RESUMEN

OBJECTIVE: Statins pleiotropically provide additional benefits in reducing atherosclerosis, but their effects on intraplaque angiogenesis (IPA) and hemorrhage (IPH) remain unclear. Therefore, we discriminated statin's lipid-lowering dependent and independent effects on IPA and IPH. APPROACH AND RESULTS: ApoE3*Leiden mice are statin-responsive due to ApoE and LDLR presence, but also allow to titrate plasma cholesterol levels by diet. Therefore, ApoE3*Leiden mice were fed a high-cholesterol-inducing-diet (HCD) with or without atorvastatin (A) or a moderate-cholesterol-inducing-diet (MCD). Mice underwent vein graft surgery to induce lesions with IPA and IPH. Cholesterol levels were significantly reduced in MCD (56%) and HCD + A (39%) compared to HCD with no significant differences between MCD and HCD + A. Both MCD and HCD + A have a similar reduction in vessel remodeling and inflammation comparing to HCD. IPA was significantly decreased by 30% in HCD + A compared to HCD or MCD. Atorvastatin treatment reduced the presence of immature vessels by 34% vs. HCD and by 25% vs. MCD, resulting in a significant reduction of IPH. Atorvastatin's anti-angiogenic capacity was further illustrated by a dose-dependent reduction of ECs proliferation and migration. Cultured mouse aortic-segments lost sprouting capacity upon atorvastatin treatment and became 30% richer in VE-Cadherin expression and pericyte coverage. Moreover, Atorvastatin inhibited ANGPT2 release and decreased VE-Cadherin(Y685)-phosphorylation in ECs. CONCLUSIONS: Atorvastatin has beneficial effects on vessel remodeling due to its lipid-lowering capacity. Atorvastatin has strong pleiotropic effects on IPA by decreasing the number of neovessels and on IPH by increasing vessel maturation. Atorvastatin improves vessel maturation by inhibiting ANGPT2 release and phospho(Y658)-mediated VE-Cadherin internalization.

Asunto(s)

Angiopoyetina 2 , Antígenos CD , Atorvastatina/farmacología , Cadherinas , Colesterol en la Dieta/efectos adversos , Neovascularización Patológica , Placa Aterosclerótica , Angiopoyetina 2/genética , Angiopoyetina 2/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Colesterol en la Dieta/farmacología , Masculino , Ratones , Ratones Mutantes , Neovascularización Patológica/inducido químicamente , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Placa Aterosclerótica/inducido químicamente , Placa Aterosclerótica/tratamiento farmacológico , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo

RESUMEN

Background: Inflammatory stimuli induced by NF-kB drive atherosclerotic lesion formation. The epigenetic P300/CBP associated factor (PCAF) post-transcriptionally acetylates FoxP3, which is required for regulatory T-cell (Treg) differentiation and immune modulation. We hypothesize that PCAF deficiency affects atherosclerosis via regulation of regulatory Tregs. Method: ApoE3*Leiden (n = 13) and ApoE3*LeidenxPCAF-/- (n = 13) were fed a high-fat diet (HFD) containing 1.25% cholesterol. Systemic FoxP3+ T cells were measured every 4 weeks by flow cytometry (n = 6). After 5-months of HFD, mice were euthanized, and hearts and blood were collected. IL-6 and TNFα concentrations were measured in plasma to identify systemic inflammatory responses. Compositional and morphometrical analyses were performed on the atherosclerotic lesions in the aortic sinuses. Results: After 5 months of HFD, plasma cholesterol concentrations were not different for ApoE3*LeidenxPCAF-/- compared to ApoE3*Leiden mice. Expression of FoxP3 by systemic CD4+ T cells decreased 1.8 fold in ApoE3*LeidenxPCAF-/- after 5 months HFD and remained significantly reduced after 5 months of HFD. Systemic TNFα and IL-6 concentrations were comparable, whereas the atherosclerotic lesion size in ApoE3*LeidenxPCAF-/- mice was increased by 28% compared to ApoE3*Leiden mice. In atherosclerotic lesions, no differences were observed in macrophage differentiation or VSMC content, although a small increase in collagen was identified. Conclusion: Our data show that PCAF deficiency resulted in a decrease in circulatory FoxP3+ regulatory T cells and ameliorated atherosclerotic lesions with no differences in systemic inflammation or macrophage differentiation in the atherosclerotic lesions. This suggests that PCAF regulates atherosclerosis via modulation of FoxP3+ regulatory T cell differentiation.

RESUMEN

Improving the efficacy of neovascularization is a promising strategy to restore perfusion of ischemic tissues in patients with peripheral arterial disease. The 14q32 microRNA cluster is highly involved in neovascularization. The Mef2a transcription factor has been shown to induce transcription of the microRNAs within this cluster. We inhibited expression of Mef2a using gene-silencing oligonucleotides (GSOs) in an in vivo hind limb ischemia model. Treatment with GSO-Mef2a clearly improved blood flow recovery within 3 days (44% recovery versus 25% recovery in control) and persisted until 14 days after ischemia induction (80% recovery versus 60% recovery in control). Animals treated with GSO-Mef2a showed increased arteriogenesis and angiogenesis in the relevant muscle tissues. Inhibition of Mef2a decreased expression of 14q32 microRNAs miR-329 (p = 0.026) and miR-494 (trend, p = 0.06), but not of other 14q32 microRNAs, nor of 14q32 microRNA precursors. Because Mef2a did not influence 14q32 microRNA transcription, we hypothesized it functions as an RNA-binding protein that influences processing of 14q32 microRNA miR-329 and miR-494. Mef2A immunoprecipitation followed by RNA isolation and rt/qPCR confirmed direct binding of MEF2A to pri-miR-494, supporting this hypothesis. Our study demonstrates a novel function for Mef2a in post-ischemic neovascularization via post-transcriptional regulation of 14q32 microRNAs miR-329 and miR-494.

RESUMEN

Neovascularisation, i. e. arteriogenesis and angiogenesis, is an inflammatory process. Therefore attraction and extravasation of leukocytes is essential for effective blood flow recovery after ischaemia. Previous studies have shown that von Willebrand factor (VWF) is a negative regulator of angiogenesis. However, it has also been shown that VWF facilitates leukocyte attraction and extravasation. We aimed to investigate the role of VWF in arteriogenesis and angiogenesis during post-ischaemic neovascularisation. Wild-type (WT) and VWF deficient (VWF-/-) C57BL/6 mice were subjected to hindlimb ischaemia via double ligation of the left femoral artery, and blood flow recovery was followed over time, using Laser Doppler Perfusion Imaging. Blood flow recovery was impaired in VWF-/- mice. After 10 days, VWF-/- mice showed a 43 ± 5 % recovery versus 68 ± 5 % in WT. Immunohistochemistry revealed that both arteriogenesis in the adductor muscles and angiogenesis in the gastrocnemius muscles were reduced in VWF-/- mice. Furthermore, leukocyte infiltration in the affected adductor muscles was reduced in VWF-/- mice. Residual paw perfusion directly after artery ligation was also reduced in VWF-/- mice, indicating a decrease in pre-existing collateral arteriole density. When we quantified collateral arterioles, we observed a 31 % decrease in the average number of collateral arterioles in the pia mater compared to WT mice (57 ± 3 in WT vs 40 ± 4 pial collaterals in VWF-/-). We conclude that VWF facilitates blood flow recovery in mice. VWF deficiency hampers both arteriogenesis and angiogenesis in a hindlimb ischaemia model. This is associated with impaired leukocytes recruitment and decreased pre-existing collateral density in the absence of VWF.

Asunto(s)

Isquemia/fisiopatología , Enfermedades de von Willebrand/fisiopatología , Animales , Arteriolas/patología , Velocidad del Flujo Sanguíneo , Circulación Colateral , Modelos Animales de Enfermedad , Arteria Femoral/fisiopatología , Miembro Posterior/irrigación sanguínea , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Flujo Sanguíneo Regional , Enfermedades de von Willebrand/genética , Factor de von Willebrand/genética

RESUMEN

BACKGROUND: In order to identify factors that stimulate arteriogenesis after ischemia, we followed gene expression profiles in two extreme models for collateral artery formation over 28 days after hindlimb ischemia, namely "good-responding" C57BL/6 mice and "poor-responding" BALB/c mice. METHODS AND RESULTS: Although BALB/c mice show very poor blood flow recovery after ischemia, most known proarteriogenic genes were upregulated more excessively and for a longer period than in C57BL/6 mice. In clear contrast, chemokine genes Ccl19, Ccl21a, and Ccl21c and the chemokine receptor CCR7 were upregulated in C57BL/6 mice 1 day after hindlimb ischemia, but not in BALB/C mice. CCL19 and CCL21 regulate migration and homing of T lymphocytes via CCR7. When subjecting CCR7-/-/LDLR-/- mice to hindlimb ischemia, we observed a 20% reduction in blood flow recovery compared with that in LDLR-/- mice. Equal numbers of α-smooth muscle actin-positive collateral arteries were found in the adductor muscles of both mouse strains, but collateral diameters were smaller in the CCR7-/-/LDLR-/-. Fluorescence-activated cell sorter analyses showed that numbers of CCR7+ T lymphocytes (both CD4+ and CD8+) were decreased in the spleen and increased in the blood at day 1 after hindlimb ischemia in LDLR-/- mice. At day 1 after hindlimb ischemia, however, numbers of activated CD4+ T lymphocytes were decreased in the draining lymph nodes of LDLR-/- mice compared with CCR7-/-/LDLR-/- mice. CONCLUSIONS: These data show that CCR7-CCL19/CCL21 axis facilitates retention CD4+ T lymphocytes at the site of collateral artery remodeling, which is essential for effective arteriogenesis.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Quimiocina CCL19/genética , Quimiocina CCL21/genética , Circulación Colateral/genética , Miembro Posterior/irrigación sanguínea , Isquemia/genética , Neovascularización Fisiológica/genética , Receptores CCR7/genética , Animales , Circulación Colateral/inmunología , Expresión Génica , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Fisiológica/inmunología , Receptores de LDL/genética , Regulación hacia Arriba

RESUMEN

Venous grafts are often used to bypass occlusive atherosclerotic lesions; however, poor patency leads to vein graft disease. Deficiency of TLR4, an inflammatory regulator, reduces vein graft disease. Here, we investigate the effects of the accessory molecule and TLR4 analogue RadioProtective 105 (RP105) on vein graft disease. RP105 deficiency resulted in a 90% increase in vein graft lesion area compared to controls. In a hypercholesterolemic setting (LDLr(-/-)/RP105(-/-) versus LDLr(-/-) mice), which is of importance as vein graft disease is usually characterized by excessive atherosclerosis, total lesion area was not affected. However we did observe an increased number of unstable lesions and intraplaque hemorrhage upon RP105 deficiency. In both setups, lesional macrophage content, and lesional CCL2 was increased. In vitro, RP105(-/-) smooth muscle cells and mast cells secreted higher levels of CCL2. In conclusion, aggravated vein graft disease caused by RP105 deficiency results from an increased local inflammatory response.

Asunto(s)

Antígenos CD/metabolismo , Aterosclerosis/metabolismo , Quimiocina CCL2/metabolismo , Macrófagos/metabolismo , Animales , Antígenos CD/genética , Aterosclerosis/genética , Células Cultivadas , Expresión Génica , Inmunohistoquímica , Mediadores de Inflamación/metabolismo , Mastocitos/metabolismo , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidores Tisulares de Metaloproteinasas/genética , Inhibidores Tisulares de Metaloproteinasas/metabolismo

RESUMEN

RATIONALE: Effective neovascularization is crucial for recovery after cardiovascular events. OBJECTIVE: Because microRNAs regulate expression of up to several hundred target genes, we set out to identify microRNAs that target genes in all pathways of the multifactorial neovascularization process. Using www.targetscan.org, we performed a reverse target prediction analysis on a set of 197 genes involved in neovascularization. We found enrichment of binding sites for 27 microRNAs in a single microRNA gene cluster. Microarray analyses showed upregulation of 14q32 microRNAs during neovascularization in mice after single femoral artery ligation. METHODS AND RESULTS: Gene silencing oligonucleotides (GSOs) were used to inhibit 4 14q32 microRNAs, miR-329, miR-487b, miR-494, and miR-495, 1 day before double femoral artery ligation. Blood flow recovery was followed by laser Doppler perfusion imaging. All 4 GSOs clearly improved blood flow recovery after ischemia. Mice treated with GSO-495 or GSO-329 showed increased perfusion already after 3 days (30% perfusion versus 15% in control), and those treated with GSO-329 showed a full recovery of perfusion after 7 days (versus 60% in control). Increased collateral artery diameters (arteriogenesis) were observed in adductor muscles of GSO-treated mice, as well as increased capillary densities (angiogenesis) in the ischemic soleus muscle. In vitro, treatment with GSOs led to increased sprout formation and increased arterial endothelial cell proliferation, as well as to increased arterial myofibroblast proliferation. CONCLUSIONS: The 14q32 microRNA gene cluster is highly involved in neovascularization. Inhibition of 14q32 microRNAs miR-329, miR-487b, miR-494, and miR-495 provides a promising tool for future therapeutic neovascularization.

Asunto(s)

Vasos Sanguíneos/metabolismo , MicroARNs/genética , Animales , Velocidad del Flujo Sanguíneo/genética , Velocidad del Flujo Sanguíneo/fisiología , Vasos Sanguíneos/fisiopatología , Proliferación Celular , Células Cultivadas , Cromosomas Humanos Par 14/genética , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica , Silenciador del Gen , Células HeLa , Miembro Posterior/irrigación sanguínea , Humanos , Isquemia/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/irrigación sanguínea , Miocitos del Músculo Liso/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos/genética

RESUMEN

AIMS: We investigated the role of the TLR4-accessory molecule RP105 (CD180) in post-ischemic neovascularization, i.e. arteriogenesis and angiogenesis. TLR4-mediated activation of pro-inflammatory Ly6Chi monocytes is crucial for effective neovascularization. Immunohistochemical analyses revealed that RP105+ monocytes are present in the perivascular space of remodeling collateral arterioles. As RP105 inhibits TLR4 signaling, we hypothesized that RP105 deficiency would lead to an unrestrained TLR4-mediated inflammatory response and hence to enhanced blood flow recovery after ischemia. METHODS AND RESULTS: RP105-/- and wild type (WT) mice were subjected to hind limb ischemia and blood flow recovery was followed by Laser Doppler Perfusion Imaging. Surprisingly, we found that blood flow recovery was severely impaired in RP105-/- mice. Immunohistochemistry showed that arteriogenesis was reduced in these mice compared to the WT. However, both in vivo and ex vivo analyses showed that circulatory pro-arteriogenic Ly6Chi monocytes were more readily activated in RP105-/- mice. FACS analyses showed that Ly6Chi monocytes became activated and migrated to the affected muscle tissues in WT mice following induction of hind limb ischemia. Although Ly6Chi monocytes were readily activated in RP105-/- mice, migration into the ischemic tissues was hampered and instead, Ly6Chi monocytes accumulated in their storage compartments, bone marrow and spleen, in RP105-/- mice. CONCLUSIONS: RP105 deficiency results in an unrestrained inflammatory response and monocyte over-activation, most likely due to the lack of TLR4 regulation. Inappropriate, premature systemic activation of pro-inflammatory Ly6Chi monocytes results in reduced infiltration of Ly6Chi monocytes in ischemic tissues and in impaired blood flow recovery.

Asunto(s)

Antígenos CD/metabolismo , Miembro Posterior/metabolismo , Isquemia/metabolismo , Monocitos/metabolismo , Neovascularización Fisiológica/genética , Receptor Toll-Like 4/metabolismo , Animales , Antígenos CD/genética , Antígenos Ly/genética , Antígenos Ly/metabolismo , Velocidad del Flujo Sanguíneo , Células de la Médula Ósea/citología , Movimiento Celular , Circulación Colateral , Modelos Animales de Enfermedad , Eliminación de Gen , Expresión Génica , Miembro Posterior/irrigación sanguínea , Miembro Posterior/patología , Isquemia/genética , Isquemia/patología , Flujometría por Láser-Doppler , Ratones , Ratones Noqueados , Monocitos/patología , Bazo/citología , Receptor Toll-Like 4/genética

RESUMEN

RATIONALE: RNA-binding proteins are critical post-transcriptional regulators of RNA and can influence pre-mRNA splicing, RNA localization, and stability. The RNA-binding protein Quaking (QKI) is essential for embryonic blood vessel development. However, the role of QKI in the adult vasculature, and in particular in vascular smooth muscle cells (VSMCs), is currently unknown. OBJECTIVE: We sought to determine the role of QKI in regulating adult VSMC function and plasticity. METHODS AND RESULTS: We identified that QKI is highly expressed by neointimal VSMCs of human coronary restenotic lesions, but not in healthy vessels. In a mouse model of vascular injury, we observed reduced neointima hyperplasia in Quaking viable mice, which have decreased QKI expression. Concordantly, abrogation of QKI attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, rendering noncontractile VSMCs with the capacity to contract. We identified that QKI localizes to the spliceosome, where it interacts with the myocardin pre-mRNA and regulates the splicing of alternative exon 2a. This post-transcriptional event impacts the Myocd_v3/Myocd_v1 mRNA balance and can be modulated by mutating the quaking response element in exon 2a of myocardin. Furthermore, we identified that arterial damage triggers myocardin alternative splicing and is tightly coupled with changes in the expression levels of distinct QKI isoforms. CONCLUSIONS: We propose that QKI is a central regulator of VSMC phenotypic plasticity and that intervention in QKI activity can ameliorate pathogenic, fibroproliferative responses to vascular injury.

Asunto(s)

Proliferación Celular , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo , Animales , Traumatismos de las Arterias Carótidas/metabolismo , Arteria Carótida Común/metabolismo , Arteria Carótida Común/patología , Movimiento Celular , Reestenosis Coronaria/metabolismo , Reestenosis Coronaria/patología , Vasos Coronarios/metabolismo , Vasos Coronarios/patología , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Hiperplasia , Ratones , Ratones Endogámicos C57BL , Ratones Quaking , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Neointima , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Interferencia de ARN , Proteínas de Unión al ARN/genética , Transactivadores/genética , Transactivadores/metabolismo , Transfección

RESUMEN

BACKGROUND: RP105 (CD180) is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown. METHODS AND RESULTS: TLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC) as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105(-/-) mice resulting in a higher proliferation rate. In RP105(-/-) mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982 ± 974 µm(2) vs.1947 ± 278 µm(2),p = 0.0014). Local LPS application augmented neointima formation in both groups, but in RP105(-/-) mice this effect was more pronounced (10316±1243 µm(2) vs.4208 ± 555 µm(2),p = 0.0002), suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500 ± 573 vs.6581 ± 1894 µm(2),p<0.05), whereas expression of the single factors RP105 or MD1 had no effect. CONCLUSION: RP105 is a potent inhibitor of post-interventional neointima formation.

Asunto(s)

Antígenos CD/metabolismo , Vasos Sanguíneos/metabolismo , Neointima/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Antígenos CD/genética , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Arteria Femoral/metabolismo , Expresión Génica , Células HEK293 , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Inmunohistoquímica , Lipopolisacáridos/farmacología , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Neointima/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Solubilidad , Receptor Toll-Like 4/genética

RESUMEN

OBJECTIVE: Therapeutic arteriogenesis, that is, expansive remodeling of preexisting collaterals, using single-action factor therapies has not been as successful as anticipated. Modulation of factors that act as a master switch for relevant gene programs may prove more effective. Transcriptional coactivator p300-CBP-associated factor (PCAF) has histone acetylating activity and promotes transcription of multiple inflammatory genes. Because arteriogenesis is an inflammation-driven process, we hypothesized that PCAF acts as multifactorial regulator of arteriogenesis. APPROACH AND RESULTS: After induction of hindlimb ischemia, blood flow recovery was impaired in both PCAF(-/-) mice and healthy wild-type mice treated with the pharmacological PCAF inhibitor Garcinol, demonstrating an important role for PCAF in arteriogenesis. PCAF deficiency reduced the in vitro inflammatory response in leukocytes and vascular cells involved in arteriogenesis. In vivo gene expression profiling revealed that PCAF deficiency results in differential expression of 3505 genes during arteriogenesis and, more specifically, in impaired induction of multiple proinflammatory genes. Additionally, recruitment from the bone marrow of inflammatory cells, in particular proinflammatory Ly6C(hi) monocytes, was severely impaired in PCAF(-/-) mice. CONCLUSIONS: These findings indicate that PCAF acts as master switch in the inflammatory processes required for effective arteriogenesis.

Asunto(s)

Arteritis/fisiopatología , Isquemia/fisiopatología , Neovascularización Fisiológica/inmunología , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/inmunología , Acetilación , Animales , Arteritis/inmunología , Arteritis/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/inmunología , Miembro Posterior/irrigación sanguínea , Histonas/metabolismo , Isquemia/inmunología , Isquemia/metabolismo , Ratones , Ratones Noqueados , Monocitos/inmunología , Monocitos/patología , Linfocitos T/inmunología , Linfocitos T/patología , Terpenos/farmacología , Transcriptoma , Factores de Transcripción p300-CBP/antagonistas & inhibidores

RESUMEN

OBJECTIVE: The role of toll-like receptors (TLRs) in vascular remodeling is well established. However, the involvement of the endosomal TLRs is unknown. Here, we study the effect of combined blocking of TLR7 and TLR9 on postinterventional remodeling and accelerated atherosclerosis. METHODS AND RESULTS: In hypercholesterolemic apolipoprotein E*3-Leiden mice, femoral artery cuff placement led to strong increase of TLR7 and TLR9 presence demonstrated by immunohistochemistry. Blocking TLR7/9 with a dual antagonist in vivo reduced neointimal thickening and foam cell accumulation 14 days after surgery by 65.6% (P=0.0079). Intima/media ratio was reduced by 64.5% and luminal stenosis by 62.8%. The TLR7/9 antagonist reduced the arterial wall inflammation, with reduced macrophage infiltration, decreased cytoplasmic high-mobility group box 1 expression, and altered serum interleukin-10 levels. Stimulation of cultured macrophages with TLR7 and TLR9 ligands enhanced tumor necrosis factor-α expression, which is decreased by TLR7/9 antagonist coadministration. Additionally, the antagonist abolished the TLR7/9-enhanced low-density lipoprotein uptake. The antagonist also reduced oxidized low-density lipoprotein-induced foam cell formation, most likely not via decreased influx but via increased efflux, because CD36 expression was unchanged whereas interleukin-10 levels were higher (36.1 ± 22.3 pg/mL versus 128.9 ± 6.6 pg/mL; P=0.008). CONCLUSIONS: Blocking TLR7 and TLR9 reduced postinterventional vascular remodeling and foam cell accumulation indicating TLR7 and TLR9 as novel therapeutic targets.

Asunto(s)

Aterosclerosis/etiología , Movimiento Celular , Vasos Coronarios/patología , Células Espumosas/fisiología , Activación de Macrófagos , Glicoproteínas de Membrana/fisiología , Receptor Toll-Like 7/fisiología , Receptor Toll-Like 9/fisiología , Angioplastia Coronaria con Balón , Animales , Citocinas/biosíntesis , Proteína HMGB1/análisis , Lipoproteínas LDL/fisiología , Glicoproteínas de Membrana/antagonistas & inhibidores , Ratones , Neointima/prevención & control , Receptor Toll-Like 7/antagonistas & inhibidores , Receptor Toll-Like 9/antagonistas & inhibidores

RESUMEN

Synthetic alkylphospholipids (APLs), such as edelfosine, miltefosine and perifosine, constitute a new class of antineoplastic compounds with various clinical applications. Here we have evaluated the antiangiogenic properties of APLs. The sensitivity of three types of vascular endothelial cells (ECs) (bovine aortic ECs, human umbilical vein ECs and human microvascular ECs) to APL-induced apoptosis was dependent on the proliferative status of these cells and correlated with the cellular drug incorporation. Although confluent, nondividing ECs failed to undergo apoptosis, proliferating ECs showed a 3-4-fold higher uptake and significant levels of apoptosis after APL treatment. These findings raised the question of whether APLs interfere with new blood vessel formation. To test the antiangiogenic properties in vitro, we studied the effect of APLs using two different experimental models. The first one tested the ability of human microvascular ECs to invade a three-dimensional human fibrin matrix and form capillary-like tubular networks. In the second model, bovine aortic ECs were grown in a collagen gel sandwich to allow tube formation. We found that all three APLs interfered with endothelial tube formation in a dose-dependent manner, with a more than 50% reduction at 25 micromol/l. Interference with the angiogenic process represents a novel mode of action of APLs and might significantly contribute to the antitumor effect of these compounds.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Endotelio Vascular/efectos de los fármacos , Fosfolípidos/farmacología , Alquilación , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/metabolismo , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Capilares/efectos de los fármacos , Capilares/crecimiento & desarrollo , Bovinos , Línea Celular Tumoral , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/citología , Endotelio Vascular/crecimiento & desarrollo , Humanos , Indicadores y Reactivos , Inhibidores de Fosfodiesterasa/farmacología , Éteres Fosfolípidos/farmacología , Fosfolípidos/química , Fosfolípidos/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacología

RESUMEN

OBJECTIVE: Emerging evidence suggests that human blood contains bone marrow (BM)-derived endothelial progenitor cells that contribute to postnatal neovascularization. Clinical trials demonstrated that administration of BM-cells can enhance neovascularization. Most studies, however, used crude cell populations. Identifying the role of different cell populations is important for developing improved cellular therapies. METHODS AND RESULTS: Effects of the hematopoietic stem cell-containing CD34+ cell population on migration, proliferation, differentiation, stimulation of, and participation in capillary-like tubule formation were assessed in an in vitro 3-dimensional matrix model using human microvascular endothelial cells. During movement over the endothelial monolayer, CD34+ cells remained stuck at sites of capillary tube formation and time- and dose-dependently formed cell clusters. Immunohistochemistry confirmed homing and proliferation of CD34+ cells in and around capillary sprouts. CD34+ cells were transduced with the LNGFR marker gene to allow tracing. LNGFR gene-transduced CD34+ cells integrated in the tubular structures and stained positive for CD31 and UEA-1. CD34+ cells alone stimulated neovascularization by 17%. Coculture with CD34- cells led to 68% enhancement of neovascularization, whereas CD34- cells displayed a variable response by themselves. Cell-cell contact between CD34+ and CD34- cells facilitated endothelial differentiation of CD34+ cells. CONCLUSIONS: Our data suggest that administration of CD34+-enriched cell populations may significantly improve neovascularization and point at an important supportive role for (endogenous or exogenous) CD34- cells.

Asunto(s)

Capilares/citología , Endotelio Vascular/citología , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Neovascularización Fisiológica/fisiología , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular/fisiología , División Celular/fisiología , Movimiento Celular/fisiología , Separación Celular , Células Cultivadas , Técnicas de Cocultivo , Células Madre Hematopoyéticas/metabolismo , Humanos

RESUMEN

The aminopeptidase inhibitor bestatin has been shown to have anti-angiogenic effects in a number of model systems. These effects are thought to result from inhibition of CD13 activity. Because tumor angiogenesis can evolve in a fibrin-rich stroma matrix we have studied for the first time the effects of bestatin on microvascular endothelial capillary-like tube formation in a fibrin matrix. Bestatin enhanced the formation of capillary-like tubes dose-dependently. Its effects were apparent at 8 micro M; the increase was 3.7-fold at 125 micro M; while high concentrations (>250 micro M), that were shown to have anti-angiogenic effects in other systems, caused extensive matrix degradation. Specific CD13-blocking antibodies WM15 and MY-7, and the aminopeptidase inhibitors amastatin and actinonin also enhanced capillary-like tube formation (maximally 1.5-fold), but these effects did not reach statistical significance. The effect of bestatin was not due to a change in uPAR availability because the relative involvement of the u-PA/u-PAR activity was not altered by bestatin. In view of the present findings we hypothesize that aminopeptidases other than CD13 predominantly contribute to the observed pro-angiogenic effect of bestatin in a fibrin matrix. The identification of this novel effect of bestatin is important in the light of the proposed use of bestatin as anti-angiogenic and/or anti-tumor agent.

Asunto(s)

Aminopeptidasas/antagonistas & inhibidores , Endotelio Vascular/efectos de los fármacos , Fibrina/metabolismo , Leucina/análogos & derivados , Leucina/farmacología , Antígenos CD13/análisis , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Humanos , Integrinas/efectos de los fármacos , Microcirculación/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Receptores de Superficie Celular/fisiología , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Venas Umbilicales , Activador de Plasminógeno de Tipo Uroquinasa/fisiología

RESUMEN

To improve current angiogenic gene therapy with a vascular endothelial growth factor (VEGF)-encoding plasmid (Baumgartner et al. Circulation 1998; 97: 1114-23 [1]; Kusumanto et al. Fifth Annual Meeting of the American Society of Gene Therapy, Boston, 2002, Abstr. 621 [2]), we have generated a combination plasmid, encoding the VEGF gene and the thymidine phosphorylase (TP, also known as platelet-derived endothelial growth factor (PD-ECGF) or gliostatin (GLS)) gene: phVEGF165-TP.MB. Upon transfection in COS-7 cells both gene products were expressed and functional as shown by Western blots, ELISAs and bioassays. Culture supernatants of COS-7 cells transfected with this plasmid were able to induce endothelial proliferation. In an in vitro angiogenesis assay with recombinant proteins, TP was able to increase VEGF-induced tube formation. The phVEGF165-TP.MB plasmid is therefore a promising candidate for in vivo angiogenesis studies.

Asunto(s)

Terapia Genética/métodos , Neovascularización Fisiológica/efectos de los fármacos , Timidina Fosforilasa/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Animales , Células COS , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Quimioterapia Combinada , Endotelio Vascular/citología , Vectores Genéticos , Humanos , Timidina Fosforilasa/genética , Timidina Fosforilasa/farmacología , Timina/biosíntesis , Transfección , Venas Umbilicales , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/farmacología

RESUMEN

Adult angiogenesis, associated with pathologic conditions, is often accompanied by the formation of a fibrinous exudate. This temporary matrix consists mainly of fibrin but is intermingled with plasma proteins and collagen fibers. The formation of capillary structures in a fibrinous matrix in vivo was mimicked by an in vitro model, in which human microvascular endothelial cells (hMVECs) seeded on top of a fibrin-10% collagen matrix form capillarylike tubular structures after stimulation with basic fibroblast growth factor/tumor necrosis factor alpha (bFGF/TNF-alpha) or vascular endothelial growth factor (VEGF)/TNF-alpha. In the fibrin-collagen matrix the metalloproteinase inhibitor BB94 inhibited tubule formation by 70% to 80%. Simultaneous inhibition of plasmin and metalloproteinases by aprotinin and BB94 caused a nearly complete inhibition of tubule formation. Adenoviral transduction of tissue inhibitor of metalloproteinases 1 (TIMP-1) and TIMP-3 into endothelial cells revealed that TIMP-3 markedly inhibited angiogenesis, whereas TIMP-1 had only a minor effect. Immunohistochemical analysis showed the presence of matrix metalloproteinase 1 (MMP-1), MMP-2, and membrane-type 1 (MT1)-MMP, whereas MMP-9 was absent. The endothelial production of these MMPs was confirmed by antigen assays and real-time polymerase chain reaction (PCR). MT1-MMP mRNA was markedly increased in endothelial cells under conditions that induced tubular structures. The presence of MMP-1, MMP-2, and MT1-MMP was also demonstrated in vivo in the newly formed vessels of a recanalized arterial mural thrombus. These data suggest that MMPs, in particular MT-MMPs, play a pivotal role in the formation of capillarylike tubular structures in a collagen-containing fibrin matrix in vitro and may be involved in angiogenesis in a fibrinous exudate in vivo.

Asunto(s)

Metaloproteinasa 1 de la Matriz/fisiología , Metaloproteinasa 2 de la Matriz/fisiología , Metaloendopeptidasas/fisiología , Neovascularización Patológica/enzimología , Neovascularización Fisiológica/fisiología , Fenilalanina/análogos & derivados , Adenoviridae/genética , Aprotinina/farmacología , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Colágeno , Medios de Cultivo/farmacología , Virus Defectuosos/genética , Sinergismo Farmacológico , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Fibrina , Factor 2 de Crecimiento de Fibroblastos/farmacología , Vectores Genéticos/genética , Cardiopatías/patología , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Linfocinas/farmacología , Metaloproteinasas de la Matriz Asociadas a la Membrana , Neovascularización Patológica/fisiopatología , Fenilalanina/farmacología , Inhibidores de Proteasas/farmacología , Tiofenos/farmacología , Trombosis/patología , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/fisiología , Inhibidor Tisular de Metaloproteinasa-3/genética , Inhibidor Tisular de Metaloproteinasa-3/fisiología , Transducción Genética , Factor de Necrosis Tumoral alfa/farmacología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular