Endocrine Abstracts

JOINT2137

Background and Aim: Cardiovascular disease (CVD) is the leading cause of death in individuals with obesity, driven largely by endothelial (EC) and smooth muscle cell (SMC) dysfunction. Obesity-induced EGFR transactivation, mediated a.o. by elevated angiotensin-II (AngII) and norepinephrine (NE) levels is supposed to contribute to vascular dysfunction and remodeling. However, the role of EC-SMC interactions in obesity-related CVD remains unclear. This study investigates the impact of AngII and NE on human primary ECs and SMCs under conditions mimicking obesity (elevated FFA and glucose).

Methods: Human primary ECs and SMCs were cultured alone or as co-culture in opposite side of cell culture insert membrane. The effects of metabolic stimuli (MS) and hormonal stimuli (HS), alone or in combination, on gene expression (DEG) were assessed by RNA-sequencing followed by gene-ontology term and functional enrichment analysis. To evaluate their impact on cell function, key parameters such as cell apoptosis/necrosis, DNA-synthesis/proliferation, glucose consumption (dglucose), lactate production (dlactate), oxidant–antioxidant balance, ATP production, mitochondrial function as well as the secretory profiles of inflammatory molecules were analyzed under each condition.

Results: In monoculture, incubation of cells with each stimulus, alone or in combination, did not affect cell apoptosis, necrosis and GSSG/GSH-ratio while proliferation and proton leak was induced in HAoEC under MS. HAoSMC metabolism was altered by MS, evidenced by elevated ATP levels. dglucose and dlactate/dglucose-ratio were enhanced by either MS or all stimuli. Incubation of cells with either or both stimuli in the presence of AG1478/EGF revealed that MS-induced cell proliferation, dglucose, dlactate and ATP production seems to be EGFR dependent. In co-culture, proton leak and GSH content were induced in HAoECs by MS and HS respectively. Transcriptomic analysis revealed that under the effect of obesity-associated stressors, number of DEG in ECs was significantly higher in co-culture. IPA highlighted the involvement of DEGs in regulation of cell cycle and inflammation. This was not the case for SMC. Co-culture also led to a significant change of the extracellular inflammatory marker profile compared to monoculture, thereby exposing cells to a different micromilieu. A potential role of EGFR in co-culture is under investigation.

Conclusion: This study indicates that the transcriptome and functional profile of cells are profoundly influenced by the cell culture type, underscoring the critical role of cell interactions in cellular behavior. The effects of MS in different vascular cells are, at least partially, mediated by EGFR. In few cases, these effects are further altered by HS.