Biology subjects

Pinheiro-Machado, E.

Publications and source records attributed to Pinheiro-Machado, E..

4 recordsLinked to original sources

bioRxiv2025

Profiling the secretome: maternal obesity impacts redox and adipogenix signaling molecules during neonatal mesenchymal stem cells' adipogenesis

Recent studies evidence an altered bioenergetic profile and higher adipogenic commitment in the MSCs from neonates of mothers with obesity. We hypothesize that these alterations may also affect the secretome of these cells. The aim of this study was to characterize the redox and adipogenic secretome of MSCs from the offspring of women with obesity compared to the ones from normal-weight women, both before and during adipogenesis. Whartons jelly-derived MSCs were isolated from newborns of normal-weight women (NW-MSC; Body Mass Index 18.5-24.5 kg/m{superscript 2}) and women with obesity (OB-MSC; Body Mass Index>30 kg/m{superscript 2}) and cultured for 0, 5 and 21 days of adipogenesis. The secretome from these cells was collected during the three timepoints and characterized through mass spectrometry. Our findings reveal fundamental difference in the secretome profiles, primarily associated with pathways involved in cellular and metabolic processes (p<0.05). Maternal obesity was found to decrease redox capacity at day 0 but subsequently triggered a compensatory increase in redox proteins during adipogenesis of OB-MSCs (p<0.05). Additionally, OB-MSCs secreted higher levels of lipid synthesis proteins and proinflammatory adipokines (p<0.05), which may contribute to the dysregulated adipogenesis observed in obesity. These results indicate that maternal obesity programs the secretome of neonatal MSCs, supporting that maternal obesity imprints early progenitor cells potentially dictating the future metabolic status of the offsprings adipocytes.

physiology↗

bioRxiv2024

Secretome analysis of human and rat pancreatic islets co-cultured with adipose-derived stromal cells reveals a signature with enhanced regenerative capacities

Pancreatic islet transplantation (PIT), a promising treatment for Type 1 Diabetes (T1D), encounters challenges in the pre- and post-transplantation phases. Co-culturing or co-transplantation of islets with mesenchymal stromal cells (MSC), known for their regenerative properties, emerged as a potential solution and was shown to increase islet function and improve PIT outcomes. This study explored the changes in the islets secretion signature (secretome) when co-cultured with adipose-derived stromal cells (ASC), an MSC subtype. The secretome profile of islets and co-cultures under various stressors, i.e., cytokines, high glucose, hypoxia, and a combination of hypoxia and high glucose, was investigated. The results shed light on the potential mechanisms through which ASC support islets functional survival. Co-culturing pancreatic islets with ASC induced substantial proteomic changes, impacting pathways crucial for energy metabolism, angiogenesis, extracellular matrix organization, and immune responses. The analysis of key signaling molecules (VEGF, PDGF, bFGF, Collagen I alpha 1, IL-1, and IL-10) revealed alterations influenced by the culturing conditions and the presence of the ASC. In vitro functional assays using the secretomes also demonstrated their potential to differentially influence angiogenic processes, enhance collagen deposition, and modulate the immune system based on the conditions in which they were generated. These findings offer valuable insights into the potential of ASC co-culturing to address challenges in PIT, paving the way for enhanced therapeutic interventions in T1D and regenerative medicine. HighlightsO_LIBoth in-silico and in-vitro data support that co-culturing pancreatic islets with ASC enhances islet function. C_LIO_LICo-culturing islets with ASC induces changes in the secretome, impacting pathways related to energy metabolism, angiogenesis, extracellular matrix organization, and immune response. C_LIO_LIKey signaling molecules, including VEGF, PDGF, bFGF, Collagen I alpha 1, IL-1, and IL-10, are differentially affected by various co-culturing conditions. C_LI

cell biology↗

bioRxiv2024

Modulating adipose-derived stromal cells' secretomes by culture conditions: effects on angiogenesis, collagen deposition, and immunomodulation.

The secretome of adipose-derived stromal cells (ASC) presents a promising avenue for cell-free therapies due to their rich mixture of bioactive molecules. Different culture conditions can modulate the composition of this mixture, but how this affects the functional properties of the secretome remains to be investigated. This study investigated the in vitro effects of normoxia, cytokines, high glucose, hypoxia, and hypoxia + high glucose-derived ASC secretomes on angiogenesis (tube formation assay), collagen deposition (Picrosirius-Red staining), and immunomodulation (One-way Mixed Lymphocyte Reaction in combination with an antibody-mediated cell-dependent cytotoxicity assay). The data showed that normoxia and hypoxia-derived secretomes consistently exhibited potent proangiogenic effects in both human and rat models. These secretomes also demonstrated positive influences on collagen deposition and immunomodulation. Interestingly, the human ASC hypoxia + high glucose-derived secretome emerged as a stimulator of collagen deposition and modulator of the immune system. Conversely, cytokines and high glucose-derived secretomes have shown less strong effects in almost all functional parameters. In conclusion, our findings indicate that modulating culturing conditions results in secretomes with different functional properties and emphasizes the multifaceted role of ASC secretomes in regenerative processes.

cell biology↗

bioRxiv2021

Defective proinsulin handling modulates the MHC I bound peptidome and activates the inflammasome in β-cells

BackgroundHow immune-tolerance is lost to pancreatic {beta}-cell peptides triggering autoimmune type 1 diabetes is enigmatic. We have shown that loss of the proinsulin ER chaperone glucose-regulated protein (GRP) 94 leads to mishandling of proinsulin, ER stress and activation of the inducible proteasome. We hypothesize that inadequate ER proinsulin folding capacity relative to biosynthetic need may lead to an altered {beta}-cell MHC-I bound peptidome and inflammasome activation, sensitizing {beta}-cells to immune attack. MethodsWe used INS-1E cells with or without GRP94 knockout (KO), or in the presence or absence of GRP94 inhibitor PU-WS13 (GRP94i, 20{micro}M), or exposed to proinflammatory cytokines interleukin (IL)-1{beta} or IFN{gamma} (15 pg/ml and 10 ng/ml, respectively) for 24 hours. RT1.A (rat MHC I) expression was evaluated using flow cytometry. The total RT1.A-bound peptidome analysis was performed on cell lysates fractionated by reverse phase high performance liquid chromatography (RP-HPLC) followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). NALP1, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (I{kappa}B), and (pro) IL-1{beta} expression and secretion were investigated by Western blotting. ResultsGRP94 KO increased RT1.A expression in {beta}-cells as did cytokine exposure compared to relevant controls. Immunopeptidome analysis showed increased RT1.A-bound peptide repertoire in GRP94 KO/i cells as well as in the cells exposed to cytokines. The GRP94 KO/cytokine exposure groups showed partial overlap in their peptide repertoire. Notably, proinsulin-derived peptides diversity increased among the total RT1.A peptidome in GRP94 KO/i along with cytokines exposure. NALP1 expression was upregulated in GRP94 deficient cells along with decreased I{kappa}B content while proIL-1{beta} cellular levels declined, coupled with an increased secretion of mature IL-1{beta}. Our results suggest that limiting {beta}-cell proinsulin chaperoning enhances RT1.A expression, alters the MHC-I peptidome including proinsulin peptides and activates inflammatory pathways, suggesting that stress impeding proinsulin handling may sensitize {beta}-cells to immune-attack.

molecular biology↗