RESUMO
CD47, a receptor for thrombospondin-1, limits two important regulatory axes: nitric oxide-cGMP signaling and cAMP signaling, both of which can promote mitochondrial biogenesis. Electron microscopy revealed increased mitochondrial densities in skeletal muscle from both CD47 null and thrombospondin-1 null mice. We further assessed the mitochondria status of CD47-null vs WT mice. Quantitative RT-PCR of RNA extracted from tissues of 3 month old mice revealed dramatically elevated expression of mRNAs encoding mitochondrial proteins and PGC-1α in both fast and slow-twitch skeletal muscle from CD47-null mice, but modest to no elevation in other tissues. These observations were confirmed by Western blotting of mitochondrial proteins. Relative amounts of electron transport enzymes and ATP/O(2) ratios of isolated mitochondria were not different between mitochondria from CD47-null and WT cells. Young CD47-null mice displayed enhanced treadmill endurance relative to WTs and CD47-null gastrocnemius had undergone fiber type switching to a slow-twitch pattern of myoglobin and myosin heavy chain expression. In 12 month old mice, both skeletal muscle mitochondrial volume density and endurance had decreased to wild type levels. Expression of myosin heavy chain isoforms and myoglobin also reverted to a fast twitch pattern in gastrocnemius. Both CD47 and TSP1 null mice are leaner than WTs, use less oxygen and produce less heat than WT mice. CD47-null cells produce substantially less reactive oxygen species than WT cells. These data indicate that loss of signaling from the TSP1-CD47 system promotes accumulation of normally functioning mitochondria in a tissue-specific and age-dependent fashion leading to enhanced physical performance, lower reactive oxygen species production and more efficient metabolism.
Assuntos
Envelhecimento/fisiologia , Antígeno CD47/metabolismo , Mitocôndrias/fisiologia , Músculo Esquelético/metabolismo , Animais , Composição Corporal/fisiologia , Peso Corporal/fisiologia , Antígeno CD47/genética , Citocromos b/genética , Citocromos c/genética , Citocromos c/metabolismo , Metabolismo Energético/fisiologia , Feminino , Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/ultraestrutura , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Rápida/ultraestrutura , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Lenta/ultraestrutura , Músculo Esquelético/ultraestrutura , Miócitos de Músculo Liso/metabolismo , Cadeias Pesadas de Miosina/genética , Fator 1 Nuclear Respiratório/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Resistência Física/fisiologia , Isoformas de Proteínas/genética , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Trombospondina 1/genética , Transativadores/genética , Fatores de Transcrição , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
Nitric oxide (NO) drives pro-survival responses in vascular cells and limits platelet adhesion, enhancing blood flow and minimizing thrombosis. The matricellular protein thrombospondin-1 (TSP1), through interaction with its receptor CD47, inhibits soluble guanylyl cyclase (sGC) activation by NO in vascular cells. In vascular smooth muscle cells (VSMCs) both intracellular cGMP and cAMP regulate adhesion, contractility, proliferation, and migration. cGMP can regulate cAMP through feedback control of hydrolysis. Inhibition of the cAMP phosphodiesterase-4 selectively interfered with the ability of exogenous TSP1 to block NO-driven VSMC adhesion but not cGMP accumulation, suggesting that cAMP also contributes to VSMC regulation by TSP1. Inhibition of phosphodiesterase-4 was sufficient to elevate cAMP levels, and inhibiting guanylyl cyclase or phosphodiesterase-3, or adding exogenous TSP1 reversed this increase in cAMP. Thus, TSP1 regulates VSMC cAMP levels in part via cGMP-dependent inhibition of phosphodiesterase-3. Additionally basal cAMP levels were consistently elevated in both VSMCs and skeletal muscle from TSP1 null mice, and treating null cells with exogenous TSP1 suppressed cAMP levels to those of wild type cells. TSP1 inhibited both forskolin and isoproterenol stimulated increases in cAMP in VSMCs. TSP1 also abrogated forskolin and isoproterenol stimulated vasodilation. Consistent with its ability to directly limit adenylyl cyclase-activated vasodilation, TSP1 also limited cAMP-induced dephosphorylation of myosin light chain-2. These findings demonstrate that TSP1 limits both cGMP and cAMP signaling pathways and functional responses in VSMCs and arteries, by both phosphodiesterase-dependent cross talk between these second messengers and by inhibition of adenylyl cyclase activation.
Assuntos
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais , Trombospondina 1/metabolismo , Vasodilatação , Actinas/metabolismo , Animais , Antígeno CD47/metabolismo , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Células Endoteliais/metabolismo , Guanilato Ciclase , Humanos , Hidrólise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Óxido Nítrico/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Ratos , Receptores Citoplasmáticos e Nucleares , Transdução de Sinais/efeitos dos fármacos , Guanilil Ciclase Solúvel , Trombospondina 1/deficiência , Trombospondina 1/genética , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
CD47 is a widely expressed cell surface receptor that serves as a counter-receptor for signal regulatory protein-α and as a receptor for the secreted matricellular protein thrombospondin-1. Thrombospondin-1 signaling through CD47 regulates cellular signaling pathways that control cell survival, growth, motility, mitochondrial biogenesis, arterial vasoactive responses to physiologic vasodilators and blood flow, and responsiveness to growth factors. Studies employing mice lacking either thrombospondin-1 or CD47 have revealed an important role for this receptor-ligand interaction in tissue responses to injury and stress. These null mice show enhanced recovery from soft tissue fixed ischemic injuries, ischemia reperfusion injuries, and radiation injuries. These studies have led to development of antisense strategies to locally or globally suppress CD47 gene expression. A translation-blocking CD47 morpholino improves tissue survival in skin flap and hindlimb fixed ischemia models, full thickness skin grafts, and a liver ischemia/reperfusion model of organ transplantation in mice. Furthermore, the benefits of morpholino treatment extend to aged mice and mice with dysregulated fat metabolism that characteristically exhibit impaired recovery from ischemic injuries. Activity of the morpholino was also demonstrated for treatment of ischemic injury in miniature pigs. Treatment with the CD47 morpholino protects mice from major effects of ionizing radiation including alopecia, deterioration of muscle function, soft tissue and cutaneous fibrosis, and loss of hematopoietic stem cells in bone marrow. Remarkably, the same treatment does not protect tumors but instead enhances their ablation by irradiation. We discuss prospects for further development of CD47 antisense therapeutics for clinical applications including reconstructive surgery, organ transplantation, angioplasty, and cancer.
RESUMO
Thrombospondin-1 (TSP1) can inhibit angiogenic responses directly by interacting with VEGF and indirectly by engaging several endothelial cell TSP1 receptors. We now describe a more potent mechanism by which TSP1 inhibits VEGF receptor-2 (VEGFR2) activation through engaging its receptor CD47. CD47 ligation is known to inhibit downstream signaling targets of VEGFR2, including endothelial nitric-oxide synthase and soluble guanylate cyclase, but direct effects on VEGFR2 have not been examined. Based on FRET and co-immunoprecipitation, CD47 constitutively associated with VEGFR2. Ligation of CD47 by TSP1 abolished resonance energy transfer with VEGFR2 and inhibited phosphorylation of VEGFR2 and its downstream target Akt without inhibiting VEGF binding to VEGFR2. The inhibitory activity of TSP1 in large vessel and microvascular endothelial cells was replicated by a recombinant domain of the protein containing its CD47-binding site and by a CD47-binding peptide derived from this domain but not by the CD36-binding domain of TSP1. Inhibition of VEGFR2 phosphorylation was lost when CD47 expression was suppressed in human endothelial cells and in murine CD47-null cells. These results reveal that anti-angiogenic signaling through CD47 is highly redundant and extends beyond inhibition of nitric oxide signaling to global inhibition of VEGFR2 signaling.
Assuntos
Antígeno CD47/metabolismo , Trombospondina 1/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Bovinos , Membrana Celular/metabolismo , Células Endoteliais/citologia , Humanos , Camundongos , Microscopia Confocal/métodos , Neovascularização Patológica , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Transdução de Sinais , Trombospondinas/metabolismoRESUMO
AIMS: Thrombospondin-1 (TSP1), via its necessary receptor CD47, inhibits nitric oxide (NO)-stimulated soluble guanylate cyclase activation in vascular smooth muscle cells, and TSP1-null mice have increased shear-dependent blood flow compared with wild-type mice. Yet, the endothelial basement membrane should in theory function as a barrier to diffusion of soluble TSP1 into the arterial smooth muscle cell layer. These findings suggested that endothelial-dependent differences in blood flow in TSP1-null mice may be the result of direct modulation of endothelial NO synthase (eNOS) activation by circulating TSP1. Here we tested the hypothesis that TSP1 inhibits eNOS activation and endothelial-dependent arterial relaxation. METHODS AND RESULTS: Acetylcholine (ACh)-stimulated activation of eNOS and agonist-driven calcium transients in endothelial cells were inhibited by TSP1. TSP1 also inhibited eNOS phosphorylation at serine(1177). TSP1 treatment of the endothelium of wild-type and TSP1-null but not CD47-null arteries inhibited ACh-stimulated relaxation. TSP1-null vessels demonstrated greater endothelial-dependent vasorelaxation compared with the wild type. Conversely, TSP1-null arteries demonstrated less vasoconstriction to phenylephrine compared with the wild type, which was corrected upon inhibition of eNOS. In TSP1-null mice, intravenous TSP1 blocked ACh-stimulated decreases in blood pressure, and both intravenous TSP1 and a CD47 agonist antibody acutely elevated blood pressure in mice. CONCLUSION: TSP1, via CD47, inhibits eNOS activation and endothelial-dependent arterial relaxation and limits ACh-driven decreases in blood pressure. Conversely, intravenous TSP1 and a CD47 antibody increase blood pressure. These findings suggest that circulating TSP1, by limiting endogenous NO production, functions as a pressor agent supporting blood pressure.
Assuntos
Pressão Sanguínea/fisiologia , Endotélio Vascular/fisiologia , Óxido Nítrico Sintase Tipo III/fisiologia , Trombospondina 1/fisiologia , Vasodilatação/fisiologia , Acetilcolina/farmacologia , Animais , Anticorpos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Antígeno CD47/imunologia , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Camundongos , Modelos Animais , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Fenilefrina/farmacologia , Trombospondina 1/genética , Trombospondina 1/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
BACKGROUND AND PURPOSE: Soluble guanylate cyclase (sGC) is the signal transduction enzyme most responsible for mediating the effects of nitric oxide (NO). Recently, NO-independent small molecule activators of sGC have been developed that have promising clinical activities. We have shown that the secreted matrix protein thrombospondin-1 (TSP-1) binds to CD47 and potently inhibits NO stimulation of sGC in endothelial and vascular smooth muscle cells (VSMCs) and platelets. Here we show that TSP-1 signalling via CD47 inhibits sGC activation by NO-independent sGC activating small molecules. EXPERIMENTAL APPROACH: Vascular smooth muscle cells and washed human platelets were pretreated with TSP-1 (2.2 nM) in the presence of haeme-dependent sGC activators (YC-1, BAY 41-2272), and a haeme-independent activator (meso-porphyrin IX), and cGMP levels were measured. The effect of sGC activators on platelet aggregation and contraction of VSMC embedded in collagen gels was also assayed in the presence and absence of TSP-1. KEY RESULTS: Thrombospondin-1 inhibited sGC activator-dependent increase in cGMP in VSMC and platelets. TSP-1 pretreatment also inhibited the ability of these agents to delay thrombin-induced platelet aggregation. TSP-1 pretreatment reduced the ability of sGC activating agents to abrogate VSMC contraction in vitro. CONCLUSIONS AND IMPLICATIONS: This work demonstrates that TSP-1 is a universal inhibitor of sGC, blocking both haeme-dependent and haeme-independent activation. These data coupled with the reported increases in TSP-1 with age, diabetes, ischaemia/reperfusion, and atherosclerosis implies that the therapeutic potential of all drugs that activate sGC could be compromised in disease states where TSP-1/CD47 signalling is elevated.
Assuntos
Plaquetas/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Trombospondina 1/farmacologia , Plaquetas/enzimologia , Células Cultivadas , GMP Cíclico/metabolismo , Ativação Enzimática , Guanilato Ciclase/metabolismo , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Agregação PlaquetáriaRESUMO
Amyloid-ß interacts with two cell surface receptors, CD36 and CD47, through which the matricellular protein thrombospondin-1 inhibits soluble guanylate cyclase activation. Here we examine whether amyloid-ß shares this inhibitory activity. Amyloid-ß inhibited both drug and nitric oxide-mediated activation of soluble guanylate cyclase in several cell types. Known cGMP-dependent functional responses to nitric oxide in platelets and vascular smooth muscle cells were correspondingly inhibited by amyloid-ß. Functional interaction of amyloid-ß with the scavenger receptor CD36 was indicated by inhibition of free fatty acid uptake via this receptor. Both soluble oligomer and fibrillar forms of amyloid-ß were active. In contrast, amyloid-ß did not compete with the known ligand SIRPα for binding to CD47. However, both receptors were necessary for amyloid-ß to inhibit cGMP accumulation. These data suggest that amyloid-ß interaction with CD36 induces a CD47-dependent signal that inhibits soluble guanylate cyclase activation. Combined with the pleiotropic effects of inhibiting free fatty acid transport via CD36, these data provides a molecular mechanism through which amyloid-ß can contribute to the nitric oxide signaling deficiencies associated with Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/metabolismo , GMP Cíclico/metabolismo , Óxido Nítrico/metabolismo , Animais , Aorta/citologia , Antígenos CD36/biossíntese , Antígeno CD47/biossíntese , Bovinos , Células Endoteliais/citologia , Humanos , Camundongos , Conformação Proteica , Pirazóis/farmacologia , Piridinas/farmacologia , Transdução de Sinais , Suínos , Veias Umbilicais/citologiaRESUMO
Thiolutin is a dithiole synthesized by Streptomyces sp. that inhibits endothelial cell adhesion and tumor growth. We show here that thiolutin potently inhibits developmental angiogenesis in zebrafish and vascular outgrowth from tissue explants in 3D cultures. Thiolutin is a potent and selective inhibitor of endothelial cell adhesion accompanied by rapid induction of HSPB1 (Hsp27) phosphorylation. The inhibitory effects of thiolutin on endothelial cell adhesion are transient, potentially due to a compensatory increase in Hsp27 protein levels. Accordingly, heat shock induction of Hsp27 limits the anti-adhesive activity of thiolutin. Thiolutin treatment results in loss of actin stress fibers, increased cortical actin as cells retract, and decreased cellular F-actin. Mass spectrometric analysis of Hsp27 binding partners following immunoaffinity purification identified several regulatory components of the actin cytoskeleton that associate with Hsp27 in a thiolutin-sensitive manner including several components of the Arp2/3 complex. Among these, ArpC1a is a direct binding partner of Hsp27. Thiolutin treatment induces peripheral localization of phosphorylated Hsp27 and Arp2/3. Hsp27 also associates with the intermediate filament components vimentin and nestin. Thiolutin treatment specifically ablates Hsp27 interaction with nestin and collapses nestin filaments. These results provide new mechanistic insights into regulation of cell adhesion and cytoskeletal dynamics by Hsp27.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Peixe-Zebra/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Citoesqueleto/efeitos dos fármacos , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Células Endoteliais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/genética , Humanos , Camundongos , Ligação Proteica , Pirrolidinonas/farmacologia , Tubulina (Proteína)/metabolismo , Peixe-Zebra/embriologiaRESUMO
Tissue ischemia and ischemia-reperfusion (I/R) remain sources of cell and tissue death. Inability to restore blood flow and limit reperfusion injury represents a challenge in surgical tissue repair and transplantation. Nitric oxide (NO) is a central regulator of blood flow, reperfusion signaling and angiogenesis. De novo NO synthesis requires oxygen and is limited in ischemic vascular territories. Nitrite (NO(2-)) has been discovered to convert to NO via heme-based reduction during hypoxia, providing a NO synthase independent and oxygen-independent NO source. Furthermore, blockade of the matrix protein thrombospondin-1 (TSP1) or its receptor CD47 has been shown to promote downstream NO signaling via soluble guanylate cyclase (sGC) and cGMP-dependant kinase. We hypothesized that nitrite would provide an ischemic NO source that could be potentiated by TSP1-CD47 blockade enhancing ischemic tissue survival, blood flow and angiogenesis. Both low dose nitrite and direct blockade of TSP1-CD47 interaction using antibodies or gene silencing increased acute blood flow and late tissue survival in ischemic full thickness flaps. Nitrite and TSP1 blockade both enhanced in vitro and in vivo angiogenic responses. The nitrite effect could be abolished by inhibition of sGC and cGMP signaling. Potential therapeutic synergy was tested in a more severe ischemic flap model. We found that combined therapy with nitrite and TSP1-CD47 blockade enhanced flap perfusion, survival and angiogenesis to a greater extent than either agent alone, providing approximately 100% flap survival. These data provide a new therapeutic paradigm for hypoxic NO signaling through enhanced cGMP mediated by TSP1-CD47 blockade and nitrite delivery.
Assuntos
Antígeno CD47/metabolismo , GMP Cíclico/metabolismo , Isquemia/tratamento farmacológico , Isquemia/metabolismo , Óxido Nítrico/metabolismo , Nitritos/farmacologia , Trombospondina 1/metabolismo , Análise de Variância , Animais , Dorso/irrigação sanguínea , Dorso/patologia , Inibidores Enzimáticos/farmacologia , Inativação Gênica , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Oxidiazóis/farmacologia , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Retalhos Cirúrgicos/irrigação sanguínea , Retalhos Cirúrgicos/patologia , Xantina Desidrogenase/metabolismoAssuntos
Neoplasias/irrigação sanguínea , Neovascularização Patológica/metabolismo , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Proteínas Angiogênicas/antagonistas & inibidores , Proteínas Angiogênicas/metabolismo , Animais , Monóxido de Carbono/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/metabolismo , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/etiologia , Óxido Nítrico/metabolismo , Oxirredução , Transdução de SinaisRESUMO
Nitric oxide (NO) locally regulates vascular resistance and blood pressure by modulating blood vessel tone. Thrombospondin-1 signaling via its receptor CD47 locally limits the ability of NO to relax vascular smooth muscle cells and increase regional blood flow in ischemic tissues. To determine whether thrombospondin-1 plays a broader role in central cardiovascular physiology, we examined vasoactive stress responses in mice lacking thrombospondin-1 or CD47. Mice lacking thrombospondin-1 exhibit activity-associated increases in heart rate, central diastolic and mean arterial blood pressure and a constant decrease in pulse pressure. CD47-deficient mice have normal central pulse pressure but elevated resting peripheral blood pressure. Both null mice show exaggerated decreases in peripheral blood pressure and increased cardiac output and ejection fraction in response to NO. Autonomic blockade also induces exaggerated hypotensive responses in awake thrombospondin-1 null and CD47 null mice. Both null mice exhibit a greater hypotensive response to isoflurane, and autonomic blockage under isoflurane anesthesia leads to premature death of thrombospondin-1 null mice. Conversely, the hypertensive response to epinephrine is attenuated in thrombospondin-1 null mice. Thus, the matricellular protein thrombospondin-1 and its receptor CD47 serve as acute physiological regulators of blood pressure and exert a vasopressor activity to maintain global hemodynamics under stress.
Assuntos
Pressão Sanguínea/fisiologia , Antígeno CD47/metabolismo , Frequência Cardíaca/fisiologia , Coração/fisiologia , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiologia , Trombospondina 1/metabolismo , Animais , Pressão Sanguínea/genética , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Ecocardiografia , Frequência Cardíaca/genética , Imunoensaio , Camundongos , Camundongos Knockout , Fluxo Sanguíneo Regional , Pele/irrigação sanguínea , Trombospondina 1/deficiênciaRESUMO
In addition to long-term regulation of angiogenesis, angiogenic growth factor signalling through nitric oxide (NO) acutely controls blood flow and haemostasis. Inhibition of this pathway may account for the hypertensive and pro-thrombotic side effects of the vascular endothelial growth factor antagonists that are currently used for cancer treatment. The first identified endogenous angiogenesis inhibitor, thrombospondin 1, also controls tissue perfusion, haemostasis and radiosensitivity by antagonizing NO signalling. We examine the role of these and other emerging activities of thrombospondin 1 in cancer. Clarifying how endogenous and therapeutic angiogenesis inhibitors regulate vascular NO signalling could facilitate development of more selective inhibitors.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Óxido Nítrico/fisiologia , Transdução de Sinais , Trombospondina 1/fisiologia , Animais , Antígenos CD36/fisiologia , GMP Cíclico/fisiologia , Hemostasia , Humanos , Neoplasias/etiologia , Tolerância a Radiação , Fluxo Sanguíneo RegionalRESUMO
Radiation-induced damage of normal tissues restricts the therapeutic doses of ionizing radiation that can be delivered to tumors and thereby limits the effectiveness of radiotherapy. Thrombospondin-1 signaling through its cell surface receptor CD47 limits recovery from several types of stress, and mice lacking either gene are profoundly resistant to radiation injury. We describe strategies to protect normal tissues from radiation damage using CD47 or thrombospondin-1 antibodies, a CD47-binding peptide, or antisense suppression of CD47. A morpholino oligonucleotide targeting CD47 confers radioresistance to human endothelial cells in vitro and protects soft tissue, bone marrow, and tumor-associated leukocytes in irradiated mice. In contrast, CD47 suppression in mice bearing melanoma or squamous lung tumors prior to irradiation result in 89% and 71% smaller tumors, respectively. Thus, inhibiting CD47 signaling maintains the viability of normal tissues following irradiation while increasing the radiosensitivity of tumors.
Assuntos
Anticorpos/imunologia , Antígeno CD47/imunologia , Tolerância a Radiação , Transdução de Sinais , Animais , Apoptose , Medula Óssea/efeitos da radiação , Antígeno CD47/metabolismo , GMP Cíclico/metabolismo , Humanos , Camundongos , Músculo Esquelético/efeitos da radiação , Óxido Nítrico/metabolismo , Trombospondina 1/imunologiaRESUMO
Thrombospondin-1 regulates nitric oxide (NO) signaling in vascular cells via CD47. Because CD47 binding motifs are conserved in the C-terminal signature domains of all five thrombospondins and indirect evidence has implied CD47 interactions with other family members, we compared activities of recombinant signature domains of thrombospondin-1, -2, and -4 to interact with CD47 and modulate cGMP signaling. Signature domains of thrombospondin-2 and -4 were less active than that of thrombospondin-1 for inhibiting binding of radiolabeled signature domain of thrombospondin-1 or SIRPalpha (signal-regulatory protein) to cells expressing CD47. Consistent with this binding selectivity, the signature domain of thrombospondin-1 was more potent than those of thrombospondin-2 or -4 for inhibiting NO-stimulated cGMP synthesis in vascular smooth muscle cells and downstream effects on cell adhesion. In contrast to thrombospondin-1- and CD47-null cells, primary vascular cells from thrombospondin-2-null mice lack enhanced basal and NO-stimulated cGMP signaling. Effects of endogenous thrombospondin-2 on NO/cGMP signaling could be detected only in thrombospondin-1-null cells. Furthermore, tissue survival of ischemic injury and acute recovery of blood flow in thrombospondin-2-nulls resembles that of wild type mice. Therefore, thrombospondin-1 is the dominant regulator of NO/cGMP signaling via CD47, and its limiting role in acute ischemic injury responses is not shared by thrombospondin-2.
Assuntos
Antígeno CD47/metabolismo , GMP Cíclico/metabolismo , Isquemia/metabolismo , Transdução de Sinais , Trombospondinas/metabolismo , Animais , Células Cultivadas , Humanos , Isquemia/genética , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Ligação Proteica , Receptores Imunológicos/metabolismo , Trombospondinas/deficiência , Trombospondinas/genéticaRESUMO
BACKGROUND: Ischemia-reperfusion (I/R) injury remains a primary complication of transplant surgery, accounting for about 80% of liver transplant failures, and is a major source of morbidity in other pathologic conditions. Activation of endothelium and inflammatory cell recruitment are central to the initiation and promulgation of I/R injury, which can be limited by the bioactive gas nitric oxide (NO). The discovery that thrombsospondin-1 (TSP1), via CD47, limits NO signaling in vascular cells and ischemic injuries in vivo suggested that I/R injury could be another important target of this signaling pathway. METHODS: Wild-type, TSP1-null, and CD47-null mice underwent liver I/R injury. Wild-type animals were pretreated with CD47 or control antibodies before liver I/R injury. Tissue perfusion via laser Doppler imaging, serum enzymes, histology, and immunohistology were assessed. RESULTS: TSP1-null and CD47-null mice subjected to subtotal liver I/R injury showed improved perfusion relative to wild-type mice. Null mice subjected to liver I/R had decreased liver enzyme release and less histologic evidence of injury. Elevated TSP1 expression in liver tissue after I/R injury suggested that preventing its interaction with CD47 could be protective. Thus, pretreatment of wild-type mice using a blocking CD47 antibody improved recovery of tissue perfusion and preserved liver integrity after I/R injury. CONCLUSIONS: Tissue survival and perfusion after liver I/R injury are limited by TSP1 and CD47. Targeting CD47 before I/R injury enhances tissue survival and perfusion in a model of liver I/R injury and suggests therapeutics for enhancing organ survival in transplantation surgery.
Assuntos
Antígeno CD47/fisiologia , Fígado/cirurgia , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/terapia , Transdução de Sinais/fisiologia , Trombospondina 1/fisiologia , Animais , Anticorpos Monoclonais/uso terapêutico , Feminino , Fatores Imunológicos/uso terapêutico , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Recuperação de Função Fisiológica , Traumatismo por Reperfusão/metabolismoRESUMO
Activation of soluble guanylate cyclase by nitric oxide (NO) controls signaling pathways that play critical roles in normal vascular physiology and in the pathogenesis of cardiovascular disease. We have identified the secreted protein thrombospondin-1 as a key regulator of NO signaling. Thrombospondin-1 limits the angiogenic activity of NO in endothelial cells, its vasodilator activity in vascular smooth muscle, and its antithrombotic activity in platelets. Loss of either thrombospondin-1 or its receptor CD47 in transgenic mice results in hyperdynamic responses to NO and reveals the importance of this pathway in normal physiology. Thrombospondin-1 and CD47 null mice show improved abilities to respond to ischemic stress, suggesting that therapeutic targeting of this pathway could benefit patients with a variety of ischemic conditions. We review the preclinical development of therapeutics targeting thrombospondin-1 or CD47 for improving survival of fixed ischemia, ischemia due to aging and peripheral vascular disease, and skin grafting.
Assuntos
Antígeno CD47/metabolismo , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Transdução de Sinais/fisiologia , Trombospondina 1/antagonistas & inibidores , Trombospondina 1/fisiologia , Animais , Antígeno CD47/genética , Antígeno CD47/fisiologia , Doenças Cardiovasculares/genética , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/fisiologia , Transdução de Sinais/genética , Trombospondina 1/genéticaRESUMO
Radiation, a primary mode of cancer therapy, acutely damages cellular macromolecules and DNA and elicits stress responses that lead to cell death. The known cytoprotective activity of nitric oxide (NO) is blocked by thrombospondin-1, a potent antagonist of NO/cGMP signaling in ischemic soft tissues, suggesting that thrombospondin-1 signaling via its receptor CD47 could correspondingly increase radiosensitivity. We show here that soft tissues in thrombospondin-1-null mice are remarkably resistant to radiation injury. Twelve hours after 25-Gy hindlimb irradiation, thrombospondin-1-null mice showed significantly less cell death in both muscle and bone marrow. Two months after irradiation, skin and muscle units in null mice showed minimal histological evidence of radiation injury and near full retention of mitochondrial function. Additionally, both tissue perfusion and acute vascular responses to NO were preserved in irradiated thrombospondin-1-null hindlimbs. The role of thrombospondin-1 in radiosensitization is specific because thrombospondin-2-null mice were not protected. However, mice lacking CD47 showed radioresistance similar to thrombospondin-1-null mice. Both thrombospondin-1- and CD47-dependent radiosensitization is cell autonomous because vascular cells isolated from the respective null mice showed dramatically increased survival and improved proliferative capacity after irradiation in vitro. Therefore, thrombospondin-1/CD47 antagonists may have selective radioprotective activity for normal tissues.
Assuntos
Antígeno CD47/metabolismo , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Trombospondina 1/metabolismo , Sobrevivência de Tecidos/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Vasos Sanguíneos/patologia , Vasos Sanguíneos/efeitos da radiação , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Células Endoteliais/patologia , Células Endoteliais/efeitos da radiação , Membro Posterior/patologia , Membro Posterior/efeitos da radiação , Hipertrofia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias/patologia , Tolerância a Radiação/efeitos da radiação , Trombospondinas/metabolismo , Raios XRESUMO
Nitric oxide (NO) plays important physiological roles in the vasculature to regulate angiogenesis, blood flow, and hemostasis. In solid tumors, NO is generally acknowledged to mediate angiogenic responses to several growth factors. This contrasts with conflicting evidence that NO can acutely increase tumor perfusion through local vasodilation or diminish perfusion by preferential relaxation of peripheral vascular beds outside the tumor. Because thrombospondin 1 (TSP1) is an important physiological antagonist of NO in vascular cells, we examined whether, in addition to inhibiting tumor angiogenesis, TSP1 can acutely regulate tumor blood flow. We assessed this activity of TSP1 in the context of perfusion responses to NO as a vasodilator and epinephrine as a vasoconstrictor. Nitric oxide treatment of wild type and TSP1 null mice decreased perfusion of a syngeneic melanoma, whereas epinephrine transiently increased tumor perfusion. Acute vasoactive responses were also independent of the level of tumor-expressed TSP1 in a melanoma xenograft, but recovery of basal perfusion was modulated by TSP1 expression. In contrast, overexpression of truncated TSP1 lacking part of its CD47 binding domain lacked this modulating activity. These data indicate that TSP1 primarily regulates long-term vascular responses in tumors, in part, because the tumor vasculature has a limited capacity to acutely respond to vasoactive agents.
Assuntos
Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/tratamento farmacológico , Trombospondina 1/farmacologia , Vasoconstritores/farmacologia , Vasodilatadores/farmacologia , Animais , Linhagem Celular Tumoral , Epinefrina/farmacologia , Feminino , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/farmacologia , Fluxo Sanguíneo Regional/efeitos dos fármacos , Trombospondina 1/deficiência , Trombospondina 1/genética , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Thiolutin is a sulfur-based microbial compound with known activity as an angiogenesis inhibitor. Relative to previously studied angiogenesis inhibitors, thiolutin is a remarkably potent inducer of heat shock protein 27 (Hsp27) phosphorylation. This phosphorylation requires p38 kinase but is independent of increased p38 phosphorylation. To elucidate how thiolutin regulates Hsp27 phosphorylation and ultimately angiogenesis, Hsp27 was immunoprecipitated using nonphosphorylated and phospho-Ser78 specific antibodies from lysates of thiolutin treated and untreated human umbilical vein endothelial cells and analyzed by LC-MS. Separate LC-MS analyses of Lys-C, Lys-C plus trypsin, and Lys-C plus Glu-C digests provided 100% sequence coverage, including the identification of a very large 13 kDa Lys-C fragment using a special sample handling procedure (4 M guanidine HCl) prior to the LC-MS analysis to improve the large peptide recovery. The analysis revealed a novel post-translational modification of Hsp27 involving truncation of the N-terminal Met and acetylation of the penultimate Thr. Analysis of a Glu-C fragment containing two phosphorylation sites, Ser78 and Ser82, and a tryptic fragment containing the other phosphorylation site, Ser15, enabled quantitative stoichiometry of Hsp27 phosphorylation by LC-MS. The strategy revealed details of Hsp27 phosphorylation, including significant di-phosphorylation at both Ser78 and Ser82, that would be difficult to obtain by traditional approaches because oligomerization of the hydrophobic N-terminal region of the molecule prevents efficient enzymatic cleavage. The combination of Western blotting, immunoprecipation, and LC-MS provides a quantitative analysis of thiolutin-stimulated Hsp27 phosphorylation and further defines the role of Hsp27 in the antiangiogenic activities of thiolutin and related dithiolethiones.
Assuntos
Células Endoteliais/efeitos dos fármacos , Proteínas de Choque Térmico/química , Sequência de Aminoácidos , Inibidores da Angiogênese/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/metabolismo , Ativação Enzimática , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Imunoprecipitação , Espectrometria de Massas , Dados de Sequência Molecular , Fosforilação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Pirrolidinonas/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Insufficient tissue perfusion underlies many acute and chronic diseases. Tissue perfusion in turn requires adequate blood flow, determined in large part by the relative state of relaxation or constriction of arterial vessels. Nitric oxide (NO) produced by vascular cells modulates blood flow and tissue perfusion by relaxing and dilating arteries. Recently, we reported that the secreted protein thrombospondin-1 (TSP1), through its cell surface receptor CD47, limits the ability of NO to relax and dilate blood vessels and thus decreases tissue perfusion. In the present study, we tested the hypothesis that blocking TSP1-CD47 signaling increases ischemic tissue survival in random cutaneous porcine flaps. METHODS: Random cutaneous flaps 2 x 10 cm2 were raised in white hairless Yucatan miniature pigs and were treated with a monoclonal antibody to TSP1, an antisense morpholino oligonucleotide to CD47 or control agents and tissue survival assessed. Primary vascular smooth muscle cell cultured from Yucatan pigs were also treated with the same agents +/- and an NO donor (DEA/NO) and cGMP quantified. RESULTS: Antibody blockade of TSP1 or morpholino suppression of CD47 dramatically enhanced survival of random tissue flaps. These responses correlated with increased blood vessel patency and tissue blood flow on vessel injection studies. NO-stimulated cGMP flux in Yucatan vascular smooth muscle cell was abrogated after antibody or morpholino treatment. CONCLUSION: Antibody ligation of TSP1 or antisense morpholino knock down of CD47 greatly increased tissue survival to ischemia. Given the similarity between porcine and human soft tissues these results suggest significant therapeutic potential for people.