RESUMO
Non-healing diabetic wounds (DW) are a serious clinical problem that remained poorly understood. We recently found that topical application of growth differentiation factor 11 (GDF11) accelerated skin wound healing in both Type 1 DM (T1DM) and genetically engineered Type 2 diabetic db/db (T2DM) mice. In the present study, we elucidated the cellular and molecular mechanisms underlying the action of GDF11 on healing of small skin wound. Single round-shape full-thickness wound of 5-mm diameter with muscle and bone exposed was made on mouse dorsum using a sterile punch biopsy 7 days following the onset of DM. Recombinant human GDF11 (rGDF11, 50 ng/mL, 10 µL) was topically applied onto the wound area twice a day until epidermal closure (maximum 14 days). Digital images of wound were obtained once a day from D0 to D14 post-wounding. We showed that topical application of GDF11 accelerated the healing of full-thickness skin wounds in both type 1 and type 2 diabetic mice, even after GDF8 (a muscle growth factor) had been silenced. At the cellular level, GDF11 significantly facilitated neovascularization to enhance regeneration of skin tissues by stimulating mobilization, migration and homing of endothelial progenitor cells (EPCs) to the wounded area. At the molecular level, GDF11 greatly increased HIF-1É expression to enhance the activities of VEGF and SDF-1É, thereby neovascularization. We found that endogenous GDF11 level was robustly decreased in skin tissue of diabetic wounds. The specific antibody against GDF11 or silence of GDF11 by siRNA in healthy mice mimicked the non-healing property of diabetic wound. Thus, we demonstrate that GDF11 promotes diabetic wound healing via stimulating endothelial progenitor cells mobilization and neovascularization mediated by HIF-1É-VEGF/SDF-1É pathway. Our results support the potential of GDF11 as a therapeutic agent for non-healing DW.
Assuntos
Diabetes Mellitus Experimental , Células Progenitoras Endoteliais , Fatores de Diferenciação de Crescimento , Cicatrização , Animais , Humanos , Camundongos , Proteínas Morfogenéticas Ósseas/metabolismo , Quimiocina CXCL12/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/patologia , Fatores de Diferenciação de Crescimento/uso terapêutico , Fatores de Diferenciação de Crescimento/metabolismo , Neovascularização Fisiológica , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismoRESUMO
Monocytes and macrophages are early sentinels of infection. The peritoneum contains two resident populations: large and small peritoneal macrophages (LPMs and SPMs). While LPMs self-renew, circulating monocytes enter the peritoneum and differentiate into SPMs. We lack information on the dynamics of monocyte-macrophage trafficking during abdominal sepsis, reflecting an important knowledge gap. In this study, we characterize the presence of LPMs, SPMs, and monocytes in the peritoneum of mice following cecal ligation and puncture (CLP)-induced sepsis and sham surgery. LPMs rapidly disappeared from the peritoneum and were scarce at 18-66 h after CLP or sham surgery. By 14 d, LPMs returned for sham mice, but they remained scarce in CLP mice. Depletion of LPMs from the peritoneum of CD11b-DTR mice greatly increased animal mortality. These data imply that LPMs are critical for sepsis survival. Monocytes rapidly infiltrated the peritoneum and were abundant at 18-66 h after CLP or sham surgery. Surprisingly, SPMs only increased at 14 d post-CLP. Therefore, monocytes may defend hosts from acute sepsis mortality without generating SPMs. More monocytes were present in mice predicted to survive sepsis versus mice predicted to die. However, altering monocyte numbers via CCR2 deficiency or adoptive transfer did not significantly affect animal survival. We reasoned that animals destined to survive sepsis may exhibit a different monocyte phenotype, rather than merely enhanced numbers. Indeed, mice predicted to survive possessed more CD31+, CXCR4hi transitional premonocytes in their abdomen. Inhibition of CXCL12-CXCR4 signaling via AMD3100 exacerbated sepsis. These data imply that recruitment of transitional premonocytes to the abdomen promotes sepsis survival.
Assuntos
Macrófagos Peritoneais/patologia , Sepse/mortalidade , Sepse/patologia , Animais , Benzilaminas/farmacologia , Quimiocina CXCL12/efeitos dos fármacos , Ciclamos/farmacologia , Modelos Animais de Doenças , Feminino , Ligadura , Macrófagos/metabolismo , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Monócitos/metabolismo , Receptores CXCR4/efeitos dos fármacos , Sepse/tratamento farmacológico , Sepse/imunologiaRESUMO
Due to lack of effective biomarkers for early diagnosis, most patients are diagnosed with advanced gastric cancer and have lower survival rates. 5-Fluorouracil (5-FU) is one of commonly used drugs for chemotherapy of gastric cancer, but drug resistance limits the wide application of agents. Retinoblastoma tumor suppressor gene 1 (RB1) is a key regulator in the progression of various human cancers, including gastric cancer. However, the effects of RB1 on chemosensitivity and the underlying mechanisms in gastric cancer (GC) are not clear. In this study, expressions of RB1 in GC cell lines were evaluated by RT-qPCR and western blot assay. CCK-8 was applied to examine the effect of 5-FU on cell viability. Meanwhile, IC50 values were calculated. The drug-resistance protein MDR1 and autophagy-related proteins were detected by western blot assay. Flow cytometry was used to detect cell cycle. The results showed that RB1 expressions were downregulated in GC cell lines and had significant differences between 5-FU resistance cell lines (SNU-620/5-FU and NUGC-3/5-FU) and non-resistance cell lines (SNU-620 and NUGC-3). Overexpression of RB1 enhanced 5-FU sensitivity of GC cells and caused cell cycle arrest in the S phase. Meanwhile, autophagy-related proteins were downregulated. Mechanistically, SDF-1/CXCR4 participated in the regulation of RB1 on cell autophagy. Autophagy activator, SDF-1 treatment and CXCR4 activation reversed the promoted effects of RB1 on 5-FU sensitivity in GC cells. In conclusion, our data revealed that RB1 was downregulated in GC cell lines. RB1 overexpression enhanced 5-FU chemosensitivity in GC cells by regulating cell autophagy via SDF-1/CXCR4 pathway. RB1 might serve as a promising therapeutic target of GC.
Assuntos
Autofagia/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Receptores CXCR4/metabolismo , Retinoblastoma/genética , Neoplasias Gástricas/patologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiocina CXCL12/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/farmacologia , Humanos , Neoplasias da Retina/genética , Neoplasias da Retina/patologia , Retinoblastoma/patologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológicoRESUMO
Intensive use of methotrexate (MTX) and/or dexamethasone (DEX) for treating childhood malignancies is known to cause chondrocyte apoptosis and growth plate dysfunction leading to bone growth impairments. However, mechanisms remain vague and it is unclear whether MTX and DEX combination treatment could have additive effects in the growth plate defects. In this study, significant cell apoptosis was induced in mature ATDC5 chondrocytes after treatment for 48 h with 10-5 M MTX and/or 10-6 M DEX treatment. PCR array assays with treated cells plus messenger RNA and protein expression confirmation analyses identified chemokine CXCL12 having the most prominent induction in each treatment group. Conditioned medium from treated chondrocytes stimulated migration of RAW264.7 osteoclast precursor cells and formation of osteoclasts, and these stimulating effects were inhibited by the neutralizing antibody for CXCL12. Additionally, while MTX and DEX combination treatment showed some additive effects on apoptosis induction, it did not have additive or counteractive effects on CXCL12 expression and its functions in enhancing osteoclastic recruitment and formation. In young rats treated acutely with MTX, there was increased expression of CXCL12 in the tibial growth plate, and more resorbing chondroclasts were found present at the border between the hypertrophic growth plate and metaphysis bone. Thus, the present study showed an association between induced chondrocyte apoptosis and stimulated osteoclastic migration and formation following MTX and/or DEX treatment, which could be potentially or at least partially linked molecularly by CXCL12 induction. This finding may contribute to an enhanced mechanistic understanding of bone growth impairments following MTX and/or DEX therapy.
Assuntos
Quimiocina CXCL12/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Dexametasona/farmacologia , Metotrexato/farmacologia , Animais , Apoptose/efeitos dos fármacos , Desenvolvimento Ósseo/efeitos dos fármacos , Condrócitos/metabolismo , Condrogênese/efeitos dos fármacos , Lâmina de Crescimento/efeitos dos fármacos , Camundongos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , RatosRESUMO
BACKGROUND: Aloe vera has been reported as a topical antibiotic and healing agent for wounds, but advantages of oral administration and mechanisms of wound healing have not been reported. Present study focuses on the evaluation of effects of oral administration of Aloe vera for excisional cutaneous wounds in Sprague Dawley rats. METHODS: Sprague Dawley (SD) rats were inflicted with excisional wounds and were either treated with Aloe vera orally (Aloe vera) or kept untreated (wound). In contrast, healthy rats were kept as control group. Wound area was measured from day 7th to day 21st. Collagen content was estimated by hydroxyproline assay. Histology was analysed by hematoxylin and eosin staining. Angiogenesis was observed by indirect ELISA for Insulin like Growth Factor (IGF-1) and Vascular Endothelial Growth Factor (VEGF) protein from skin, serum and bone marrow. Chemotaxis was evaluated by RT-PCR analysis for Stromal cell-Derived Factor-1 (SDF-1) and C-X-C chemokine receptor type 4 (CXCR-4) from skin and bone marrow. RESULTS: Aloe vera healed wounds earlier than untreated rats with gradual improvement in wound areas and collagen content. Aloe vera also improved the expression of IGF-1 and VEGF in skin and bone marrow indicating an improvement in angiogenesis. RT- PCR analysis showed increased expression of genes for chemotaxis (SDF-1 and CXCR-4) in skin and bone marrow. CONCLUSION: Aloe vera improves healing by increasing collagen content, improving angiogenesis and chemotaxis.
Assuntos
Aloe , Neovascularização Fisiológica/efeitos dos fármacos , Extratos Vegetais/farmacologia , Cicatrização/efeitos dos fármacos , Administração Oral , Animais , Quimiocina CXCL12/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Colágeno/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores CXCR4/efeitos dos fármacos , Pele/efeitos dos fármacos , Somatomedinas/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: Chronic rejection induces the occurrence of orthotopic allograft transplantation (OAT) vasculopathy, which results in failure of the donor organ. Numerous studies have demonstrated that in addition to regulating blood sugar homeostasis, dipeptidyl peptidase-4 (DPP-4) inhibitors can also provide efficacious therapeutic and protective effects against cardiovascular diseases. However, their effects on OAT-induced vasculopathy remain unknown. Thus, the aim of this study was to investigate the direct effects of sitagliptin on OAT vasculopathy in vivo and in vitro. METHODS: The PVG/Seac rat thoracic aorta graft to ACI/NKyo rat abdominal aorta model was used to explore the effects of sitagliptin on vasculopathy. Human endothelial progenitor cells (EPCs) were used to investigate the possible underlying mechanisms. RESULTS: We demonstrated that sitagliptin decreases vasculopathy in OAT ACI/NKyo rats. Treatment with sitagliptin decreased BNP and HMGB1 levels, increased GLP-1 activity and stromal cell-derived factor 1α (SDF-1α) expression, elevated the number of circulating EPCs, and improved the differentiation possibility of mononuclear cells to EPCs ex vivo. However, in vitro studies showed that recombinant B-type natriuretic peptide (BNP) and high mobility group box 1 (HMGB1) impaired EPC function, whereas these phenomena were reversed by glucagon-like peptide 1 (GLP-1) receptor agonist treatment. CONCLUSIONS: We suggest that the mechanisms underlying sitagliptin-mediated inhibition of OAT vasculopathy probably occur through a direct increase in GLP-1 activity. In addition to the GLP-1-dependent pathway, sitagliptin may regulate SDF-1α levels and EPC function to reduce OAT-induced vascular injury. This study may provide new prevention and treatment strategies for DPP-4 inhibitors in chronic rejection-induced vasculopathy.
Assuntos
Aorta Torácica/transplante , Inibidores da Dipeptidil Peptidase IV/farmacologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Fosfato de Sitagliptina/farmacologia , Doenças Vasculares/fisiopatologia , Animais , Quimiocina CXCL12/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/efeitos dos fármacos , Proteína HMGB1/efeitos dos fármacos , Masculino , Peptídeo Natriurético Encefálico/efeitos dos fármacos , Ratos , Ratos Endogâmicos ACI , Transplante HomólogoRESUMO
BACKGROUND: Prenatal exposure to ethanol (EtOH) has lasting effects on neuropeptide and neuroimmune systems in the brain alongside detrimental alcohol-related behaviors. At low-to-moderate doses, prenatal EtOH stimulates neurogenesis in lateral hypothalamus (LH) and increases neurons that express the orexigenic peptides hypocretin/orexin (Hcrt/OX) and melanin-concentrating hormone (MCH), and the proinflammatory chemokine CCL2, which through its receptor CCR2 stimulates cell differentiation and movement. Our recent studies demonstrated that CCL2 and CCR2 colocalize with MCH neurons and are involved in EtOH's stimulatory effect on their development but show no relation to Hcrt/OX. Here, we investigated another chemokine, CXCL12, and its receptor, CXCR4, which promote neurogenesis and neuroprogenitor cell proliferation, to determine if they also exhibit peptide specificity in their response to EtOH exposure. METHODS: Pregnant rats were intraorally administered a moderate dose of EtOH (2 g/kg/d) from embryonic day 10 (E10) to E15. Their embryos and postnatal offspring were examined using real-time quantitative PCR and immunofluorescence histochemistry, to determine if EtOH affects CXCL12 and CXCR4 and the colocalization of CXCR4 with Hcrt/OX and MCH neurons in the LH and with radial glia neuroprogenitor cells in the hypothalamic neuroepithelium (NEP). RESULTS: Prenatal EtOH strongly stimulated CXCL12 and CXCR4 in LH neurons of embryos and postnatal offspring. This stimulation was significantly stronger in Hcrt/OX than MCH neurons in LH and also occurred in radial glia neuroprogenitor cells dense in the NEP. These effects were sexually dimorphic, consistently stronger in females than males. CONCLUSIONS: While showing prenatal EtOH exposure to have a sexually dimorphic, stimulatory effect on CXCL12 and CXCR4 in LH similar to CCL2 and its receptor, these results reveal their distinct relationship to the peptide neurons, with the former closely related to Hcrt/OX and the latter to MCH, and they link EtOH's actions in LH to a stimulatory effect on neuroprogenitor cells in the NEP.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Quimiocina CXCL12/efeitos dos fármacos , Células Ependimogliais/efeitos dos fármacos , Etanol/farmacologia , Região Hipotalâmica Lateral/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores CXCR4/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Embrião de Mamíferos , Células Ependimogliais/metabolismo , Região Hipotalâmica Lateral/citologia , Região Hipotalâmica Lateral/metabolismo , Hormônios Hipotalâmicos/metabolismo , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Imuno-Histoquímica , Melaninas/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/metabolismo , Orexinas/metabolismo , Hormônios Hipofisários/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptores CXCR4/metabolismoRESUMO
The dipeptidyl peptidase-4 inhibitor saxagliptin is a widely used antihyperglycemic agent in patients with type 2 diabetes. The purpose of this study was to evaluate the effects of saxagliptin on endothelial function in patients with type 2 diabetes. This was a prospective, multicenter, interventional study. A total of 34 patients with type 2 diabetes were enrolled at four university hospitals in Japan. Treatment of patients was initially started with saxagliptin at a dose of 5 mg daily. Assessment of endothelial function assessed by flow-mediated vasodilation (FMD) and measurement of stromal cell-derived factor-1α (SDF-1α) were conducted at baseline and at 3 months after treatment with saxagliptin. A total of 31 patients with type 2 diabetes were included in the analysis. Saxagliptin significantly increased FMD from 3.1 ± 3.1% to 4.2 ± 2.4% (P = 0.032) and significantly decreased total cholesterol from 190 ± 24 mg/dL to 181 ± 25 mg/dL (P = 0.002), glucose from 160 ± 53 mg/dL to 133 ± 25 mg/dL (P < 0.001), HbA1c from 7.5 ± 0.6% to 7.0 ± 0.6% (P < 0.001), urine albumin-to-creatinine ratio from 63.8 ± 134.2 mg/g to 40.9 ± 83.0 mg/g (P = 0.043), and total SDF-1α from 2108 ± 243 pg/mL to 1284 ± 345 pg/mL (P < 0.001). These findings suggest that saxagliptin is effective for improving endothelial function.
Assuntos
Adamantano/análogos & derivados , Quimiocina CXCL12/metabolismo , Dipeptídeos/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Adamantano/metabolismo , Adamantano/uso terapêutico , Idoso , Glicemia/efeitos dos fármacos , Quimiocina CXCL12/efeitos dos fármacos , Diabetes Mellitus Tipo 2/fisiopatologia , Dipeptídeos/metabolismo , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Células Endoteliais/metabolismo , Feminino , Hemoglobinas Glicadas/efeitos dos fármacos , Humanos , Hipoglicemiantes/uso terapêutico , Japão , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Vasodilatação/efeitos dos fármacosRESUMO
INTRODUCTION: A number of studies indicate that endothelin-1 (ET-1) may act as an inflammatory cell "gatekeeper," by regulating the influx of neutrophils following pulmonary injury. To further examine the role of ET-1 in modulating lung inflammation, hamsters were treated with an endothelin receptor antagonist (ERA), HJP272, either 1 h prior to intratracheal instillation of amiodarone (AM) or 24 h afterwards. METHODS: In both cases, the extent of lung injury and repair was determined by (1) histopathological changes; (2) neutrophil content in bronchoalveolar lavage fluid (BALF); (3) lung collagen content; (4) tumor necrosis factor receptor 1 expression by BALF macrophages; (5) BALF levels of (a) transforming growth factor beta-1, (b) stromal cell-derived factor 1 (commonly referred to as CXCL12), and (c) platelet-derived growth factor BB; (6) alveolar septal cell apoptosis. RESULTS: For each parameter, pretreatment with HJP272 resulted in a significant reduction compared to AM alone, whereas post-treatment was either ineffective or produced only a marginally significant change, suggesting that the course of lung inflammation and repair is programmed at a very early stage. CONCLUSIONS: This finding may explain why ERAs are not an effective treatment for human pulmonary fibrosis. Nevertheless, they may be useful as an adjunct to therapeutic regimens involving drugs that have fibrogenic potential.
Assuntos
Amiodarona/toxicidade , Antagonistas dos Receptores de Endotelina/farmacologia , Endotelina-1/antagonistas & inibidores , Hidroxiquinolinas/farmacologia , Pulmão/efeitos dos fármacos , Fibrose Pulmonar/induzido quimicamente , Vasodilatadores/toxicidade , Animais , Apoptose/efeitos dos fármacos , Becaplermina/efeitos dos fármacos , Becaplermina/metabolismo , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Quimiocina CXCL12/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Colágeno/efeitos dos fármacos , Colágeno/metabolismo , Feminino , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Mesocricetus , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Receptores Tipo I de Fatores de Necrose Tumoral/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Crescimento Transformador beta1/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The angiotensin (ANG)-(1-7)/Mas receptor (MasR) pathway activates vascular repair-relevant functions of bone marrow progenitor cells. We tested the effects of ANG-(1-7) on mobilization and vasoreparative functions of progenitor cells that are impaired in diabetes. The study was performed in streptozotocin-induced diabetic (db/db) mice. Diabetes resulted in a decreased number of Lineage-Sca-1+c-Kit+ (LSK) cells in the circulation, which was normalized by ANG-(1-7). Diabetes-induced depletion of LSK cells in the bone marrow was reversed by ANG-(1-7). ρ-Kinase (ROCK) activity was increased specifically in bone marrow LSK cells by ANG-(1-7) in diabetes, and the beneficial effects of ANG-(1-7) were prevented by fasudil. ANG-(1-7) increased Slit3 levels in the bone marrow supernatants, which activated ROCK in LSK cells and sensitized them for stromal-derived factor-1α (SDF)-induced migration. Diabetes prevented the mobilization of LSK cells in response to ischemia and impaired the recovery of blood flow, both of which were reversed by ANG-(1-7) in both models of diabetes. Genetic ablation of MasR prevented ischemia-induced mobilization of LSK cells and impaired blood flow recovery, which was associated with decreased proliferation and migration of LSK cells in response to SDF or vascular endothelial growth factor. These results suggest that MasR is a promising target for the treatment of diabetic bone marrow mobilopathy and vascular disease.
Assuntos
Angiotensina I/farmacologia , Vasos Sanguíneos/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/fisiopatologia , Neovascularização Fisiológica/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Células-Tronco/efeitos dos fármacos , Vasodilatadores/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Vasos Sanguíneos/fisiopatologia , Medula Óssea/efeitos dos fármacos , Medula Óssea/fisiopatologia , Linhagem da Célula , Quimiocina CXCL12/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Isquemia , Masculino , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Receptores Acoplados a Proteínas G/genética , Recuperação de Função Fisiológica/efeitos dos fármacos , Regeneração , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Quinases Associadas a rho/efeitos dos fármacos , Quinases Associadas a rho/metabolismoRESUMO
BACKGROUND: Endothelial progenitor cell (EPC) functions are impaired in the presence of diabetes mellitus. Aliskiren is a direct renin inhibitor, which is expected to modify proangiogenic cells. This study aimed to investigate whether and how aliskiren could improve the function of EPCs from patients with type II diabetes (T2DM). MATERIALS AND METHODS: Endothelial progenitor cells fibronectin adhesion assay, chamber assay and in vitro tube formation assay were used to estimate the degree of EPC adhesion, migration and tube formation abilities. EPC protein and mRNA expressions were evaluated by Western blot and quantitative RT-PCR, respectively. EPC vascular endothelial growth factor (VEGF) and (pro)renin receptor ((P)RR) expression was knocked down by VEGF and (P)RR siRNA. RESULTS: Aliskiren (0·1 or 10 µM) dose-dependently improved functions and increased both VEGF and stromal cell-derived factor-1α (SDF-1α) expression of EPCs from patients with T2DM or EPCs from healthy volunteers and treated with high glucose. Transfection with VEGF siRNA significantly reduced the aliskiren-induced SDF-1α expression. Furthermore, (P)RR siRNA transfection impaired the aliskiren-induced VEGF and SDF-1 expression. CONCLUSIONS: The results show that aliskiren improved EPC function from patients with T2DM in a dose-dependent manner probably via the (P)RR and VEGF/SDF-1α-related mechanisms.
Assuntos
Amidas/farmacologia , Anti-Hipertensivos/farmacologia , Diabetes Mellitus Tipo 2 , Células Progenitoras Endoteliais/efeitos dos fármacos , Fumaratos/farmacologia , RNA Mensageiro/efeitos dos fármacos , Western Blotting , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocina CXCL12/efeitos dos fármacos , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Células Progenitoras Endoteliais/metabolismo , Humanos , Hidralazina/farmacologia , Imidazóis/farmacologia , Técnicas In Vitro , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetrazóis/farmacologia , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
In spite of high complete remission rates in Acute Myeloid Leukemia (AML), little progress has been made in the long-term survival of relapsing AML patients, urging for the development of novel therapies. The CXCR4/SDF-1 axis is a potential therapeutic target in AML to reduce the enhanced survival and proliferation of leukemic cells, with current drug development efforts focusing on antagonists and blocking antibodies. The RNAi technology mediated by siRNA is a promising alternative; however, further development of clinically relevant siRNA carriers is needed since siRNA on its own is an incompetent silencing agent. Here, we report on lipid-substituted polymeric carriers for siRNA delivery to AML cells, specifically targeting CXCR4. Our results demonstrate an effective suppression of CXCR4 protein with the polymeric siRNA delivery in AML THP-1 cells. The suppression of CXCR4 as well as its ligand, SDF-1 (CXCL12), decreased THP-1 cell numbers due to reduced cell proliferation. The reduced proliferation was also observed in the presence of human bone marrow stromal cells (hBMSC), suggesting that our approach would be effective in the protective bone marrow microenvironment. The combination of CXCR4 silencing and cytarabine treatment resulted in more effective cytotoxicity when the cells were co-incubated with hBMSC. We observed a decrease in the toxicity of the lipopolymer/siRNA complexes when THP-1 cells were treated in the presence of hBMSC but this effect did not negatively affect CXCR4 silencing. In addition, siRNA delivery to mononuclear cells derived from AML patients led to significant CXCR4 silencing in 2 out of 5 samples, providing a proof-of-concept for clinical translation. We conclude that decreasing CXCR4 expression via lipopolymer/siRNA complexes is a promising option for AML therapy and could provide an effective alternative to current CXCR4 inhibition strategies.
Assuntos
Quimiocina CXCL12/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/uso terapêutico , Receptores CXCR4/efeitos dos fármacos , Antimetabólitos/administração & dosagem , Antimetabólitos/uso terapêutico , Células da Medula Óssea , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Quimiocina CXCL12/genética , Citarabina/administração & dosagem , Citarabina/uso terapêutico , Sistemas de Liberação de Medicamentos , Inativação Gênica , Humanos , Lipídeos/química , Polímeros , Receptores CXCR4/genética , Células EstromaisRESUMO
The aim of this study was to assess the in vivo effects of the extract of the medicinal mushroom, Lactarius deterrimus, when administered (60 mg/kg, i.p.) daily for four weeks to streptozotocin- (STZ-) induced diabetic rats. Diabetic rats treated with the L. deterrimus extract displayed several improved biochemical parameters in the circulation: reduced hyperglycemia, lower triglyceride concentration and reduced glycated hemoglobin, glycated serum protein, and advanced glycation end product (AGE) levels. This treatment also adjusted the diabetes-induced redox imbalance. Thus, higher activities of the antioxidative enzymes, superoxide dismutase, and catalase in the circulation were accompanied by increased levels of free intracellular thiols and glutathionylated proteins after treatment with the L. deterrimus extract. In addition to a systemic antioxidant effect, the administration of the extract to diabetic rats also had a positive localized effect on pancreatic islets where it decreased AGE formation, and increased the expression of chemokine CXCL12 protein that mediates the restoration of ß-cell population through the activation of the serine/threonine-specific Akt protein kinase prosurvival pathway. As a result, the numbers of proliferating cell nuclear antigen- (PCNA-) and insulin-positive ß-cells were increased. These results show that the ability of the L. deterrimus extract to alleviate oxidative stress and increase ß-cell mass represents a therapeutic potential for diabetes management.
Assuntos
Agaricales , Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Glicemia/metabolismo , Catalase , Quimiocina CXCL12/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Hemoglobinas Glicadas/efeitos dos fármacos , Hemoglobinas Glicadas/metabolismo , Produtos Finais de Glicação Avançada/efeitos dos fármacos , Produtos Finais de Glicação Avançada/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos Wistar , Compostos de Sulfidrila , Superóxido Dismutase/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Triglicerídeos/metabolismoRESUMO
BACKGROUND: Poor marrow cellularity alone cannot explain poor hematopoietic progenitor cell (HPC) mobilization. This study assessed the role of CD8+ T cells in HPC cell mobilization and engraftment. STUDY DESIGN AND METHODS: Mobilization and engraftment were assessed in 192 autologous HPC donors. CD34+, CD4+, and CD8+ T-cell contents in apheresis products were evaluated. Using a chemotaxis assay, we assessed the effect of purified autologous CD8+ T cells from low and high mobilizers on HPC migration from high to low stromal cell-derived factor (SDF-1α) concentration gradients. We also assessed CD8+ T-cell content association with days to neutrophil engraftment. RESULTS: The median number of CD34+ cells/kg was 4.7 × 10(6) . Patients were categorized according to their total CD34+ cell collection quartile distribution into low, moderate, and high mobilizers. We found that HPC products from low mobilizers contained more CD8+ T cells than HPC products from moderate and high mobilizers. Chemotaxis assays showed depletion of CD8+ T cells enhances HPC mobilization independent of SDF-1α concentration. Neutrophil engraftment analysis showed that the higher the CD8+ T-cell content per unit CD34+ cell, the faster the rate of engraftment. CONCLUSION: Our findings suggest CD8+ T cells inhibit HPC mobilization and facilitate homing and engraftment.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Quimiotaxia/imunologia , Sobrevivência de Enxerto/imunologia , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Leucaférese , Adolescente , Adulto , Idoso , Autoenxertos , Linfócitos T CD8-Positivos/metabolismo , Quimiocina CXCL12/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
AIM: We hypothesized that platelet inactivation induced by drugs might interfere with periodontal repair in experimental periodontitis by suppressing the release of biological mediators from platelets at the site of injury. MATERIAL AND METHODS: Sixty rats were randomly assigned to six groups (n = 10) and ligatures were placed around lower first molars of three groups. The other three groups were used as negative controls. Ligatures were removed after 10 days of periodontitis induction and all groups were submitted to treatment with aspirin (Asp) (30 mg/kg), clopidogrel (Clop) (75 mg/kg) or NaCl 0.9% intra-gastrically once daily for 3 days. Periodontal tissue was assessed by the measurement of CXCL12, CXCL4, CCL5 and platelet-derived growth factor (PDGF) by enzyme-linked immunosorbent assay; histomorphometrical analysis of polymorphonuclear (PMN) infiltration, attachment loss, bone loss and osteoclast numbers and quantification of blood vessels by imunnohistochemistry. RESULTS: During periodontal repair and treatment with NaCl 0.9%, CCL5 was decreased and CXCL12 increased when compared with negative control groups. Asp and Clop did not affect CCL5 expression, decreased CXCL12 but only Clop decreased CXCL4 and PDGF content compared with saline-treated animals. Clop increased blood vessel number, reduced PMN count and decreased attachment and bone loss, also decreased osteoclast number in animals submitted or not to periodontal repair. CONCLUSION: Systemic administration of Clop for 3 days improved the repair process associated with experimental periodontal disease, suggesting that it may have therapeutic value under situations where tissues undergo a transition from inflammation to repair.
Assuntos
Periodontite/tratamento farmacológico , Periodonto/efeitos dos fármacos , Inibidores da Agregação Plaquetária/uso terapêutico , Ticlopidina/análogos & derivados , Perda do Osso Alveolar/tratamento farmacológico , Animais , Aspirina/administração & dosagem , Aspirina/uso terapêutico , Contagem de Células , Quimiocina CCL5/efeitos dos fármacos , Quimiocina CXCL12/efeitos dos fármacos , Clopidogrel , Infusões Parenterais , Masculino , Microvasos/efeitos dos fármacos , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Perda da Inserção Periodontal/tratamento farmacológico , Inibidores da Agregação Plaquetária/administração & dosagem , Fator Plaquetário 4/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Cloreto de Sódio , Ticlopidina/administração & dosagem , Ticlopidina/uso terapêuticoRESUMO
INTRODUCTION: Production of chemokines by tissue resident cells is one of the main mechanisms involved in the inflammatory infiltrate formation during inflammation. The specific ability of fibroblasts from different oral tissues such as gingiva, periodontal ligament, and dental pulp from permanent and deciduous teeth in producing the chemokines CCL3 and CXCL12 under stimulation by bacterial products commonly found in endodontic infections was investigated. METHODS: Cultures of fibroblasts from gingiva and periodontal ligament as well as from dental pulp from permanent and deciduous teeth were established by using an explant technique and stimulated with increasing concentrations of Escherichia coli lipopolysaccharide (EcLPS) and Enterococcus faecalis lipoteichoic acid (EfLTA) for 1, 6, and 24 hours. Supernatants were tested for CCL3 and CXCL12 by enzyme-linked immunosorbent assay. RESULTS: In general, CCL3 production was induced by EcLPS in the 4 fibroblast subtypes and by EfLTA in fibroblasts from gingiva and periodontal ligament. Constitutive CXCL12 synthesis decreased in all fibroblast subtypes especially under stimulation with EcLPS. Fibroblast from permanent deciduous teeth was the cell type presenting the most expressive reduction in CXCL12 release by both stimuli. On the basis of computational matching of CXCL12 mRNA with the microRNAs miR-141 and miR-200a, their expression was also investigated. Although detected in the fibroblasts, these molecules remained unaltered by bacterial by-product stimulation. CONCLUSIONS: EcLPS and EfLTA induced the production of CCL3 and unbalanced the synthesis of CXCL12 in a manner dependent on the specific tissue origin of fibroblasts.
Assuntos
Quimiocina CCL3/efeitos dos fármacos , Quimiocina CXCL12/efeitos dos fármacos , Polpa Dentária/citologia , Fibroblastos/efeitos dos fármacos , Gengiva/citologia , Lipopolissacarídeos/farmacologia , Ligamento Periodontal/citologia , Ácidos Teicoicos/farmacologia , Adolescente , Adulto , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Criança , Corantes , Polpa Dentária/efeitos dos fármacos , Enterococcus faecalis , Escherichia coli , Gengiva/efeitos dos fármacos , Humanos , MicroRNAs/análise , Ligamento Periodontal/efeitos dos fármacos , Fenótipo , Sais de Tetrazólio , Tiazóis , Dente Decíduo/citologia , Adulto JovemAssuntos
Células da Medula Óssea/citologia , Terapia Baseada em Transplante de Células e Tecidos , Quimiocina CXCL12/efeitos dos fármacos , Heparina/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Infarto do Miocárdio/terapia , Receptores CXCR4/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Feminino , HumanosRESUMO
Chemokine stromal cell-derived factor-1 (SDF-1) signals via binding to its primary transmembrane receptor, cysteine (C)-X-C chemokine receptor-4 (CXCR4). We previously reported that SDF-1 regulates osteogenic differentiation of mesenchymal stem/progenitor cells (MPCs) induced by bone morphogenetic protein-2 (BMP2). Although BMP2 is also capable of inducing chondrogenic differentiation of MPCs, the involvement of SDF-1 signaling in this function of BMP2 remains unknown. In this study, we aimed to test the role of SDF-1 signaling involved in BMP2-induced chondrogenic differentiation, using ATDC5 chondroprogenitors and mouse bone marrow-derived mesenchymal stromal cells (BMSCs). Our data showed that blocking of the SDF-1/CXCR4 pathway inhibits the differentiation of these cells into the chondrocytic lineages in response to BMP2 stimulation, evidenced by the reduced expression of type II collagen α1 (Col2α1), aggrecan, and type X collagen α1 (Col10α1), markers for chondrogenic differentiation. This effect of blocking SDF-1 signaling on BMP2-chondrogenic differentiation is associated with suppressed Sox9 and Runx2 expression (key transcription factors required for early and late stages of chondrogenic differentiation, respectively) and mediated via inhibiting intracellular Smad and Erk activation. Moreover, we found that addition of exogenous SDF-1 protein synergistically enhances the BMP2-induced chondrogenic differentiation in a dose-dependent manner. Collectively, our results demonstrated a novel role of SDF-1 signaling in regulating BMP2-induced chondrogenic differentiation in vitro. These data provide new insights into molecular mechanisms underlying BMP2-osteo/chondrogenesis, and may lead to the identification of new therapeutic targets and strategies to improve cartilage repair and regeneration in broad orthopaedic situations.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Diferenciação Celular/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Condrogênese/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Quimiocina CXCL12/efeitos dos fármacos , Condrogênese/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores CXCR4/metabolismo , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas Smad/metabolismoRESUMO
Macrophage migration inhibitory factor (MIF), a structurally and functionally unique pleiotropic mediator in inflammation and immune processes, was identified decades ago. There is now strong evidence that MIF promotes revascularization and is involved in wound healing processes. However, its exact role in wound healing is still a matter of debate. A cohort of 33 patients was recruited, including 14 patients with acute and 19 patients with chronic wounds. Both serum and wound fluid samples were collected from each patient, and MIF and cortisol concentrations were determined. To functionally underscore MIF's potential role in wound revascularization, a chemotaxis assay was adapted to test whether and to what extent serum samples and wound fluids of each group promote the chemotactic migration of endothelial progenitor cells (EPCs). MIF serum levels were significantly higher in chronic wound patients than in acute wound patients. Wound exudates of chronic wounds, however, contained a significantly lower concentration of MIF. In chronic wound patients, EPC migration might be delayed, as suggested by in vitro chemotaxis experiments. Despite the overall descriptive nature of this study, we conclude that MIF is correlated with occurrence of chronic wound. The increased MIF levels in the serum of chronic wound patients might be due to MIF's systemic effect of its proinflammatory activities, while its locally decreased levels in chronic wound exudates might be responsible for impaired recruitment of EPCs. Additional prospective data and detailed in vivo models are needed for a more comprehensive understanding of the role of MIF in chronic wound healing.