RESUMO
The signal regulatory protein α (SIRPα)/CD47 axis has emerged as an important innate immune checkpoint that enables cancer cell escape from macrophage phagocytosis. SIRPα expression is limited to macrophages, dendritic cells, and neutrophils-cells enriched in the tumor microenvironment. In this study, we present novel anti-SIRP Abs, SIRP-1 and SIRP-2, as an approach to targeting the SIRPα/CD47 axis. Both SIRP-1 and SIRP-2 bind human macrophage SIRPα variants 1 and 2, the most common variants in the human population. SIRP-1 and SIRP-2 are differentiated among reported anti-SIRP Abs in that they induce phagocytosis of solid and hematologic tumor cell lines by human monocyte-derived macrophages as single agents. We demonstrate that SIRP-1 and SIRP-2 disrupt SIRPα/CD47 interaction by two distinct mechanisms: SIRP-1 directly blocks SIRPα/CD47 and induces internalization of SIRPα/Ab complexes that reduce macrophage SIRPα surface levels and SIRP-2 acts via disruption of higher-order SIRPα structures on macrophages. Both SIRP-1 and SIRP-2 engage FcγRII, which is required for single-agent phagocytic activity. Although SIRP-1 and SIRP-2 bind SIRPγ with varying affinity, they show no adverse effects on T cell proliferation. Finally, both Abs also enhance phagocytosis when combined with tumor-opsonizing Abs, including a highly differentiated anti-CD47 Ab, AO-176, currently being evaluated in phase 1 clinical trials, NCT03834948 and NCT04445701 SIRP-1 and SIRP-2 are novel, differentiated SIRP Abs that induce in vitro single-agent and combination phagocytosis and show no adverse effects on T cell functionality. These data support their future development, both as single agents and in combination with other anticancer drugs.
Assuntos
Apresentação de Antígeno , Antígenos de Diferenciação/imunologia , Antineoplásicos Imunológicos/imunologia , Macrófagos/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Fagocitose , Receptores Imunológicos/imunologia , Linfócitos T/imunologia , Humanos , Células Jurkat , Células THP-1 , Células U937RESUMO
OBJECTIVE: Comprehensive understanding of the mechanisms regulating angiogenesis might provide new strategies for angiogenic therapies for treating diverse physiological and pathological ischemic conditions. The E-twenty six (ETS) factor Ets variant 2 (ETV2; aka Ets-related protein 71) is essential for the formation of hematopoietic and vascular systems. Despite its indispensable function in vessel development, ETV2 role in adult angiogenesis has not yet been addressed. We have therefore investigated the role of ETV2 in vascular regeneration. APPROACH AND RESULTS: We used endothelial Etv2 conditional knockout mice and ischemic injury models to assess the role of ETV2 in vascular regeneration. Although Etv2 expression was not detectable under steady-state conditions, its expression was readily observed in endothelial cells after injury. Mice lacking endothelial Etv2 displayed impaired neovascularization in response to eye injury, wounding, or hindlimb ischemic injury. Lentiviral Etv2 expression in ischemic hindlimbs led to improved recovery of blood perfusion with enhanced vessel formation. After injury, fetal liver kinase 1 (Flk1), aka VEGFR2, expression and neovascularization were significantly upregulated by Etv2, whereas Flk1 expression and vascular endothelial growth factor response were significantly blunted in Etv2-deficient endothelial cells. Conversely, enforced Etv2 expression enhanced vascular endothelial growth factor-mediated endothelial sprouting from embryoid bodies. Lentiviral Flk1 expression rescued angiogenesis defects in endothelial Etv2 conditional knockout mice after hindlimb ischemic injury. Furthermore, Etv2(+/-); Flk1(+/-) double heterozygous mice displayed a more severe hindlimb ischemic injury response compared with Etv2(+/-) or Flk1(+/-) heterozygous mice, revealing an epistatic interaction between ETV2 and FLK1 in vascular regeneration. CONCLUSIONS: Our study demonstrates a novel obligatory role for the ETV2 in postnatal vascular repair and regeneration.
Assuntos
Proteínas Angiogênicas/metabolismo , Células Endoteliais/metabolismo , Isquemia/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Regeneração , Fatores de Transcrição/metabolismo , Proteínas Angiogênicas/deficiência , Proteínas Angiogênicas/genética , Animais , Células Cultivadas , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/fisiopatologia , Modelos Animais de Doenças , Células Endoteliais/patologia , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Heterozigoto , Membro Posterior , Isquemia/genética , Isquemia/patologia , Isquemia/fisiopatologia , Isquemia/terapia , Lentivirus/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Fenótipo , Recuperação de Função Fisiológica , Transdução de Sinais , Pele/irrigação sanguínea , Fatores de Tempo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , CicatrizaçãoRESUMO
A biodegradable positron-emitting dendritic nanoprobe targeted at alpha(v)beta(3) integrin, a biological marker known to modulate angiogenesis, was developed for the noninvasive imaging of angiogenesis. The nanoprobe has a modular multivalent core-shell architecture consisting of a biodegradable heterobifunctional dendritic core chemoselectively functionalized with heterobifunctional polyethylene oxide (PEO) chains that form a protective shell, which imparts biological stealth and dictates the pharmacokinetics. Each of the 8 branches of the dendritic core was functionalized for labeling with radiohalogens. Placement of radioactive moieties at the core was designed to prevent in vivo dehalogenation, a potential problem for radiohalogens in imaging and therapy. Targeting peptides of cyclic arginine-glycine-aspartic acid (RGD) motifs were installed at the terminal ends of the PEO chains to enhance their accessibility to alpha(v)beta(3) integrin receptors. This nanoscale design enabled a 50-fold enhancement of the binding affinity to alpha(v)beta(3) integrin receptors with respect to the monovalent RGD peptide alone, from 10.40 nM to 0.18 nM IC(50). Cell-based assays of the (125)I-labeled dendritic nanoprobes using alpha(v)beta(3)-positive cells showed a 6-fold increase in alpha(v)beta(3) receptor-mediated endocytosis of the targeted nanoprobe compared with the nontargeted nanoprobe, whereas alpha(v)beta(3)-negative cells showed no enhancement of cell uptake over time. In vivo biodistribution studies of (76)Br-labeled dendritic nanoprobes showed excellent bioavailability for the targeted and nontargeted nanoprobes. In vivo studies in a murine hindlimb ischemia model for angiogenesis revealed high specific accumulation of (76)Br-labeled dendritic nanoprobes targeted at alpha(v)beta(3) integrins in angiogenic muscles, allowing highly selective imaging of this critically important process.
Assuntos
Dendrímeros , Integrina alfaVbeta3/metabolismo , Nanotecnologia , Neovascularização Fisiológica , Tomografia por Emissão de Pósitrons/métodos , Animais , Membro Posterior/irrigação sanguínea , Isquemia/diagnóstico por imagem , Isquemia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligopeptídeos/metabolismo , Polietilenoglicóis/química , Distribuição TecidualRESUMO
Despite their divergent developmental ancestry, plasma cells and gastric zymogenic (chief) cells share a common function: high-capacity secretion of protein. Here we show that both cell lineages share increased expression of a cassette of 269 genes, most of which regulate endoplasmic reticulum (ER) and Golgi function, and they both induce expression of the transcription factors X-box binding protein 1 (Xbp1) and Mist1 during terminal differentiation. XBP1 is known to augment plasma cell function by establishing rough ER, and MIST1 regulates secretory vesicle trafficking in zymogenic cells. We examined morphology and function of plasma cells in wild-type and Mist1(-/-) mice and found subtle differences in ER structure but no overall defect in plasma cell function, suggesting that Mist1 may function redundantly in plasma cells. We next reasoned that MIST1 might be useful as a novel and reliable marker of plasma cells. We found that MIST1 specifically labeled normal plasma cells in mouse and human tissues, and, moreover, its expression was also characteristic of plasma cell differentiation in a cohort of 12 human plasma cell neoplasms. Overall, our results show that MIST1 is enriched upon plasma cell differentiation as a part of a genetic program facilitating secretory cell function and also that MIST1 is a novel marker of normal and neoplastic plasma cells in mouse and human tissues.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Plasmócitos/citologia , Plasmócitos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Anticorpos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Celulas Principais Gástricas/citologia , Celulas Principais Gástricas/efeitos dos fármacos , Celulas Principais Gástricas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Membranas Intracelulares/metabolismo , Leucemia Plasmocitária/metabolismo , Leucemia Plasmocitária/patologia , Lipopolissacarídeos/farmacologia , Camundongos , Plasmócitos/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Fatores de Transcrição de Fator Regulador X , Sindecana-1/metabolismo , Fatores de Transcrição/genética , Proteína 1 de Ligação a X-BoxRESUMO
The development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDH(hi)) activity, a progenitor cell function conserved between several lineages. BM ALDH(hi) cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDH(hi) cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDH(hi) cells, mice transplanted with purified ALDH(hi) cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDH(hi) cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue, suggesting that transient ALDH(hi) cell engraftment stimulated endogenous revascularization. Thus, human BM ALDH(hi) cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans.
Assuntos
Aldeído Desidrogenase/metabolismo , Transplante de Medula Óssea/métodos , Extremidades/irrigação sanguínea , Neovascularização Fisiológica , Animais , Técnicas de Cultura de Células , Extremidades/patologia , Humanos , Camundongos , Camundongos SCID , Células-Tronco Multipotentes/fisiologia , Regeneração , Transplante HeterólogoRESUMO
Inhibitors of adaptive immune checkpoints have shown promise as cancer treatments. CD47 is an innate immune checkpoint receptor broadly expressed on normal tissues and overexpressed on many tumors. Binding of tumor CD47 to signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells triggers a "don't eat me" signal that inhibits phagocytosis enabling escape of innate immune surveillance. Blocking CD47/SIRPα interaction promotes phagocytosis reducing tumor burden in numerous xenograft and syngeneic animal models. We have developed a next-generation humanized anti-CD47 antibody, AO-176, that not only blocks the CD47/SIRPα interaction to induce tumor cell phagocytosis, but also induces tumor cytotoxicity in hematologic and solid human tumor cell lines, but not normal noncancerous cells, by a cell autonomous mechanism (not ADCC). AO-176 also binds preferentially to tumor versus many normal cell types. In particular, AO-176 binds negligibly to RBCs in contrast to tumor cells, even at high concentrations up to 200 µg/mL and does not agglutinate RBCs up to 1 mg/mL in vitro These properties are expected not only to decrease the antigen sink, but also to minimize on-target clinical adverse effects observed following treatment with other reported RBC-binding anti-CD47 antibodies. When tested in cynomolgus monkeys, AO-176 was well tolerated with no adverse effects. Finally, we show that AO-176 demonstrates dose-dependent antitumor activity in tumor xenograft models. Taken together, the unique properties and antitumor activity of our next-generation anti-CD47 antibody, AO-176, distinguishes it from other CD47/SIRPα axis targeting agents in clinical development.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antígeno CD47/antagonistas & inibidores , Eritrócitos/metabolismo , Imunidade Inata/imunologia , Neoplasias/tratamento farmacológico , Fagocitose , Receptores Imunológicos/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Antígenos de Diferenciação/imunologia , Apoptose , Antígeno CD47/imunologia , Proliferação de Células , Feminino , Humanos , Imunidade Inata/efeitos dos fármacos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/imunologia , Neoplasias/patologia , Receptores Imunológicos/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The cellular composition and morphology of the stomach epithelium have been described in detail; however, the molecular mechanisms that regulate the differentiation of the various cell lineages as well as the function of mature gastric cells are far less clear. Recently, dissection of the molecular anatomy of the stomach has been boosted by the advent of functional genomics, which allows investigators to determine patterns of gene expression across virtually the entire cellular transcriptome. In this review, we discuss the impact of functional genomic studies on the understanding of gastric epithelial physiology. We show how functional genomic studies have uncovered genes that are useful as new cell lineage-specific markers of differentiation and provide new insights into cell physiology. For example, vascular endothelial growth factor B (Vegfb) has been identified as a parietal cell-specific marker that may allow parietal cells to regulate the mucosal vascular network. We also discuss how functional genomics has identified aberrantly expressed genes in disease states. Human epididymis 4 (HE4), for example, was recently identified as a metaplasia-induced gene product in mice based on microarray analysis. Finally, we will examine how analysis of higher-order patterns of gene expression can go beyond simply identifying individual genes to show how cells work as integrated systems. Specifically, we show how application of a Gene Ontology (GO) analysis of gene expression patterns from multiple tissues identifies the gastric parietal cell as an outlier, unlike other differentiated cell lineages in the stomach or elsewhere in the body.
Assuntos
Células Epiteliais/metabolismo , Genômica/métodos , Células Parietais Gástricas/metabolismo , Linhagem da Célula/genética , Células Epiteliais/citologia , Mucosa Gástrica/citologia , Mucosa Gástrica/metabolismo , Perfilação da Expressão Gênica , Humanos , Modelos Biológicos , Células Parietais Gástricas/citologia , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
There is accumulating evidence that delivery of bone marrow cells to sites of ischemia by direct local injection or mobilization into the blood can stimulate angiogenesis. This has stimulated tremendous interest in the translational potential of angiogenic cell population(s) in the bone marrow to mediate therapeutic angiogenesis. However, the mechanisms by which these cells stimulate angiogenesis are unclear. Herein, we show that the inflammatory subset of monocytes is selectively mobilized into blood after surgical induction of hindlimb ischemia in mice and is selectively recruited to ischemic muscle. Adoptive-transfer studies show that delivery of a small number of inflammatory monocytes early (within 48 h) of induction of ischemia results in a marked increase in the local production of MCP-1, which in turn, is associated with a secondary, more robust wave of monocyte recruitment. Studies of mice genetically deficient in MCP-1 or CCR2 indicate that although not required for the early recruitment of monocytes, the secondary wave of monocyte recruitment and subsequent stimulation of angiogenesis are dependent on CCR2 signaling. Collectively, these data suggest a novel role for MCP-1 in the inflammatory, angiogenic response to ischemia.
Assuntos
Quimiocina CCL2/fisiologia , Membro Posterior/irrigação sanguínea , Inflamação/imunologia , Isquemia/fisiopatologia , Monócitos/fisiologia , Neovascularização Fisiológica , Transferência Adotiva , Animais , Medula Óssea/imunologia , Medula Óssea/patologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CCR2/fisiologia , ReperfusãoRESUMO
Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival times. The efficacy of current systemic therapies for HCC is limited. In this study, we used xenograft tumor models to investigate the use of antibodies that block CD47 and inhibit HCC tumor growth. Immunostaining of tumor tissue and HCC cell lines demonstrated CD47 over-expression in HCC as compared to normal hepatocytes. Macrophage phagocytosis of HCC cells was increased after treatment with CD47 antibodies (CD47mAbs) that block CD47 binding to SIRPα. Further, CD47 blockade inhibited tumor growth in both heterotopic and orthotopic models of HCC, and promoted the migration of macrophages into the tumor mass. Our results demonstrate that targeting CD47 by specific antibodies has potential immunotherapeutic efficacy in human HCC.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno CD47/imunologia , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígeno CD47/biossíntese , Carcinoma Hepatocelular/imunologia , Movimento Celular/imunologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos SCID , Fagocitose/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Ischemia-reperfusion injury (IRI) significantly contributes to delayed graft function and inflammation, leading to graft loss. Ischemia-reperfusion injury is exacerbated by the thrombospondin-1-CD47 system through inhibition of nitric oxide signaling. We postulate that CD47 blockade and prevention of nitric oxide inhibition reduce IRI in organ transplantation. METHODS: We used a syngeneic rat renal transplantation model of IRI with bilaterally nephrectomized recipients to evaluate the effect of a CD47 monoclonal antibody (CD47mAb) on IRI. Donor kidneys were flushed with CD47mAb OX101 or an isotype-matched control immunoglobulin and stored at 4°C in University of Wisconsin solution for 6 hr before transplantation. RESULTS: CD47mAb perfusion of donor kidneys resulted in marked improvement in posttransplant survival, lower levels of serum creatinine, blood urea nitrogen, phosphorus and magnesium, and less histological evidence of injury. In contrast, control groups did not survive more than 5 days, had increased biochemical indicators of renal injury, and exhibited severe pathological injury with tubular atrophy and necrosis. Recipients of CD47mAb-treated kidneys showed decreased levels of plasma biomarkers of renal injury including Cystatin C, Osteopontin, Tissue Inhibitor of Metalloproteinases-1 (TIMP1), ß2-Microglobulin, Vascular Endothelial Growth Factor A (VEGF-A), and clusterin compared to the control group. Furthermore, laser Doppler assessment showed higher renal blood flow in the CD47mAb-treated kidneys. CONCLUSION: These results provide strong evidence for the use of CD47 antibody-mediated blockade to reduce IRI and improve organ preservation for renal transplantation.
Assuntos
Antígeno CD47/fisiologia , Transplante de Rim , Rim/irrigação sanguínea , Traumatismo por Reperfusão/prevenção & controle , Animais , Anticorpos Monoclonais/uso terapêutico , Antígeno CD47/imunologia , Sobrevivência de Enxerto , Rim/patologia , Masculino , Ratos , Ratos Endogâmicos Lew , Resultado do TratamentoRESUMO
After cell fate specification, differentiating cells must amplify the specific subcellular features required for their specialized function. How cells regulate such subcellular scaling is a fundamental unanswered question. Here, we show that the E3 ubiquitin ligase Mindbomb 1 (MIB1) is required for the apical secretory apparatus established by gastric zymogenic cells as they differentiate from their progenitors. When Mib1 was deleted, death-associated protein kinase-1 (DAPK1) was rerouted to the cell base, microtubule-associated protein 1B (MAP1B) was dephosphorylated, and the apical vesicles that normally support mature secretory granules were dispersed. Consequently, secretory granules did not mature. The transcription factor MIST1 bound the first intron of Mib1 and regulated its expression. We further showed that loss of MIB1 and dismantling of the apical secretory apparatus was the earliest quantifiable aberration in zymogenic cells undergoing transition to a precancerous metaplastic state in mouse and human stomach. Our results reveal a mechanistic pathway by which cells can scale up a specific, specialized subcellular compartment to alter function during differentiation and scale it down during disease.
Assuntos
Diferenciação Celular , Celulas Principais Gástricas/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Crescimento Celular , Polaridade Celular , Celulas Principais Gástricas/enzimologia , Sequência Conservada , Proteínas Quinases Associadas com Morte Celular , Humanos , Neoplasias Intestinais/enzimologia , Neoplasias Intestinais/patologia , Metaplasia/induzido quimicamente , Metaplasia/enzimologia , Metaplasia/patologia , Camundongos , Camundongos Knockout , Microtúbulos/genética , Microtúbulos/metabolismo , Transporte Proteico , Estômago/patologia , Tamoxifeno , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Chronic inflammation is a major risk factor for cancer, including gastric cancers and other gastrointestinal cancers. For example, chronic inflammation caused by autoimmune gastritis (AIG) is associated with an increased risk of gastric polyps, gastric carcinoid tumors, and possibly adenocarcinomas. In this study, we characterized the progression of gastric cancer in a novel mouse model of AIG. In this model, disease was caused by CD4(+) T cells expressing a transgenic T-cell receptor specific for a peptide from the H(+)/K(+) ATPase proton pump, a protein expressed by parietal cells in the stomach. AIG caused epithelial cell aberrations that mimicked most of those seen in progression of human gastric cancers, including chronic gastritis followed by oxyntic atrophy, mucous neck cell hyperplasia, spasmolytic polypeptide-expressing metaplasia, dysplasia, and ultimately gastric intraepithelial neoplasias. Our work provides the first direct evidence that AIG supports the development of gastric neoplasia and provides a useful model to study how inflammation drives gastric cancer.
Assuntos
Doenças Autoimunes/etiologia , Linfócitos T CD4-Positivos/imunologia , Gastrite/complicações , ATPase Trocadora de Hidrogênio-Potássio/imunologia , Inflamação/complicações , Receptores de Antígenos de Linfócitos T/fisiologia , Neoplasias Gástricas/etiologia , Adenocarcinoma/etiologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Western Blotting , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Citometria de Fluxo , Imunofluorescência , Mucosa Gástrica/imunologia , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Gastrite/imunologia , Gastrite/patologia , Humanos , Técnicas Imunoenzimáticas , Inflamação/imunologia , Inflamação/patologia , Interferon gama/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/patologia , Metaplasia/complicações , Metaplasia/imunologia , Metaplasia/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
There is considerable interest in the potential of cell-based approaches to mediate therapeutic angiogenesis for acute and chronic vascular syndromes. Using a mouse model of HLI, we showed previously that adoptive transfer of a small number of donor monocytes enhanced revascularization significantly. Herein, we provide data suggesting that the BM resident monocytes sense systemic signals that influence their future functional capacity. Specifically, following induction of distant ischemia, the angiogenic capacity of BM resident monocytes is reduced markedly. We provide evidence that G-CSF and IL-6 represent such "conditioning" signals. Systemic levels of G-CSF and IL-6 are increased significantly following induction of HLI. Accordingly, BM resident monocytes from ischemic mice exhibited increased pSTAT3 and STAT3 target gene expression. Finally, G-CSFR(-/-) and IL-6(-/-) mice were resistant to the deleterious effects of ischemic conditioning on monocyte angiogenic potential. RNA expression profiling suggested that ischemia-conditioned monocytes in the BM up-regulate the well-described M2 polarization markers Chi3l4 and Lrg1. Consistent with this observation, M2-skewed monocytes from SHIP(-/-) mice also had impaired angiogenic capacity. Collectively, these data show that G-CSF and IL-6 provide signals that determine the angiogenic potential of BM resident monocytes.
Assuntos
Células da Medula Óssea/fisiologia , Fator Estimulador de Colônias de Granulócitos/fisiologia , Interleucina-6/fisiologia , Monócitos/fisiologia , Neovascularização Fisiológica , Transferência Adotiva , Animais , Membro Posterior/irrigação sanguínea , Isquemia , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição STAT3/metabolismoRESUMO
There is compelling evidence that circulating angiogenic cells exist that are able to home to sites of vascular injury and stimulate angiogenesis. However, the number of angiogenic cells in the blood is low, limiting their delivery to sites of ischemia. Treatment with certain cytokines may mobilize angiogenic cells into the blood, potentially circumventing this limitation. Herein, we show that treatment with granulocyte colony-stimulating factor (G-CSF) or AMD3100, a novel CXCR4 antagonist, significantly stimulated angiogenesis in a murine model of acute hindlimb ischemia. The kinetics of angiogenic-cell mobilization by these agents appears to be distinct, with more rapid revascularization observed in AMD3100-treated mice. Combination treatment with G-CSF and AMD3100 resulted in the earliest and most complete recovery in blood flow to the ischemic hindlimb. Adoptive transfer of mobilized blood mononuclear cells, while potently stimulating angiogenesis, did not result in the significant incorporation of donor cells into the neoendothelium. Cell-fractionation studies showed that it is the monocyte population in the blood that mediates angiogenesis in this model. Collectively, these data suggest that monocytes mobilized into the blood by G-CSF or AMD3100 stimulate angiogenesis at sites of ischemia through a paracrine mechanism.
Assuntos
Indutores da Angiogênese/farmacologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Compostos Heterocíclicos/metabolismo , Monócitos/citologia , Neovascularização Patológica , Comunicação Parácrina , Animais , Benzilaminas , Antígeno CD11b/biossíntese , Ciclamos , Citocinas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Receptores CXCR4/metabolismoRESUMO
OBJECTIVES: The objective of this study was to determine whether the number of endothelial progenitor cells (EPCs) and circulating angiogenic cells (CACs) in peripheral blood was associated with the presence and severity of coronary artery disease (CAD) in patients undergoing coronary angiography. BACKGROUND: Previous studies have suggested an inverse relationship between levels of circulating EPCs/CACs and the presence of CAD or cardiovascular risk factors, whereas other studies have observed increased numbers of EPCs in the setting of acute ischemia. However, the criteria used to identify specific angiogenic cell subpopulations and methods of evaluating CAD varied in these studies. In the present study, we used rigorous criteria to identify EPCs and CACs in the blood of patients undergoing coronary angiography. METHODS: The number of EPCs and CACs were measured in the blood of 48 patients undergoing coronary angiography. Patients with acute coronary syndromes were excluded. RESULTS: Compared with patients without angiographically significant CAD, the number of EPCs was increased (1.11 +/- 2.50 vs. 4.01 +/- 3.70 colonies/well, p = 0.004) and the number of CACs trended higher (175 +/- 137 vs. 250 +/- 160 cells per mm(2), p = 0.09) among patients with significant CAD. The highest levels of EPCs were isolated from patients subsequently selected for revascularization (5.03 +/- 4.10 colonies/well). CONCLUSIONS: In patients referred for coronary angiography, higher numbers of EPCs, and a trend toward higher numbers of CACs, were associated with the presence of significant CAD, and EPC number correlated with maximum angiographic stenosis severity. Endothelial progenitor cell levels were highest in patients with CAD selected for revascularization.
Assuntos
Angiografia Coronária , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/patologia , Células Endoteliais/patologia , Células-Tronco/patologia , Contagem de Células , Células Cultivadas , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/fisiopatologia , Humanos , Neovascularização Fisiológica , Índice de Gravidade de DoençaRESUMO
Circulating endothelial progenitor cells (EPCs) are thought to contribute to angiogenesis following vascular injury, stimulating interest in their ability to mediate therapeutic angiogenesis. However, the number of EPCs in the blood is low, limiting endogenous repair, and a method to rapidly mobilize EPCs has not been reported. In this study, healthy donors were mobilized sequentially with the CXCR4 antagonist, AMD3100, and G-CSF. The number of EPCs and circulating angiogenic cells (CACs) in the blood and pheresis product was determined and the angiogenic capacity of each cell population assessed. Compared with baseline, treatment with AMD3100 or G-CSF increased the number of blood CACs 10.0-fold +/- 4.4-fold and 8.8-fold +/- 3.7-fold, respectively. The number of EPCs in the blood increased 10.2-fold +/- 3.3-fold and 21.8-fold +/- 5.4-fold, respectively. On a percell basis, CACs harvested from G-CSF-mobilized blood displayed increased in vivo angiogenic potential compared with AMD3100-mobilized CACs. Mobilized EPCs displayed a greater proliferative capacity than EPCs isolated from baseline blood. Both CACs and EPCs were efficiently harvested by leukapheresis. Cryopreserved CACs but not EPCs retained functional activity after thawing. These data show that AMD3100 is a potent and rapid mobilizer of angiogenic cells and demonstrate the feasibility of obtaining and storing large numbers of angiogenic cells by leukapheresis.