RESUMO
Thrombocytopenia is a critical problem that occurs in many hematologic diseases, as well as after cancer therapy and radiation exposure. Platelet transfusion is the most commonly used therapy but has limitations of alloimmunization, availability, and expense. Thus, the development of safe, small, molecules to enhance platelet production would be advantageous for the treatment of thrombocytopenia. Herein, we report that an important lipid mediator and a peroxisome proliferator-activated receptor gamma (PPARgamma) ligand called 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), increases Meg-01 maturation and platelet production. 15d-PGJ(2) also promotes platelet formation from culture-derived mouse and human megakaryocytes and accelerates platelet recovery after in vivo radiation-induced bone marrow injury. Interestingly, the platelet-enhancing effects of 15d-PGJ(2) in Meg-01 cells are independent of PPARgamma, but dependent on reactive oxygen species (ROS) accumulation; treatment with antioxidants such as glutathione ethyl ester (GSH-EE); or N-acetylcysteine (NAC) attenuate 15d-PGJ(2)-induced platelet production. Collectively, these data support the concept that megakaryocyte redox status plays an important role in platelet generation and that small electrophilic molecules may have clinical efficacy for improving platelet numbers in thrombocytopenic patients.
Assuntos
Antineoplásicos/farmacologia , Plaquetas/metabolismo , Megacariócitos/metabolismo , Prostaglandina D2/análogos & derivados , Trombocitopenia/metabolismo , Acetilcisteína/farmacologia , Animais , Antineoplásicos/uso terapêutico , Antioxidantes/farmacologia , Medula Óssea/metabolismo , Linhagem Celular , Feminino , Glutationa/análogos & derivados , Glutationa/farmacologia , Humanos , Masculino , Camundongos , PPAR gama/metabolismo , Transfusão de Plaquetas , Prostaglandina D2/farmacologia , Prostaglandina D2/uso terapêutico , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/terapia , Espécies Reativas de Oxigênio/metabolismo , Trombocitopenia/terapiaRESUMO
OBJECTIVE: Platelets are crucial for hemostasis and are vital regulators of inflammation. Foxp3 is a key transcription factor for T regulatory cell development. Humans with IPEX (immune dysregulation, polyendocrinopathy, enteropathy, x-linked) and the scurfy (Foxp3(sf)) mouse have mutations in the Foxp3 gene that lead to a host of pathologies including autoimmunity and skin diseases. Scurfy mice and some humans with IPEX are also thrombocytopenic. The purpose of this study was to determine whether the absence of functional Foxp3 leads to defects in megakaryocytes and platelets. METHODS AND RESULTS: We discovered that human and mouse megakaryocytes express Foxp3 mRNA and protein. Using shRNA and Foxp3(sf) mice, we demonstrated that Foxp3-deficient mouse and human megakaryocyte progenitors exhibited proliferation defects. Striking platelet abnormalities were observed in both an IPEX patient and Foxp3(sf) mice. Impaired platelet spreading and release of TGF-beta and CD40 ligand (CD40L), and abnormal levels of plasma CD40L were observed in a case of IPEX syndrome. Foxp3(sf) mice were thrombocytopenic and had increased platelet volume and altered serum levels of CD40L, TXB(2), and TGF-beta. CONCLUSIONS: These findings provide compelling new evidence that Foxp3 is needed for proper megakaryopoiesis and plays a role in regulating platelet function including spreading and release.
Assuntos
Plaquetas/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Megacariócitos/metabolismo , Trombopoese/fisiologia , Animais , Plaquetas/citologia , Western Blotting , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Humanos , Masculino , Megacariócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Probabilidade , RNA Mensageiro/análise , Especificidade da EspécieRESUMO
Widely known for its role in adipogenesis and energy metabolism, PPARγ also plays a role in platelet function. To further understand functions of platelet-derived PPARγ, we produced rabbit polyclonal (PoAbs) and mouse monoclonal (MoAbs) antibodies against PPARγ 14mer/19mer peptide-immunogens. Unexpectedly, our work produced two key findings. First, MoAbs but not PoAbs produced against PPARγ peptide-immunogens displayed antigenic crossreactivity with highly conserved PPARα and PPARß/δ. Similarly, Santa Cruz PoAb sc-7196 was monospecific for PPARγ while MoAb sc-7273 crossreacted with PPARα and PPARß/δ. Second, immunized rabbits and mice exhibited unusual pathology including cachexia, excessive bleeding, and low platelet counts leading to thrombocytopenia. Spleens from immunized mice were fatty, hemorrhagic and friable. Although passive administration of anti-PPARγ PoAbs failed to induce experimental thrombocytopenia, megakaryocytopoiesis was induced 4-8-fold in mouse spleens. Similarly, marrow megakaryocytopoiesis was enhanced 1.8-4-fold in immunized rabbits. These peptide-immunogens are 100% conserved in human, rabbit and mouse; thus, immune-mediated platelet destruction via crossreactivity with platelet-derived PPARγ likely caused bleeding, thrombocytopenia, and compensatory megakaryocytopoiesis. Such overt pathology would cause significant problems for large-scale production of anti-PPARγ PoAbs. Furthermore, a major pitfall associated with MoAb production against closely related molecules is that monoclonicity does not guarantee monospecificity, an issue worth further scientific scrutiny.
Assuntos
Anticorpos/metabolismo , Homeostase/fisiologia , PPAR gama/fisiologia , Peptídeos/imunologia , Trombocitopenia , Trombopoese , Animais , Anticorpos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Caquexia , Mapeamento de Epitopos , Feminino , Histocitoquímica , Humanos , Intestinos/patologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Ovário/patologia , PPAR gama/antagonistas & inibidores , PPAR gama/química , PPAR gama/imunologia , PPAR gama/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Isoformas de Proteínas , Coelhos , Baço/patologia , Trombocitopenia/patologia , Trombocitopenia/fisiopatologiaRESUMO
The differentiation of preadipocyte fibroblasts to adipocytes is a crucial process to many disease states including obesity, cardiovascular, and autoimmune diseases. In Graves' disease, the orbit of the eye can become severely inflamed and infiltrated with T lymphocytes as part of the autoimmune process. The orbital fibroblasts convert to fat-like cells causing the eye to protrude, which is disfiguring and can lead to blindness. Recently, the transcription factor peroxisome proliferator activated receptor (PPAR)-gamma and its natural (15d-PGJ2) and synthetic (thiazolidinedione-type) PPAR-gamma agonists have been shown to be crucial to the in vitro differentiation of preadipocyte fibroblasts to adipocytes. We show herein several novel findings. First, that activated T lymphocytes from Graves' patients drive the differentiation of PPAR-gamma-expressing orbital fibroblasts to adipocytes. Second, this adipogenic differentiation is blocked by nonselective small molecule cyclooxygenase (Cox)-1/Cox-2 inhibitors and by Cox-2 selective inhibitors. Third, activated, but not naïve, human T cells highly express Cox-2 and synthesize prostaglandin D2 and related prostaglandins that are PPAR-gamma ligands. These provocative new findings provide evidence for how activated T lymphocytes, through production of PPAR-gamma ligands, profoundly influence human fibroblast differentiation to adipocytes. They also suggest the possibility that, in addition to the orbit, T lymphocytes influence the deposition of fat in other tissues.