RESUMO
We identified murine miR-322, orthologous to human miR-424, as a new regulator of insulin receptor, IGF-1 receptor and sirtuin 4 mRNA in vitro and in vivo in the heart and found that miR-322/424 is highly expressed in the heart of mice. C57Bl/6N mice fed 10weeks of high fat diet (HFD) presented signs of cardiomyopathy and a stable miR-322 cardiac level while cardiac function was slightly affected in 11week-old ob/ob which overexpressed miR-322. We thus hypothesized that mmu-miR-322 could be protective against cardiac consequences of hyperinsulinemia and hyperlipidemia. We overexpressed or knocked-down mmu-miR-322 using AAV9 and monitored cardiac function in wild-type C57Bl/6N mice fed a control diet (CD) or a HFD and in ob/ob mice. The fractional shortening progressively declined while the left ventricle systolic diameter increased in HFD mice infected with an AAVcontrol or with an AAVsponge (decreasing miR-322 bioavailability) but also in ob/ob mice infected with AAVsponge. Similar observations were also found in CD-fed mice infected with AAVsponge. On the contrary over-expressing miR-322 with AAVmiR-322 was efficient in protecting the heart from HFD effects in C57Bl/6N mice. This cardioprotection could be associated with the regulation of identified targets IGF1R, INSR and CD1, a decrease in insulin signaling pathway and an enrichment of genes involved in mitochondrial function and fatty acid oxidation as demonstrated by transcriptome analysis. Altogether, these results emphasize miR-322 as a new potential therapeutic target against cardiac consequences of metabolic syndrome, which represents an increasing burden in the western countries.
Assuntos
Cardiopatias/metabolismo , Insulina/metabolismo , Síndrome Metabólica/metabolismo , MicroRNAs/biossíntese , Transdução de Sinais , Animais , Dependovirus , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Vetores Genéticos , Cardiopatias/genética , Cardiopatias/patologia , Cardiopatias/terapia , Humanos , Hiperinsulinismo/genética , Hiperinsulinismo/metabolismo , Hiperinsulinismo/patologia , Hiperinsulinismo/terapia , Hiperlipidemias/genética , Hiperlipidemias/metabolismo , Hiperlipidemias/patologia , Hiperlipidemias/terapia , Insulina/genética , Masculino , Síndrome Metabólica/genética , Síndrome Metabólica/patologia , Síndrome Metabólica/terapia , Camundongos , Camundongos Obesos , MicroRNAs/genética , Ratos , Ratos Wistar , Transdução GenéticaRESUMO
Vascular endothelial growth factor (VEGF)-induced blood vessel growth is involved in both physiological and pathological angiogenesis and requires integrin-mediated signaling. We now show that an integrin-binding protein initially described in milk-fat globule, MFG-E8 (also known as lactadherin), is expressed in and around blood vessels and has a crucial role in VEGF-dependent neovascularization in the adult mouse. Using neutralizing antibodies and lactadherin-deficient animals, we show that lactadherin interacts with alphavbeta3 and alphavbeta5 integrins and alters both VEGF-dependent Akt phosphorylation and neovascularization. In the absence of VEGF, lactadherin administration induced alphavbeta3- and alphavbeta5-dependent Akt phosphorylation in endothelial cells in vitro and strongly improved postischemic neovascularization in vivo. These results show a crucial role for lactadherin in VEGF-dependent neovascularization and identify lactadherin as an important target for the modulation of neovascularization.
Assuntos
Indutores da Angiogênese/metabolismo , Antígenos de Superfície/metabolismo , Proteínas do Leite/metabolismo , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Análise de Variância , Animais , Southern Blotting , Cruzamentos Genéticos , Feminino , Vetores Genéticos , Humanos , Integrina alfaVbeta3/metabolismo , Isquemia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-aktRESUMO
Loss-of-function mutations in the cardiac Na+ channel α-subunit Nav1.5, encoded by SCN5A, cause Brugada syndrome (BrS), a hereditary disease characterized by sudden cardiac death due to ventricular fibrillation. We previously evidenced in vitro the dominant-negative effect of the BrS Nav1.5-R104W variant, inducing retention of wild-type (WT) channels and leading to a drastic reduction of the resulting Na+ current (I Na ). To explore this dominant-negative effect in vivo, we created a murine model using adeno-associated viruses (AAVs). METHODS: Due to the large size of SCN5A, a dual AAV vector strategy was used combining viral DNA recombination and trans-splicing. Mice were injected with two AAV serotypes capsid 9: one packaging the cardiac specific troponin-T promoter, the 5' half of hSCN5A cDNA, a splicing donor site and a recombinogenic sequence; and another packaging the complementary recombinogenic sequence, a splicing acceptor site, the 3' half of hSCN5A cDNA fused to the gfp gene sequence, and the SV40 polyA signal. Eight weeks after AAV systemic injection in wild-type (WT) mice, echocardiography and ECG were recorded and mice were sacrificed. The full-length hSCN5A-gfp expression was assessed by western blot and immunohistochemistry in transduced heart tissues and the Na+ current was recorded by the patch-clamp technique in isolated adult GFP-expressing heart cells. RESULTS: Almost 75% of the cardiomyocytes were transduced in hearts of mice injected with hNav1.5 and â¼30% in hNav1.5-R104W overexpressing tissues. In ventricular mice cardiomyocytes expressing R104W mutant channels, the endogenous I Na was significantly decreased. Moreover, overexpression of R104W channels in normal hearts led to a decrease of total Nav1.5 expression. The R104W mutant also induced a slight dilatation of mice left ventricles and a prolongation of RR interval and P-wave duration in transduced mice. Altogether, our results demonstrated an in vivo dominant-negative effect of defective R104W channels on endogenous ones. CONCLUSION: Using a trans-splicing and viral DNA recombination strategy to overexpress the Na+ channel in mouse hearts allowed us to demonstrate in vivo the dominant-negative effect of a BrS variant identified in the N-terminus of Nav1.5.
RESUMO
Inflammatory cell infiltration is a feature of postischemic neovascularization. However, mechanisms leading to leukocyte attraction to the site of neovascularization are still undefined. We hypothesized that the CXC chemokine receptor 3 (CXCR3) may contribute to leukocyte accumulation and subsequently to blood vessel growth in the ischemic area. Ischemia induced by femoral artery ligature improved the number of CXCR3-expressing cells and the level of its ligand, CXCL10. Angiographic score, blood flow recovery measurement, and capillary density analysis showed a significant decrease of ischemic/nonischemic leg ratio in CXCR3-deficient mice when compared with controls (P<0.05), at day 21 after ischemia. Interestingly, this impairment was as important as that observed in mice deficient for the well known CC-chemokine monocyte chemoattractant protein-1 (MCP-1). At day 7 of ischemic injury, the number of CD3-positive T cells and Mac-3-positive monocytes/macrophages was 38% and 45% lower, respectively, in the ischemic leg of CXCR3-deficient mice compared with the control group (P<0.05), suggesting an important role for CXCR3 in leukocyte recruitment into the ischemic area. VEGF protein content, a classical proangiogenic factor, was also markedly reduced (80% reduction) in ischemic leg of CXCR3-deficient mice (P<0.01). Injection of bone marrow-derived mononuclear cells (BM-MNCs) isolated from wild-type animals restored the neovascularization reaction in CXCR3-deficient mice whereas BM-MNCs from CXCR3-deficient mice was ineffective. In conclusion, CXCR3 plays a key role in neovascularization and provides novel information on the mechanisms leading to leukocyte infiltration in the vessel growth area.
Assuntos
Membro Posterior/irrigação sanguínea , Isquemia/fisiopatologia , Neovascularização Fisiológica/fisiologia , Receptores de Quimiocinas/fisiologia , Animais , Arteríolas/metabolismo , Transplante de Medula Óssea , Capilares/metabolismo , Quimiocina CCL2/deficiência , Quimiocina CCL2/fisiologia , Quimiocina CXCL10 , Quimiocina CXCL9 , Quimiocinas CXC/biossíntese , Quimiocinas CXC/genética , Quimiotaxia de Leucócito/fisiologia , Artéria Femoral , Isquemia/terapia , Fluxometria por Laser-Doppler , Ligadura , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/genética , Receptores CXCR3 , Receptores de Quimiocinas/deficiência , Receptores de Quimiocinas/genética , Subpopulações de Linfócitos T/fisiologia , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
OBJECTIVE: We analyzed the involvement of thromboxane (TX) A2/prostaglandin (PG) H2 (TP) receptor in ischemia-induced neovascularization in mice. METHODS AND RESULTS: Unilateral hindlimb ischemia was induced by right femoral artery ligature in male C57BL/6J mice (n=7 per group). Animals were then treated with or without TP receptor antagonist (S18886, 5 or 10 mg/kg per day; ramatroban, 10 mg/kg per day) or aspirin (30 mg/kg per day) in drinking water for 21 days. Hindlimb ischemia raised plasma level of TXB2, the stable metabolite of TXA2, by 4.7-fold. This increase was blocked by aspirin treatment whereas S18886 (5 or 10 mg/kg per day) had no effect. However, neither S 18886 nor aspirin affected postischemic neovascularization. We next assessed the putative involvement of TXA2 signaling in angiotensin II (Ang II) proangiogenic pathway. Ang II (0.3 mg/kg per day) enhanced TXB2 plasma levels by 2.6-fold over that of control (P<0.01). Ang II-induced TXB2 upregulation was reduced by cotreatment with Ang II type I receptor antagonist (candesartan, 20 mg/kg per day). Angiographic score, capillary number, and foot perfusion were improved by 1.7-, 1.7-, and 1.4-fold, respectively, in Ang II-treated mice compared with controls (P<0.05). Ang II proangiogenic effect was associated with a 1.6-fold increase in VEGF-A protein content (P<0.05) and a 1.4-fold increase in the number of Mac-3-positive cells (ie, macrophages) in ischemic areas (P<0.05). Interestingly, treatments with TP receptor antagonists or aspirin hampered the proangiogenic effects of Ang II. CONCLUSIONS: Endogenous activation of TXA2 receptor by eicosanoids did not modulate spontaneous neovascularization in the setting of ischemia. Conversely, TXA2 signaling is involved in Ang II-induced AT1-dependent vessel growth.
Assuntos
Angiotensina II/sangue , Isquemia/metabolismo , Neovascularização Fisiológica/fisiologia , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Vasoconstritores/sangue , Angiotensina II/farmacologia , Animais , Capilares/fisiologia , Membro Posterior/irrigação sanguínea , Isquemia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Naftalenos/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Propionatos/farmacologia , Receptores de Tromboxano A2 e Prostaglandina H2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tromboxano A2/sangue , Tromboxano B2/sangue , Vasculite/metabolismo , Vasculite/fisiopatologia , Vasoconstritores/farmacologiaRESUMO
Vascular endothelial growth factors (VEGFs) and their receptors have emerged as central regulators of the angiogenic process. However, involvement of VEGF-B, one of these factors, in angiogenesis remains obscure. Mice received subcutaneous injection of Matrigel alone or Matrigel with human recombinant protein rhVEGF-B167 or with rhVEGF-A165. After 14 days, cell ingrowth in the Matrigel plug was increased by 2.0- and 2.5-fold in rhVEGF-B167-treated and rhVEGF-A165-treated mice, respectively (P<0.01), in association with a raise in phospho-Akt/Akt (1.8-fold, P<0.01) and endothelial NO synthase (eNOS) (1.80- and 1.60-fold, respectively; P<0.05) protein levels measured by Western blot. VEGF-B-induced cell ingrowth was impaired by treatment with NOS inhibitor (NG-nitro-l-arginine methyl ester; L-NAME, 10 mg/kg per day). Treatment with neutralizing antibody directed against the VEGF-B receptor VEGF-R1 (anti-VEGFR1, 10 microg) completely abrogated VEGF-B-related effects. Proangiogenic effect of VEGF-B was confirmed in a mouse model of surgically induced hindlimb ischemia. Plasmids containing human form of VEGF-A (phVEGF-A165) or VEGF-B (phVEGF-B167 or phVEGF-B186) were administered by in vivo electrotransfer. Angiographic score at day 28 showed significant improvement in ischemic/nonischemic leg ratio by 1.4- and 1.5-fold in mice treated with phVEGF-B167 and phVEGF-B186, respectively (P<0.05). Laser Doppler perfusion data also evidenced a 1.5-fold increase in phVEGF-B167-treated and phVEGF-B186-treated mice (P<0.05). Such an effect was associated with an upregulation of phospho-Akt/Akt and eNOS protein levels in the ischemic legs and was hampered by treatment with anti-VEGFR1. This study demonstrates for the first time that VEGF-B, in part through its receptor VEGF-R1, promotes angiogenesis in association with an activation of Akt and eNOS-related pathways.
Assuntos
Fatores de Crescimento Endotelial/fisiologia , Neovascularização Fisiológica/fisiologia , Proteínas Serina-Treonina Quinases , Animais , Arteríolas/efeitos dos fármacos , Bioensaio , Capilares/efeitos dos fármacos , Contagem de Células , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Colágeno/metabolismo , Combinação de Medicamentos , Fatores de Crescimento Endotelial/farmacologia , Feminino , Membro Posterior/irrigação sanguínea , Membro Posterior/fisiopatologia , Isquemia/fisiopatologia , Laminina/metabolismo , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Fosforilação , Proteoglicanas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator B de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Identification of factors that may stimulate ischemia-induced neovascularization without increasing atherosclerotic plaque progression is of major therapeutic importance. We hypothesized that interleukin-18 binding protein (IL-18BP), a major antiinflammatory protein with plaque-stabilizing activities, may affect the neovascularization in mice ischemic hindlimb. Ischemia was produced by artery femoral occlusion in mice that were subjected to in vivo intramuscular electrotransfer of either an empty plasmid or a murine IL-18BP plasmid. Angiographic score, capillary density (CD31 staining), and laser Doppler perfusion data at day 28 showed significant improvement in ischemic/nonischemic leg ratio by respectively 1.6-, 1.4-, and 1.5-fold in IL-18BP-treated mice compared with controls (P<0.01). This was associated with a significant 2-fold increase in both vascular endothelial growth factor (VEGF) and phospho-Akt protein content in the ischemic hindlimb of IL-18BP-treated mice (P<0.05). Similar results were obtained in IL-18-deficient mice. Because bone marrow-derived endothelial progenitor cells (BM-EPCs) are involved in postnatal vasculogenesis, EPCs were isolated and cultivated from bone marrow mononuclear cells. IL-18BP treatment led to a significant 1.8-fold increase in the percentage of BM-EPCs characterized as cells positive for both AcLDL-Dil and von Willebrand factor (P<0.001). In conclusion, IL-18BP stimulates ischemia-induced neovascularization in association with an activation of VEGF/Akt signaling and an increase in BM-EPCs mobilization and differentiation. Our findings strongly suggest a major antiangiogenic role of endogenous IL-18 in postischemic injury.
Assuntos
Glicoproteínas/fisiologia , Membro Posterior/irrigação sanguínea , Interleucina-18/fisiologia , Isquemia/fisiopatologia , Neovascularização Patológica/fisiopatologia , Proteínas Serina-Treonina Quinases , Angiografia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/fisiologia , Linhagem Celular , Fatores de Crescimento Endotelial/metabolismo , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Glicoproteínas/genética , Membro Posterior/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Interleucina-18/genética , Interleucina-18/farmacologia , Fluxometria por Laser-Doppler , Linfocinas/metabolismo , Linfocinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Fosforilação , Plasmídeos/administração & dosagem , Plasmídeos/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
OBJECTIVE: We analyzed the beneficial therapeutic effect of angiotensin converting enzyme inhibitor (ACEI) on both retinal and hind limb neovascularization in diabetic mice. METHODS AND RESULTS: Diabetic mice (streptozotocin, 40 mg/kg) were treated with or without ACEI (Perindopril, 3 mg/kg per day) or AT1 receptor blocker (Candesartan, 20 mg/kg) for 4 months. Hind limb ischemia was then induced by right femoral artery ligature for 1 additional month. In the ischemic leg, angiographic score, capillary density, and foot perfusion were increased by 2.7, 2.0-fold, and 1.6-fold, respectively, in ACEI-treated diabetic mice compared with untreated diabetic animals (P<0.01). ACEI also raised vascular endothelial growth factor (VEGF) protein level by 1.4-fold in ischemic diabetic leg. This ACEI pro-angiogenic effect was totally blunted in diabetic bradykinin B2 receptor-deficient animals, suggesting that it was mediated by the bradykinin pathway. In the diabetic retina, angiotensinogen and ACE mRNA levels were increased by 2.8-fold and 4.1-fold, respectively (P<0.01 versus nondiabetic mice), highlighting a local activation of renin-angiotensin system. Diabetes also raised VEGF protein level by 1.5-fold (P<0.05 versus nondiabetic mice). Treatments with ACEI and AT1 receptor blocker hampered diabetes-induced VEGF upregulation and retinal neovascularization. CONCLUSIONS: ACE inhibition improved neovascularization in the diabetic ischemic leg through activation of bradykinin signaling, whereas it reduced vessel growth in the diabetic retina through inhibition of overacting Ang II pathway.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Diabetes Mellitus Tipo 1/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Angiografia/métodos , Animais , Peso Corporal , Bradicinina/metabolismo , Capilares/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/induzido quimicamente , Membro Posterior/irrigação sanguínea , Membro Posterior/efeitos dos fármacos , Isquemia/metabolismo , Fluxometria por Laser-Doppler/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Mensageiro/metabolismo , Retina/química , Retina/metabolismo , Retina/patologia , Vasos Retinianos/efeitos dos fármacos , EstreptozocinaRESUMO
BACKGROUND: Angiogenesis gene therapy with human tissue kallikrein (hTK) has shown promise for ischemic disease. The present study was undertaken to (1) assess an optimal gene transfer modality, (2) clarify hTK angiogenic pathways, and (3) discount possible side effects. METHODS AND RESULTS: The hTK gene was transferred to murine adductors by increasing doses of an adenovirus (Ad.hTK). Heterologous protein production was evaluated by ELISA and immunohistochemistry. Structural and functional characteristics of hTK-induced neovascularization were assessed. Muscular endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF)-A mRNA and protein content were evaluated by real-time polymerase chain reaction and Western blotting. The ability of hTK to phosphorylate-activate Akt/protein kinase B (Akt-B) and VEGF receptor 2 (VEGF-R2) was also determined. Implication of the aforementioned mechanisms in Ad.hTK-induced neovascularization was challenged by blocking Akt-B with a dominant-negative Akt construct; NOS with N(G)-nitro-L-arginine methyl ester; and VEGF-A with neutralizing antibody, VEGF-R2 antagonist, or Ad carrying soluble VEGF-R1 gene. We found that 10(7) PFU Ad.hTK led to peak increases in capillary and arteriole density. Newly developed arterioles persisted for up to 8 weeks. Ad.hTK did not change microvascular permeability. Ad.hTK upregulated eNOS mRNA and protein and activated Akt-B through Ser-473 phosphorylation. Inhibitory studies documented that these biochemical events were instrumental to Ad.hTK-induced neovascularization. In contrast, Ad.hTK neither affected VEGF-A and VEGF-R2 levels nor increased VEGF-R2 phosphorylation. Consistently, Ad.hTK-induced neovascularization was not disturbed by any of the different approaches used to block VEGF-A. CONCLUSIONS: Our findings provide new information on the pathway involved in hTK-induced neoangiogenesis and represent an advancement toward clinical applications with Ad.hTK.
Assuntos
Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico Sintase/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Calicreínas Teciduais/fisiologia , Adenoviridae/genética , Animais , Western Blotting , Divisão Celular , Células Endoteliais/citologia , Endotélio Vascular/citologia , Indução Enzimática/efeitos dos fármacos , Exsudatos e Transudatos , Genes Dominantes , Vetores Genéticos/farmacologia , Humanos , Camundongos , Microcirculação , Músculo Esquelético/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Neovascularização Fisiológica/genética , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Fosforilação , Fosfosserina/análise , Reação em Cadeia da Polimerase , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Calicreínas Teciduais/genética , Transdução Genética , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/análiseRESUMO
BACKGROUND: Bone marrow-derived mononuclear cells (BM-MNCs) enhance postischemic neovascularization, and their therapeutic use is currently under clinical investigation. We evaluated the safety of BM-MNC-based therapy in the setting of atherosclerosis. METHODS AND RESULTS: Apolipoprotein E (apoE)-knockout (KO) mice were divided into 4 groups: 20 nonischemic mice receiving intravenous injection of either saline (n=10) or 10(6) BM-MNCs from wild-type animals (n=10) and 20 mice with arterial femoral ligature receiving intravenous injection of either saline (n=10) or 10(6) BM-MNCs from wild-type animals (n=10) at the time of ischemia induction. Animals were monitored for 4 additional weeks. Atherosclerosis was evaluated in the aortic sinus. BM-MNC transplantation improved tissue neovascularization in ischemic hind limbs, as revealed by the 210% increase in angiography score (P<0.0001), the 33% increase in capillary density (P=0.01), and the 65% increase in tissue Doppler perfusion score (P=0.0002). Hindlimb ischemia without BM-MNC transplantation or BM-MNC transplantation without ischemia did not affect atherosclerotic plaque size. However, transplantation of 10(6) BM-MNCs into apoE-KO mice with hindlimb ischemia induced a significant 48% to 72% increase in lesion size compared with the other 3 groups (P=0.0025), despite similar total cholesterol levels. Transplantation of 10(5) BM-MNCs produced similar results, whereas transplantation of 10(6) apoE-KO-derived BM-MNCs had neither proangiogenic nor proatherogenic effects. There was no difference in plaque composition between groups. CONCLUSIONS: BM-MNC therapy is unlikely to affect atherosclerotic plaque stability in the short term. However, it may promote further atherosclerotic plaque progression in an ischemic setting.
Assuntos
Apolipoproteínas E/deficiência , Arteriosclerose/terapia , Transplante de Medula Óssea/efeitos adversos , Membro Posterior/irrigação sanguínea , Isquemia/terapia , Neovascularização Fisiológica , Seio Aórtico/patologia , Animais , Apolipoproteínas E/genética , Arteriosclerose/metabolismo , Quimiocina CCL2/sangue , Colesterol/sangue , Progressão da Doença , Artéria Femoral , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Segurança , Falha de Tratamento , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
BACKGROUND: Adipose tissue development and remodeling are closely associated with the growth of vascular network. We hypothesized that adipose tissue may contain progenitor cells with angiogenic potential and that therapy based on adipose tissue-derived progenitor cells administration may constitute a promising cell therapy in patients with ischemic disease. METHODS AND RESULTS: In mice, cultured stromal-vascular fraction (SVF) cells from adipose tissue have a great proangiogenic potential, comparable to that of bone marrow mononuclear cells in the mouse ischemic hindlimb model. Similarly, cultured human SVF cells differentiate into endothelial cells, incorporate into vessels, and promote both postischemic neovascularization in nude mice and vessel-like structure formation in Matrigel plug. In vitro, these cells represent a homogeneous population of CD34- and CD13-positive cells, which can spontaneously express the endothelial cell markers CD31 and von Willebrand factor when cultured in semisolid medium. Interestingly, dedifferentiated mature human adipocytes have the potential to rapidly acquire the endothelial phenotype in vitro and to promote neovascularization in ischemic tissue and vessel-like structure formation in Matrigel plug, suggesting that cells of endothelial and adipocyte phenotypes may have a common precursor. CONCLUSIONS: This study demonstrates, for the first time, that adipocytes and endothelial cells have a common progenitor. Such adipose lineage cells participate in vascular-like structure formation in Matrigel plug and enhance the neovascularization reaction in ischemic tissue. These results also highlight the concept that adipose lineage cells represent a suitable new cell source for therapeutic angiogenesis in ischemic disease.
Assuntos
Tecido Adiposo/citologia , Endotélio Vascular/citologia , Isquemia/terapia , Neovascularização Fisiológica , Transplante de Células-Tronco , Adipócitos/citologia , Animais , Biomarcadores/análise , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Humanos , Isquemia/diagnóstico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Músculo Esquelético/irrigação sanguínea , Fenótipo , Células-Tronco/citologia , Células-Tronco/metabolismo , Células Estromais/metabolismo , Células Estromais/transplanteRESUMO
UNLABELLED: The ATP-binding cassette transporter MRP4 (encoded by ABCC4) regulates membrane cyclic nucleotides concentrations in arterial cells including smooth muscle cells. MRP4/ABCC4 deficient mice display a reduction in smooth muscle cells proliferation and a prevention of pulmonary hypertension in response to hypoxia. We aimed to study gene transfer of a MRP4/ABCC4 silencing RNA via intratracheal delivery of aerosolized adeno-associated virus 1 (AAV1.shMRP4 or AAV1.control) in a monocrotaline-induced model of pulmonary hypertension in rats. Gene transfer was performed at the time of monocrotaline administration and the effect on the development of pulmonary vascular remodeling was assessed 35 days later. AAV1.shMRP4 dose-dependently reduced right ventricular systolic pressure and hypertrophy with a significant reduction with the higher doses (i.e., >10(11) DRP/animal) as compared to AAV1. CONTROL: The higher dose of AAV1.shMRP4 was also associated with a significant reduction in distal pulmonary arteries remodeling. AAV1.shMRP4 was finally associated with a reduction in the expression of ANF, a marker of cardiac hypertrophy. Collectively, these results support a therapeutic potential for downregulation of MRP4 for the treatment of pulmonary artery hypertension.
RESUMO
After acute ischemia of tissues, neovascularization must be sufficient and fast enough to preserve tissue integrity and organ function, and may thus be considered as a therapeutic strategy. This study examined the possible role of the very-low-dose combination of perindopril (angiotensin-converting enzyme inhibitor) and indapamide (diuretic), used first-line in the treatment of essential hypertension, on ischemia-induced angiogenesis. Ischemia was produced by artery femoral occlusion in rats treated or not with the very-low-dose combination (perindopril 0.76 mg/kg/day + indapamide 0.24 mg/kg/day) or each component given alone at the same dosage for 3 and 28 days. At day 3, angiographic vessel density and laser Doppler perfusion data showed significant improvement in ischemic/nonischemic leg ratio by, respectively, 1.9-fold and 1.5-fold in rats treated with the very-low-dose combination when compared with controls (p < 0.05). This was associated with an increase in vascular endothelial growth factor (VEGF; 2.2-fold) and endothelial nitric-oxide synthase (1.6-fold) protein content in the ischemic hindlimb, assessed by Western blot. At day 28, the very-low-dose combination (3- and 1.6-fold) and perindopril alone (1.8- and 1.4-fold) and indapamide alone (2.0- and 1.4-fold) increased the angiograhic score and blood flow perfusion, respectively, in reference to controls (p < 0.05). Furthermore, addition of VEGF-neutralizing antibody (2.5 microg/kg twice a week) or NOS inhibitor (N(G)-nitro-L-arginine methyl ester, 10 mg/kg/day) prevented the pro-angiogenic effect induced by the perindopril/indapamide combination. The very-low-dose combination of perindopril and indapamide induces an early and sustained effect on the revascularization process observed in ischemic tissue and may provide a favorable therapeutic neovascularization after ischemia.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Diuréticos/administração & dosagem , Indapamida/administração & dosagem , Isquemia/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Perindopril/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Esquema de Medicação , Quimioterapia Combinada , Fatores de Crescimento Endotelial/metabolismo , Membro Posterior/irrigação sanguínea , Membro Posterior/efeitos dos fármacos , Membro Posterior/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Isquemia/metabolismo , Linfocinas/metabolismo , Masculino , Neovascularização Fisiológica/fisiologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III , Ratos , Ratos Wistar , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Mechanisms that hinder ischemia-induced neovascularization in diabetes remain poorly understood. We hypothesized that endogenous bone marrow mononuclear cell (BM-MNC) dysfunction may contribute to the abrogated postischemic revascularization reaction associated with diabetes. We first analyzed the effect of diabetes (streptozotocin, 40 mg/kg) on BM-MNC pro-angiogenic potential in a model of surgically induced hindlimb ischemia. In nondiabetic animals, transplantation of BM-MNCs isolated from nondiabetic animals raised the ischemic/nonischemic angiographic score, capillary number, and blood flow recovery by 1.8-, 2.7-, and 2.2-fold, respectively, over that of PBS-injected nondiabetic animals (P < 0.05). Administration of diabetic BM-MNCs also improved the neovascularization reaction in ischemic hindlimbs of nondiabetic mice but to a lesser extent from that observed with nondiabetic BM-MNC transplantation. In diabetic mice, injection of nondiabetic BM-MNCs was still more efficient than that of diabetic BM-MNCs. Such BM-MNC dysfunction was associated with the impairment of diabetic BM-MNC capacity to differentiate into endothelial progenitor cells (EPCs) in vitro and to participate in vascular-like structure formation in a subcutaneous Matrigel plug. Placenta growth factor (PlGF) administration improved by sixfold the number of EPCs differentiated from diabetic BM-MNCs in vitro and enhanced ischemic/nonischemic angiographic score, capillary number and blood flow recovery by 1.9-, 1.5- and 1.6-fold, respectively, over that of untreated diabetic animals (P < 0.01). Endogenous BM-MNC pro-angiogenic potential was affected in diabetes. Therapeutic strategy based on PlGF administration restored such defects and improved postischemic neovascularization in diabetic mice.