RESUMO
Adipose tissue stromal cells (ADSCs) are prone to functional decline and senescence during metabolic disturbances. In diabetes mellitus (DM), the pathogenic microenvironment induces oxidative stress causing ADSCs to senesce. The senescence associated secretory phenotype (SASP) in turn drives disease progression. The pathogenesis of DM is thus both a cause and consequence of senescence. Therapeutically preventing the onset of senescence in ADSCs may play a significant role in preventing disease progression and directly impact the onset of comorbidities. The purpose of this study was to establish an in vitro model that mimic the DM micro-environment to use as a screening tool to assess the therapeutic efficacy of preventative and restorative agents. Exposing ADSCs (Assuntos
Tecido Adiposo
, Senescência Celular
, Senescência Celular/efeitos dos fármacos
, Tecido Adiposo/citologia
, Tecido Adiposo/metabolismo
, Células Estromais/metabolismo
, Humanos
, Animais
, Metformina/farmacologia
, Diabetes Mellitus/metabolismo
, Diabetes Mellitus/patologia
, Células Cultivadas
, Fenótipo Secretor Associado à Senescência
, Espécies Reativas de Oxigênio/metabolismo
, Fator de Necrose Tumoral alfa/metabolismo
, Glucose/metabolismo
RESUMO
Diabetes is a complex multifactorial disorder associated with hyperglycemia, oxidative stress, and inflammation. The pathological microenvironment impairs mesenchymal stem cell (MSC) viability and dysregulates their proregenerative and immune-modulatory function causing maladaptive tissue damage. Targeting stem cells to protect them against impairment could thus delay the onset of complications and enhance the quality of life in diabetes mellitus patients. The aim of this study was to investigate the efficacy of N-acetylcysteine (NAC) and ascorbic-acid-2-phosphate (AAP) oral supplementation as preventative measure against MSC impairment. Healthy wild-type control (C57BL/6J) (male, n = 24) and obese diabetic (B6.C-Lepob/J) (ob/ob) (male, n = 24) mice received either placebo or antioxidant (NAC/AAP) supplementation for a period of 6 weeks. Metabolic parameters (weight and blood glucose) and the oxidative status (serum total serum antioxidant capacity, malondialdehyde) of animals were assessed. At the end of the 6-week supplementation period, bone marrow MSCs were isolated and their functionality (growth rate, viability, adipogenesis, and osteogenesis) assessed ex vivo. Real time quantitative polymerase chain reaction microarray analysis was also performed to assess the expression of 84 genes related to oxidative stress in MSCs. Despite no change in the metabolic profile, NAC/AAP supplementation improved the antioxidant status of diabetic animals and reduced lipid peroxidation, which is indicative of cellular damage. NAC/AAP also improved the population doubling time of MSCs (first 6-days postisolation) and significantly downregulated the expression of two genes (Nox1 and Rag2) associated with oxidative stress compared to placebo treatment. Taken together, this study has shown reduced oxidative stress and improvements in MSC function following in vivo antioxidant supplementation in healthy control and type 2 diabetic mice.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Células-Tronco Mesenquimais , Acetilcisteína/farmacologia , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/patologia , Suplementos Nutricionais , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Fosfatos , Qualidade de VidaRESUMO
Disease-associated impairment/dysfunction of stem cell populations is prominent in chronic metabolic and inflammatory diseases, such as type 2 diabetes mellitus (DM) where the multifunctional properties (viability, proliferation, paracrine secretion, multilineage differentiation) of bone marrow resident mesenchymal stem cells (MSCs) can be affected. The growth and viability impairments make it difficult to study the underlying molecular mechanisms related to the dysfunction of these cells in vitro. We have consequently optimized the isolation and culture conditions for impaired/dysfunctional bone marrow MSCs from B6.Cg-Lepob/J obese prediabetic mice. The method described here permits ex vivo investigations into disease-associated functional impairments and the dysregulated molecular mechanisms in these primary MSCs through direct comparisons with their healthy wild-type C57BL6/J control mouse counterparts.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Doenças Metabólicas/patologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Doença Crônica , Diabetes Mellitus Tipo 2/patologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Estado Pré-Diabético/patologiaRESUMO
Obesity-associated inflammatory mechanisms play a key role in the pathogenesis of metabolic-related diseases. Failure of anti-inflammatory control mechanisms within adipose tissue and peripheral blood mononuclear cells (PBMCs) have been implicated in disease progression. This study investigated the efficacy of allogeneic adipose tissue-derived mesenchymal stem cells conditioned media (ADSC-CM) to counteract persistent inflammation by inducing an anti-inflammatory phenotype and cytokine response within PBMCs derived from patients with and without metabolic syndrome. Forty six (n=46) mixed ancestry females (18 - 45 years) were subdivided into a) healthy lean (HL) (n=10) (BMI < 25 kg/m2), b) overweight/obese (OW/OB) (BMI ≥ 25 kg/m2, < 3 metabolic risk factors) (n=22) and c) metabolic syndrome (MetS) (visceral adiposity , ≥ 3 metabolic risk factors) (n=14) groups. Body composition (DXA scan), metabolic (cholesterol, HDL, LDL, triglycerides, blood glucose) and inflammatory profiles (38-Plex cytokine panel) were determined. PBMCs were isolated from whole blood and treated ex vivo with either i) autologous participant-derived serum ii) ADSCs-CM or iii) a successive treatment regime. The activation status (CD11b+) and intracellular cytokine (IL6, IL10, TNFa) expression were determined in M1 (CD68+CD206-CD163-) and M2 (CD68+CD163+ CD206+) macrophage populations using flow cytometry. ADSC-CM treatment, promoted a M2 macrophage phenotype and induced IL10 expression, this was most pronounced in the OW/OB group. This response is likely mediated by multiple complementing factors within ADSC-CM, yet to be identified. This study is the first to demonstrate the therapeutic potential of ADSC-CM to restore the inflammatory balance in immune compromised obese individuals.