Abstract P186: Chemical And Mechanical Activation Of The Mechanosensor Piezo1 Alters Adipogenesis In Pvat Preadipocytes

During hypertension, vascular remodeling allows the blood vessel to withstand high blood pressure (BP). This process is well characterized in the media and intima layers of the vessel. In the perivascular adipose tissue (PVAT) there is evidence for fibrosis development during hypertension; but PVAT remodeling is poorly understood. In stem cells (i.e., adipocyte progenitors) from non-PVAT depots, mechanical forces affect adipogenesis' commitment and lipogenic stages. The mechanism involves PIEZO1, a mechanosensor that boosts the differentiation of preadipocytes towards osteogenic and fibroblastic lineages. However, PVAT's particular anatomical location continuously exposes it to forces generated by blood flow that could affect adipogenesis during normotensive and hypertensive states. Our objective was to evaluate PIEZO1's role in the adipogenic potential of preadipocytes. We hypothesize that activation of PIEZO1 reduces Adipogenesis in PVAT preadipocytes. Aortic (APVAT) was collected from male SD rats at 10 weeks of age (n=15) to harvest preadipocytes by Liberase™ digestion. Nonselective cationic channel PIEZO1 activity was evaluated with Ca 2+ indicator Fluo-4AM. Piezo1 was reduced with siRNA. Preadipocytes were differentiated for 4 d in adipogenic media containing PIEZO1 agonist Yoda1 (CON=0; YODA1=10μM). Mechanical strain (MS) was applied with FlexCell System at 12%, half-sine at 1 Hz for 4 d (MS+; MS-). Adipogenesis was evaluated by quantification of adipogenic gene network expression using PCR; lipid accumulation using lipophilic stains (Bodipy, siRNA experiments) or Oil Red O (FlexCell experiments). Adipogenesis efficiency is reported as Adipocyte/Total cells as measured in the IncuCyte Live-Cell ® system. Yoda1 reduced adipogenesis by 33% compared with CON and as expected, increased cytoplasmic Ca 2+ . In si Piezo1 cells, the anti-adipogenic effect of Yoda1 was reversed. MS+ reduced adipogenesis efficiency (0.15±0.06) compared with MS- (0.22±0.1). These data demonstrate that Piezo1 activation in PVAT may be an adaptive or pathogenic mechanism by which adipocyte populations are reduced, thus minimizing their secretion of vasoactive adipokines, and enhancing the deleterious impact of hypertension on PVAT function.

Anti-Adipogenic Effect of Alchemilla monticola is Mediated Via PI3K/AKT Signaling Inhibition in Human Adipocytes

Obesity is a persistent and continuously expanding social health concern. Excessive fat mass accumulation is associated with increased risk of chronic diseases including diabetes, atherosclerosis, non-alcoholic steatohepatitis, reproductive dysfunctions and certain types of cancer. Alchemilla monticola Opiz. is a perennial plant of the Rosaceae family traditionally used to treat inflammatory conditions and as a component of weight loss herbal mixtures. In the search for bioactive leads with potential anti-adipogenic effect from A. monticola extract (ALM), we have employed nuclear magnetic resonance (NMR) based metabolomics to obtain data for the phytochemical profile of the extract. Further, molecular docking simulation was performed against key adipogenic targets for selected pure compounds, present in the ALM extract. Evaluation of the biological activity was done in human adipocytes exposed to ALM (5, 10 and 25 μg/ml), pure astragalin (AST) or quercitrin (QUE) both at the concentrations of 5, 10 and 25 μM. Investigation of the molecular pathways involved was performed through real-time quantitative PCR and Western blot analyses. According to the docking predictions strong putative affinity was revealed for both AST and QUE towards peroxisome proliferator-activated receptor gamma (PPARγ) and phosphoinositide 3-kinase (PI3K). Assessment of the intracellular lipid accumulation revealed anti-adipogenic activity of ALM. Correspondingly, the expression of the adipogenic genes CCAAT/enhancer-binding protein alpha (CEBPA) and PPARG was downregulated upon ALM and AST treatment. The Western blotting results exposed protein kinase B (AKT), PI3K and PPARγ as targets for the inhibitory effect of ALM and AST on adipogenesis. Collectively, we provide a broader insight of the phytochemical composition of A. monticola. Additionally, we demonstrate the anti-adipogenic effect of ALM and its active compound AST in human adipocytes. Furthermore, PI3K/AKT signaling pathway is identified to mediate the ALM anti-adipogenic action. Hence, the ALM extract and its secondary metabolite AST are worth further exploration as potentially active agents in obesity management.

Dexamethasone Induces Changes in Osteogenic Differentiation of Human Mesenchymal Stromal Cells via SOX9 and PPARG, but Not RUNX2

Despite the huge body of research on osteogenic differentiation and bone tissue engineering, the translation potential of in vitro results still does not match the effort employed. One reason might be that the protocols used for in vitro research have inherent pitfalls. The synthetic glucocorticoid dexamethasone is commonly used in protocols for trilineage differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). However, in the case of osteogenic commitment, dexamethasone has the main pitfall of inhibiting terminal osteoblast differentiation, and its pro-adipogenic effect is well known. In this work, we aimed to clarify the role of dexamethasone in the osteogenesis of hBMSCs, with a particular focus on off-target differentiation. The results showed that dexamethasone does induce osteogenic differentiation by inhibiting SOX9 expression, but not directly through RUNX2 upregulation as it is commonly thought. Rather, PPARG is concomitantly and strongly upregulated, leading to the formation of adipocyte-like cells within osteogenic cultures. Limiting the exposure to dexamethasone to the first week of differentiation did not affect the mineralization potential. Gene expression levels of RUNX2, SOX9, and PPARG were simulated using approximate Bayesian computation based on a simplified theoretical model, which was able to reproduce the observed experimental trends but with a different range of responses, indicating that other factors should be integrated to fully understand how dexamethasone influences cell fate. In summary, this work provides evidence that current in vitro differentiation protocols based on dexamethasone do not represent a good model, and further research is warranted in this field.

Piceatannol Is Superior to Resveratrol at Suppressing Adipogenesis in Human Visceral Adipose-Derived Stem Cells

Resveratrol (3,4′,5-trans-trihydroxystilbene) and piceatannol (3,3′,4′,5-trans-tetraphydroxystilbene) are major stilbene compounds that are predominantly present in various natural foods, such as berries and fruits. Both phytochemical compounds are consumed as dietary supplements to prevent various metabolic diseases and for their anti-aging properties. Adipose-derived stem cells from human visceral adipose tissue (vASCs) are a useful in vitro model for evaluating their adipogenic effect. Treatment with resveratrol and piceatannol significantly inhibited lipid accumulation in vASCs. Their effective concentrations were 5, 10, and 20 μM for inhibiting adipogenesis of vASCs. Interestingly, despite the similar chemical structures of the two compounds, piceatannol showed a higher anti-adipogenic effect at 20 μM than resveratrol in vASCs. Moreover, the inhibitory capacity of lipid droplet generation was higher for piceatannol at 20 μM than that of resveratrol. Piceatannol significantly attenuated the expression level of adipogenic markers (e.g., CCAAT/enhanced binding protein α (C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ), and adipocyte fatty acid binding protein (aP2)) compared to resveratrol at the mRNA and protein levels. These results suggest that piceatannol is a superior anti-adipogenic compound compared to resveratrol in the vASC model of visceral obesity.