obesity increases the risk of developing cancer and worsens treatment outcome. Our lab has shown that adipose tissue contributes to B-cell acute lymphoblastic leukemia (B-ALL) resistance to chemotherapy both in vitro and in vivo. However, the mechanisms by which adipose tissue causes ALL cell resistance are complex and not completely understood. We therefore implanted obese mice (n=3) with syngeneic BCR/ABL B-ALL cells and performed single-cell RNA sequencing (scRNAseq) of ALL collected from epididymal adipose tissue vs. bone marrow. Our data uncovered a strong adipose microenvironment effect on the ALL transcriptome, identifying 577 differentially expressed genes using a log2 fold threshold of 0.25 and Bonferroni corrected p-values <0.05. Notably, we found that ALL cells in adipose tissue upregulate expression of Tnfsf11, the gene that encodes receptor activator of nuclear factor kappa beta ligand (RANKL). 93% of ALL cells in adipose tissue express RANKL compared to 33% in marrow, along with a log2 fold expression increase of 2.12 (p<1 × 10−100, Wilcoxon test). Because ALL expression of RANKL has been linked to bone resorption and CNS invasion, both of which have increased incidence in obese patients, we elected to explore this further. Ex vivo culture with human visceral adipose tissue validated ALL cell RANKL upregulation, demonstrating a 1.63 and 17.5-fold increase in gene expression after 48 hours in BV173 and RS4;11 B-ALL cell lines, respectively (n=4-6, both p<0.01, paired t test). Soluble RANKL was not detected in media from ALL cells cultured alone, detectable in media conditioned by human adipose tissue (0.018 pM), but increased in media from adipose-ALL cocultures (RS4;11: 0.065 pM, BV173: 0.317 pM, n=1). One potential candidate signal to stimulate ALL cell RANKL expression is the inflammatory mediator prostaglandin E2 (PGE2), which is known to be released by adipose tissue. Physiological concentrations of PGE2 (200 ng/mL) caused a similar increase in ALL cell RANKL gene expression as adipose tissue in both cell lines (n=6). PGE2 treatment also caused a mild resistance of ALL cells to daunorubicin chemotherapy in vitro, increasing the EC50 from 27±4 to 53±12 nM in RS4;11 (n=6, p<0.05), and from 39±11 to 62±19 nM in BV173 (n=6, p=0.06). Adipose tissue induction of RANKL expression was partially blocked by the addition of PGE2 receptor EP2 and EP4 blockers. Taken together, our results suggest that PGE2 produced by the adipose microenvironment activates ALL cell RANKL expression, which could potentially contribute to CNS invasion, bone breakdown, and chemoresistance. Citation Format: Michael Cohen, Jia Tan, Tyler Kuk, Blesscamal Nadeak, In Sook Ahn, Xia Yang, Etan Orgel, Steven D. Mittelman. Adipose tissue upregulates expression of RANKL in B-cell acute lymphoblastic leukemia through prostaglandin E2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 178.

Abstract 761: The relationship between ANGPTL4 and epidermal malignant transformation

Abstract High body mass index (BMI) is a risk factor for several types of cancers. Although epidemiology suggests obesity is negatively associated with non-melanoma skin cancer (NMSC), bariatric surgery has been shown to reduce the risk, suggesting a possible role of adipose tissue in the initiation and promotion of skin cancer. To gain mechanistic insight into how adipose tissue drives cutaneous carcinogenesis, we cultured non-tumorigenic mouse keratinocytes (JB6 P+ cells) with or without factors released from murine visceral adipose tissue (mFTF) and performed RNA-Sequencing. Published work demonstrated mFTF induces anchorage-independent cell growth, a surrogate marker for malignant transformation. Transcriptomic analysis demonstrated a significant induction of Angptl4 mRNA in JB6 P+ cells cultured with mFTF for 3 and 8 hours compared to the vehicle-treated cells. ANGPTL4 is an endogenous inhibitor of lipoprotein lipase that modulates free fatty acid delivery to adipose tissue and oxidative tissues such as muscle and liver. The C-terminal protein demonstrates tumor-associated activities such as angiogenesis, metastasis, protection against anoikis, and enhancement of cell survival. Therefore, we hypothesized that the induction of this gene promotes malignant transformation. RNA-seq data was validated in multiple cell models, including primary keratinocytes with human adipose tissue. Immunohistochemistry was used to assess ANGPTL4 expression in 37 actinic keratosis (pre-malignant) and cutaneous squamous cell carcinoma (cSCC) from patients with different BMIs (18.6 - 49.5). Notably, ANGPTL4 was only observed in the suprabasal epidermal layers in normal skin. For samples containing both lesional and perilesional skin, there was an increase in ANGPTL4 expression in the lesional epidermis relative to the normal perilesional skin. Furthermore, ANGPTL4 expression increased from transition to actinic keratosis to cSCC in situ, suggesting that ANGTL4 is induced during malignant transformation as observed in our pre-clinical models. Interestingly, once cSCC developed an invasive component, ANGTPL4 expression was lost in the invasive keratinocytes but persisted in the in situ component. There was no association between ANGPTL4 expression and BMI in lesional tissue. These data suggest that targeting ANTPLT4 may be a novel therapy for the prevention of skin cancer and that ANGPTL4-high patients or cancer patients with obesity may be a critical demographic for potential risk assessment or therapeutic intervention. Future studies will determine the impact of knocking out ANGTPL4 in our models of malignant transformation. Citation Format: Nat Ato Yawson, Jonathan D. Diedrich, Karen T. Liby, Jesse Veenstra, Jamie Jenna Bernard. The relationship between ANGPTL4 and epidermal malignant transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 761.

Cornea-Specific Human Adipose Stem Cell-Derived Extracellular Matrix for Corneal Stroma Tissue Engineering.

Utilizing tissue-specific extracellular matrices (ECMs) is vital for replicating the composition of native tissues and developing biologically relevant biomaterials. Human- or animal-derived donor tissues and organs are the current gold standard for the source of these ECMs. To overcome the several limitations related to these ECM sources, including the highly limited availability of donor tissues, cell-derived ECM offers an alternative approach for engineering tissue-specific biomaterials, such as bioinks for three-dimensional (3D) bioprinting. 3D bioprinting is a state-of-the-art biofabrication technology that addresses the global need for donor tissues and organs. In fact, there is a vast global demand for human donor corneas that are used for treating corneal blindness, often resulting from damage in the corneal stromal microstructure. Human adipose tissue is one of the most abundant tissues and easy to access, and adipose tissue-derived stem cells (hASCs) are a highly advantageous cell type for tissue engineering. Furthermore, hASCs have already been studied in clinical trials for treating corneal stromal pathologies. In this study, a corneal stroma-specific ECM was engineered without the need for donor corneas by differentiating hASCs toward corneal stromal keratocytes (hASC-CSKs). Furthermore, this ECM was utilized as a component for corneal stroma-specific bioink where hASC-CSKs were printed to produce corneal stroma structures. This cost-effective approach combined with a clinically relevant cell type provides valuable information on developing more sustainable tissue-specific solutions and advances the field of corneal tissue engineering.