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For over a century, fasting regimens have improved health, lifespan and tissue regeneration in diverse organisms, including humans1-6. However, how fasting and post-fast refeeding affect adult stem cells and tumour formation has yet to be explored in depth. Here we demonstrate that post-fast refeeding increases intestinal stem cell (ISC) proliferation and tumour formation; post-fast refeeding augments the regenerative capacity of Lgr5+ ISCs, and loss of the tumour suppressor gene Apc in post-fast-refed ISCs leads to a higher tumour incidence in the small intestine and colon than in the fasted or ad libitum-fed states, demonstrating that post-fast refeeding is a distinct state. Mechanistically, we discovered that robust mTORC1 induction in post-fast-refed ISCs increases protein synthesis via polyamine metabolism to drive these changes, as inhibition of mTORC1, polyamine metabolite production or protein synthesis abrogates the regenerative or tumorigenic effects of post-fast refeeding. Given our findings, fast-refeeding cycles must be carefully considered and tested when planning diet-based strategies for regeneration without increasing cancer risk, as post-fast refeeding leads to a burst in stem-cell-driven regeneration and tumorigenicity.
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Objective: The extent of collateral artery enlargement determines the risk of limb loss due to peripheral arterial disease. Hypercholesterolemia impairs collateral artery enlargement, but the underlying mechanism remains poorly characterized. This study tests the hypothesis that hypercholesterolemia impairs collateral artery enlargement through a ten-eleven translocation 1 (Tet1)-dependent hematopoietic stem cell (HSC)-autonomous mechanism that increases their differentiation into proinflammatory Ly6Chi monocytes and restricts their conversion into proangiogenic Ly6Clow monocytes. Methods: To test our hypothesis, we induced limb ischemia and generated chimeric mouse models by transplanting HSCs from either wild-type (WT) mice or hypercholesterolemic mice into lethally irradiated WT recipient mice. Results: We found that the lethally irradiated WT recipient mice reconstituted with HSCs from hypercholesterolemic mice displayed lower blood flow recovery and collateral artery enlargement that was nearly identical to that observed in hypercholesterolemic mice, despite the absence of hypercholesterolemia and consistent with an HSC-autonomous mechanism. We showed that hypercholesterolemia impairs collateral artery enlargement by a Tet1-dependent mechanism that increases HSC differentiation toward proinflammatory Ly6Chi monocytes and restricts the conversion of Ly6Chi monocytes into proangiogenic Ly6Clow monocytes. Moreover, Tet1 epigenetically reprograms monocyte gene expression within the HSCs. Restoration of Tet1 expression in HSCs of hypercholesterolemic mice restores WT collateral artery enlargement and blood flow recovery after induction of hindlimb ischemia. Conclusions: These results show that hypercholesterolemia impairs collateral artery enlargement by a novel Tet1-dependent HSC-autonomous mechanism that epigenetically reprograms monocyte gene expression within the HSCs.
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PDGFRA-expressing mesenchyme supports intestinal stem cells. Stomach epithelia have related niche dependencies, but their enabling mesenchymal cell populations are unknown, in part because previous studies pooled the gastric antrum and corpus. Our high-resolution imaging, transcriptional profiling, and organoid assays identify regional subpopulations and supportive capacities of purified mouse corpus and antral PDGFRA+ cells. Sub-epithelial PDGFRAHi myofibroblasts are principal sources of BMP ligands and two molecularly distinct pools distribute asymmetrically along antral glands but together fail to support epithelial growth in vitro. In contrast, PDGFRALo CD55+ cells strategically positioned beneath gastric glands promote epithelial expansion in the absence of other cells or factors. This population encompasses a small fraction expressing the BMP antagonist Grem1. Although Grem1+ cell ablation in vivo impairs intestinal stem cells, gastric stem cells are spared, implying that CD55+ cell activity in epithelial self-renewal derives from other subpopulations. Our findings shed light on spatial, molecular, and functional organization of gastric mesenchyme and the spectrum of signaling sources for epithelial support.
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Mucosa Gástrica , Estômago , Camundongos , Animais , Células-Tronco , Intestinos , Antro Pilórico , Receptores Proteína Tirosina Quinases , Células EpiteliaisRESUMO
Signals from the surrounding niche drive proliferation and suppress differentiation of intestinal stem cells (ISCs) at the bottom of intestinal crypts. Among sub-epithelial support cells, deep sub-cryptal CD81+ PDGFRAlo trophocytes capably sustain ISC functions ex vivo. Here, we show that mRNA and chromatin profiles of abundant CD81- PDGFRAlo mouse stromal cells resemble those of trophocytes and that both populations provide crucial canonical Wnt ligands. Mesenchymal expression of key ISC-supportive factors extends along a spatial and molecular continuum from trophocytes into peri-cryptal CD81- CD55hi cells, which mimic trophocyte activity in organoid co-cultures. Graded expression of essential niche factors is not cell-autonomous but dictated by the distance from bone morphogenetic protein (BMP)-secreting PDGFRAhi myofibroblast aggregates. BMP signaling inhibits ISC-trophic genes in PDGFRAlo cells near high crypt tiers; that suppression is relieved in stromal cells near and below the crypt base, including trophocytes. Cell distances thus underlie a self-organized and polar ISC niche.
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Mucosa Intestinal , Nicho de Células-Tronco , Animais , Camundongos , Mucosa Intestinal/metabolismo , Intestinos , Transdução de Sinais , Diferenciação Celular , Proliferação de CélulasRESUMO
Wnt and Rspondin (RSPO) signaling drives proliferation, and bone morphogenetic protein inhibitors (BMPi) impede differentiation, of intestinal stem cells (ISCs). Here, we identify the mouse ISC niche as a complex, multi-layered structure that encompasses distinct mesenchymal and smooth muscle populations. In young and adult mice, diverse sub-cryptal cells provide redundant ISC-supportive factors; few of these are restricted to single cell types. Niche functions refine during postnatal crypt morphogenesis, in part to oppose the dense aggregation of differentiation-promoting BMP+ sub-epithelial myofibroblasts at crypt-villus junctions. Muscularis mucosae, a specialized muscle layer, first appears during this period and supplements neighboring RSPO and BMPi sources. Components of this developing niche are conserved in human fetuses. The in vivo ablation of mouse postnatal smooth muscle increases BMP signaling activity, potently limiting a pre-weaning burst of crypt fission. Thus, distinct and progressively specialized mesenchymal cells together create the milieu that is required to propagate crypts during rapid organ growth and to sustain adult ISCs.
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Intestinos , Nicho de Células-Tronco , Humanos , Camundongos , Animais , Nicho de Células-Tronco/fisiologia , Mucosa Intestinal/metabolismo , Diferenciação Celular , Proteínas Morfogenéticas Ósseas/metabolismo , Músculo LisoRESUMO
PDGFRA-expressing mesenchyme provides a niche for intestinal stem cells. Corresponding compartments are unknown in the stomach, where corpus and antral glandular epithelia have similar niche dependencies but are structurally distinct from the intestine and from each other. Previous studies considered antrum and corpus as a whole and did not assess niche functions. Using high-resolution imaging and sequencing, we identify regional subpopulations and niche properties of purified mouse corpus and antral PDGFRA + cells. PDGFRA Hi sub-epithelial myofibroblasts are principal sources of BMP ligands in both gastric segments; two molecularly distinct groups distribute asymmetrically along antral glands but together fail to support epithelial organoids in vitro . In contrast, strategically positioned PDGFRA Lo cells that express CD55 enable corpus and antral organoid growth in the absence of other cellular or soluble factors. Our study provides detailed insights into spatial, molecular, and functional organization of gastric mesenchyme and the spectrum of signaling sources for stem cell support.
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Hypercholesterolemia accelerates the phenotypes of aging in hematopoietic stem cells (HSCs). As yet, little is known about the underlying mechanism. We found that hypercholesterolemia downregulates Ten eleven translocation 1 (Tet1) in HSCs. The total HSC population was increased, while the long-term (LT) population, side population and reconstitution capacity of HSCs were significantly decreased in Tet1-/- mice. Expression of the Tet1 catalytic domain in HSCs effectively restored the LT population and reconstitution capacity of HSCs isolated from Tet1-/- mice. While Tet1 deficiency upregulated the expression of p19 and p21 in HSCs by decreasing the H3K27me3 modification, the restoration of Tet1 activity reduced the expression of p19, p21 and p27 by restoring the H3K27me3 and H3K36me3 modifications on these genes. These results indicate that Tet1 plays a critical role in maintaining the quiescence and reconstitution capacity of HSCs and that hypercholesterolemia accelerates HSC aging phenotypes by decreasing Tet1 expression in HSCs.
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Envelhecimento/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Hipercolesterolemia/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Feminino , Histonas/genética , Histonas/metabolismo , Humanos , Hipercolesterolemia/genética , Masculino , Metilação , Camundongos , Camundongos Knockout , Fenótipo , Proteínas Proto-Oncogênicas/genéticaRESUMO
People with type 2 diabetes mellitus (T2DM) have a 25-fold higher risk of limb loss than non-diabetics due in large part to impaired wound healing. Here, we show that the impaired wound healing phenotype found in T2D mice is recapitulated in lethally irradiated wild type recipients, whose hematopoiesis is reconstituted with hematopoietic stem cells (HSCs) from T2D mice, indicating an HSC-autonomous mechanism. This impaired wound healing phenotype of T2D mice is due to a Nox-2-dependent increase in HSC oxidant stress that decreases microRNA let-7d-3p, which, in turn, directly upregulates Dnmt1, leading to the hypermethylation of Notch1, PU.1, and Klf4. This HSC-autonomous mechanism reduces the number of wound macrophages and skews their polarization towards M1 macrophages. These findings reveal a novel inflammatory mechanism by which a metabolic disorder induces an epigenetic mechanism in HSCs, which predetermines the gene expression of terminally differentiated inflammatory cells that controls their number and function.
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Diferenciação Celular , Diabetes Mellitus Tipo 2/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Macrófagos/metabolismo , Cicatrização/genética , Animais , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA , Células-Tronco Hematopoéticas/citologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Macrófagos/citologia , Camundongos , MicroRNAs/genética , NADPH Oxidase 2/metabolismo , Estresse Oxidativo , Proteínas Proto-Oncogênicas/genética , Receptor Notch1/genética , Transativadores/genéticaRESUMO
Obesity will soon surpass smoking as the most preventable cause of cancer. Hypercholesterolemia, a common comorbidity of obesity, has been shown to increase cancer risk, especially colorectal cancer. However, the mechanism by which hypercholesterolemia or any metabolic disorder increases cancer risk remains unknown. In this study, we show that hypercholesterolemia increases the incidence and pathologic severity of colorectal neoplasia in two independent mouse models. Hypocholesterolemia induced an oxidant stress-dependent increase in miR101c, which downregulated Tet1 in hematopoietic stem cells (HSC), resulting in reduced expression of genes critical to natural killer T cell (NKT) and γδ T-cell differentiation. These effects reduced the number and function of terminally differentiated NKT and γδ T cells in the thymus, the colon submucosa, and during early tumorigenesis. These results suggest a novel mechanism by which a metabolic disorder induces epigenetic changes to reduce lineage priming of HSC toward immune cells, thereby compromising immunosurveillance against cancer. Cancer Res; 77(9); 2351-62. ©2017 AACR.
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Neoplasias Colorretais/genética , Hipercolesterolemia/genética , MicroRNAs/genética , Oxigenases de Função Mista/genética , Obesidade/genética , Proteínas Proto-Oncogênicas/genética , Animais , Carcinogênese , Diferenciação Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/patologia , Células-Tronco Hematopoéticas/patologia , Humanos , Hipercolesterolemia/complicações , Hipercolesterolemia/patologia , Ativação Linfocitária/genética , Camundongos , MicroRNAs/biossíntese , Oxigenases de Função Mista/biossíntese , Células T Matadoras Naturais/patologia , Obesidade/complicações , Obesidade/patologia , Estresse Oxidativo/genética , Proteínas Proto-Oncogênicas/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Oxidized low-density lipoprotein (oxLDL) is an important risk factor in the development of atherosclerosis. oxLDL has been shown to decrease endothelial progenitor cell (EPC) number by inducing apoptosis. p38 mitogen-activated protein kinase (MAPK) was shown to be activated by oxLDL and participated in the regulation of EPC number and function. However, the role of p38 remains unknown. Here, we show that oxLDL-induced p38 phosphorylation in EPCs is time and dose dependent. Treatment with antioxidant N-acetyl cysteine restored oxLDL-induced p38 phosphorylation to basal levels. LOX-1-blocking antibody also significantly decreased oxLDL-induced p38 phosphorylation. Interestingly, TUNEL staining showed that pretreatment with the p38 inhibitor SB203580 further increased oxLDL-induced apoptosis in EPCs. In accordance with these findings, pretreatment with SB203580 further attenuated Akt phosphorylation in EPCs challenged with oxLDL, indicating an interaction between Akt and p38 MAPK pathways. In agreement, inhibition of p38 MAPK further attenuated Akt phosphorylation and increased apoptosis in EPCs isolated from hypercholesterolemic ApoE-/- mice. In conclusion, p38 MAPK serves as an anti-apoptotic pathway by supporting Akt activity when EPCs are challenged with oxLDL.
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Apoptose/efeitos dos fármacos , Células Progenitoras Endoteliais/efeitos dos fármacos , Hipercolesterolemia/enzimologia , Lipoproteínas LDL/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Progenitoras Endoteliais/enzimologia , Células Progenitoras Endoteliais/patologia , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Depuradores Classe E/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Clinical studies suggest that hypercholesterolemia may cause ageing in hematopoietic stem cells (HSCs) because ageing-associated alterations were found in peripheral blood cells and their bone marrow residing precursors in patients with advanced atherosclerosis. We hypothesized that hypercholesterolemia induces oxidant stress in hematopoietic stems cells that accelerates their ageing. METHODS AND RESULTS: Here we show that HSCs from ApoE(-/-) mice, as well as HSCs from C57Bl/6 mice fed a high cholesterol diet (HCD) accumulated oxLDL and had greater ROS levels. In accordance, the expression pattern of the genes involved in ROS metabolism changed significantly in HSCs from ApoE(-/-) mice. Hypercholesterolemia caused a significant reduction in phenotypically defined long-term HSC compartment, telomere length, and repopulation capacity of KTLS cells, indicating accelerated ageing in these cells. Gene array analysis suggested abnormal cell cycle status, and the key cell cycle regulators including p19(ARF), p27(Kip1) and p21(Waf1) were upregulated in KTLS cells from hypercholesterolemic mice. These effects were p38-dependent and reversed in vivo by treatment of hypercholesterolemic mice with antioxidant N-acetylcysteine. The oxidant stress also caused aberrant expression of Notch1 that caused loss of quiescence and proliferation leading to the expansion of KTLS compartment in hypercholesterolemic mice. CONCLUSION: Taken together, we provide evidence that hypercholesterolemia can cause oxidant stress that accelerates the ageing and impairs the reconstitution capacity of HSCs.
Assuntos
Senescência Celular , Células-Tronco Hematopoéticas/metabolismo , Hipercolesterolemia/metabolismo , Estresse Oxidativo , Animais , Antioxidantes/farmacologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Proliferação de Células , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Hipercolesterolemia/genética , Hipercolesterolemia/patologia , Lipoproteínas LDL/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Receptor Notch1/metabolismo , Encurtamento do Telômero , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Mesenchymal stem cells (MSCs), due to their paracrine, transdifferentiation, and immunosuppressive effects, hold great promise as a therapy for peripheral arterial disease. Diabetes is an important risk factor for peripheral arterial disease; however, little is known of how type II diabetes affects the therapeutic function of MSCs. This review summarizes the current status of preclinical and clinical studies that have been performed to determine the efficacy of MSCs in the treatment of peripheral arterial disease. We also present findings from our laboratory regarding the impact of type II diabetes on the therapeutic efficacy of MSCs neovascularization after the induction of hindlimb ischemia. In our studies, we documented that experimental type II diabetes in db/db mice impaired MSCs' therapeutic function by favoring their differentiation towards adipocytes, while limiting their differentiation towards endothelial cells. Moreover, type II diabetes impaired the capacity of MSCs to promote neovascularization in the ischemic hindlimb. We further showed that these impairments of MSC function and multipotency were secondary to hyperinsulinemia-induced, Nox4-dependent oxidant stress in db/db MSCs. Should human MSCs display similar oxidant stress-induced impairment of function, these findings might permit greater leverage of the potential of MSC transplantation, particularly in the setting of diabetes or other cardiovascular risk factors, as well as provide a therapeutic approach by reversing the oxidant stress of MSCs prior to transplantation.
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Terapia Baseada em Transplante de Células e Tecidos , Diabetes Mellitus Tipo 2/complicações , Transplante de Células-Tronco Mesenquimais , Doença Arterial Periférica/terapia , Animais , Transdiferenciação Celular , Membro Posterior/irrigação sanguínea , Membro Posterior/fisiopatologia , Humanos , Células-Tronco Mesenquimais , Camundongos , Neovascularização Fisiológica , Estresse OxidativoRESUMO
Nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) is a potent vasodilator and signaling molecule that plays essential roles in neovascularization. During limb ischemia, decreased NO bioavailability occurs secondary to increased oxidant stress, decreased L-arginine and tetrahydrobiopterin. This study tested the hypothesis that dietary cosupplementation with tetrahydrobiopterin (BH4), L-arginine and vitamin C acts synergistically to decrease oxidant stress, increase NO and thereby increase blood flow recovery after hindlimb ischemia. Rats were fed normal chow, chow supplemented with BH4 or L-arginine (alone or in combination) or chow supplemented with BH4 + L-arginine + vitamin C for 1 wk before induction of hindlimb ischemia. In the is-chemic hindlimb, cosupplementation with BH4 + L-arginine resulted in greater eNOS and phospho-eNOS (P-eNOS) expression, Ca(2+)-dependent NOS activity and NO concentration in the ischemic calf region (gastrocnemius), as well as greater NO concentration in the region of collateral arteries (gracilis). Rats receiving cosupplementation of BH4 + L-arginine led to greater recovery of foot perfusion and greater collateral enlargement than did rats receiving either agent separately. The addition of vitamin C to the BH4 + L-arginine regimen further increased these dependent variables. In addition, rats given all three supplements showed significantly less Ca(2+)-independent activity, less nitrotyrosine accumulation, greater glutathione (GSH)-to-glutathione disulfide (GSSG) ratio and less gastrocnemius muscle necrosis, on both macroscopic and microscopic levels. In conclusion, co-supplementation with BH4 + L-arginine + vitamin C significantly increased blood flow recovery after hindlimb ischemia by reducing oxidant stress, increasing NO bioavailability, enlarging collateral arteries and reducing muscle necrosis. Oral cosupplementation of BH4, L-arginine and vitamin C holds promise as a biological therapy to induce collateral artery enlargement.
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Arginina/farmacologia , Ácido Ascórbico/farmacologia , Biopterinas/análogos & derivados , Membro Posterior/irrigação sanguínea , Isquemia/patologia , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Administração Oral , Animais , Arginina/administração & dosagem , Arginina/uso terapêutico , Ácido Ascórbico/administração & dosagem , Ácido Ascórbico/uso terapêutico , Biopterinas/administração & dosagem , Biopterinas/farmacologia , Biopterinas/uso terapêutico , Cálcio/metabolismo , Sinergismo Farmacológico , Membro Posterior/efeitos dos fármacos , Membro Posterior/patologia , Membro Posterior/fisiopatologia , Isquemia/tratamento farmacológico , Isquemia/fisiopatologia , Masculino , Modelos Biológicos , Músculos/efeitos dos fármacos , Músculos/enzimologia , Músculos/patologia , Músculos/fisiopatologia , Nitratos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Nitritos/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Fluxo Sanguíneo Regional/efeitos dos fármacosRESUMO
Nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) is a potent vasodilator and signaling molecule that plays an essential role in vascular remodeling of collateral arteries and perfusion recovery in response to hindlimb ischemia. In ischemic conditions, decreased NO bioavailability was observed because of increased oxidative stress, decreased L-arginine and tetrahydrobiopterin. This study tested the hypothesis that dietary cosupplementation with tetrahydrobiopterin (BH4), L-arginine, and vitamin C acts synergistically to decrease oxidative stress, increase nitric oxide and improve blood flow in response to acute hindlimb ischemia. Rats were fed normal chow, chow supplemented with BH4 or L-arginine (alone or in combination) or chow supplemented with BH4 + L-arginine + vitamin C for 1 wk before induction of unilateral hindlimb ischemia. Cosupplementation with BH4 + L-arginine resulted in greater eNOS expression, Ca²âº-dependent NOS activity and NO concentration in gastrocnemius from the ischemic hindlimb, as well as greater recovery of foot perfusion and more collateral artery enlargement than did rats receiving either agent separately. The addition of vitamin C to the BH4 + L-arginine regimen did further increase these dependent variables, although only the increase in eNOS expression reached statistical significances. In addition, rats given all three supplements demonstrated significantly less Ca²âº-independent activity, less nitrotyrosine accumulation, greater glutathione:glutathione disulfide (GSH:GSSG) ratio and less gastrocnemius muscle necrosis, on both macroscopic and microscopic levels. In conclusion, cosupplementation with BH4 + L-arginine + vitamin C significantly increased vascular perfusion after hindlimb ischemia by increasing eNOS activity and reducing oxidative stress and tissue necrosis. Oral cosupplementation of L-arginine, BH4 and vitamin C holds promise as a biological therapy to induce collateral artery enlargement.
Assuntos
Antioxidantes/farmacologia , Arginina/farmacologia , Ácido Ascórbico/farmacologia , Biopterinas/análogos & derivados , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fluxo Sanguíneo Regional/efeitos dos fármacos , Administração Oral , Animais , Antioxidantes/administração & dosagem , Arginina/administração & dosagem , Ácido Ascórbico/administração & dosagem , Biopterinas/administração & dosagem , Biopterinas/farmacologia , Cálcio/metabolismo , Sinergismo Farmacológico , Ativação Enzimática , Membro Posterior/irrigação sanguínea , Membro Posterior/efeitos dos fármacos , Isquemia/tratamento farmacológico , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Necrose , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: This study tested the hypothesis that type 2 diabetes restricts multipotency of db/db mesenchymal stem cells (MSCs), promotes their terminal differentiation into adipocytes rather than endothelial cells, thereby promotes adipocytic infiltration into ischemic muscles, and reduces their capacity to participate in postischemic neovascularization. METHODS AND RESULTS: To test this hypothesis, we transplanted MSCs from db/db or wild-type (WT) mice into WT recipients after induction of hind limb ischemia. WT recipients of db/db MSCs demonstrated adipocyte infiltration of ischemic muscle and impaired neovascularization; WT recipients of WT MSCs showed no intramuscular adipocyte infiltration and had significantly enhanced neovascularization (P<0.05; n=6). Confocal microscopy showed that the percentage of MSCs that differentiated into an adipocyte phenotype was greater and into an endothelial cell was less in WT recipients transplanted with db/db MSCs than those transplanted with WT MSCs (P<0.05; n=6). In vitro, db/db MSCs exhibited greater oxidant stress, greater adipocyte differentiation, and less endothelial differentiation than WT MSCs, and these differences were reversed by treatment with N-acetylcysteine or Nox4 siRNA (P<0.05; n=6). Insulin increased Nox4 expression, oxidant stress, and adipocyte differentiation in WT MSCs, and these insulin-induced effects were reversed by Nox4 siRNA (P<0.05; n=6). Reversal of db/db MSC oxidant stress by in vivo pretreatment with Nox4 siRNA before transplantation reversed their impaired capacity to augment postischemic neovascularization. CONCLUSIONS: Type 2 diabetes-induced oxidant stress restricts the multipotency of MSCs and impairs their capacity to increase blood flow recovery after the induction of hind-limb ischemia. Reversal of MSC oxidant stress might permit greater leverage of the therapeutic potential of MSC transplantation in the setting of diabetes.
Assuntos
Adipócitos/fisiologia , Diabetes Mellitus Tipo 2/fisiopatologia , Isquemia/fisiopatologia , Células-Tronco Mesenquimais/fisiologia , Neovascularização Fisiológica/fisiologia , Animais , Diferenciação Celular , Movimento Celular , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Expressão Gênica , Membro Posterior/irrigação sanguínea , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Isquemia/etiologia , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/fisiologia , NADPH Oxidase 4 , NADPH Oxidases/efeitos dos fármacos , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , RNA Interferente Pequeno/fisiologiaRESUMO
We tested the hypothesis that oral supplementation with the endothelial nitric oxide synthase (eNOS) cofactor tetrahydrobiopterin (BH(4)) improved the therapeutic efficacy of eNOS gene transfer in the ischemic rat hindlimb. BH(4) or vehicle were begun 1 week before induction of hindlimb ischemia, whereas recombinant adenovirus containing bovine eNOS cDNA (AdeNOS) or vehicle [phosphate-buffered saline (PBS)] was infused intra-arterially into the ischemic hindlimb 10 days after induction of ischemia. Rats receiving co-treatment with dietary BH(4) and eNOS gene transfer (the [eNOS, +BH(4)] group) had greater eNOS expression, phospho-eNOS expression (Ser(1177)), Ca(2+)-dependent NOS activity, and nitrite + nitrate concentrations in the ischemic gastrocnemius than did rats receiving AdeNOS alone. The [eNOS, +BH(4)] group demonstrated less nitrotyrosine and a higher ratio of reduced:oxidized glutathione (GSH:GSSG) in the ischemic gastrocnemius muscle than did rats receiving AdeNOS alone. The [eNOS, +BH(4)] group had greater flow recovery and a higher capillary:myocyte ratio in the ischemic hindlimb than did rats receiving AdeNOS alone. Finally, the [eNOS,+BH(4)] group had less necrosis of hindlimb muscles than rats given AdeNOS alone. We conclude that adjunctive dietary therapy with BH(4) increases the beneficial effects of eNOS gene transfer within the ischemic gastrocnemius muscle, as evidenced by increased nitric oxide (NO) production, diminished oxidative stress, enhanced flow recovery, and reduced necrosis.
Assuntos
Adenoviridae/genética , Biopterinas/análogos & derivados , Isquemia/tratamento farmacológico , Isquemia/terapia , Extremidade Inferior/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Biopterinas/uso terapêutico , Western Blotting , Glutationa/metabolismo , Imuno-Histoquímica , Isquemia/metabolismo , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Óxido Nítrico Sintase Tipo III/genética , Ratos , Ratos Sprague-DawleyRESUMO
We tested the hypothesis that oxidized low-density lipoprotein (oxLDL)-induced inactivation of Akt within endothelial progenitor cells (EPCs) is mediated at the level of phosphoinositide 3-kinase (PI3K), specifically by nitrosylation of the p85 subunit of PI3K, and that this action is critical in provoking oxLDL-induced EPC apoptosis. Hypercholesterolemic ApoE null mice had a significant reduction of the phosphorylated Akt (p-Akt)/Akt ratio in EPCs, as well as a greater percentage of apoptosis in these cells than EPCs isolated from wild-type (WT) C57Bl/6 mice. EPCs were isolated from WT spleen and exposed to oxLDL in vitro. oxLDL increased O2â» and H2O2 in these cells and induced a dose- and time-dependent reduction in the p-Akt/Akt ratio and increase in EPC apoptosis. These effects were significantly reduced by the antioxidants superoxide dismutase, L-NAME, epicatechin and FeTPPs. oxLDL also induced nitrosylation of the p85 subunit of PI3K and subsequent dissociation of the p85 and p110 subunits, an effect significantly reduced by all the antioxidant agents tested. EPC transfection with a constitutively active Akt isoform (Ad-myrAkt) significantly reduced oxLDL-induced apoptosis of WT EPCs. The present findings indicate that oxLDL disrupts the PI3K/Akt signaling pathway at the level of p85 in EPCs. This dysfunction can be reversed by ex vivo antioxidant therapy.
Assuntos
Apoptose , Células Endoteliais/enzimologia , Hipercolesterolemia/enzimologia , Lipoproteínas LDL/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Células-Tronco/enzimologia , Animais , Antioxidantes/farmacologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Apoptose/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Ativação Enzimática , Peróxido de Hidrogênio/metabolismo , Hipercolesterolemia/genética , Hipercolesterolemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Superóxidos/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
OBJECTIVE: The goals of this study were to determine if endothelial nitric oxide synthase (eNOS) affects both early and late collateral arterial adaptation and blood flow recovery after severe limb ischemia in a mouse model and to determine if eNOS-derived NO is necessary for recruitment of chemokine (C-X-C motif) receptor 4 (CXCR4)(+) vascular endothelial growth factor receptor-1 (VEGFR1)(+) hemangiocytes to the site of ischemia. METHODS: Two studies were completed. In the first, hind limb ischemia was induced by unilateral femoral artery excision in three groups: C57Bl6 (wild-type), eNOS(-/-), and C57Bl/6 mice treated with N(G)-nitro-L-arginine methyl ester (L-NAME) from 1 day before excision through day 3 after excision (early L-NAME group). These groups were studied on day 3 after induction of ischemia. In the second study, hind limb ischemia was induced in C57Bl/6 mice (wild-type) and C57Bl/6 mice treated with L-NAME from days 3 through 28 after induction of ischemia. These groups were studied day 28 after ischemia induction. Dependent variables included hind limb perfusion, collateral artery diameter, and the number and location of hemangiocytes within the ischemic hind limb. RESULTS: In the first study, toe gangrene developed in the eNOS(-/-) and early L-NAME treatment groups by day 2. These groups demonstrated less blood flow recovery and smaller collateral artery diameter than the wild-type group. Hemangiocytes were present within the adventitia of collateral arteries in the wild-type group but were only sparsely present, in a random pattern, in the eNOS(-/-) and early L-NAME treatment groups. In the second study, the late L-NAME group showed less blood flow recovery and smaller collateral artery diameter on day 28 of ischemia than the wild-type group. Hemangiocytes were present in a pericapillary distribution in the wild-type group, but were present only sparsely in the late L-NAME treatment group. CONCLUSION: Early (day 3) and late (day 28) adaptive responses to hind limb ischemia both require eNOS-derived NO. NO is necessary for normal hemangiocyte recruitment to the ischemic tissue.
Assuntos
Circulação Colateral , Isquemia/enzimologia , Músculo Esquelético/irrigação sanguínea , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Fluxo Sanguíneo Regional , Animais , Quimiotaxia , Circulação Colateral/efeitos dos fármacos , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Gangrena , Membro Posterior , Isquemia/patologia , Isquemia/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Receptores CXCR4/metabolismo , Recuperação de Função Fisiológica , Fluxo Sanguíneo Regional/efeitos dos fármacos , Células-Tronco/metabolismo , Fatores de Tempo , Dedos do Pé/patologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: We sought to directly compare the effects of type 1 and type 2 diabetes on postischemic neovascularization and evaluate the mechanisms underlying differences between these groups. We tested the hypothesis that type 2 diabetic mice have a greater reduction in endothelial nitric oxide synthase (eNOS) expression, a greater increase in oxidative stress, and reduced arteriogenesis and angiogenesis, resulting in less complete blood flow recovery than type 1 diabetic mice after induction of hind limb ischemia. METHODS: Hind limb ischemia was generated by femoral artery excision in streptozotocin-treated mice (model of type 1 diabetes), in Lepr(db/db) mice (model of type 2 diabetes), and in control (C57BL/6) mice. Dependent variables included eNOS expression and markers of arteriogenesis, angiogenesis, and oxidative stress. RESULTS: Postischemia recovery of hind limb perfusion was significantly less in type 2 than in type 1 diabetic mice; however, neither group demonstrated a significant increase in collateral artery diameter or collateral artery angioscore in the ischemic hind limb. The capillary/myofiber ratio in the gastrocnemius muscle decreased in response to ischemia in control or type 1 diabetic mice but remained the same in type 2 diabetic mice. Gastrocnemius muscle eNOS expression was lower in type 1 and 2 diabetic mice than in control mice. This expression decreased after induction of ischemia in type 2 but not in type 1 diabetic mice. The percentage of endothelial progenitor cells (EPC) in the peripheral blood failed to increase in either diabetic group after induction of ischemia, whereas this variable significantly increased in the control group in response to ischemia. EPC eNOS expression decreased after induction of ischemia in type 1 but not in type 2 diabetic mice. EPC nitrotyrosine accumulation increased after induction of ischemia in type 2 but not in type 1 diabetic mice. EPC migration in response to vascular endothelial growth factor was reduced in type 1 and type 2 diabetic mice vs control mice. EPC incorporation into tubular structures was less effective in type 2 diabetic mice. Extensive fatty infiltration was present in ischemic muscle of type 2 but not in type 1 diabetic mice. CONCLUSION: Type 2 diabetic mice displayed a significantly less effective response to hind limb ischemia than type 1 diabetic mice.