RESUMO
Cardiovascular complications are included among the systemic effects of tyrosine kinase inhibitor (TKI)-based therapeutic strategies. To test the hypothesis that inhibition of Kit tyrosine kinase that promotes cardiac progenitor cell (CPC) survival and function may be one of the triggering mechanisms of imatinib mesylate (IM)-related cardiovascular effects, the anatomical, structural and ultrastructural changes in the heart of IM-treated rats were evaluated. Cardiac anatomy in IM-exposed rats showed a dose-dependent, restrictive type of remodeling and depressed hemodynamic performance in the absence of remarkable myocardial fibrosis. The effects of IM on rat and human CPCs were also assessed. IM induced rat CPC depletion, reduced growth and increased cell death. Similar effects were observed in CPCs isolated from human hearts. These results extend the notion that cardiovascular side effects are driven by multiple actions of IM. The identification of cellular mechanisms responsible for cardiovascular complications due to TKIs will enable future strategies aimed at preserving concomitantly cardiac integrity and anti-tumor activity of advanced cancer treatment.
Assuntos
Cardiomiopatias/induzido quimicamente , Mesilato de Imatinib/toxicidade , Miocárdio/patologia , Células-Tronco/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Hemodinâmica/efeitos dos fármacos , Humanos , Masculino , Miocárdio/ultraestrutura , RatosRESUMO
c-Kit(pos) cardiac progenitor cells (CPCs) represent a successful approach in healing the infarcted heart and rescuing its mechanical function, but electrophysiological consequences are uncertain. CPC mobilization promoted by hepatocyte growth factor (HGF) and IGF-1 improved electrogenesis in myocardial infarction (MI). We hypothesized that locally delivered CPCs supplemented with HGF + IGF-1 (GFs) can concur in ameliorating electrical stability of the regenerated heart. Adult male Wistar rats (139 rats) with 4-wk-old MI or sham conditions were randomized to receive intramyocardial injection of GFs, CPCs, CPCs + GFs, or vehicle (V). Enhanced green fluorescent protein-tagged CPCs were used for cell tracking. Vulnerability to stress-induced arrhythmia was assessed by telemetry-ECG. Basic cardiac electrophysiological properties were examined by epicardial multiple-lead recording. Hemodynamic function was measured invasively. Hearts were subjected to anatomical, morphometric, immunohistochemical, and molecular biology analyses. Compared with V and at variance with individual CPCs, CPCs + GFs approximately halved arrhythmias in all animals, restoring cardiac anisotropy toward sham values. GFs alone reduced arrhythmias by less than CPCs + GFs, prolonging ventricular refractoriness without affecting conduction velocity. Concomitantly, CPCs + GFs reactivated the expression levels of Connexin-43 and Connexin-40 as well as channel proteins of key depolarizing and repolarizing ion currents differently than sole GFs. Mechanical function and anatomical remodeling were equally improved by all regenerative treatments, thus exhibiting a divergent behavior relative to electrical aspects. Conclusively, we provided evidence of distinctive antiarrhythmic action of locally injected GF-supplemented CPCs, likely attributable to retrieval of Connexin-43, Connexin-40, and Cav1.2 expression, favoring intercellular coupling and spread of excitation in mended heart.
Assuntos
Antiarrítmicos/uso terapêutico , Fator de Crescimento de Hepatócito/uso terapêutico , Fator de Crescimento Insulin-Like I/uso terapêutico , Infarto do Miocárdio/terapia , Células-Tronco , Animais , Conexina 43/metabolismo , Masculino , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Ratos , Ratos WistarRESUMO
Secretion of proteins and neurotransmitters from large dense core vesicles (LDCVs) is a highly regulated process. Adrenal LDCV formation involves the granin proteins chromogranin A (CgA) and chromogranin B (CgB); CgA- and CgB-derived peptides regulate catecholamine levels and blood pressure. We investigated function of the granin VGF (nonacronymic) in LDCV formation and the regulation of catecholamine levels and blood pressure. Expression of exogenous VGF in nonendocrine NIH 3T3 fibroblasts resulted in the formation of LDCV-like structures and depolarization-induced VGF secretion. Analysis of germline VGF-knockout mouse adrenal medulla revealed decreased LDCV size in noradrenergic chromaffin cells, increased adrenal norepinephrine and epinephrine content and circulating plasma epinephrine, and decreased adrenal CgB. These neurochemical changes in VGF-knockout mice were associated with hypertension. Germline knock-in of human VGF1-615 into the mouse Vgf locus rescued the hypertensive knockout phenotype, while knock-in of a truncated human VGF1-524 that lacks several C-terminal peptides, including TLQP-21, resulted in a small but significant increase in systolic blood pressure compared to hVGF1-615 mice. Finally, acute and chronic administration of the VGF-derived peptide TLQP-21 to rodents decreased blood pressure. Our studies establish a role for VGF in adrenal LDCV formation and the regulation of catecholamine levels and blood pressure.
Assuntos
Pressão Sanguínea , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Vesículas Secretórias/metabolismo , Medula Suprarrenal/metabolismo , Angiotensina Amida/sangue , Animais , Células Cromafins/metabolismo , Cromogranina A/metabolismo , Citoplasma/metabolismo , Epinefrina/sangue , Técnicas de Introdução de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Fatores de Crescimento Neural , Neurotransmissores/metabolismo , Fragmentos de Peptídeos/metabolismo , FenótipoRESUMO
AIM: To assess the role of Notch activation in predicting bevacizumab efficacy in colorectal cancer (CRC). MATERIALS & METHODS: Notch activation was evaluated by immunohistochemistry (IHC) on 65 CRC enrolled within randomized clinical trials assessing first-line bevacizumab-based chemotherapy and on 21 CRC treated with chemotherapy alone. RESULTS: Strong Notch (IHC 3+) activation was negatively associated with response (18 vs 62% in low Notch cases [IHC 0, 1, 2+]; p = 0.016), progression-free survival (4.9 vs 12.1 months; p = 0.002) and overall survival (19.3 vs 30.4 months; p = 0.039). No correlation was found between Notch activation and clinical outcome in CRC treated with chemotherapy alone. CONCLUSION: A potential role of Notch activation in the antitumor activity of bevacizumab could be hypothesized.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Receptores Notch/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Idoso de 80 Anos ou mais , Bevacizumab/administração & dosagem , Biomarcadores , Proteínas de Ligação ao Cálcio , Estudos de Casos e Controles , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Retratamento , Resultado do TratamentoRESUMO
The increasing population of cancer survivors faces considerable morbidity and mortality due to late effects of the antineoplastic therapy. Cardiotoxicity is a major limiting factor of therapy with doxorubicin (DOXO), the most effective anthracycline, and is characterized by a dilated cardiomyopathy that can develop even years after treatment. Studies in animals have proposed the cardiac progenitor cells (CPCs) as the cellular target responsible for DOXO-induced cardiomyopathy but the relevance of these observations to clinical settings is unknown. In this study, the analysis of the DOXO-induced cardiomyopathic human hearts showed that the majority of human CPCs (hCPCs) was senescent. In isolated hCPCs, DOXO triggered DNA damage response leading to apoptosis early after exposure, and telomere shortening and senescence at later time interval. Functional properties of hCPCs, such as migration and differentiation, were also negatively affected. Importantly, the differentiated progeny of DOXO-treated hCPCs prematurely expressed the senescence marker p16(INK4a). In conclusion, DOXO exposure severely affects the population of hCPCs and permanently impairs their function. Premature senescence of hCPCs and their progeny can be responsible for the decline in the regenerative capacity of the heart and may represent the cellular basis of DOXO-induced cardiomyopathy in humans.
Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Cardiomiopatia Dilatada/induzido quimicamente , Senescência Celular/efeitos dos fármacos , Doxorrubicina/efeitos adversos , Mioblastos Cardíacos/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Adulto , Antibióticos Antineoplásicos/uso terapêutico , Western Blotting , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Morte Celular/efeitos dos fármacos , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Doxorrubicina/uso terapêutico , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-kit/metabolismo , Homeostase do Telômero , beta-Galactosidase/metabolismoRESUMO
OBJECTIVE: Despite a well-documented association between stress and depression with cardiac morbidity and mortality, there is no satisfactory explanation for the mechanisms linking affective and cardiac disorders. This study investigated cardiac electrophysiological properties in an animal model of depression. METHODS: Depression-relevant physiological and behavioral parameters were measured in adult male wild-type rats during and after a period of intermittent social defeat stress (n = 12) or empty cage exposure (control, n = 11). Nine days after the last defeat/empty cage exposure, high-definition epicardial mapping was performed under anesthesia. RESULTS: Stressed animals versus controls displayed a larger reduction in the circadian amplitude of heart rate (-32% [3%] versus -13 [2%]; p = .001) and body temperature (-33% [4%] versus -5% [2%]; p = .001) rhythms, had smaller body weight gain (+11% [1%] versus +17% [1%]; p < .001), and showed a larger reduction in sucrose solution intake (-19% [6%] versus -7% [4%]; p = .006). Epicardial mapping analysis revealed a decrease in the transversal conduction velocity of the wavefront (0.23 [0.0] versus 0.27 [0.1] m/s; p = .02) and a shortening of the effective refractory period (86.8 [2.1] versus 95.9 [3.0] milliseconds; p = .01) in stressed animals. Upon killing, moderate left ventricular fibrosis was observed in the stressed group. CONCLUSIONS: Intermittent social stress procedure is associated with depression-like symptoms and altered myocardial electrical stability in a potentially proarrhythmic manner. In particular, reduced myocardial refractoriness and impaired conduction, which are considered major determinants of arrhythmogenesis, represent possible mechanisms underlying cardiac vulnerability.
Assuntos
Arritmias Cardíacas/fisiopatologia , Depressão/fisiopatologia , Coração/fisiopatologia , Miocárdio/patologia , Remodelação Ventricular/fisiologia , Animais , Arritmias Cardíacas/psicologia , Comportamento Animal/fisiologia , Temperatura Corporal/fisiologia , Peso Corporal/fisiologia , Transtornos Cronobiológicos/fisiopatologia , Transtornos Cronobiológicos/psicologia , Ritmo Circadiano/fisiologia , Depressão/patologia , Modelos Animais de Doenças , Eletrocardiografia , Mapeamento Epicárdico , Fibrose , Frequência Cardíaca/fisiologia , Masculino , Ratos , Restrição Física , Estresse Psicológico/patologia , Estresse Psicológico/fisiopatologiaRESUMO
The peptides encoded by the VGF gene are gaining biomedical interest and are increasingly being scrutinized as biomarkers for human disease. An endocrine/neuromodulatory role for VGF peptides has been suggested but never demonstrated. Furthermore, no study has demonstrated so far the existence of a receptor-mediated mechanism for any VGF peptide. In the present study, we provide a comprehensive in vitro, ex vivo and in vivo identification of a novel pro-lipolytic pathway mediated by the TLQP-21 peptide. We show for the first time that VGF-immunoreactivity is present within sympathetic fibres in the WAT (white adipose tissue) but not in the adipocytes. Furthermore, we identified a saturable receptor-binding activity for the TLQP-21 peptide. The maximum binding capacity for TLQP-21 was higher in the WAT as compared with other tissues, and selectively up-regulated in the adipose tissue of obese mice. TLQP-21 increases lipolysis in murine adipocytes via a mechanism encompassing the activation of noradrenaline/ß-adrenergic receptors pathways and dose-dependently decreases adipocytes diameters in two models of obesity. In conclusion, we demonstrated a novel and previously uncharacterized peripheral lipolytic pathway encompassing the VGF peptide TLQP-21. Targeting the sympathetic nerve-adipocytes interaction might prove to be a novel approach for the treatment of obesity-associated metabolic complications.
Assuntos
Neuropeptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Composição Corporal , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/metabolismo , Masculino , Camundongos , Células NIH 3T3 , Fatores de Crescimento Neural , Obesidade/induzido quimicamente , Obesidade/metabolismo , Ligação Proteica , Transporte Proteico , Receptores de Superfície CelularRESUMO
BACKGROUND: The epidermal growth factor receptor (EGFR) is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. RESULTS: In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. CONCLUSION: Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Análise de Variância , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/química , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cetuximab , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estabilidade Proteica/efeitos dos fármacos , Quinazolinas/administração & dosagem , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Trastuzumab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RATIONALE: Nerve growth factor (NGF) promotes angiogenesis and cardiomyocyte survival, which are both desirable for postinfarction myocardial healing. Nonetheless, the NGF potential for cardiac repair has never been investigated. OBJECTIVE: To define expression and localization of NGF and its high-affinity receptor TrkA (tropomyosin-related receptor A) in the human infarcted heart and to investigate the cardiac roles of both endogenous and engineered NGF using a mouse model of myocardial infarction (MI). METHODS AND RESULTS: Immunostaining for NGF and TrkA was performed on heart samples from humans deceased of MI or unrelated pathologies. To study the post-MI functions of endogenous NGF, a NGF-neutralizing antibody (Ab-NGF) or nonimmune IgG (control) was given to MI mice. To investigate the NGF therapeutic potential, human NGF gene or control (empty vector) was delivered to the murine periinfarct myocardium. Results indicate that NGF is present in the infarcted human heart. Both cardiomyocytes and endothelial cells (ECs) possess TrkA, which suggests NGF cardiovascular actions in humans. In MI mice, Ab-NGF abrogated native reparative angiogenesis, increased EC and cardiomyocyte apoptosis and worsened cardiac function. Conversely, NGF gene transfer ameliorated EC and cardiomyocyte survival, promoted neovascularization and improved myocardial blood flow and cardiac function. The prosurvival/proangiogenic Akt/Foxo pathway mediated the therapeutic benefits of NGF transfer. Moreover, NGF overexpression increased stem cell factor (the c-kit receptor ligand) expression, which translated in higher myocardial abundance of c-kit(pos) progenitor cells in NGF-engineered hearts. CONCLUSIONS: NGF elicits pleiotropic beneficial actions in the post-MI heart. NGF should be considered as a candidate for therapeutic cardiac regeneration.
Assuntos
Células Endoteliais/metabolismo , Terapia Genética , Infarto do Miocárdio/terapia , Miocárdio/metabolismo , Fator de Crescimento Neural/biossíntese , Regeneração , Idoso , Animais , Apoptose , Autopsia , Estudos de Casos e Controles , Células Cultivadas , Circulação Coronária , Modelos Animais de Doenças , Células Endoteliais/patologia , Feminino , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Neovascularização Fisiológica , Fator de Crescimento Neural/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Receptor trkA/metabolismo , Proteínas Recombinantes/biossíntese , Regeneração/genética , Transdução de Sinais , Fator de Células-Tronco/metabolismo , Células-Tronco/metabolismo , Células-Tronco/patologia , Fatores de Tempo , Transfecção , Função Ventricular EsquerdaRESUMO
The mechanisms underlying the success of propranolol in the treatment of infantile hemangioma (IH) remain elusive and do not fully explain the rapid regression of hemangiomatous lesions following drug administration. As autophagy is critically implicated in vascular homeostasis, we determined whether ß-blockers trigger the autophagic flux on infantile hemangioma-derived endothelial cells (Hem-ECs) in vitro. MATERIAL AND METHODS: Fresh tissue specimens, surgically removed for therapeutic purpose to seven children affected by proliferative IH, were subjected to enzymatic digestion. Cells were sorted with anti-human CD31 immunolabeled magnetic microbeads. Following phenotypic characterization, expanded Hem-ECs, at P2 to P6, were exposed to different concentrations (50 µM to 150 µM) of propranolol, atenolol or metoprolol alone and in combination with the autophagy inhibitor Bafilomycin A1. Rapamycin, a potent inducer of autophagy, was also used as control. Autophagy was assessed by Lysotracker Red staining, western blot analysis of LC3BII/LC3BI and p62, and morphologically by transmission electron microscopy. RESULTS: Hem-ECs treated with either propranolol, atenolol or metoprolol displayed positive LysoTracker Red staining. Increased LC3BII/LC3BI ratio, as well as p62 modulation, were documented in ß-blockers treated Hem-ECs. Abundant autophagic vacuoles and multilamellar bodies characterized the cytoplasmic ultrastructural features of autophagy in cultured Hem-ECs exposed in vitro to ß-blocking agents. Importantly, similar biochemical and morphologic evidence of autophagy were observed following rapamycin while Bafilomycin A1 significantly prevented the autophagic flux promoted by ß-blockers in Hem-ECs. CONCLUSION: Our data suggest that autophagy may be ascribed among the mechanisms of action of ß-blockers suggesting new mechanistic insights on the potential therapeutic application of this class of drugs in pathologic conditions involving uncontrolled angiogenesis.
Assuntos
Hemangioma , Propranolol , Antagonistas Adrenérgicos beta/farmacologia , Aminas , Atenolol/farmacologia , Atenolol/uso terapêutico , Autofagia , Proliferação de Células , Criança , Células Endoteliais , Hemangioma/patologia , Humanos , Macrolídeos , Metoprolol/uso terapêutico , Propranolol/farmacologia , Propranolol/uso terapêutico , Sirolimo/farmacologiaRESUMO
In addition to long-term regulation of blood pressure (BP), in the kidney resides the initial trigger for hypertension development due to an altered capacity to excrete sodium and water. Betaine is one of the major organic osmolytes, and its betaine/gamma-aminobutyric acid transporter (BGT-1) expression in the renal medulla relates to interstitial tonicity and urinary osmolality and volume. This study investigated altered water and sodium balance as well as changes in antidiuretic hormone (ADH) activity in female spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats from their 3-5 weeks of age (prehypertensive phase) to SHR's 28-30 weeks of age (established hypertension-organ damage). Young prehypertensive SHRs showed a reduced daily urine output, an elevated urine osmolarity, and higher immunostaining of tubule BGT-1, alpha-1-Na-K ATPase in the outer medulla vs. age-matched WKY. ADH circulating levels were not different between young prehypertensive SHR and WKY, but the urine aquaporin2 (AQP2)/creatinine ratio and labeling of AQP2 in the collecting duct were increased. At 28-30 weeks, hypertensive SHR with moderate renal failure did not show any difference in urinary osmolarity, urine AQP2/creatinine ratio, tubule BGT-1, and alpha-1-Na-K ATPase as compared with WKY. These results suggest an increased sensitivity to ADH in prehypertensive female SHR. On this basis, a second series of experiments were set to study the role of ADH V1 and V2 receptors in the development of hypertension, and a group of female prehypertensive SHRs were treated from the 25th to 49th day of age with either V1 (OPC21268) or V2 (OPC 41061) receptor antagonists to evaluate the BP time course. OPC 41061-treated SHRs had a delayed development of hypertension for 5 weeks without effect in OPC 21268-treated SHRs. In prehypertensive female SHR, an increased renal ADH sensitivity is crucial for the development of hypertension by favoring a positive water balance. Early treatment with selective V2 antagonism delays future hypertension development in young SHRs.
RESUMO
We evaluated the healing potential of human fetal aorta-derived CD133(+) progenitor cells and their conditioned medium (CD133(+) CCM) in a new model of ischemic diabetic ulcer. Streptozotocin-induced diabetic mice underwent bilateral limb ischemia and wounding. One wound was covered with collagen containing 2x10(4) CD133(+) or CD133(-) cells or vehicle. The contralateral wound, covered with only collagen, served as control. Fetal CD133(+) cells expressed high levels of wingless (Wnt) genes, which were downregulated following differentiation into CD133(-) cells along with upregulation of Wnt antagonists secreted frizzled-related protein (sFRP)-1, -3, and -4. CD133(+) cells accelerated wound closure as compared with CD133(-) or vehicle and promoted angiogenesis through stimulation of endothelial cell proliferation, migration, and survival by paracrine effects. CD133(+) cells secreted high levels of vascular endothelial growth factor (VEGF)-A and interleukin (IL)-8. Consistently, CD133(+) CCM accelerated wound closure and reparative angiogenesis, with this action abrogated by co-administering the Wnt antagonist sFRP-1 or neutralizing antibodies against VEGF-A or IL-8. In vitro, these effects were recapitulated following exposure of high-glucose-primed human umbilical vein endothelial cells to CD133(+) CCM, resulting in stimulation of migration, angiogenesis-like network formation and induction of Wnt expression. The promigratory and proangiogenic effect of CD133(+) CCM was blunted by sFRP-1, as well as antibodies against VEGF-A or IL-8. CD133(+) cells stimulate wound healing by paracrine mechanisms that activate Wnt signaling pathway in recipients. These preclinical findings open new perspectives for the cure of diabetic ulcers.
Assuntos
Diabetes Mellitus Experimental/complicações , Pé Diabético/cirurgia , Células-Tronco Fetais/transplante , Isquemia/complicações , Extremidade Inferior/irrigação sanguínea , Neovascularização Fisiológica , Transplante de Células-Tronco , Proteínas Wnt/metabolismo , Cicatrização , Antígeno AC133 , Animais , Antígenos CD/análise , Aorta/embriologia , Diferenciação Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Experimental/cirurgia , Pé Diabético/etiologia , Pé Diabético/metabolismo , Pé Diabético/fisiopatologia , Células-Tronco Fetais/imunologia , Células-Tronco Fetais/metabolismo , Glicoproteínas/análise , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interleucina-8/metabolismo , Isquemia/metabolismo , Isquemia/fisiopatologia , Isquemia/cirurgia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Comunicação Parácrina , Peptídeos/análise , Transdução de Sinais , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: The impact of diabetes on the bone marrow (BM) microenvironment was not adequately explored. We investigated whether diabetes induces microvascular remodeling with negative consequence for BM homeostasis. METHODS AND RESULTS: We found profound structural alterations in BM from mice with type 1 diabetes with depletion of the hematopoietic component and fatty degeneration. Blood flow (fluorescent microspheres) and microvascular density (immunohistochemistry) were remarkably reduced. Flow cytometry verified the depletion of MECA-32(+) endothelial cells. Cultured endothelial cells from BM of diabetic mice showed higher levels of oxidative stress, increased activity of the senescence marker beta-galactosidase, reduced migratory and network-formation capacities, and increased permeability and adhesiveness to BM mononuclear cells. Flow cytometry analysis of lineage(-) c-Kit(+) Sca-1(+) cell distribution along an in vivo Hoechst-33342 dye perfusion gradient documented that diabetes depletes lineage(-) c-Kit(+) Sca-1(+) cells predominantly in the low-perfused part of the marrow. Cell depletion was associated to increased oxidative stress, DNA damage, and activation of apoptosis. Boosting the antioxidative pentose phosphate pathway by benfotiamine supplementation prevented microangiopathy, hypoperfusion, and lineage(-) c-Kit(+) Sca-1(+) cell depletion. CONCLUSIONS: We provide novel evidence for the presence of microangiopathy impinging on the integrity of diabetic BM. These discoveries offer the framework for mechanistic solutions of BM dysfunction in diabetes.
Assuntos
Medula Óssea/irrigação sanguínea , Medula Óssea/fisiopatologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/fisiopatologia , Angiopatias Diabéticas/fisiopatologia , Animais , Antígenos Ly/metabolismo , Medula Óssea/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Homeostase/fisiologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos , Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fluxo Sanguíneo Regional/fisiologia , EstreptozocinaRESUMO
Background: Pheochromocytoma (PHEO) clinical manifestations generally mirror excessive catecholamines secretion; rarely the clinical picture may reflect secretion of other hormones. Watery diarrhea, hypokalemia and achlorhydria (WDHA) is a rare syndrome related to excessive secretion of vasoactive intestinal peptide (VIP). Clinical Case: A 73-year-old hypotensive man affected by adrenal PHEO presented with weight loss and watery diarrhea associated with hypokalemia, hyperchloremic metabolic acidosis (anion gap 15 mmol/l) and a negative urinary anion gap. Abdominal computed tomography scan showed a right adrenal PHEO, 8.1 cm in maximum diameter, with tracer uptake on 68GaDOTA-octreotate positron emission tomography. Metastasis in lumbar region and lung were present. Both chromogranin A and VIP levels were high (more than10 times the normal value) with slightly elevated urine normetanephrine and metanephrine excretion. Right adrenalectomy was performed and a somatostatin analogue therapy with lanreotide started. Immunostaining showed chromogranin A and VIP co-expression, with weak somatostatin-receptor-2A positivity. In two months, patient clinical conditions deteriorated with severe WDHA and multiple liver and lung metastasis. Metabolic acidosis and hypokalemia worsened, leading to hemodynamic shock and exitus. Conclusions: A rare case of WDHA syndrome caused by malignant VIP-secreting PHEO was diagnosed. High levels of circulating VIP were responsible of the rapidly evolving clinical picture with massive dehydration and weight loss along with severe hyperchloremic metabolic acidosis and hypokalemia due to the profuse untreatable diarrhea. The rescue treatment with lanreotide was unsuccessful because of the paucity of somatostatin-receptor-2A on VIP-secreting PHEO chromaffin cells.
Assuntos
Acidose/diagnóstico , Diarreia/diagnóstico , Hipopotassemia/diagnóstico , Feocromocitoma/fisiopatologia , Peptídeo Intestinal Vasoativo/química , Acidose/complicações , Neoplasias das Glândulas Suprarrenais/complicações , Neoplasias das Glândulas Suprarrenais/diagnóstico , Adrenalectomia , Idoso , Células Cromafins/citologia , Diarreia/complicações , Humanos , Hipopotassemia/complicações , Masculino , Peptídeos Cíclicos/uso terapêutico , Neoplasias do Sistema Nervoso Periférico , Cintilografia , Receptores de Somatostatina/uso terapêutico , Somatostatina/análogos & derivados , Somatostatina/uso terapêutico , Síndrome , Tomografia Computadorizada por Raios X , Redução de PesoRESUMO
The use of injectable scaffolds to repair the infarcted heart is receiving great interest. Thermosensitive polymers, in situ polymerization, in situ cross-linking, and self-assembling peptides are the most investigated approaches to obtain injectability.Aim of the present work was the preparation and characterization of a novel bioactive scaffold, in form of injectable microspheres, for cardiac repair. Gellan/gelatin microspheres were prepared by a water-in-oil emulsion and loaded by adsorption with Insulin-like growth factor 1 to promote tissue regeneration. Obtained microspheres underwent morphological, physicochemical and biological characterization, including cell culture tests in static and dynamic conditions and in vivo tests. Morphological analysis of the microspheres showed a spherical shape, a microporous surface and an average diameter of 66 ± 17µm (under dry conditions) and 123 ± 24 µm (under wet conditions). Chemical Imaging analysis pointed out a homogeneous distribution of gellan, gelatin and Insulin-like growth factor-1 within the microsphere matrix. In vitro cell culture tests showed that the microspheres promoted rat cardiac progenitor cells adhesion, and cluster formation. After dynamic suspension culture within an impeller-free bioreactor, cells still adhered to microspheres, spreading their cytoplasm over microsphere surface. Intramyocardial administration of microspheres in a cryoinjury rat model attenuated chamber dilatation, myocardial damage and fibrosis and improved cell homing.Overall, the findings of this study confirm that the produced microspheres display morphological, physicochemical, functional and biological properties potentially adequate for future applications as injectable scaffold for cardiac tissue engineering.
Assuntos
Coração/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/administração & dosagem , Microesferas , Miocárdio/patologia , Alicerces Teciduais , Animais , Materiais Biocompatíveis , Reatores Biológicos , Adesão Celular , Meios de Cultura , Injeções , Insulina/metabolismo , Cinética , Masculino , Microfluídica , Microscopia Eletrônica de Varredura , Infarto do Miocárdio/terapia , Polímeros/química , Ratos , Ratos Wistar , Regeneração , Células-Tronco/citologia , Engenharia Tecidual/métodosRESUMO
Diabetes impairs endothelial function and reparative neovascularization. The p75 receptor of neurotrophins (p75(NTR)), which is scarcely present in healthy endothelial cells (ECs), becomes strongly expressed by capillary ECs after induction of peripheral ischemia in type-1 diabetic mice. Here, we show that gene transfer-induced p75(NTR) expression impairs the survival, proliferation, migration, and adhesion capacities of cultured ECs and endothelial progenitor cells (EPCs) and inhibits angiogenesis in vitro. Moreover, intramuscular p75(NTR) gene delivery impairs neovascularization and blood flow recovery in a mouse model of limb ischemia. These disturbed functions are associated with suppression of signaling mechanisms implicated in EC survival and angiogenesis. In fact, p75(NTR) depresses the VEGF-A/Akt/eNOS/NO pathway and additionally reduces the mRNA levels of ITGB1 [beta (1) integrin], BIRC5 (survivin), PTTG1 (securin) and VEZF1. Diabetic mice, which typically show impaired postischemic muscular neovascularization and blood perfusion recovery, have these defects corrected by intramuscular gene transfer of a dominant negative mutant form of p75(NTR). Collectively, our data newly demonstrate the antiangiogenic action of p75(NTR) and open new avenues for the therapeutic use of p75(NTR) inhibition to combat diabetes-induced microvascular liabilities.
Assuntos
Apoptose/fisiologia , Angiopatias Diabéticas/metabolismo , Endotélio Vascular/fisiologia , Isquemia/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica/fisiologia , Receptor de Fator de Crescimento Neural/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Modelos Animais de Doenças , Endotélio Vascular/patologia , Humanos , Isquemia/etiologia , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Fator de Crescimento Neural/genética , Transdução de Sinais/fisiologia , Estreptozocina , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
In recent years, there has been an increasing interest toward the covalent binding of bioactive peptides from extracellular matrix proteins on scaffolds as a promising functionalization strategy in the development of biomimetic matrices for tissue engineering. A totally new approach for scaffold functionalization with peptides is based on Molecular Imprinting technology. In this work, imprinted particles with recognition properties toward laminin and fibronectin bioactive moieties were synthetized and used for the functionalization of biomimetic sponges, which were based on a blend of alginate, gelatin, and elastin. Functionalized sponges underwent a complete morphological, physicochemical, mechanical, functional, and biological characterization. Micrographs of functionalized sponges showed a highly porous structure and a quite homogeneous distribution of imprinted particles on their surface. Infrared and thermal analyses pointed out the presence of interactions between blend components. Biodegradation and mechanical properties appeared adequate for the aimed application. The results of recognition tests showed that the deposition on sponges did not alter the specific recognition and binding behavior of imprinted particles. In vitro biological characterization with cardiac progenitor cells showed that early cell adherence was promoted. In vivo analysis showed that developed scaffolds improved cardiac progenitor cell adhesion and differentiation toward myocardial phenotypes.
RESUMO
BACKGROUND: Mutations of genes related to Krebs cycle enzymes, kinases or to pseudohypoxic signaling pathways, including Von-Hippel-Lindau (VHL) and transmembrane-protein-127 predispose to pheochromocytoma and paraganglioma development. Homozygous loss of function mutation of VHL (VHL 598C>T) gene can associate with polycythemia because of an altered hypoxia sensing. PATIENT: A 19-year-old normotensive man presented with headache, fatigue associated with severe erythrocytosis (hematocrit 76%), high hemoglobin (25.3âg/dl) in normoxic condition. Bone marrow biopsy showed marked hyperplasia of erythroid series. The Janus kinase 2 (V617F) mutation was absent. Abdominal computed tomography scan showed a 8-mm left adrenal pheochromocytoma with tracer uptake on GaDOTA-octreotate PET. Twenty-four-hour urinary metanephrine excretion was slightly increased, while normetanephrine, 3-methoxytyramine were normal. Adrenal veins sampling showed high left-side erythropoietin secretion. RESULTS: Next-generation sequencing genetic analysis evidenced two concurrent heterozygous mutation of VHL598C>T and of transmembrane-protein-127 c.268G>A. Left side adrenalectomy improved symptoms, erythrocytosis, hemoglobin, and erythropoietin circulating levels. Adrenal histologic sections showed a pheochromocytoma with extensive immunostaining for erythropoietin, but also coexpression of chromogranin A, a marker of chromaffin tissue. CONCLUSION: Congenital polycythemia was clinically diagnosed, mimicking Chuvash polycythemia. Chuvash polycythemia is an autosomal recessive disorder that usually harbors a homozygous mutation of VHL598C>T but not predispose to pheochromocytoma development; in contrast our patient showed for the first time that the concurrent heterozygous VHL and TMEM mutations, resulted in a clinical phenotype of a normotensive patient with polycythemia due to erythropoietin-secreting pheochromocytoma that improved after adrenalectomy.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Eritropoetina/metabolismo , Heterozigoto , Mutação , Feocromocitoma/genética , Policitemia/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Neoplasias das Glândulas Suprarrenais/complicações , Genótipo , Humanos , Masculino , Fenótipo , Feocromocitoma/complicações , Policitemia/complicações , Adulto JovemRESUMO
Prenatal environment exerts profound influences on the development of an organism and stressful events during pregnancy can bring about long-term physiological/behavioral alterations in the offspring. Epidemiological evidence points to a relationship between intrauterine growth restriction (IUGR), body weight at birth, and adult cardiovascular disease. Experimental research employed different models of IUGR, including altered maternal nutrition, exposure to elevated glucocorticoids, and reduced placental perfusion, all of which can program, when acting during sensitive temporal windows of foetal life, alterations in cardiovascular regulation and stress sensitivity. Original data are presented indicating that prenatal psychological stress (intermittent restraint) does not induce in the rat adult offspring changes of plasma corticosterone levels, cardiac autonomic modulation, and circadian rhythmicity of heart rate (HR), body temperature (T) and physical activity (Act) at rest. However, prenatally stressed rats--when further stimulated in adulthood--exhibit prolonged adrenocortical stress responsivity, disturbed circadian rhythmicity of HR, T, and Act, and increased adrenal weight. This evidence supports the idea that prenatal stress per se does not change dramatically a given structure or function, but it affects resilience and renders the animal more susceptible to pathophysiological outcomes when further insults occur during adulthood.