RESUMEN
Background: The intimal hyperplasia (IH) and vascular remodelling that follows endovascular injury, for instance after post-angioplasty re-stenosis, results in downstream ischaemia and progressive end organ damage. Interferon gamma (IFNγ) is known to play a critical role in this process. In mouse models we have previously shown that fibrocytes expressing tissue factor (TF) are recruited early to the site of injury. Through thrombin generation and protease activated receptor-1 (PAR-1) activation, fibrocytes secrete angiopoietin-2, stimulate neointimal cell proliferation, inhibit apoptosis and induce CXCL-12 production, all of which contribute to the progressive IH that then develops. In this study we investigated the relationship between TF, angiopoietin-2 and IFNγ. Methods and results: IH developing in carotid arteries of wild-type mice 4 weeks after endoluminal injury contained a significant proportion of IFNγ+ fibrocytes and macrophages, which we show, using a previously defined adoptive transfer model, were derived from circulating CD34+ cells. IH did not develop after injury in IFNγ-deficient mice, except after transplantation of WT bone marrow or adoptive transfer of WT CD34+ cells. In vitro, CD34+ cells isolated from post-injury mice did not express IFNγ, but this was induced when provided with FVIIa and FX, and enhanced when prothrombin was also provided: In both cases IFNγ secretion was TF-dependent and mediated mainly through protease activated PAR-1. IFNγ was predominantly expressed by fibrocytes. In vivo, all IFNγ+ neointimal cells in WT mice co-expressed angiopoietin-2, as did the small numbers of neointimal cells recruited in IFNγ-/- mice. Adoptively transferred WT CD34+ cells treated with either an anti-TIE-2 antibody, or with siRNA against angiopoetin-2 inhibited the expression of IFNγ and the development of IH. Conclusion: TF-dependent angiopoietin-2 production by newly recruited fibrocytes, and to a lesser extent macrophages, switches on IFNγ expression, and this is necessary for the IH to develop. These novel findings enhance our understanding of the pathophysiology of IH and expose potential targets for therapeutic intervention.
Asunto(s)
Angiopoyetina 2 , Hiperplasia , Interferón gamma , Macrófagos , Ratones Noqueados , Neointima , Tromboplastina , Animales , Ratones , Interferón gamma/metabolismo , Angiopoyetina 2/metabolismo , Neointima/patología , Neointima/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Tromboplastina/metabolismo , Tromboplastina/genética , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Masculino , Fibroblastos/metabolismo , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/metabolismoRESUMEN
Circulating sca1+/flk1+ cells are hypothesized to be endothelial progenitor cells (EPCs) in mice that contribute to atheroprotection by replacing dysfunctional endothelial cells. Decreased numbers of circulating sca1+/flk1+ cells correlate with increased atherosclerotic lesions and impaired reendothelialization upon electric injury of the common carotid artery. However, legitimate doubts remain about the identity of the putative EPCs and their contribution to endothelial restoration. Hence, our study aimed to establish a phenotype for sca1+/flk1+ cells to gain a better understanding of their role in atherosclerotic disease. In wild-type mice, sca1+/flk1+ cells were mobilized into the peripheral circulation by granulocyte-colony stimulating factor (G-CSF) treatment and this movement correlated with improved endothelial regeneration upon carotid artery injury. Multicolor flow cytometry analysis revealed that sca1+/flk1+ cells predominantly co-expressed surface markers of conventional B cells (B2 cells). In RAG2-deficient mice and upon B2 cell depletion, sca1+/flk1+ cells were fully depleted. In the absence of monocytes, sca1+/flk1+ cell levels were unchanged. A PCR array focused on cell surface markers and next-generation sequencing (NGS) of purified sca1+/flk1+ cells confirmed their phenotype to be predominantly that of B cells. Finally, the depletion of B2 cells, including sca1+/flk1+ cells, in G-CSF-treated wild-type mice partly abolished the endothelial regenerating effect of G-CSF, indicating an atheroprotective role for sca1+/flk1+ B2 cells. In summary, we characterized sca1+/flk1+ cells as a subset of predominantly B2 cells, which are apparently involved in endothelial regeneration.
Asunto(s)
Antígenos Ly/metabolismo , Aterosclerosis/metabolismo , Subgrupos de Linfocitos B/metabolismo , Traumatismos de las Arterias Carótidas/metabolismo , Arteria Carótida Común/metabolismo , Proliferación Celular , Células Progenitoras Endoteliales/metabolismo , Proteínas de la Membrana/metabolismo , Repitelización , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Antígenos Ly/genética , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/patología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/patología , Arteria Carótida Común/inmunología , Arteria Carótida Común/patología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Células Progenitoras Endoteliales/inmunología , Células Progenitoras Endoteliales/patología , Femenino , Depleción Linfocítica , Masculino , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
AIMS: Defects in efficient endothelial healing have been associated with complication of atherosclerosis such as post-angioplasty neoatherosclerosis and plaque erosion leading to thrombus formation. However, current preventive strategies do not consider re-endothelialization in their design. Here, we investigate mechanisms linking immune processes and defect in re-endothelialization. We especially evaluate if targeting phosphoinositide 3-kinase γ immune processes could restore endothelial healing and identify immune mediators responsible for these defects. METHODS AND RESULTS: Using in vivo model of endovascular injury, we showed that both ubiquitous genetic inactivation of PI3Kγ and hematopoietic cell-specific PI3Kγ deletion improved re-endothelialization and that CD4+ T-cell population drives this effect. Accordingly, absence of PI3Kγ activity correlates with a decrease in local IFNγ secretion and its downstream interferon-inducible chemokine CXCL10. CXCL10 neutralization promoted re-endothelialization in vivo as the same level than those observed in absence of PI3Kγ suggesting a role of CXCL10 in re-endothelialization defect. Using a new established ex vivo model of carotid re-endothelialization, we showed that blocking CXCL10 restore the IFNγ-induced inhibition of endothelial healing and identify smooth muscle cells as the source of CXCL10 secretion in response to Th1 cytokine. CONCLUSION: Altogether, these findings expose an unforeseen cellular cross-talk within the arterial wall whereby a PI3Kγ-dependent T-cell response leads to CXCL10 production by smooth muscle cells which in turn inhibits endothelial healing. Therefore, both PI3Kγ and the IFNγ/CXCL10 axis provide novel strategies to promote endothelial healing.
Asunto(s)
Linfocitos T CD4-Positivos/enzimología , Traumatismos de las Arterias Carótidas/enzimología , Quimiocina CXCL10/metabolismo , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Células Endoteliales/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Cicatrización de Heridas , Animales , Linfocitos T CD4-Positivos/inmunología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/patología , Proliferación Celular , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase Ib/deficiencia , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Modelos Animales de Enfermedad , Células Endoteliales/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Interferón gamma/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/inmunología , Miocitos del Músculo Liso/patología , Comunicación Paracrina , Repitelización , Transducción de SeñalRESUMEN
The innate immunity system plays a critical role in vascular repair and restenosis development. Liposomes encapsulating bisphosphonates (LipBPs), but not free BPs, suppress neointima formation following vascular injury mediated in part by monocytes. The objective of this study was to elucidate the role of monocyte subpopulations on vascular healing following LipBP treatment. The potency- and dose-dependent treatment effect of clodronate (CLOD) and alendronate (ALN) liposomes on restenosis inhibition, total monocyte depletion, and monocytes subpopulation was studied. Rats subjected to carotid injury were treated by a single IV injection of LipBPs at the time of injury. Low- and high-dose LipALN treatment (3 and 10 mg/kg, respectively) resulted in a dose-dependent effect on restenosis development after 30 days. Both doses of LipALN resulted in a dose-dependent inhibition of restenosis, but only high dose of LipALN depleted monocytes (-60.1 ± 4.4%, 48 h post injury). Although LipCLOD treatment (at an equivalent potency to 3 mg/kg alendronate) significantly reduced monocyte levels (72.1 ± 6%), no restenosis inhibition was observed. The major finding of this study is the correlation found between monocyte subclasses and restenosis inhibition. Non-classical monocyte (NCM) levels were found higher in LipALN-treated rats, but lower in LipCLOD-treated rats, 24 h after injury and treatment. We suggest that the inhibition of circulating monocyte subpopulations is the predominant mechanism by which LipBPs prevent restenosis. The effect of LipBP treatment on the monocyte subpopulation correlates with the dose and potency of LipBPs.
Asunto(s)
Alendronato/administración & dosificación , Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Ácido Clodrónico/administración & dosificación , Reestenosis Coronaria/prevención & control , Monocitos/inmunología , Lesiones del Sistema Vascular/tratamiento farmacológico , Animales , Traumatismos de las Arterias Carótidas/inmunología , Liposomas , Masculino , Ratas , Lesiones del Sistema Vascular/inmunologíaRESUMEN
Inflammatory cells play key roles in restenosis upon vascular surgical procedures such as bypass grafts, angioplasty and stent deployment but the molecular mechanisms by which these cells affect restenosis remain unclear. The p110δ isoform of phosphoinositide 3-kinase (PI3K) is mainly expressed in white blood cells. Here, we have investigated whether p110δ PI3K is involved in the pathogenesis of restenosis in a mouse model of carotid injury, which mimics the damage following arterial grafts. We used mice in which p110δ kinase activity has been disabled by a knockin (KI) point mutation in its ATP-binding site (p110δD910A/D910A PI3K mice). Wild-type (WT) and p110δD910A/D910A mice were subjected to longitudinal carotid injury. At 14 and 30 days after carotid injury, mice with inactive p110δ showed strongly decreased infiltration of inflammatory cells (including T lymphocytes and macrophages) and vascular smooth muscle cells (VSMCs), compared with WT mice. Likewise, PI-3065, a p110δ-selective PI3K inhibitor, almost completely prevented restenosis after artery injury. Our data showed that p110δ PI3K plays a main role in promoting neointimal thickening and inflammatory processes during vascular stenosis, with its inhibition providing significant reduction in restenosis following carotid injury. p110δ-selective inhibitors, recently approved for the treatment of human B-cell malignancies, therefore, present a new therapeutic opportunity to prevent the restenosis upon artery injury.
Asunto(s)
Traumatismos de las Arterias Carótidas/enzimología , Estenosis Carotídea/enzimología , Fosfatidilinositol 3-Quinasa Clase I/inmunología , Inflamación/enzimología , Animales , Arterias Carótidas/enzimología , Arterias Carótidas/inmunología , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/patología , Estenosis Carotídea/genética , Estenosis Carotídea/inmunología , Estenosis Carotídea/patología , Fosfatidilinositol 3-Quinasa Clase I/genética , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Masculino , Ratones Endogámicos C57BL , Neointima/enzimología , Neointima/genética , Neointima/inmunología , Neointima/patología , Mutación PuntualRESUMEN
BACKGROUND/AIMS: Neointimal formation following vascular injury remains a major mechanism of restenosis, whereas the precise sources of neointimal cells are still uncertain. We tested the hypothesis that both injured arterial cells and non-arterial cells contribute to intimal hyperplasia. METHODS: Following allograft transplantation of the balloon-injured carotid common artery (n = 3-6), the cellular composition of the transplant grafts and the origins of neointimal cells were measured by immunohistochemistry and immunofluorescence staining. RESULTS: Smooth muscle actin (SMA)-positive and CD68-positive cells were clearly observed 14 days later in the neointima after allograft transplantation of the balloon-injured carotid common artery, where re-endothelialization was not yet complete. Green fluorescent protein (GFP) and wild-type (WT) allograft transplantation revealed that the majority of the neointima cells were apparently from the recipient (≈85%) versus the donor (≈15%). Both monocyte chemotactic protein-1 (MCP-1)/CCR2 and stromal cell-derived factor-1 (SDF-1)/CXCR4 signaling were involved in intimal hyperplasia, with bone marrow-derived cells also playing a role. CONCLUSION: These data support the hypothesis that intimal hyperplasia could develop in our novel rat allograft transplantation model of arterial injury, where neointima is attributable not only to local arterial cells but also non-arterial cells including the bone marrow.
Asunto(s)
Células de la Médula Ósea/patología , Traumatismos de las Arterias Carótidas/patología , Arteria Carótida Común/patología , Células Endoteliales/patología , Neointima/patología , Trasplante de Tejidos , Túnica Íntima/lesiones , Actinas/genética , Actinas/inmunología , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/inmunología , Células de la Médula Ósea/inmunología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/cirugía , Arteria Carótida Común/inmunología , Arteria Carótida Común/cirugía , Linaje de la Célula/inmunología , Rastreo Celular , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Quimiocina CXCL12/genética , Quimiocina CXCL12/inmunología , Células Endoteliales/inmunología , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Neointima/genética , Neointima/inmunología , Neointima/cirugía , Ratas , Ratas Endogámicas Lew , Ratas Transgénicas , Receptores CCR2/genética , Receptores CCR2/inmunología , Receptores CXCR4/genética , Receptores CXCR4/inmunología , Transducción de Señal , Trasplante Homólogo , Túnica Íntima/inmunología , Túnica Íntima/cirugíaRESUMEN
Percutaneous coronary intervention is widely adopted to treat patients with coronary artery disease. However, restenosis remains an unsolved clinical problem after vascular interventions. The role of the systemic and local immune response in the development of restenosis is not fully understood. Hence, the aim of the current study was to investigate the role of the human immune system on subsequent neointima formation elicited by vascular injury in a humanized mouse model. Immunodeficient NOD.Cg-PrkdcscidIL2rgtm1Wjl(NSG) mice were reconstituted with human (h)PBMCs immediately after both carotid wire and femoral cuff injury were induced in order to identify how differences in the severity of injury influenced endothelial regeneration, neointima formation, and homing of human inflammatory and progenitor cells. In contrast to non-reconstituted mice, hPBMC reconstitution reduced neointima formation after femoral cuff injury whereas hPBMCs promoted neointima formation after carotid wire injury 4 weeks after induction of injury. Neointimal endothelium and smooth muscle cells in the injured arteries were of mouse origin. Our results indicate that the immune system may differentially respond to arterial injury depending on the severity of injury, which may also be influenced by the intrinsic properties of the arteries themselves, resulting in either minimal or aggravated neointima formation.
Asunto(s)
Traumatismos de las Arterias Carótidas/inmunología , Arteria Femoral/inmunología , Oclusión de Injerto Vascular/inmunología , Leucocitos Mononucleares/inmunología , Lesiones del Sistema Vascular/inmunología , Animales , Traumatismos de las Arterias Carótidas/parasitología , Traumatismos de las Arterias Carótidas/terapia , Modelos Animales de Enfermedad , Arteria Femoral/lesiones , Arteria Femoral/trasplante , Oclusión de Injerto Vascular/fisiopatología , Humanos , Leucocitos Mononucleares/trasplante , Ratones , Ratones SCID/inmunología , Ratones SCID/lesiones , Miocitos del Músculo Liso/inmunología , Miocitos del Músculo Liso/patología , Neointima/inmunología , Neointima/fisiopatología , Lesiones del Sistema Vascular/fisiopatología , Lesiones del Sistema Vascular/terapiaRESUMEN
Interleukin-8 (IL8) is highly expressed by injured arteries in a variety of diseases and is a chemoattractant for neutrophils which express IL8 receptors IL8RA and RB (IL8RA/B) on their membranes. Neutrophils interact with the damaged endothelium and initiate an inflammatory cascade at the site of injury. We have generated a novel translational targeted cell therapy for acute vascular injury using adenoviral vectors to overexpress IL8RA/B and green fluorescent protein (GFP) on the surface of endothelial cells (ECs) derived from human induced pluripotent stem cells (HiPS-IL8RA/B-ECs). We hypothesize that HiPS-IL8RA/B-ECs transfused intravenously into rats with balloon injury of the carotid artery will target to the injured site and compete with neutrophils, thus inhibiting inflammation and neointima formation. Young adult male Sprague-Dawley rats underwent balloon injury of the right carotid artery and received intravenous transfusion of saline vehicle, 1.5 × 10(6) HiPS-ECs, 1.5 × 10(6) HiPS-Null-ECs, or 1.5 × 10(6) HiPS-IL8RA/B-ECs immediately after endoluminal injury. Tissue distribution of HiPS-IL8RA/B-ECs was analyzed by a novel GFP DNA qPCR method. Cytokine and chemokine expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 h postinjury by ELISA and immunohistochemistry, respectively. Neointimal, medial areas, and reendothelialization were measured 14 days postinjury. HiPS-IL8RA/B-ECs homed to injured arteries, inhibited inflammatory mediator expression and inflammatory cell infiltration, accelerated reendothelialization, and attenuated neointima formation after endoluminal injury while control HiPS-ECs and HiPS-Null-ECs did not. HiPS-IL8RA/B-ECs transfused into rats with endoluminal carotid artery injury target to the injured artery and provide a novel strategy to treat vascular injury.
Asunto(s)
Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/terapia , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Madre Pluripotentes Inducidas/trasplante , Neointima/prevención & control , Receptores de Interleucina-8/inmunología , Animales , Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/patología , Células Endoteliales , Ensayo de Inmunoadsorción Enzimática , Proteínas Fluorescentes Verdes/genética , Humanos , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Inflamación , Masculino , Neointima/inmunología , Neointima/patología , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Receptores de Interleucina-8/genéticaRESUMEN
Whilst innate B1-B cells are atheroprotective, adaptive B2-B cells are considered pro-atherogenic. Different subsets of B regulatory cells (B(reg)) have been described. In experimental arthritis and lupus-like disease, B(reg) are contained within the CD21(hi)CD23(hi)CD24(hi) B cell pool. The existence and role of B(reg) in vascular disease is not known. We sought to investigate the existence, identity and location of B(reg) in vascular disease. The representation of B2-B cell subsets in the spleens and lymph nodes (LNs) of Apolipoprotein E(-/-) (ApoE(-/-)) mice compared to controls was characterised by flow cytometry. Additionally, we utilised a model of neointima formation based on the placement of a perivascular collar around the carotid artery in ApoE(-/-) mice to ascertain whether B cells and B cell subsets confer protection against lesion development. Adoptive transfer of B cells was performed from wild type or genetically modified mice. We showed that CD21(hi)CD23(hi)CD24(hi) B cells are unexpectedly increased in the draining LNs of ApoE(-/-) mice. Adoptive transfer of LN-derived B2-B cells or purified CD21(hi)CD23(hi)CD24(hi) B cells to syngeneic mice reduced lesion size and inflammation without changing serum cholesterol levels. Follicular B2-B cells did not confer protection. IL-10 blockade or transfer of IL10-deficient B cells prevented LN-derived B cell-mediated protection. This is the first identification of a specific LN-derived B2-B(reg) subset that confers IL-10 mediated protection from neointima formation. This may open the way for immune modulatory approaches in cardiovascular disease.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Enfermedades de las Arterias Carótidas/prevención & control , Traumatismos de las Arterias Carótidas/prevención & control , Proliferación Celular , Hipercolesterolemia/inmunología , Interleucina-10/inmunología , Ganglios Linfáticos/inmunología , Activación de Linfocitos , Inmunidad Adaptativa , Traslado Adoptivo , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Subgrupos de Linfocitos B/metabolismo , Subgrupos de Linfocitos B/trasplante , Antígeno CD24/inmunología , Antígeno CD24/metabolismo , Antígenos CD40/inmunología , Antígenos CD40/metabolismo , Arterias Carótidas/inmunología , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/inmunología , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Genotipo , Hipercolesterolemia/complicaciones , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Ganglios Linfáticos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Neointima , Fenotipo , Factores Protectores , Receptores de Complemento 3d/inmunología , Receptores de Complemento 3d/metabolismo , Receptores de IgE/inmunología , Receptores de IgE/metabolismo , Transducción de Señal , Linfocitos T Reguladores/inmunología , Factores de TiempoRESUMEN
OBJECTIVE: This study was aimed to investigate the effects of carbon monoxide releasing molecule (CORM-2), a novel carbon monoxide carrier, on the reendothelialization of carotid artery in rat endothelial denudation model. METHODS: Male rats subjected to carotid artery balloon injury were treated with CORM-2, inactive CORM-2 (iCORM-2) or dimethyl sulfoxide (DMSO). The reendothelialization capacity was evaluated by Evans Blue dye and the immunostaining with anti-CD31 antibody. The number of circulating endothelial progenitor cells (EPCs) was detected by flow cytometry. The proliferation, migration, and adhesion of human umbilical vein endothelial cells (HUVECs) were assessed by using [3H]thymidine, Boyden chamber and human fibronectin respectively. The expressions of protein were detected by using western blot analysis. RESULTS: CORM-2 remarkably accelerated the re-endothelialization 5 d later and inhibited neointima formation 28 d later. In addition, the number of peripheral EPCs significantly increased in CORM-2-treated rats than that in iCORM-2 or DMSO-treated rats after 5 d later. In vitro experiments, CORM-2 significantly enhanced the proliferation, migration and adhesion of HUVECs. The levels of Akt, eNOS phosphorylation, and NO generation in HUVECs were also much higher in CORM-2 treated group. Blocking of PI3K/Akt/eNOS signaling pathway markedly suppressed the enhanced migration and adhesion of HUVECs induced by CORM-2. CONCLUSION: CORM-2 could promote endothelial repair, and inhibit neointima formation after carotid artery balloon injury, which might be associated with the function changes of HUVECs regulated by PI3K/Akt/eNOS pathway.
Asunto(s)
Monóxido de Carbono/farmacología , Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Arteria Carótida Común/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/metabolismo , Animales , Monóxido de Carbono/metabolismo , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Arteria Carótida Común/inmunología , Arteria Carótida Común/metabolismo , Arteria Carótida Común/patología , Adhesión Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Humanos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Dihydroaustrasulfone alcohol is the synthetic precursor of austrasulfone, which is a marine natural product, isolated from the Taiwanese soft coral Cladiella australis. Dihydroaustrasulfone alcohol has anti-inflammatory, neuroprotective, antitumor and anti-atherogenic properties. Although dihydroaustrasulfone alcohol has been shown to inhibit neointima formation, its effect on human vascular smooth muscle cells (VSMCs) has not been elucidated. We examined the effects and the mechanisms of action of dihydroaustrasulfone alcohol on proliferation, migration and phenotypic modulation of human aortic smooth muscle cells (HASMCs). Dihydroaustrasulfone alcohol significantly inhibited proliferation, DNA synthesis and migration of HASMCs, without inducing cell death. Dihydroaustrasulfone alcohol also inhibited platelet-derived growth factor (PDGF)-induced expression of cyclin-dependent kinases (CDK) 2, CDK4, cyclin D1 and cyclin E. In addition, dihydroaustrasulfone alcohol inhibited PDGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whereas it had no effect on the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/(Akt). Moreover, treatment with PD98059, a highly selective ERK inhibitor, blocked PDGF-induced upregulation of cyclin D1 and cyclin E and downregulation of p27kip1. Furthermore, dihydroaustrasulfone alcohol also inhibits VSMC synthetic phenotype formation induced by PDGF. For in vivo studies, dihydroaustrasulfone alcohol decreased smooth muscle cell proliferation in a rat model of restenosis induced by balloon injury. Immunohistochemical staining showed that dihydroaustrasulfone alcohol noticeably decreased the expression of proliferating cell nuclear antigen (PCNA) and altered VSMC phenotype from a synthetic to contractile state. Our findings provide important insights into the mechanisms underlying the vasoprotective actions of dihydroaustrasulfone alcohol and suggest that it may be a useful therapeutic agent for the treatment of vascular occlusive disease.
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Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos/farmacología , Butanonas/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Ciclo Celular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Sulfonas/farmacología , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/uso terapéutico , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Aorta/citología , Butanonas/administración & dosificación , Butanonas/uso terapéutico , Fármacos Cardiovasculares/administración & dosificación , Fármacos Cardiovasculares/farmacología , Fármacos Cardiovasculares/uso terapéutico , Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Arteria Carótida Común/efectos de los fármacos , Arteria Carótida Común/inmunología , Arteria Carótida Común/metabolismo , Arteria Carótida Común/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inyecciones Intraperitoneales , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Ratas Sprague-Dawley , Sulfonas/administración & dosificación , Sulfonas/uso terapéuticoRESUMEN
OBJECTIVE: Atherosclerosis, the cause of 50% of deaths in westernized societies, is widely regarded as a chronic vascular inflammatory disease. Vascular smooth muscle cell (VSMC) inflammatory activation in response to local proinflammatory stimuli contributes to disease progression and is a pervasive feature in developing atherosclerotic plaques. Therefore, it is of considerable therapeutic importance to identify mechanisms that regulate the VSMC inflammatory response. APPROACH AND RESULTS: We report that myocardin, a powerful myogenic transcriptional coactivator, negatively regulates VSMC inflammatory activation and vascular disease. Myocardin levels are reduced during atherosclerosis, in association with phenotypic switching of smooth muscle cells. Myocardin deficiency accelerates atherogenesis in hypercholesterolemic apolipoprotein E(-/-) mice. Conversely, increased myocardin expression potently abrogates the induction of an array of inflammatory cytokines, chemokines, and adhesion molecules in VSMCs. Expression of myocardin in VSMCs reduces lipid uptake, macrophage interaction, chemotaxis, and macrophage-endothelial tethering in vitro, and attenuates monocyte accumulation within developing lesions in vivo. These results demonstrate that endogenous levels of myocardin are a critical regulator of vessel inflammation. CONCLUSIONS: We propose myocardin as a guardian of the contractile, noninflammatory VSMC phenotype, with loss of myocardin representing a critical permissive step in the process of phenotypic transition and inflammatory activation, at the onset of vascular disease.
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Aterosclerosis/metabolismo , Traumatismos de las Arterias Carótidas/metabolismo , Inflamación/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/patología , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Quimiocinas/metabolismo , Quimiotaxis , Citocinas/metabolismo , Modelos Animales de Enfermedad , Genotipo , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Metabolismo de los Lípidos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/inmunología , Monocitos/metabolismo , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/inmunología , Miocitos del Músculo Liso/patología , Neointima , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Fenotipo , Interferencia de ARN , Ratas Wistar , Factores de Tiempo , Transactivadores/deficiencia , Transactivadores/genética , TransfecciónRESUMEN
Interventional strategies to treat atherosclerosis, such as transluminal angioplasty and stent implantation, often cause vascular injury. This leads to intimal hyperplasia (IH) formation that induces inflammatory and fibroproliferative processes and ultimately restenosis. We show that phosphoinositide 3-kinase γ (PI3Kγ) is a key player in IH formation and is a valid therapeutic target in its prevention/treatment. PI3Kγ-deficient mice and mice expressing catalytically inactive PI3Kγ (PI3Kγ KD) showed reduced arterial occlusion and accumulation of monocytes and T cells around sites of vascular lesion. The transfer of PI3Kγ KD CD4(+) T cells into Rag2-deficient mice greatly reduced vascular occlusion compared with WT cells, clearly demonstrating the involvement of PI3Kγ in CD4(+) T cells during IH formation. In addition we found that IH is associated with increased levels of Th1 and Th17 cytokines. A specific decrease in the Th1 response was observed in the absence of PI3Kγ activity, leading to decreased CXCL10 and RANTES production by smooth muscle cells. Finally, we show that treatment with the PI3Kγ inhibitor AS-605240 is sufficient to decrease IH in both mouse and rat models, reinforcing the therapeutic potential of PI3Kγ inhibition. Altogether, these findings demonstrate a new role for PI3Kγ activity in Th1-controlled IH development.
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Neointima/enzimología , Neointima/inmunología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Células TH1/efectos de los fármacos , Células TH1/inmunología , Animales , Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Traumatismos de las Arterias Carótidas/enzimología , Traumatismos de las Arterias Carótidas/inmunología , Fosfatidilinositol 3-Quinasa Clase Ib/deficiencia , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Inhibidores Enzimáticos/farmacología , Arteria Femoral/enzimología , Arteria Femoral/inmunología , Arteria Femoral/lesiones , Marcación de Gen , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neointima/tratamiento farmacológico , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Tiazolidinedionas/farmacologíaRESUMEN
OBJECTIVE: 17ß-Estradiol (E2) offers cardiovascular protection in young female animals and postmenopausal women. In contrast, randomized trials of menopausal hormones performed in older women have shown harm or no cardiovascular benefit. We hypothesize that E2 effects on vascular inflammation are age dependent. APPROACH AND RESULTS: Young (10 weeks) and aged (52 weeks) female C57BL/6 mice were used as source for primary cultures of bone marrow-derived macrophages (BMMs) and vascular smooth muscle cells (VSMCs). E2 pretreatment of cells derived from young mice attenuated C-reactive protein (CRP)-induced expression of inflammatory mediators. In contrast, E2 pretreatment of cells from aged mice did not alter (BMMs) or paradoxically exaggerated (VSMCs) inflammatory mediator response to CRP. Using E2 receptor (ER) knockout mice, we demonstrated that E2 regulates inflammatory response to CRP in BMMs via ERα and in VSMCs via ERß. BMMs derived from aged (versus young) mice expressed significantly less ERα mRNA and protein. A selective ligand of the novel ER GPR30 reproduced the E2 effects in BMMs and VSMCs. Unlike in young mice, E2 did not reduce neointima formation in ligated carotid arteries of aged CRP transgenic mice. CONCLUSIONS: E2 attenuates inflammatory response to CRP in BMMs and VSMCs derived from young but not aged mice and reduces neointima formation in injured carotid arteries of young but not aged CRP transgenic mice. ERα expression in BMMs is greatly diminished with aging. These data suggest that vasoprotective effects of E2 are age dependent and may explain the vasotoxic effects of E2 seen in clinical trials of postmenopausal women.
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Antiinflamatorios/farmacología , Estradiol/farmacología , Receptor alfa de Estrógeno/agonistas , Receptor beta de Estrógeno/agonistas , Inflamación/prevención & control , Macrófagos/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Factores de Edad , Animales , Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Células Cultivadas , Modelos Animales de Enfermedad , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/inmunología , Miocitos del Músculo Liso/metabolismo , Neointima , ARN Mensajero/metabolismo , Receptores de Estrógenos , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
PURPOSE: Restenosis caused by intimal hyperplasia (IH) remains a significant drawback for vascular interventions. It is crucial to understand the molecular mechanisms that control activation of smooth muscle cells (SMCs) after the injury in order to develop strategies to prevent IH. The purpose of the present study was to investigate the early alterations in arterial-wall gene expression after balloon injury in the rat carotid artery with focus on the induction of an inflammatory response. MATERIALS AND METHODS: Twenty-four male Sprague-Dawley rats were subjected to injury of the left common carotid artery by using a 2-F Fogarty catheter. The arteries were harvested 5, 10, and 20 hours after injury. Uninjured arteries from an additional eight rats were used as controls. RNA was isolated and used for genome-wide microarray expression analysis, followed by validation of selected genes with quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemistry was performed on the cross-sectioned vessels. RESULTS: Analysis of gene expression by microarrays showed that the most differentially expressed genes were primarily associated with inflammation, cell proliferation, migration, and adhesion. As confirmed by qRT-PCR, microarray data showed a significant (P < .005) upregulation of cytokines and chemokines (IL-6, CCL2, CXCL1, AIMP1, and CD44) just 5 hours after injury. Immunohistochemistry demonstrated that CCL2 and the adhesion receptor CD44 were expressed by SMCs in the early response to injury and in the absence of leukocyte infiltration. CONCLUSIONS: Arterial injury is followed by an early induction of inflammatory genes in the vessel wall that appears to be confined to SMCs.
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Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/inmunología , Citocinas/inmunología , Regulación de la Expresión Génica/inmunología , Mediadores de Inflamación/inmunología , Músculo Liso Vascular/inmunología , Miocitos del Músculo Liso/inmunología , Animales , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/patología , Masculino , Músculo Liso Vascular/patología , Ratas , Ratas Sprague-DawleyRESUMEN
RATIONALE: Platelets are the most important cells in the primary prevention of blood loss after injury. In addition, platelets are at the interface between circulating leukocytes and the (sub)endothelium regulating inflammatory responses. OBJECTIVE: Our aim was to study the dynamic process that leads to the formation of procoagulant and proinflammatory platelets under physiological flow. METHODS AND RESULTS: In the present study, we describe the formation of extremely long, negatively charged membrane strands that emerge from platelets adhered under flow. These flow-induced protrusions (FLIPRs) are formed in vitro on different physiological substrates and are also detected in vivo in a mouse carotid injury model. FLIPRs are formed downstream the adherent and activated platelets and reach lengths of 250 µm. FLIPR formation is shear-dependent and requires cyclophilin D, calpain, and Rac1 activation. It is accompanied by a disassembly of the F-actin and microtubule organization. Monocytes and neutrophils roll over FLIPRs in a P-selectin/P-selectin glycoprotein ligand-1-dependent manner, retrieving fragments of FLIPRs as microparticles on their surface. Consequently, monocytes and neutrophils become activated, as demonstrated by increased CD11b expression and L-selectin shedding. CONCLUSIONS: The formation of long platelet membrane extensions, such as the ones presented in our flow model, may pave the way to generate an increased membrane surface for interaction with monocytes and neutrophils. Our study provides a mechanistic model for platelet membrane transfer and the generation of monocyte/neutrophil-microparticle complexes. We propose that the formation of FLIPRs in vivo contributes to the well-established proinflammatory function of platelets and platelet-derived microparticles.
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Plaquetas/citología , Plaquetas/inmunología , Traumatismos de las Arterias Carótidas/inmunología , Micropartículas Derivadas de Células/inmunología , Monocitos/inmunología , Neutrófilos/inmunología , Animales , Calcio/metabolismo , Traumatismos de las Arterias Carótidas/patología , Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Voluntarios Sanos , Humanos , Ratones , Monocitos/citología , Neutrófilos/citología , Activación Plaquetaria/inmunología , Flujo Sanguíneo Regional/inmunología , Estrés MecánicoRESUMEN
OBJECTIVE: The N-terminal lectin-like domain (domain 1 [D1]) of thrombomodulin (TM) is known to have an anti-inflammatory function. We previously showed that recombinant TM domain 1 (rTMD1) interacts with a carbohydrate molecule, Lewis Y (Le(y)), which is found to be expressed on adhesion molecules and involved in cell adhesion. Here, we tested the effect of rTMD1/Le(y) interaction on leukocyte recruitment in inflammation. APPROACH AND RESULTS: The expression of Le(y) on the surface of human umbilical vein endothelial cells was increased by tumor necrosis factor-α stimulation. Direct binding of rTMD1 to Le(y) on the cell surface was observed. rTMD1 inhibited Le(y)-mediated leukocyte adhesion on the Le(y)-immobilized flow chamber and activated endothelium under a shear flow. The following leukocyte transmigration to endothelium was also reduced by rTMD1 through binding Le(y). In vivo, treatment of rTMD1 reduced leukocyte recruitment to the inflammatory sites in carotid ligation injury and thioglycollate-induced peritonitis. rTMD1 administration in apolipoprotein E-deficient mice effectively suppressed atherosclerotic plaque formation and macrophage infiltration in atherosclerotic lesions. Increased Le(y) expression, as well as administered rTMD1, was observed in inflamed vessels. CONCLUSIONS: rTMD1 suppresses vascular inflammation by inhibiting leukocyte recruitment to endothelium through attenuating Le(y)-mediated adhesion and further protects against atherosclerosis progression. The present study provides a mechanism showing that rTMD1 can inhibit inflammation by binding to its carbohydrate ligand Le(y).
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Antiinflamatorios/administración & dosificación , Aterosclerosis/prevención & control , Quimiotaxis de Leucocito/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Antígenos del Grupo Sanguíneo de Lewis/metabolismo , Neutrófilos/efectos de los fármacos , Trombomodulina/administración & dosificación , Vasculitis/prevención & control , Animales , Antiinflamatorios/metabolismo , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Antígenos del Grupo Sanguíneo de Lewis/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/inmunología , Neutrófilos/metabolismo , Placa Aterosclerótica , Estructura Terciaria de Proteína , Proteínas Recombinantes/administración & dosificación , Trombomodulina/metabolismo , Migración Transendotelial y Transepitelial/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Vasculitis/inmunología , Vasculitis/metabolismo , Vasculitis/patologíaRESUMEN
OBJECTIVE: Reendothelialization after vascular injury (ie, balloon angioplasty or stent implantation) is clinically extremely relevant to promote vascular healing. We here investigated the therapeutic potential of the toll-like receptor 2/6 agonist macrophage-activating lipopeptide (MALP)-2 on reendothelialization and neointima formation in a murine model of vascular injury. APPROACH AND RESULTS: The left common carotid artery was electrically injured, and reendothelialization was quantified by Evans blue staining after 3 days. A single injection of MALP-2 (1 or 10 µg, IV) after vascular injury accelerated reendothelialization (P<0.001). Proliferation of endothelial cells at the wound margins determined by 5-ethynyl-2'-deoxyuridine incorporation was significantly higher in MALP-2-treated animals (P<0.05). Furthermore, wire injury-induced neointima formation of the left common carotid artery was completely prevented by a single injection of MALP-2 (10 µg, IV). In vitro, MALP-2 induced proliferation (BrdU incorporation) and closure of an artificial wound of endothelial cells (P<0.05) but not of smooth muscle cells. Protein array and ELISA analysis of isolated primary endothelial cells and ex vivo stimulated carotid segments revealed that MALP-2 stimulated the release of multiple growth factors and cytokines predominantly from endothelial cells. MALP-2 induced a strong activation of the mitogen-activated protein kinase cascade in endothelial cells, which was attenuated in smooth muscle cells. Furthermore, MALP-2 significantly enhanced circulating monocytes and hematopoietic progenitor cells. CONCLUSIONS: The toll-like receptor 2/6 agonist MALP-2 promotes reendothelialization and inhibits neointima formation after experimental vascular injury via enhanced proliferation and migration of endothelial cells. Thus, MALP-2 represents a novel therapeutic option to accelerate reendothelialization after vascular injury.
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Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Arteria Carótida Común/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Lipopéptidos/farmacología , Neointima , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 6/agonistas , Lesiones del Sistema Vascular/tratamiento farmacológico , Animales , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Arteria Carótida Común/inmunología , Arteria Carótida Común/metabolismo , Arteria Carótida Común/patología , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Ensayo de Inmunoadsorción Enzimática , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/inmunología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Agregación Plaquetaria/efectos de los fármacos , Análisis por Matrices de Proteínas , Factores de Tiempo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 6/metabolismo , Lesiones del Sistema Vascular/inmunología , Lesiones del Sistema Vascular/metabolismo , Lesiones del Sistema Vascular/patología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Activated platelets release many inflammatory molecules with important roles in accelerating vascular inflammation. Much is known about platelet and platelet-derived mediator interactions with endothelial cells and leukocytes, but few studies have examined the effects of platelets on components of the vascular wall. Vascular smooth muscle cells (VSMCs) undergo phenotypic changes in response to injury including the production of inflammatory molecules, cell proliferation, cell migration, and a decline in the expression of differentiation markers. In this study, we demonstrate that the platelet-derived chemokine platelet factor 4 (PF4/CXCL4) stimulates VSMC injury responses both in vitro and in vivo in a mouse carotid ligation model. PF4 drives a VSMC inflammatory phenotype including a decline in differentiation markers, increased cytokine production, and cell proliferation. We also demonstrate that PF4 effects are mediated, in part, through increased expression of the transcription factor Krüppel-like factor 4. Our data indicate an important mechanistic role for platelets and PF4 in VSMC injury responses both in vitro and in vivo.
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Plaquetas/inmunología , Traumatismos de las Arterias Carótidas/inmunología , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/lesiones , Factor Plaquetario 4/metabolismo , Vasculitis/inmunología , Animales , Arterias Carótidas/citología , Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ligadura , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/citología , Vasculitis/patología , Vasculitis/fisiopatologíaRESUMEN
BACKGROUND: Cell transplantation has great potential for promoting endothelial repair and reducing the complications of percutaneous coronary intervention (PCI). The aim of this study was to investigate the effect of transplantation of human umbilical cord blood endothelial progenitor cells (EPCs) on injured arteries. METHODS: Umbilical cord blood mononuclear cells were obtained from post-partum lying-in women, and EPCs were isolated, cultured, expanded and identified by immunofluorescence. The carotid arterial endothelium of New Zealand white rabbits was injured by dilatation with a 3F balloon, and the EPCs were injected into the lumen of the injured artery in the transplanted group (n = 16), while an equal volume of phosphated buffered saline (PBS) was injected into the control group after balloon injury (n = 16). The animals were sacrificed after either 2 or 4 weeks, and the grafted cells were identified by double immunofluorescence staining with human nuclear antigen (HNA) and CD31 antibodies. Arterial cross sections were analyzed by pathology, immunohistochemistry and morphometry to evaluate the reparative effects of EPCs. Proliferating cell nuclear antigen (PCNA) and transforming growth factor (TGF)-ß1 mRNA expression were detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Fluorescence-labeled EPCs were found in the neointima. The neointimal area and the neointimal/medial area ratio were significantly lower in the transplanted group than in the control group (P < 0.05). von Willebrand factor (vWF) immunohistostaining showed more VWF-positive cells in the transplanted animals than in the controls (8.75 ± 2.92 vs. 4.50 ± 1.77, P < 0.05). Compared with the control group, the transplanted group had lower expression of PCNA mRNA (0.67 ± 0.11 vs. 1.25 ± 0.40, P < 0.01) and higher expression of TGF-ß1 mRNA (1.10 ± 0.21 vs. 0.82 ± 0.07, P < 0.05). CONCLUSIONS: EPCs derived from human umbilical cord blood were successfully transplanted into injured vessels. The transplanted EPCs inhibited neointimal hyperplasia and promoted vascular re-endothelialization.