RESUMO
RATIONALE: The impact of diabetes mellitus on bone marrow (BM) structure is incompletely understood. OBJECTIVE: Investigate the effect of type-2 diabetes mellitus (T2DM) on BM microvascular and hematopoietic cell composition in patients without vascular complications. METHODS AND RESULTS: Bone samples were obtained from T2DM patients and nondiabetic controls (C) during hip replacement surgery and from T2DM patients undergoing amputation for critical limb ischemia. BM composition was assessed by histomorphometry, immunostaining, and flow cytometry. Expressional studies were performed on CD34(pos) immunosorted BM progenitor cells (PCs). Diabetes mellitus causes a reduction of hematopoietic tissue, fat deposition, and microvascular rarefaction, especially when associated with critical limb ischemia. Immunohistochemistry documented increased apoptosis and reduced abundance of CD34(pos)-PCs in diabetic groups. Likewise, flow cytometry showed scarcity of BM PCs in T2DM and T2DM+critical limb ischemia compared with C, but similar levels of mature hematopoietic cells. Activation of apoptosis in CD34(pos)-PCs was associated with upregulation and nuclear localization of the proapoptotic factor FOXO3a and induction of FOXO3a targets, p21 and p27(kip1). Moreover, microRNA-155, which regulates cell survival through inhibition of FOXO3a, was downregulated in diabetic CD34(pos)-PCs and inversely correlated with FOXO3a levels. The effect of diabetes mellitus on anatomic and molecular end points was confirmed when considering background covariates. Furthermore, exposure of healthy CD34(pos)-PCs to high glucose reproduced the transcriptional changes induced by diabetes mellitus, with this effect being reversed by forced expression of microRNA-155. CONCLUSIONS: We provide new anatomic and molecular evidence for the damaging effect of diabetes mellitus on human BM, comprising microvascular rarefaction and shortage of PCs attributable to activation of proapoptotic pathway.
Assuntos
Células da Medula Óssea/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Angiopatias Diabéticas/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Células-Tronco Hematopoéticas/metabolismo , MicroRNAs/metabolismo , Microvasos/metabolismo , Transdução de Sinais , Nicho de Células-Tronco , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD34/metabolismo , Apoptose , Biomarcadores/metabolismo , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Exame de Medula Óssea , Estudos de Casos e Controles , Linhagem da Célula , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Citometria de Fluxo , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/patologia , Humanos , Imuno-Histoquímica , Isquemia/genética , Isquemia/metabolismo , Isquemia/patologia , Masculino , MicroRNAs/genética , Microvasos/imunologia , Microvasos/patologia , Pessoa de Meia-Idade , Doença Arterial Periférica/genética , Doença Arterial Periférica/metabolismo , Doença Arterial Periférica/patologia , TransfecçãoRESUMO
OBJECTIVE: The p75 neurotrophin receptor (p75(NTR)) contributes to diabetes mellitus-induced defective postischemic neovascularization. The interleukin-33 receptor ST2 is expressed as transmembrane (ST2L) and soluble (sST2) isoforms. Here, we studied the following: (1) the impact of p75(NTR) in the healing of ischemic and diabetic calf wounds; (2) the link between p75(NTR) and ST2; and (3) circulating sST2 levels in critical limb ischemia (CLI) patients. METHODS AND RESULTS: Diabetes mellitus was induced in p75(NTR) knockout (p75KO) mice and wild-type (WT) littermates by streptozotocin. Diabetic and nondiabetic p75KO and WT mice received left limb ischemia induction and a full-thickness wound on the ipsilateral calf. Diabetes mellitus impaired wound closure and angiogenesis and increased ST2 expression in WT, but not in p75KO wounds. In cultured endothelial cells, p75(NTR) promoted ST2 (both isoforms) expression through p38(MAPK)/activating transcription factor 2 pathway activation. Next, sST2 was measured in the serum of patients with CLI undergoing either revascularization or limb amputation and in the 2 nondiabetic groups (with CLI or nonischemic individuals). Serum sST2 increased in diabetic patients with CLI and was directly associated with higher mortality at 1 year from revascularization. CONCLUSIONS: p75(NTR) inhibits the healing of ischemic lower limb wounds in diabetes mellitus and promotes ST2 expression. Circulating sST2 predicts mortality in diabetic CLI patients.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus/mortalidade , Isquemia/fisiopatologia , Extremidade Inferior/irrigação sanguínea , Proteínas do Tecido Nervoso/fisiologia , Receptores de Superfície Celular/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Fator de Crescimento Neural/fisiologia , Fator 2 Ativador da Transcrição/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores/metabolismo , Células Cultivadas , Complicações do Diabetes/complicações , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Isquemia/etiologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/farmacologia , Valor Preditivo dos Testes , Receptores de Fator de Crescimento Neural/deficiência , Receptores de Fator de Crescimento Neural/genética , Estreptozocina/efeitos adversos , Cicatrização/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Critical limb ischemia (CLI), foot ulcers, former amputation, and impaired regeneration are independent risk factors for limb amputation in subjects with diabetes. The present work investigates whether and by which mechanism diabetes negatively impacts on functional properties of muscular pericytes (MPs), which are resident stem cells committed to reparative angiomyogenesis. We obtained muscle biopsy samples from patients with diabetes who were undergoing major limb amputation and control subjects. Diabetic muscles collected at the rim of normal tissue surrounding the plane of dissection showed myofiber degeneration, fat deposition, and reduction of MP vascular coverage. Diabetic MPs (D-MPs) display ultrastructural alterations, a differentiation bias toward adipogenesis at the detriment of myogenesis and an inhibitory activity on angiogenesis. Furthermore, they have an imbalanced redox state, with downregulation of the antioxidant enzymes superoxide dismutase 1 and catalase, and activation of the pro-oxidant protein kinase C isoform ß-II (PKCßII)-dependent p66Shc signaling pathway. A reactive oxygen species scavenger or, even more effectively, clinically approved PKCßII inhibitors restore D-MP angiomyogenic activity. Inhibition of the PKCßII-dependent p66Shc signaling pathway could represent a novel therapeutic approach for the promotion of muscle repair in individuals with diabetes.