RESUMEN
Peripheral artery disease is an atherosclerotic occlusive disease that causes limb ischemia and has few effective noninterventional treatments. Stem cell therapy is promising, but concomitant diabetes may limit its effectiveness. We evaluated the therapeutic potential of skeletal muscle pericytes to augment postischemic neovascularization in wild-type and type 2 diabetic (T2DM) mice. Wild-type C57BL/6J and leptin receptor spontaneous mutation db/db T2DM mice underwent unilateral femoral artery excision to induce limb ischemia. Twenty-four hours after ischemia induction, CD45-CD34-CD146+ skeletal muscle pericytes or vehicle controls were transplanted into ischemic hindlimb muscles. At postoperative day 28, pericyte transplantation augmented blood flow recovery in wild-type mice (79.3 ± 5% vs. 61.9 ± 5%; P = 0.04), but not in T2DM mice (48.6% vs. 46.3 ± 5%; P = 0.51). Pericyte transplantation augmented collateral artery enlargement in wild-type (26.7 ± 2 µm vs. 22.3 ± 1 µm, P = 0.03), but not T2DM mice (20.4 ± 1.4 µm vs. 18.5 ± 1.2 µm, P = 0.14). Pericyte incorporation into collateral arteries was higher in wild-type than in T2DM mice ( P = 0.002). Unexpectedly, pericytes differentiated into Schwann cells in vivo. In vitro, Insulin increased Nox2 expression and decreased tubular formation capacity in human pericytes. These insulin-induced effects were reversed by N-acetylcysteine antioxidant treatment. In conclusion, T2DM impairs the ability of pericytes to augment neovascularization via decreased collateral artery enlargement and impaired engraftment into collateral arteries, potentially via hyperinsulinemia-induced oxidant stress. While pericytes show promise as a unique form of stem cell therapy to increase postischemic neovascularization, characterizing the molecular mechanisms by which T2DM impairs their function is essential to achieve their therapeutic potential.
Asunto(s)
Diabetes Mellitus Tipo 2/patología , Isquemia/cirugía , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Pericitos/trasplante , Animales , Diferenciación Celular , Células Cultivadas , Circulación Colateral , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Modelos Animales de Enfermedad , Humanos , Insulina/farmacología , Isquemia/metabolismo , Isquemia/patología , Isquemia/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Pericitos/patología , Fenotipo , Receptores de Leptina/genética , Flujo Sanguíneo Regional , Remodelación VascularRESUMEN
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.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Progenitoras Endoteliales/efectos de los fármacos , Hipercolesterolemia/enzimología , Lipoproteínas LDL/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Células Progenitoras Endoteliales/enzimología , Células Progenitoras Endoteliales/patología , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Depuradores de Clase E/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Arginina/farmacología , Ácido Ascórbico/farmacología , Biopterinas/análogos & derivados , Miembro Posterior/irrigación sanguínea , Isquemia/patología , Óxido Nítrico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Administración Oral , Animales , Arginina/administración & dosificación , Arginina/uso terapéutico , Ácido Ascórbico/administración & dosificación , Ácido Ascórbico/uso terapéutico , Biopterinas/administración & dosificación , Biopterinas/farmacología , Biopterinas/uso terapéutico , Calcio/metabolismo , Sinergismo Farmacológico , Miembro Posterior/efectos de los fármacos , Miembro Posterior/patología , Miembro Posterior/fisiopatología , Isquemia/tratamiento farmacológico , Isquemia/fisiopatología , Masculino , Modelos Biológicos , Músculos/efectos de los fármacos , Músculos/enzimología , Músculos/patología , Músculos/fisiopatología , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Nitritos/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacos , Flujo Sanguíneo Regional/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antioxidantes/farmacología , Arginina/farmacología , Ácido Ascórbico/farmacología , Biopterinas/análogos & derivados , Óxido Nítrico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Flujo Sanguíneo Regional/efectos de los fármacos , Administración Oral , Animales , Antioxidantes/administración & dosificación , Arginina/administración & dosificación , Ácido Ascórbico/administración & dosificación , Biopterinas/administración & dosificación , Biopterinas/farmacología , Calcio/metabolismo , Sinergismo Farmacológico , Activación Enzimática , Miembro Posterior/irrigación sanguínea , Miembro Posterior/efectos de los fármacos , Isquemia/tratamiento farmacológico , Masculino , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Necrosis , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Adenoviridae/genética , Biopterinas/análogos & derivados , Isquemia/tratamiento farmacológico , Isquemia/terapia , Extremidad Inferior/patología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Animales , Biopterinas/uso terapéutico , Western Blotting , Glutatión/metabolismo , Inmunohistoquímica , Isquemia/metabolismo , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Óxido Nítrico Sintasa de Tipo III/genética , Ratas , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Apoptosis , Células Endoteliales/enzimología , Hipercolesterolemia/enzimología , Lipoproteínas LDL/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Células Madre/enzimología , Animales , Antioxidantes/farmacología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Apoptosis/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Activación Enzimática , Peróxido de Hidrógeno/metabolismo , Hipercolesterolemia/genética , Hipercolesterolemia/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Estrés Oxidativo , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/efectos de los fármacos , Células Madre/efectos de los fármacos , Células Madre/patología , Superóxidos/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
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.
Asunto(s)
Circulación Colateral , Isquemia/enzimología , Músculo Esquelético/irrigación sanguínea , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Flujo Sanguíneo Regional , Animales , Quimiotaxis , Circulación Colateral/efectos de los fármacos , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Gangrena , Miembro Posterior , Isquemia/patología , Isquemia/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/deficiencia , Óxido Nítrico Sintasa de Tipo III/genética , Receptores CXCR4/metabolismo , Recuperación de la Función , Flujo Sanguíneo Regional/efectos de los fármacos , Células Madre/metabolismo , Factores de Tiempo , Dedos del Pie/patología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
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.
Asunto(s)
Envejecimiento/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre Hematopoyéticas/metabolismo , Hipercolesterolemia/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Femenino , Histonas/genética , Histonas/metabolismo , Humanos , Hipercolesterolemia/genética , Masculino , Metilación , Ratones , Ratones Noqueados , Fenotipo , Proteínas Proto-Oncogénicas/genéticaRESUMEN
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.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Células Endoteliales/enzimología , Isquemia/fisiopatología , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Óxido Nítrico Sintasa de Tipo III/metabolismo , Células Madre/enzimología , Animales , Glucemia/metabolismo , Peso Corporal , Quimiotaxis , Circulación Colateral , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/genética , Miembro Posterior , Isquemia/complicaciones , Isquemia/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Estrés Oxidativo , Receptores de Leptina/genética , Recuperación de la Función , Flujo Sanguíneo Regional , Factores de Tiempo , Tirosina/análogos & derivados , Tirosina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Yak is a unique domestic animal of Qinghai-Tibetan Plateau. Its unique adaptability to the high altitude environment has been hypothesized due to special pulmonary microvasculature. However, the anatomical evidence is still less. The present study characterized the subpleural pulmonary microvascular architecture of newborn yak by vascular corrosion cast and the scanning electron microscopy. The results showed the dense vascular network occurred in subpleural area in newborn yak. Subpleural vascular network was found in most of observed areas, while the sparse vascular network crept onto the subpleural network in some fields of view. The subpleural microvessels and their branches made up of the subpleural microvascular network. According to the branching sequence of vessels, the subpleural arterioles could be divided into four grades: the arteriole, terminal arteriole, precapillary arteriole and capillary. The subpleural capillary network in the local area could be classified into three different forms, including sheetlike vascular network, wrinkled vascular network and weblike vascular network. It was the specific characteristics on the cast surface of the microvessels that was the adaptable peculiarities. Anastomoses were found between the pleural microvessels and the interlobular capillaries, or between the pleural microvessels and the subpleural capillaries, or between the interlobular capillaries and the subpleural capillaries. Therefore, there was significant difference on the subpleural pulmonary microvasculature between newborn yak and other adult mammals.
Asunto(s)
Bovinos/anatomía & histología , Pulmón/irrigación sanguínea , Animales , Animales Recién Nacidos , Femenino , Masculino , Microscopía Electrónica de Rastreo/veterinaria , Microvasos/ultraestructuraRESUMEN
BACKGROUND: Most current animal models of hindlimb ischemia use acute arterial occlusion that does not accurately reflect the pathogenesis of gradual arterial occlusion in humans. We, therefore, developed the first mouse model of gradual arterial occlusion and tested the hypothesis that the mechanisms regulating blood flow recovery are critically dependent on the rate of arterial occlusion. METHODS: Gradual arterial occlusion was induced by placing ameroid constrictors on the proximal and distal left femoral artery, and ligating the femoral arterial branches (n = 36). Acute arterial occlusion was accomplished by excising the left femoral artery (n = 36). The blood flow recovery was studied by laser Doppler imaging. Differential gene expression between these two models was assessed by quantitative real-time polymerase chain reactions (PCR). Inflammatory and progenitor cells recruitment were determined by immunohistochemistry. RESULTS: We found that hypoxia-related genes increased significantly in the calf, but not in the thigh, after gradual and acute femoral arterial occlusion (P < .05). Shear-stress dependent genes and inflammatory genes were upregulated immediately in the thigh only after acute femoral arterial occlusion (P < .05). These differences in gene expression were consistent with increased SDF-1alpha expression, recruitment of macrophages and hemangiocytes, and higher blood flow recovery after acute arterial occlusion than after gradual arterial occlusion (P < .05). CONCLUSION: This is the first study to show the mechanisms that regulate blood flow recovery are critically dependent on the rate of arterial occlusion. This novel model of gradual arterial occlusion may more closely resemble the human diseases, and may provide more accurate mechanistic insights for creating novel molecular therapies.
Asunto(s)
Arteriopatías Oclusivas/fisiopatología , Velocidad del Flujo Sanguíneo/fisiología , Quimiocina CXCL12/genética , Arteria Femoral/fisiología , Expresión Génica , ARN Mensajero/genética , Recuperación de la Función/fisiología , Enfermedad Aguda , Animales , Arteriopatías Oclusivas/genética , Arteriopatías Oclusivas/metabolismo , Quimiocina CXCL12/biosíntesis , Enfermedad Crónica , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Inmunohistoquímica , Flujometría por Láser-Doppler , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Reacción en Cadena de la PolimerasaRESUMEN
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.
Asunto(s)
Diferenciación Celular , Diabetes Mellitus Tipo 2/metabolismo , Células Madre Hematopoyéticas/metabolismo , Macrófagos/metabolismo , Cicatrización de Heridas/genética , Animales , ADN (Citosina-5-)-Metiltransferasa 1/genética , Metilación de ADN , Células Madre Hematopoyéticas/citología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Macrófagos/citología , Ratones , MicroARNs/genética , NADPH Oxidasa 2/metabolismo , Estrés Oxidativo , Proteínas Proto-Oncogénicas/genética , Receptor Notch1/genética , Transactivadores/genéticaRESUMEN
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.
Asunto(s)
Neoplasias Colorrectales/genética , Hipercolesterolemia/genética , MicroARNs/genética , Oxigenasas de Función Mixta/genética , Obesidad/genética , Proteínas Proto-Oncogénicas/genética , Animales , Carcinogénesis , Diferenciación Celular/genética , Línea Celular Tumoral , Linaje de la Célula/genética , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/patología , Células Madre Hematopoyéticas/patología , Humanos , Hipercolesterolemia/complicaciones , Hipercolesterolemia/patología , Activación de Linfocitos/genética , Ratones , MicroARNs/biosíntesis , Oxigenasas de Función Mixta/biosíntesis , Células T Asesinas Naturales/patología , Obesidad/complicaciones , Obesidad/patología , Estrés Oxidativo/genética , Proteínas Proto-Oncogénicas/biosíntesis , Receptores de Antígenos de Linfocitos T gamma-delta , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Senescencia Celular , Células Madre Hematopoyéticas/metabolismo , Hipercolesterolemia/metabolismo , Estrés Oxidativo , Animales , Antioxidantes/farmacología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Proliferación Celular , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/patología , Hipercolesterolemia/genética , Hipercolesterolemia/patología , Lipoproteínas LDL/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés Oxidativo/efectos de los fármacos , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Receptor Notch1/metabolismo , Acortamiento del Telómero , Factores de Tiempo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
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.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Diabetes Mellitus Tipo 2/complicaciones , Trasplante de Células Madre Mesenquimatosas , Enfermedad Arterial Periférica/terapia , Animales , Transdiferenciación Celular , Miembro Posterior/irrigación sanguínea , Miembro Posterior/fisiopatología , Humanos , Células Madre Mesenquimatosas , Ratones , Neovascularización Fisiológica , Estrés OxidativoRESUMEN
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.
Asunto(s)
Adipocitos/fisiología , Diabetes Mellitus Tipo 2/fisiopatología , Isquemia/fisiopatología , Células Madre Mesenquimatosas/fisiología , Neovascularización Fisiológica/fisiología , Animales , Diferenciación Celular , Movimiento Celular , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Expresión Génica , Miembro Posterior/irrigación sanguínea , Hipoglucemiantes/farmacología , Insulina/farmacología , Isquemia/etiología , Masculino , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/fisiología , NADPH Oxidasa 4 , NADPH Oxidasas/efectos de los fármacos , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Estrés Oxidativo/efectos de los fármacos , ARN Interferente Pequeño/fisiologíaRESUMEN
AIM: To study the influence of the expression of human alpha galactosidase and alpha1,2 fucosyltransferase on Gal alpha 1,3 Gal and consequent xenoreactivity in NIH3T3 cells. METHODS: The expression levels of G antigen and H antigen and binding of human natural antibodies (IgG and IgM) and complement (C3c) to NIH3T3 cells were analyzed by flow cytometry. Western blot was employed to further determine the expression of glycoproteins of G antigen. Cytolysis assay with normal human serum was performed by MTT assay. RESULTS: Western blot showed that glycoproteins with molecular weight of 107 kDa, 98 kDa, 88 kDa, 56 kDa, 40 kDa, and 37 kDa were inhibited and even abrogated totally in alpha galactosidase transfectants and alpha 1,2 fucosyltransferase transfectants. The combined transfection of the two enzymes led to a much stronger inhibition of the glycoproteins. The binding of GS-IB4 was decreased by 57.4 % in alpha galactosidase transfectants, 28.8 % in alpha 1,2 fucosyltransferase transfectants, and 72.1 % in combined transfectants, respectively. In contrast, UEA-1 binding was increased about 6.7-fold, 6.0-fold, and 8.0-fold respectively. The xenoreactivity with human IgG was also reduced by 61.4 %, 67.0 %, and 73.4 %, respectively in the three kinds of transfectants. The resistance to cytolysis mediated by human serum was enhanced by 42.4 % in alpha galactosidase transfectants, 51.9 % in alpha 1,2 fucosyltranferase, and even 65.5 % in the combined transfectants. CONCLUSION: Although alpha galactosidase and alpha 1,2 fucosyltransferase had different biochemical properties, they could inhibit the expression of Gal alpha 1,3 Gal synergistically, leading to stronger resistance of xenograft against cytolysis.
Asunto(s)
Disacáridos/metabolismo , Fucosiltransferasas/genética , alfa-Galactosidasa/genética , Células 3T3 , Animales , Complemento C3c/metabolismo , Fucosiltransferasas/biosíntesis , Inmunoglobulina G/metabolismo , Inmunoglobulina M/metabolismo , Ratones , Transfección , Trasplante Heterólogo , alfa-Galactosidasa/biosíntesis , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
AIM: To examine the effects of the expression of alpha-galactosidase on the expression of the major xenoepitope Galalpha(1,3) Gal (G antigen) in NIH 3T3 cell. METHODS: The expression levels of G antigen and H antigen and binding of human natural antibodies (IgG and IgM) and complement (C3c) to NIH 3T3 cells were analyzed by flow cytometry. Western blot was employed to further determine the expression of glycoproteins of G antigen. Cytolysis assay with normal human serum was performed by MTT assay. RESULTS: In transfectants, Western blot showed that the binding of human IgG to glycosylated proteins located on the cell membrane was decreased, even abrogated totally. Together with the reduced binding of Gs-IB4 (Griffonia simplicifolia) to transfectants, the stable expression of human alpha-galactosidase effectively inhibited Galalpha(1,3) Gal, Gal epitope synthesis in NIH 3T3 cell. As a result, the xenoreactivities of human IgG, IgM, and C3c were reduced by 73.4 %, 22.3 % and 47.9 %, respectively, while the cell lysis mediated by human XNA and complements was decreased by 42.4 %. CONCLUSION: The stable expression of human alpha-galactosidase in NIH 3T3 cell strongly inhibits the expression of Gal epitopes, resulting in abrupt reduction in xenorejection induced by human serum.