RESUMO
BACKGROUND: Stem cells are used as an alternative treatment option for patients with congenital heart disease (CHD) due to their regenerative potential, but they are subject to low retention rate in the injured myocardium. Also, the diseased microenvironment in the injured myocardium may not provide healthy cues for optimal stem cell function. OBJECTIVE: In this study, we prepared a novel human-derived cardiac scaffold to improve the functional behaviors of stem cells. METHODS: Decellularized extracellular matrix (ECM) scaffolds were fabricated by removing cells of human-derived cardiac appendage tissues. Then, bone marrow c-kit+ progenitor cells from patients with congenital heart disease were seeded on the cardiac ECM scaffolds. Cell adhesion, survival, proliferation and cardiac differentiation on human cardiac decellularized ECM scaffold were evaluated in vitro. Label-free mass spectrometry was applied to analyze cardiac ECM proteins regulating cell behaviors. RESULTS: It was shown that cardiac ECM scaffolds promoted stem cell adhesion and proliferation. Importantly, bone marrow c-kit+ progenitor cells cultured on cardiac ECM scaffold for 14 days differentiated into cardiomyocyte-like cells without supplement with any inducible factors, as confirmed by the increased protein level of Gata4 and upregulated gene levels of Gata4, Nkx2.5, and cTnT. Proteomic analysis showed the proteins in cardiac ECM functioned in multiple biological activities, including regulation of cell proliferation, regulation of cell differentiation, and cardiovascular system development. CONCLUSION: The human-derived cardiac scaffold constructed in this study may help repair the damaged myocardium and hold great potential for tissue engineering application in pediatric patients with CHD.
Assuntos
Cardiopatias Congênitas , Células-Tronco Mesenquimais , Células da Medula Óssea , Criança , Matriz Extracelular Descelularizada , Matriz Extracelular , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/terapia , Humanos , Proteômica , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
PURPOSE: Oestrogen receptor ß is believed to exert a cardioprotective effect against ischaemic injury. Nonetheless, the mechanism underlying its protective action remains to be fully elucidated. Recently, increased attention has been focused on Notch1 signalling for ameliorating cardiac ischaemic injury. Here, we hypothesised that oestrogen receptor ß activation attenuates myocardial infarction (MI)-induced cardiac damage by modulating the Notch1 signalling pathway. METHODS: Male C57BL/6 mice were used to establish an MI model through the ligation of the anterior descending branch of the left coronary artery. Two chemical drugs, 2,3-Bis(4-hydroxyphenyl)-propionitrile (DPN) and N-[N-(3,5-difluorophenacetyl)-l-alanyl]-s-phenylglycine t-butyl ester (DAPT), a specific inhibitor of Notch1 signalling) were administered via intraperitoneal injection to change oestrogen receptor ß and Notch1 activities. Immunohistochemistry, western blot analysis, enzyme-linked immunosorbent assay (Elisa) assessment and echocardiography were used in this study to analyse cardiac oxidative stress, apoptosis, infraction volume, fibrosis and cardiac function. RESULTS: DPN-mediated oestrogen receptor ß activation effectively protected cardiomyocytes from MI-induced oxidative damage and apoptosis. Furthermore, oestrogen receptor ß activation reduced the infarct size and lowered the levels of myocardial enzymes in the serum, thereby leading to greater overall cardiac function improvement. Ischaemic injury-induced myocardial fibrosis was attenuated by oestrogen receptor ß activation. Nevertheless, all of these cardioprotective effects of oestrogen receptor ß activation were almost abrogated by DAPT administration, i.e. DAPT attenuated the anti-oxidative and anti-apoptotic effects and the decrease in infarct and fibrotic areas and reversed cardiac functional recovery. The levels of phospho-phosphatidylinositol-3-kinase (PI3K) and phospho-protein kinase B (Akt) were increased after DPN administration, and this change was reversed after DAPT was administered. CONCLUSIONS: All of these new findings indicate that oestrogen receptor ß activation is effective in ameliorating MI-induced cardiac dysfunction by enhancing Notch1 signalling and that PI3K/Akt signalling is the downstream mediator.
Assuntos
Receptor beta de Estrogênio/agonistas , Estrogênios/farmacologia , Infarto do Miocárdio/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Nitrilas/farmacologia , Receptor Notch1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Receptor beta de Estrogênio/metabolismo , Fibrose , Masculino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
Bone marrow-derived stem/progenitor cells have been utilized for cardiac or vascular repair after ischemic injury, but they are subject to apoptosis and immune rejection in the ischemic site. Multiple scaffolds were used as delivery tools to transplant stem/progenitor cells; however, these scaffolds did not show intrinsically antiapoptotic or anti-inflammatory properties. Decellularized aortic scaffolds that facilitate cell delivery and tissue repair were prepared by removing cells of patient-derived aortic tissues. Scanning electron microscopy (SEM) showed cells attached well to the scaffold after culturing for 5 days. Live/dead staining showed most seeded cells survived at day 7 on a decellularized aortic scaffold. Ki67 staining demonstrated that decellularized aortic scaffold promoted proliferation of bone marrow-derived CD34+ progenitor cells. Apoptosis of CD34+ progenitor cells induced by H2O2 at high concentration was significantly alleviated in the presence of decellularized aortic scaffolds, demonstrating a protective effect against oxidative stress-induced apoptosis. Furthermore, decellularized aortic scaffolds significantly reduced the expression of proinflammatory cytokines (IL-8, GM-CSF, MIP-1ß, GRO-α, Entoxin, and GRO) concurrently with an increase in anti-inflammatory cytokines (IL-2 and TGF-ß) released from CD34+ progenitor cells when exposed to H2O2 at low concentration. Finally, neovascularization was observed by H&E and immunohistochemical staining 14 days after the decellularized aortic scaffolds were subcutaneously implanted in nude mice. This preclinical study demonstrates that the use of a decellularized aortic scaffold possessing antiapoptotic and anti-inflammatory properties may represent a promising strategy for cardiovascular repair after ischemic injury.
Assuntos
Inflamação/terapia , Neovascularização Fisiológica/genética , Transplante de Células-Tronco , Alicerces Teciduais , Cicatrização/genética , Animais , Antígenos CD34/genética , Aorta/citologia , Aorta/transplante , Apoptose/genética , Proliferação de Células/genética , Células Endoteliais/transplante , Matriz Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Peróxido de Hidrogênio/toxicidade , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/patologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Camundongos , Camundongos Nus , Microscopia Eletrônica de Varredura , Neovascularização Fisiológica/fisiologia , Células-Tronco/imunologia , Células-Tronco/ultraestrutura , Engenharia Tecidual/métodosRESUMO
Peptidyl arginine deiminase-4 (PAD4), a PAD enzyme family member, catalyzes the posttranslational conversion of arginine residues to citrulline in target proteins. Although PAD4 is believed to play a crucial role in various pathological conditions such as infectious diseases, autoimmune diseases, and ischemic conditions, the effect of PAD4 in myocardial infarction (MI)-induced cardiac injury remains to be examined. Here, we hypothesize that PAD4 contributes to cardiac ischemic injury by exacerbating the inflammatory response and promoting neutrophil extracellular trap (NET) formation after MI. Permanent left coronary artery ligation, a condition that mimics MI, was performed on male C57BL/6 mice. [(3S,4R)-3-amino-4-hydroxy-1-piperidinyl] [2-[1-(cyclopropylmethyl)-1H-indol-2-yl]-7-methoxy-1-methyl-1H-benzimidazol-5-yl]-methanone (GSK484), an inhibitor of PAD4, was delivered via intraperitoneal injection to inhibit PAD4 activity. Cardiac PAD4 expression, tissue injury scoring, neutrophil infiltration, cit-H3 expression, NET formation, inflammatory cytokine secretion, apoptosis, and cardiac function were analyzed. In the current study, we discovered the protective effect of PAD4 inhibition using the PAD4-specific inhibitor GSK484 in cardiomyocytes challenged by MI. GSK484-mediated PAD4 inhibition can moderately preserve ventricle histological structure and myocardium integrity after MI, thereby reducing the infarct size and decreasing myocardial enzyme levels in serum. PAD4 inhibition also effectively protects cardiomyocytes from MI-induced NET formation and inflammatory cytokine secretion, in turn alleviating cardiac ischemia-induced apoptosis of cardiomyocytes. Collectively, these findings demonstrate the efficacy of specific PAD4 inhibition in reducing MI-induced neutrophil infiltration, NET formation, inflammatory reaction, and cardiomyocyte apoptosis, thereby increasing overall cardiac function improvement. These results provide novel insights for the development of new strategies to treat cardiovascular dysfunction in MI patients.
Assuntos
Cardiotônicos/farmacologia , Infarto do Miocárdio/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Proteína-Arginina Desiminase do Tipo 4/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Cardiotônicos/uso terapêutico , Modelos Animais de Doenças , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Ventrículos do Coração/imunologia , Ventrículos do Coração/patologia , Humanos , Masculino , Camundongos , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/imunologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/imunologia , Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/imunologia , Infiltração de Neutrófilos/efeitos dos fármacos , Proteína-Arginina Desiminase do Tipo 4/imunologia , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Remodelação Ventricular/efeitos dos fármacos , Remodelação Ventricular/imunologiaRESUMO
Vascular patches are commonly applied in tissue repair and reconstruction in congenital cardiac surgery. However, the currently available patch materials are inappropriate to be used in the pediatric population due to their lack of supporting tissue growth potential. In our study an active patch material was developed by seeding pediatric patient's bone marrow stem cells on a decellularized aortic extracellular matrix (ECM) scaffold. The patch was then implanted to repair abdominal aorta defects of nude rats. Two months after implantation, tissue remodeling, vascular cell regeneration, and cellular integration were investigated using histology and fluorescent staining. Histology demonstrated infiltration of host cells and formation of organized cell layers as well as intact collagen and elastic fibers inside the patch material. Immunofluorescence indicated regeneration of endothelial and smooth muscle cells. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified multiple vascularization-promoting components and growth factors in decellularized aortic ECM scaffold. These results demonstrated growth potential and suitability of human derived tissue-engineered patch for vascular reconstruction, and thus, it might be considered in the future as treatment option in pediatric patients.
RESUMO
Synthetic or biologic materials are usually used to repair vascular malformation in congenital heart defects; however, non-autologous materials show both mismatch compliance and antigenicity, as well as a lack of recellularization on its surface. Here, we constructed a tissue-engineered vascular patch (TEVP) using decellularized extracellular matrix (ECM) scaffold obtained from excised human aorta during surgery, which was seeded with patient-derived bone marrow CD34-positive (CD34+) progenitor cells. While cellular components were removed, the decellularized ECM scaffold retained native ECM composition, similar mechanical performance to undecellularized aortic tissue, and supported the adhesion, survival and proliferation of CD34+ progenitor cells. Interestingly, after in vitro seeding of decellularized aortic ECM scaffold for 21 d, CD34+ progenitor cells differentiated into mature vascular endothelial cells without addition of any growth factors, as confirmed by the increased levels of endothelial surface markers (CD31, Von Willebrand factor (VWF), VE-cadherin and ICAM-2) and upregulated gene levels (CD31, VWF and eNOS) concurrently with decreased expression of stem cell markers (CD133 and CD34), thus, resulting in surface endothelialization of decellularized ECM scaffold. Consequently, the patient-specific TEVP constructed in this study holds great potential for clinical use in pediatric patients with vascular malformation.
Assuntos
Aorta/citologia , Células Endoteliais/citologia , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Antígeno AC133/metabolismo , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Aorta/metabolismo , Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Proliferação de Células , Células Cultivadas , Células Endoteliais/metabolismo , Matriz Extracelular/química , Humanos , Óxido Nítrico Sintase Tipo III/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Fator de von Willebrand/metabolismoRESUMO
Cardiac tissue engineering using biomaterials with or without combination of stem cell therapy offers a new option for repairing infarcted heart. However, the bioactivity of biomaterials remains to be optimized because currently available biomaterials do not mimic the biochemical components as well as the structural properties of native myocardial extracellular matrix. Here we hypothesized that human heart valve-derived scaffold (hHVS), as a clinically relevant novel biomaterial, may provide the proper microenvironment of native myocardial extracellular matrix for cardiac repair. In this study, human heart valve tissue was sliced into 100 µm tissue sheet by frozen-sectioning and then decellularized to form the hHVS. Upon anchoring onto the hHVS, post-infarct murine BM c-kit+ cells exhibited an increased capacity for proliferation and cardiomyogenic differentiation in vitro. When used to patch infarcted heart in a murine model of myocardial infarction, either implantation of the hHVS alone or c-kit+ cell-seeded hHVS significantly improved cardiac function and reduced infarct size; while c-kit+ cell-seeded hHVS was even superior to the hHVS alone. Thus, we have successfully developed a hHVS for cardiac repair. Our in vitro and in vivo observations provide the first clinically relevant evidence for translating the hHVS-based biomaterials into clinical strategies to treat myocardial infarction.
Assuntos
Células da Medula Óssea/fisiologia , Valva Mitral/fisiologia , Infarto do Miocárdio/terapia , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Materiais Biocompatíveis , Células da Medula Óssea/ultraestrutura , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Humanos , Masculino , Camundongos Endogâmicos C57BL , Valva Mitral/ultraestruturaRESUMO
Adult heart suffering from increased workload will undergo myocardial hypertrophy, subsequent cardiomyocyte (CM) death, and eventually heart failure. However, the effect of increasing afterload on the neonatal heart remains unknown. We performed ascending aortic constriction (AAC) in neonatal rats 8-12 hours after birth (P0, P indicates postpartum). Seven days after surgery, in vivo heart function was evaluated using cardiac ultrasonography. Haematoxylineosin and Masson staining were used to assess CM diameter and collagen deposition. Moreover, expression of both EdU and Ki67 were evaluated to determine DNA synthesis levels, and pH3 and aurora B as markers for mitosis in CMs. CM isolation was performed by heart perfusion at P0, P3, P5, and P7, respectively. CM number on P0 was 1.01 ± 0.29 × 106. We found that CM cell cycle activation was significantly increased among constricted hearts, as demonstrated by increased Ki67, EdU, pH3, and aurora B positive cells/1000 CMs. At day 7 (P7), constriction group hearts manifested increased wall thickness (0.55 ± 0.05 mm versus 0.85 ± 0.10 mm, P < 0.01, n = 6), and improved hemodynamics as well as left ventricular ejection fraction (65.5 ± 3.7% versus 77.7 ± 4.8%, P < 0.01, n = 6). Of note, the population of CMs was also markedly increased in the constriction group (2.92 ± 0.27 × 106 versus 3.41 ± 0.40 × 106, P < 0.05, n = 6). In summary, we found that during the first week after birth significant numbers of neonatal CMs can reenter the cell cycle. Ascending aortic constriction promotes neonatal rat CM proliferation resulting in 16.7% more CMs in the heart.
Assuntos
Proliferação de Células , Coração/fisiologia , Miócitos Cardíacos/fisiologia , Regeneração , Animais , Animais Recém-Nascidos , Aorta , Cardiomegalia , Constrição , Modelos Animais de Doenças , Feminino , Masculino , RatosRESUMO
Although the bone marrow mononuclear cell (BMMNC) is known as an ideal cell type for cell-based therapy for MI treatment, the effective subpopulation still remains unknown. Our study aimed at identifying the optimal subset of BMMNCs suited for cardiac regeneration. In this study, we observed that MI led to (i) a significant increase of the c-kit(+)AT2R(+) BMMNC subpopulation in mice and (ii) a modest increase of AT2R(+) BMMNCs in humans. c-kit(+)AT2R(+) and c-kit(+)AT2R(-) BMMNC subpopulations were obtained from mice after MI. Then, we cocultured cardiac H9C2 cells with c-kit(+)AT2R(+), c-kit(+)AT2R(-), and unfractionated BMMNCs; finally, we found that the c-kit(+)AT2R(+) subset is superior to the c-kit(+)AT2R(-) subset in improving cardiomyocyte protection in vitro. Of note, c-kit(+)AT2R(+) BMMNCs showed a more robust migration capacity than c-kit(+)AT2R(-) and unfractionated BMMNCs in vitro and in vivo. Additionally, compared to c-kit(+)AT2R(-) and unfractionated BMMNCs, intravenous transplantation of c-kit(+)AT2R(+) BMMNC resulted in smaller infarct size and lower levels of inflammatory reactions in heart tissue, leading to a higher global heart function improvement. In conclusion, our results indicate that the c-kit(+)AT2R(+) BMMNC subpopulation exerts a protective effect against MI and shows promising therapeutic possibilities with regard to the treatment of ischemic heart disease.
RESUMO
BACKGROUND/AIMS: Adipose-derived stem cells (ASCs) belong to mesenchymal stem cells and may play a potential role as seeding cells in stem cell transplantation. To be able to exploit stem cells as therapeutic tool, their defects in some important cellular functions, such as low survival rate and cellular activity, should be considered. This is especially the case for stem cells that are intended for transplantation. Of note, stem cell responses to hormones should be considered since estrogen is known to play a critical role in stem cell behavior. However, different impacts of the estrogen receptor (ER) types α and ß have not been fully determined in ASC function. In this study, we investigated effects of ERα and ERß on ASC proliferation, migration, as well as in adipogenesis. METHODS: ASCs obtained from mice were cultured with 100nM ERα or ERß agonist PPT and DPN, respectively. The ERα and ERß antagonist ICI 182,780 (100nM) was used as control. RESULTS: Compared to ERß, ERα appears more potent in improving ASC proliferation and migration. Investigation of adipogenesis revealed that ERß played a significant role in suppressing ASC-mediated brown tissue adipogenesis which is in contrast to ERα. These results correlated with reduced mRNA expression of UCP-1, PGC-1α and PPAR-x03B3;. CONCLUSIONS: ERα plays a more critical role in promoting ASC proliferation and migration while ERß is more potent in suppressing ASC brown adipose tissue differentiation mediated by decreased UCP-1, PGC-1α and PPAR-x03B3; expression.
Assuntos
Adipogenia , Tecido Adiposo Marrom/citologia , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Células-Tronco/citologia , Tecido Adiposo Marrom/metabolismo , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco/metabolismoRESUMO
Electrospun nanofibrous sheets get increasing attention in myocardial infarction (MI) treatment due to their good cytocompatibility to deliver transplanted stem cells to infarcted areas and due to mechanical characteristics to support damaged tissue. Cardiac extracellular matrix is essential for implanted cells since it provides the cardiac microenvironment. In this study, we hypothesized high concentrations of cardiac nature protein (NP), namely elastin and collagen, in hybrid polycaprolactone (PCL) electrospun nanofibrous sheets could be effective as cardiac-mimicking patch. Optimal ratio of elastin and collagen with PCL in electrospun sheets (80% NP/PCL) was selected based on cytocompatibility and mechanical characteristics. Bone-marrow (BM) c-kit(+) cells anchoring onto NP/PCL sheets exhibited increased proliferative capacity compared with those seeded on PCL in vitro. Moreover, we examined the improvement of cardiac function in MI mice by cell-seeded cardiac patch. Green Fluorescent Protein (GFP)-labeled BM c-kit(+) cells were loaded on 80% NP/PCL sheets which was transplanted into MI mice. Both 80% NP/PCL and c-kit(+)-seeded 80% NP/PCL effectively improved cardiac function after 4 weeks of transplantation, with reduced infarction area and restricted LV remodeling. C-kit(+)-seeded 80% NP/PCL was even superior to the 80% NP/PCL alone and both superior to PCL. GFP(+) cells were identified both in the sheets and local infarcted area where transplanted cells underwent cardiac differentiation after 4 weeks. To the best of our knowledge, this is the first report that sheets with high concentrations of nature proteins loaded with BM c-kit(+) cells might be a novel promising candidate for tissue-engineered cardiac patch to improve cardiac repair after MI.
RESUMO
The renin-angiotensin system is involved in hypertension and, thus, in cardiac and vascular injury. In general, angiotensin II is considered as the main mediator of this system but angiotensin IIderived peptides were also shown to exert effects in such diseases. Moreover, it became obvious that different cell and corresponding tissue types are characterized by their own renin-angiotensin system. This system is composed of various peptidic derivatives of the precursor angiotensinogen. Those angiotensinogen-derived peptides can be processed further by peptidases and can bind corresponding receptors. Various clinical trials were initiated considering inhibition of the renin-angiotensin system at different stages in cardiac injuries. Recently, a phase 3 trial using infused angiotensin II (LJPC-501) as treatment option in catecholamine-resistent hypotension was established (ClinicalTrials.gov identifier NCT02338843) although it might be that an influence of AngII-derived peptides is not considered. In general, more intense research on AngII-derived peptides should result in novel strategies and therapeutic options in treatment of cardiac and vascular injuries since these peptides exert actions by themselves, some may interfere with AngII-mediated effects, and some can bind different receptors as well. Consequently, they may also become new promising therapeutics in clinical settings in the future. This short review introduces all currently known angiotensins at once, their production and role related to cardiac and vascular injury, which immune cells show renin-angiotensin system components, and how immune cells containing such components might be involved in such diseases as well.
Assuntos
Angiotensina II/uso terapêutico , Doenças Cardiovasculares/etiologia , Sistema Renina-Angiotensina/fisiologia , Angiotensinogênio/química , Angiotensinogênio/metabolismo , Angiotensinas/fisiologia , Doenças Cardiovasculares/tratamento farmacológico , Ensaios Clínicos Fase III como Assunto , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/etiologiaRESUMO
BACKGROUND: Pre- and early clinical studies on patients with autoimmune diseases suggested that induction of regulatory T(T(reg)) cells may contribute to the immunosuppressive effects of glucocorticoids (GCs). OBJECTIVE: We readdressed the influence of GC therapy on T(reg) cells in immunocompetent human subjects and naïve mice. METHODS: Mice were treated with increasing doses of intravenous dexamethasone followed by oral taper, and T(reg) cells in spleen and blood were analyzed by FACS. Sixteen patients with sudden hearing loss but without an inflammatory disease received high-dose intravenous prednisolone followed by stepwise dose reduction to low oral prednisolone. Peripheral blood T(reg) cells were analyzed prior and after a 14 day GC therapy based on different markers. RESULTS: Repeated GC administration to mice for three days dose-dependently decreased the absolute numbers of T(reg) cells in blood (100 mg dexamethasone/kg body weight: 2.8±1.8×10(4) cells/ml vs. 33±11×10(4) in control mice) and spleen (dexamethasone: 2.8±1.9×10(5)/spleen vs. 95±22×10(5)/spleen in control mice), which slowly recovered after 14 days taper in spleen but not in blood. The relative frequency of FOXP3(+) T(reg) cells amongst the CD4(+) T cells also decreased in a dose dependent manner with the effect being more pronounced in blood than in spleen. The suppressive capacity of T(reg) cells was unaltered by GC treatment in vitro. In immunocompetent humans, GCs induced mild T cell lymphocytosis. However, it did not change the relative frequency of circulating T(reg) cells in a relevant manner, although there was some variation depending on the definition of the T(reg) cells (FOXP3(+): 4.0±1.5% vs 3.4±1.5%*; AITR(+): 0.6±0.4 vs 0.5±0.3%, CD127(low): 4.0±1.3 vs 5.0±3.0%* and CTLA4+: 13.8±11.5 vs 15.6±12.5%; * p<0.05). CONCLUSION: Short-term GC therapy does not induce the hitherto supposed increase in circulating T(reg) cell frequency, neither in immunocompetent humans nor in mice. Thus, it is questionable that the clinical efficacy of GCs is achieved by modulating T(reg) cell numbers.
Assuntos
Glucocorticoides/farmacologia , Imunossupressores/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Adulto , Animais , Contagem de Células , Relação Dose-Resposta a Droga , Feminino , Glucocorticoides/administração & dosagem , Humanos , Imunossupressores/administração & dosagem , Masculino , Camundongos , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Fatores de TempoRESUMO
Adrenocortical carcinoma is a rare but highly malignant neoplasm with still limited treatment options. Epidermal growth factor receptor (EGFR) has been shown to be overexpressed in many solid tumors, but its expression in adrenocortical carcinoma has been studied only in a limited number of cases. Therefore, we analyzed the expression of EGFR in 169 adrenocortical carcinoma samples and compared it with 31 adrenocortical adenomas. Additionally, in 30 cases of adrenocortical carcinoma, exons 18-21 of the EGFR gene were cloned and sequenced. EGFR expression was found in 128 of 169 adrenocortical carcinoma samples (76%), and in 60 of these samples (=36%) strong membrane staining was detected. However, there was no significant correlation with clinical outcome. In addition, all 30 sequenced cases revealed unmutated EGFR genes. In contrast, only 1 out of 31 adrenocortical adenomas weakly expressed the EGFR (3%). In summary, EGFR was overexpressed in more than three-quarters of adrenocortical carcinoma cases of this series. However, no mutations of the EGFR gene were found and EGFR expression was not of prognostic relevance. As EGFR is hardly expressed in adrenocortical adenomas, our results suggest that its expression in adrenocortical tumors indicates a malignant phenotype, which may be used in the differential diagnosis between adrenocortical adenomas and carcinomas.
Assuntos
Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Adenoma Adrenocortical/metabolismo , Adenoma Adrenocortical/patologia , Receptores ErbB/biossíntese , Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Análise Serial de TecidosRESUMO
CONTEXT: No immunohistochemical marker has been established to reliably differentiate adrenocortical tumors from other adrenal masses. A panel of markers like melan-A and inhibin-α is currently used for this purpose but suffers from limited diagnostic accuracy. We hypothesized that expression of steroidogenic factor-1 (SF-1), a transcription factor involved in adrenal development, is of value for the differential diagnosis of adrenal masses and predicts prognosis in adrenocortical carcinoma (ACC). PATIENTS AND METHODS: SF-1 protein expression was assessed by immunohistochemistry on tissue samples from 167 ACC, 52 adrenocortical adenomas (ACA), six normal adrenal glands, six normal ovaries and 73 neoplastic nonsteroidogenic tissues. In an independent cohort of 33 ACC and 58 ACA, SF-1 mRNA expression was analyzed. SF-1 expression was correlated with clinical outcome in patients with ACC. RESULTS: SF-1 protein staining was detectable in 158 of 161 (98%) evaluable ACC samples including 49 (30%) with strong SF-1 staining and in all normal and benign steroidogenic tissues. In addition, SF-1 mRNA expression was present in all 91 analyzed adrenocortical tumors. In contrast, SF-1 expression was absent in all nonsteroidogenic tumors. Strong SF-1 protein expression significantly correlated with poor clinical outcome: tumor stage-adjusted hazard ratio for death 2.46 [95% confidence interval (CI) = 1.30-4.64] and for recurrence 3.91 (95% CI = 1.71-8.94). Similar results were obtained in the independent cohort using RNA analysis [tumor stage-adjusted hazard ratio for death 4.69 (95% CI = 1.44-15.30)]. CONCLUSION: SF-1 is a highly valuable immunohistochemical marker to determine the adrenocortical origin of an adrenal mass with high sensitivity and specificity. In addition, SF-1 expression is of stage-independent prognostic value in patients with ACC.