RESUMO
Ischemic preconditioning (IPC) describes a phenomenon wherein brief ischemia of the heart induces a potent cardioprotective mechanism against succeeding ischemic insult. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostanoid biosynthesis, is upregulated in the ischemic heart and contributes to IPC. Prostaglandin E2 (PGE2) protects the heart from ischemia-reperfusion (I/R) injury via its receptor subtype EP4. We sought to clarify the role of the PGE2/EP4 system in the late phase of IPC. Mice were subjected to four IPC treatment cycles, consisting of 5 min of occlusion of the left anterior descending coronary artery (LAD). We found that COX-2 mRNA was significantly upregulated in wild-type hearts at 6 h after IPC treatment. Cardiac PGE2 levels at 24 h after IPC treatment were significantly increased in both wild-type mice and mice lacking EP4 (EP4-/-). At 24 h after IPC treatment, I/R injury was induced by 30 min of LAD occlusion followed by 2 h of reperfusion and the cardiac infarct size was determined. The infarct size was significantly reduced by IPC treatment in wild-type mice; a reduction was not observed in EP4-/- mice. AE1-329, an EP4 agonist, significantly reduced infarct size and significantly ameliorated deterioration of cardiac function in wild-type mice subjected to I/R without IPC treatment. Furthermore, AE1-329 significantly enhanced the I/R-induced activation of Akt, a pro-survival kinase. We demonstrated that the PGE2/EP4 system in the heart plays a critical role in the late phase of IPC, partly by augmenting Akt-mediated signaling. These findings clarify the mechanism of IPC and may contribute to the development of therapeutic strategies for ischemic heart disease.
Assuntos
Precondicionamento Isquêmico Miocárdico , Infarto do Miocárdio , Traumatismo por Reperfusão Miocárdica , Camundongos , Animais , Proteínas Proto-Oncogênicas c-akt/uso terapêutico , Ciclo-Oxigenase 2 , Prostaglandinas/uso terapêuticoRESUMO
Tumors induce angiogenesis to acquire oxygen and nutrition from their adjacent microenvironment. Tumor angiogenesis has been believed to be induced primarily by the secretion of vascular endothelial growth factor-A (VEGF-A) from various tumors. VEGF-A binds to VEGF receptor 2 (VEGFR2), resulting in subsequent activation of cellular substances regulating cell proliferation, survival, and angiogenesis. Antiangiogenic therapies targeting the VEGF-A/VEGFR2 axis, including bevacizumab and ramucirumab, humanized monoclonal antibodies against VEGF-A and VEGFR2, respectively, have been proposed as a promising strategy aimed at preventing tumor growth, invasion, and metastasis. Phase III clinical trials using bevacizumab and ramucirumab have shown that not all tumor patients benefit from such antiangiogenic agents, and that some patients who initially benefit subsequently become less responsive to these antibodies, suggesting the possible existence of VEGF-independent angiogenic factors. In this review, we focus on VEGF-independent and VEGFR2-dependent tumor angiogenesis, as well as VEGFR2-independent tumor angiogenesis. Additionally, we discuss VEGF-independent angiogenic factors which have been reported in previous studies. Various molecular targeting drugs are currently being evaluated as potential antitumor therapies. We expect that precision medicine will permit the development of innovative antiangiogenic therapies targeting individual angiogenic factors selected on the basis of the genetic screening of tumors.
Assuntos
Neoplasias , Fator A de Crescimento do Endotélio Vascular , Indutores da Angiogênese , Inibidores da Angiogênese/uso terapêutico , Bevacizumab/farmacologia , Bevacizumab/uso terapêutico , Humanos , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Adventitial abnormalities including enhanced vasa vasorum malformation are associated with development and vulnerability of atherosclerotic plaque. However, the mechanisms of vasa vasorum malformation and its role in vascular remodeling have not been fully clarified. We recently reported that ninjurin-1 (Ninj1) is a crucial adhesion molecule for pericytes to form matured neovessels. The purpose is to examine if Ninj1 regulates adventitial angiogenesis and affects the vascular remodeling of injured vessels using pericyte-specific Ninj1 deletion mouse model. Mouse femoral arteries were injured by insertion of coiled wire. Four weeks after vascular injury, fixed arteries were decolorized. Vascular remodeling, including intimal hyperplasia and adventitial microvessel formation were estimated in a three-dimensional view. Vascular fragility, including blood leakiness was estimated by extravasation of fluorescein isothiocyanate (FITC)-lectin or FITC-dextran from microvessels. Ninj1 expression was increased in pericytes in response to vascular injury. NG2-CreER/Ninj1loxp mice were treated with tamoxifen (Tam) to induce deletion of Ninj1 in pericyte (Ninj1 KO). Tam-treated NG2-CreER or Tam-nontreated NG2-CreER/Ninj1loxp mice were used as controls. Intimal hyperplasia was significantly enhanced in Ninj1 KO compared with controls. Vascular leakiness was significantly enhanced in Ninj1 KO. In Ninj1 KO, the number of infiltrated macrophages in adventitia was increased, along with the expression of inflammatory cytokines. In conclusion, deletion of Ninj1 in pericytes induces the immature vasa vasorum formation of injured vasculature and exacerbates adventitial inflammation and intimal hyperplasia. Thus, Ninj1 contributes to the vasa vasorum maturation in response to vascular injury and to reduction of vascular remodeling.NEW & NOTEWORTHY Although abnormalities of adventitial vasa vasorum are associated with vascular remodeling such as atherosclerosis, the mechanisms of vasa vasorum malformation and its role in vascular remodeling have not been fully clarified. The present study provides a line of novel evidence that ninjurin-1 contributes to adventitial microvascular maturation during vascular injury and regulates vascular remodeling.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Artéria Femoral/metabolismo , Neointima/genética , Fatores de Crescimento Neural/genética , Pericitos/metabolismo , Vasa Vasorum/metabolismo , Remodelação Vascular/genética , Túnica Adventícia/metabolismo , Túnica Adventícia/patologia , Animais , Artéria Femoral/lesões , Artéria Femoral/patologia , Técnicas de Inativação de Genes , Hiperplasia/genética , Inflamação/genética , Inflamação/metabolismo , Macrófagos/patologia , Camundongos , Neointima/patologia , Neovascularização Fisiológica/genética , Transcriptoma , Túnica Íntima/metabolismo , Túnica Íntima/patologia , Vasa Vasorum/patologia , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/patologiaRESUMO
Ninjurin 1 (Ninj1) is identified as a peripheral nerve injury-induced protein. However, the role of Ninj1 in nerve regeneration is unclear. Schwann cells (SCs) and microvasculature are critical for peripheral nerve regeneration. SCs precursors and microvascular pericytes (PCs), which are nerve/glial antigen 2 (NG2)-positive cells are observed in peripheral nervous system. In this study, we investigated the role of Ninj1 in peripheral nerve regeneration using NG2+cell-specific inducible deletion of Ninj1 mouse model. The number of NG2+cells, which were associated with and without microvessels was increased after sciatic nerve crush injury. There was a significant increase in the expression of Ninj1 and EphA7 in the injured nerve tissue. This increase was mostly observed in NG2+cells. Genetic tracing of NG2+cells was performed using tamoxifen (Tam) treatment on NG2CreERT:R26R-tdTomato mice. The sciatic nerve was injured following the Tam-treatment, then tdTomato-expressing SCs were mostly observed in regenerated SCs at 21 days after nerve injury. Ninj1 gene knockout (Ninj1 KO) in NG2+cells was induced using NG2CreERT:Ninj1loxp mice. Tam-treated-NG2CreERT or Tam-nontreated NG2CreERT:Ninj1loxp mice were used as controls. Following Tam-treatment, the sciatic nerve in each group was injured. Ninj1KO significantly attenuated the expression of the myelin binding protein (MBP) as well as the number of myelinated axons. The expression of MBP in cultured SCs was significantly reduced by SiRNA-mediated Ninj1 knockdown (KD). Ninj1KD also attenuated the differentiation of SCs by isolated EphA7+multipotent PCs. The current data indicate that Ninj1 plays a vital role in peripheral nerve regeneration. This is observed particularly in the myelination process of NG2+cells including SCs precursors and multipotent PCs.
Assuntos
Antígenos/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Fatores de Crescimento Neural/metabolismo , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/fisiopatologia , Proteoglicanas/metabolismo , Células de Schwann/metabolismo , Animais , Antígenos/genética , Moléculas de Adesão Celular Neuronais/genética , Linhagem Celular , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Fatores de Crescimento Neural/genética , Pericitos/citologia , Pericitos/metabolismo , Traumatismos dos Nervos Periféricos/genética , Traumatismos dos Nervos Periféricos/metabolismo , Proteoglicanas/genéticaRESUMO
Objective- Angiogenesis, entire step from endothelial cells (ECs) sprouts to vascular maturation, is a critical response to ischemia. To form functional mature vessels, interactions between ECs and pericytes are essential. Ninj1 (ninjurin1) is an adhesion molecule that contributes to the pathogenesis of neuroinflammation. We recently demonstrated that Ninj1 is expressed in pericytes during angiogenesis. However, the role of Ninj1 in angiogenesis under pathophysiological ischemic conditions has not yet been elucidated. Approach and Results- Ninj1 was detected in microvessels, and its expression was enhanced in ischemic tissues after mouse hindlimb ischemia. Knockdown of Ninj1 was performed by injection of biodegradable microspheres releasing Ninj1-small interfering RNA into muscle tissues. Alternatively, pericyte-specific Ninj1 knockout was induced by tamoxifen treatment of NG2-CreERT/Ninj1-flox mice. Ninj1 knockdown/knockout reduced the formation of blood-circulating functional vessels among total CD31+ microvessels within ischemic tissues and subsequently attenuated color Doppler-assessed blood flow recovery. Ninj1 overexpression enhanced expression of Anpt (angiopoietin) 1, whereas Ninj1 knockdown enhanced the endogenous Anpt1 antagonist, Anpt2 expression in pericytes and inhibited the association of pericytes with ECs and subsequent formation of capillary-like structure, that is, EC tube surrounded with pericytes in 3-dimensional gel culture. Conclusions- Our data demonstrate that Ninj1 is involved in the formation of functional matured vessels through the association between pericytes and ECs, resulting in blood flow recovery from ischemia. These findings further the current our understanding of vascular maturation and may support the development of therapeutics for ischemic diseases.
Assuntos
Moléculas de Adesão Celular Neuronais/deficiência , Células Endoteliais/metabolismo , Deleção de Genes , Isquemia/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Fatores de Crescimento Neural/deficiência , Pericitos/metabolismo , Angiopoietina-1/metabolismo , Angiopoietina-2/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/genética , Comunicação Celular , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Membro Posterior , Isquemia/genética , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/genética , Recuperação de Função Fisiológica , Fluxo Sanguíneo Regional , Transdução de SinaisRESUMO
BACKGROUND: Capillary pericytes (cPCs), the mural cells of microvessels, play an important role in the formation and maintenance of microvessels; however, little is known about the mechanisms of how cPCs regulate angiogenesis. To identify factors that modulate cPC function, genes whose levels were altered in cPCs during neovessel formation were identified through a microarray screen. METHODS AND RESULTS: Ninjurin1 (nerve injury-induced protein, Ninj1) was selected as a candidate factor for angiogenesis regulation. Ninj1 was expressed in capillary cells including endothelial cells (cECs) and was expressed at a higher level in cPCs. Hypoxia induced the gene expression of Ninj1 in addition of vascular endothelial growth factor (VEGF) in cPCs. When cPCs were co-incubated with a thoracic aorta in a three-dimensional Matrigel system, the length of the EC-tubes sprouting from the aorta was increased. Small interfering RNA-mediated downregulation of Ninj1 in cPCs enhanced these cPCs-mediated angiogenic effects, whereas overexpression of Ninj1 attenuated their effects. The production of angiogenic growth factors, such as VEGF and angiopoietin 1, by cPCs was enhanced by the downregulation of Ninj1, and reduced by the overexpression of Ninj1. CONCLUSIONS: Ninj1 is a novel regulator for the angiogenic effect of PCs. Specifically, Ninj1 negatively regulates the formation of neovessels, that is, the EC-tube, by reducing the trophic effects of cPCs.
Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Células Endoteliais/citologia , Neovascularização Fisiológica/fisiologia , Fatores de Crescimento Neural/fisiologia , Pericitos/citologia , Animais , Aorta Torácica , Capilares , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Moléculas de Adesão Celular Neuronais/biossíntese , Moléculas de Adesão Celular Neuronais/genética , Técnicas de Cultura de Células , Hipóxia Celular , Linhagem Celular Transformada , Linhagem da Célula , Técnicas de Cocultura , Colágeno , Combinação de Medicamentos , Perfilação da Expressão Gênica , Genes Reporter , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Isquemia/patologia , Laminina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese , Miócitos de Músculo Liso , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/genética , Técnicas de Cultura de Órgãos , Proteoglicanas , Interferência de RNA , RNA Interferente Pequeno/farmacologiaRESUMO
Adventitial microvessels, vasa vasorum in the vessel walls, have an active role in the vascular remodeling, although its mechanisms are still unclear. It has been reported that microvascular pericytes (PCs) possess mesenchymal plasticity. Therefore, microvessels would serve as a systemic reservoir of stem cells and contribute to the tissues remodeling. However, most aspects of the biology of multipotent PCs (mPCs), in particular of pathological microvessels are still obscure because of the lack of appropriate methods to detect and isolate these cells. In order to examine the characteristics of mPCs, we established immortalized cells residing in adventitial capillary growing at the injured vascular walls. We recently developed in vivo angiogenesis to observe adventitial microvessels using collagen-coated tube (CCT), which also can be used as an adventitial microvessel-rich tissue. By using the CCT, CD146- or NG2-positive cells were isolated from the adventitial microvessels in the injured arteries of mice harboring a temperature-sensitive SV40 T-antigen gene. Several capillary-derived endothelial cells (cECs) and PCs (cPCs) cell lines were established. cECs and cPCs maintain a number of key endothelial and PC features. Co-incubation of cPCs with cECs formed capillary-like structure in Matrigel. Three out of six cPC lines, termed capillary mPCs demonstrated both mesenchymal stem cell- and neuronal stem cell-like phenotypes, differentiating effectively into adipocytes, osteoblasts, as well as schwann cells. mPCs differentiated to ECs and PCs, and formed capillary-like structure on their own. Transplanted DsRed-expressing mPCs were resident in the capillary and muscle fibers and promoted angiogenesis and myogenesis in damaged skeletal muscle. Adventitial mPCs possess transdifferentiation potential with unique phenotypes, including the reconstitution of capillary-like structures. Their phenotype would contribute to the pathological angiogenesis associated with vascular remodeling. These cell lines also provide a reproducible cellular tool for high-throughput studies on angiogenesis, vascular remodeling, and regeneration as well.
Assuntos
Capilares/patologia , Pericitos/fisiologia , Regeneração/fisiologia , Vasa Vasorum/citologia , Remodelação Vascular , Animais , Antígenos , Diferenciação Celular , Separação Celular , Células Endoteliais/fisiologia , Camundongos , Camundongos SCID , Neovascularização Fisiológica , Proteoglicanas , Células-Tronco/fisiologia , TranscriptomaRESUMO
An immature vasa vasorum in the adventitia of arteries has been implicated in induction of the formation of unstable atherosclerotic plaques. Normalization/maturation of the vasa vasorum may be an attractive therapeutic approach for arteriosclerotic diseases. Nerve growth factor (NGF) is a pleotropic molecule with angiogenic activity in addition to neural growth effects. However, whether NGF affects the formation of microvessels in addition to innervation during pathological angiogenesis is unclear. In the present study, we show a new role for NGF in neovessels around injured arterial walls using a novel in vivo angiogenesis assay. The vasa vasorum around arterial walls was induced to grow using wire-mediated mouse femoral arterial injury. When collagen-coated tube (CCT) was placed beside the injured artery for 7-14 days, microvessels grew two-dimensionally in a thin layer on the CCT (CCT-membrane) in accordance with the development of the vasa vasorum. The perivascular nerve was found at not only arterioles but also capillaries in the CCT-membrane. Biodegradable hydrogels containing VEGF and NGF were applied around the injured artery/CCT. VEGF significantly increased the total length and instability of microvessels within the CCT-membrane. In contrast, NGF induced regeneration of the peripheral nerve around the microvessels and induced the maturation and stabilization of microvessels. In an ex vivo nerve-free angiogenesis assay, although NGF potentially stimulated vascular sprouting from aorta tissues, no effects of NGF on vascular maturation were observed. These data demonstrated that NGF had potent angiogenic effects on the microvessels around the injured artery, and especially induced the maturation/stabilization of microvessels in accordance with the regeneration of perivascular nerves.
Assuntos
Artéria Femoral/efeitos dos fármacos , Artéria Femoral/lesões , Microvasos/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Vasa Vasorum/fisiologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Indutores da Angiogênese/farmacologia , Animais , Artéria Femoral/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/inervação , Microvasos/fisiologia , Neovascularização Fisiológica/fisiologia , Vasa Vasorum/inervaçãoRESUMO
We describe three cases of J-wave syndrome in which ventricular fibrillation (VF) was probably induced by corticosteroid therapy. The patients involved were being treated with prednisolone for concomitant bronchial asthma. One of the three patients had only one episode of VF during her long follow-up period (14 years). Two patients had hypokalemia during their VF episodes. Corticosteroids have been shown to induce various types of arrhythmia and to modify cardiac potassium channels. We discuss the possible association between corticosteroid therapy and VF in J-wave syndrome based on the cases we have encountered.
Assuntos
Antiarrítmicos/administração & dosagem , Reanimação Cardiopulmonar/métodos , Desfibriladores Implantáveis , Glucocorticoides/efeitos adversos , Hipopotassemia , Fibrilação Ventricular , Adulto , Asma/tratamento farmacológico , Eletrocardiografia/métodos , Feminino , Glucocorticoides/administração & dosagem , Humanos , Hipopotassemia/induzido quimicamente , Hipopotassemia/complicações , Hipopotassemia/diagnóstico , Masculino , Síndrome , Resultado do Tratamento , Fibrilação Ventricular/diagnóstico , Fibrilação Ventricular/etiologia , Fibrilação Ventricular/terapiaRESUMO
MicroRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. However, the physiological functions of these non-coding RNAs in renal interstitial mesenchymal cells remain unclear. To conclusively evaluate the role of miRNAs, we generated conditional knockout (cKO) mice with platelet-derived growth factor receptor-ß (PDGFR-ß)-specific inactivation of the key miRNA pathway gene Dicer. The cKO mice were subjected to unilateral ureteral ligation, and renal interstitial fibrosis was quantitatively evaluated using real-time polymerase chain reaction and immunofluorescence staining. Compared with control mice, cKO mice had exacerbated interstitial fibrosis exhibited by immunofluorescence staining and mRNA expression of PDGFR-ß. A microarray analysis showed decreased expressions of miR-9-5p, miR-344g-3p, and miR-7074-3p in cKO mice compared with those in control mice, suggesting an association with the increased expression of PDGFR-ß. An analysis of the signaling pathways showed that the major transcriptional changes in cKO mice were related to smooth muscle cell differentiation, regulation of DNA metabolic processes and the actin cytoskeleton, positive regulation of fibroblast proliferation and Ras protein signal transduction, and focal adhesion-PI3K/Akt/mTOR signaling pathways. Depletion of Dicer in mesenchymal cells may downregulate the signaling pathway related to miR-9-5p, miR-344g-3p, and miR-7074-3p, which can lead to the progression of chronic kidney disease. These findings highlight the possibility for future diagnostic or therapeutic developments for renal fibrosis using miR-9-5p, miR-344g-3p, and miR-7074-3p.
Assuntos
Fibrose , Rim , Células-Tronco Mesenquimais , Camundongos Knockout , MicroRNAs , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Ribonuclease III , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Camundongos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Rim/patologia , Rim/metabolismo , Células-Tronco Mesenquimais/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Transdução de Sinais , Nefropatias/genética , Nefropatias/patologia , Nefropatias/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , MasculinoRESUMO
Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that processes DNA-repair function and controls cellular response to oxidative stress. Endothelial progenitor cells (EPCs) are recruited to oxidative stress-rich injured vascular walls and positively contribute to vascular repair and endothelialization. We hypothesized that APE1 functions for EPCs-mediated inhibition of neointima formation in injured vasculature. EPCs isolated from bone marrow cells of C57BL6 mice (12-16 wk old) were able to survive in the presence of hydrogen peroxide (H2O2; up to 1,000 µM) due to the highly expressed reactive oxygen species (ROS) scavengers. However, adhesion capacity of EPCs was significantly inhibited by H2O2 (100 µM) even though an intracellular ROS was retained at small level. An APE1-selective inhibitor or RNA interference-mediated knockdown of endogenous APE1 in EPCs aggravated the H2O2-mediated inhibition of EPCs-adhesion. In contrast, when APE1 was overexpressed in EPCs using an adenovirus harboring the APE1 gene (APE-EPCs), adhesion was significantly improved during oxidative stress. To examine in vivo effects of APE1 in EPCs, APE-EPCs were transplanted via the tail vein after wire-mediated injury of the mouse femoral artery. The number of adherent EPCs at injured vascular walls and the vascular repair effect of EPCs were enhanced in APE-EPCs compared with control EPCs. Among the cellular functions of EPCs, adhesion is especially sensitive to oxidative stress. APE1 enhances in vivo vascular repair effects of EPCs in part through the maintenance of adhesion properties of EPCs. APE1 may be a novel and useful target gene for effective cellular transplantation therapy.
Assuntos
Adesão Celular/fisiologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/fisiologia , Células Endoteliais/fisiologia , Neointima/fisiopatologia , Células-Tronco/fisiologia , Animais , Vasos Sanguíneos/lesões , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Células Endoteliais/transplante , Sequestradores de Radicais Livres , Humanos , Peróxido de Hidrogênio/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxidantes/farmacologia , Espécies Reativas de OxigênioRESUMO
BACKGROUND: Prostacyclin (PGI2) enhances angiogenesis, especially in cooperation with bone marrow (BM)-derived endothelial progenitor cells (EPCs). However, the mechanisms of PGI2 in EPC-mediated angiogenesis in vivo remain unclear. The purpose of this study was to clarify the role of PGI2 in EPC-mediated angiogenesis using BM-specific IP deletion mice. METHODS AND RESULTS: Hind limb ischemia (HLI) was induced in wild-type (WT) mice transplanted with IP-deleted BM (WT/BM(IP(-/-)). Recovery of blood flow (RBF) in WT/BM(IP(-/-)) was impaired for 28 days after HLI, whereas RBF in IP(-/-)/BM(WT) was attenuated for up to 7 days compared with WT/BM(WT). The impaired RBF in WT/BM(IP(-/-)) was completely recovered by intramuscular injection of WT EPCs but not IP(-/-) EPCs. The impaired effects of IP(-/-) EPCs were in accordance with reduced formation of capillary and arterioles in ischemic muscle. An ex vivo aortic ring assay revealed that microvessel formation was enhanced by accumulation/adhesion of EPCs to perivascular sites as pericytes. IP(-/-)EPCs, in which expression of integrins was decreased, had impaired production of angiogenic cytokines, adhesion to neovessels and their angiogenic effects. The small-interfering RNA (siRNA)-mediated knockdown of integrin ß1 in WT EPCs attenuated adhesion to microvessels and their in vivo and in vitro angiogenic effects. CONCLUSIONS: PGI2 may induce persistent angiogenic effects in HLI through adhesion of EPCs to perivascular sites of neovessels via integrins in addition to paracrine effects.
Assuntos
Transplante de Medula Óssea , Células Endoteliais/metabolismo , Epoprostenol/metabolismo , Isquemia/terapia , Microcirculação , Neovascularização Fisiológica , Células-Tronco/metabolismo , Animais , Adesão Celular , Modelos Animais de Doenças , Células Endoteliais/patologia , Epoprostenol/genética , Membro Posterior/irrigação sanguínea , Membro Posterior/metabolismo , Membro Posterior/patologia , Isquemia/genética , Isquemia/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pericitos/metabolismo , Pericitos/patologia , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismo , Células-Tronco/patologiaRESUMO
BACKGROUND: Skeletal muscle comprises almost 40% of the human body and is essential for movement, structural support and metabolic homeostasis. Size of multinuclear skeletal muscle is stably maintained under steady conditions with the sporadic fusion of newly produced myocytes to compensate for the muscular turnover caused by daily wear and tear. It is becoming clear that microvascular pericytes (PCs) exhibit myogenic activity. However, whether PCs act as myogenic stem cells for the homeostatic maintenance of skeletal muscles during adulthood remains uncertain. METHODS: We utilized PC-fused myofibers using PC-specific lineage tracing mouse (NG2-CreERT/Rosa-tdTomato) to observe whether muscle resident PCs have myogenic potential during daily life. Genetic PC deletion mouse model (NG2-CreERT/DTA) was used to test whether PC differentiates to myofibers for maintenance of muscle structure and function under homeostatic condition. RESULTS: Under steady breeding conditions, tdTomato-expressing PCs were infused into myofibers, and subsequently, PC-derived nuclei were incorporated into myofibers. Especially in type-I slow-type myofibers such as the soleus, tdTomato+ myofibers were already observed 3 days after PC labeling; their ratio reached a peak (approximately 80%) within 1 month and was maintained for more than 1 year. Consistently, the NG2+ PC-specific deletion induced muscular atrophy in a slow-type myofiber-specific manner under steady breeding conditions. The number of myonucleus per volume of each myofiber was constant during observation period. CONCLUSIONS: These findings demonstrate that the turnover of myonuclei in slow-type myofibers is relatively fast, with PCs acting as myogenic stem cells-the suppliers of new myonuclei under steady conditions-and play a vital role in the homeostatic maintenance of slow-type muscles.
Assuntos
Músculo Esquelético , Pericitos , Animais , Humanos , Camundongos , Núcleo Celular , Homeostase , Atrofia MuscularRESUMO
Inflammatory responses in the kidney lead to tubulointerstitial fibrosis, a common feature of chronic kidney diseases. Here we examined the role of prostaglandin E(2) (PGE(2)) in the development of tubulointerstitial fibrosis. In the kidneys of wild-type mice, unilateral ureteral obstruction leads to progressive tubulointerstitial fibrosis with macrophage infiltration and myofibroblast proliferation. This was accompanied by an upregulation of COX-2 and PGE(2) receptor subtype EP(4) mRNAs. In the kidneys of EP(4) gene knockout mice, however, obstruction-induced histological alterations were significantly augmented. In contrast, an EP(4)-specific agonist significantly attenuated these alterations in the kidneys of wild-type mice. The mRNAs for macrophage chemokines and profibrotic growth factors were upregulated in the kidneys of wild-type mice after ureteral obstruction. This was significantly augmented in the kidneys of EP(4)-knockout mice and suppressed by the EP(4) agonist but only in the kidneys of wild-type mice. Notably, COX-2 and MCP-1 proteins, as well as EP(4) mRNA, were localized in renal tubular epithelial cells after ureteral obstruction. In cultured renal fibroblasts, another EP(4)-specific agonist significantly inhibited PDGF-induced proliferation and profibrotic connective tissue growth factor production. Hence, an endogenous PGE(2)-EP(4) system in the tubular epithelium limits the development of tubulointerstitial fibrosis by suppressing inflammatory responses.
Assuntos
Dinoprostona/metabolismo , Células Epiteliais/metabolismo , Nefropatias/prevenção & controle , Túbulos Renais/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Fibrose , Ácido Fólico , Regulação da Expressão Gênica , Heptanoatos/farmacologia , Nefropatias/etiologia , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miofibroblastos/metabolismo , Miofibroblastos/patologia , RNA Mensageiro/metabolismo , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/deficiência , Receptores de Prostaglandina E Subtipo EP4/genética , Transdução de Sinais , Fatores de Tempo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Obstrução Ureteral/complicaçõesRESUMO
Arg324 of sarcoplasmic reticulum Ca2+-ATPase forms electrostatic interactions with the phosphate moiety of phospholipids in most reaction states, and a hydrogen bond with Tyr122 in other states. Using site-directed mutagenesis, we explored the functional roles of Arg324 interactions, especially those with lipids, which at first glance might seem too weak to modulate the function of such a large membrane protein. The hydrogen bond forms transiently and facilitates Ca2+ binding from the cytoplasmic side. The contributions of the electrostatic interactions to the reaction steps were quantified using a rate vs activity coefficient plot. We found that the interaction between Arg324 and lipids decreases the affinity for luminal Ca2+. The transformation rate of the phosphoenzyme intermediate is facilitated by the electrostatic interactions, and the function of these interactions depends not only on the type but also on the composition of the phospholipids. The properties observed in microsomes could not be reproduced with any single phospholipid, but with a mixture of phospholipids that mimics the native membrane. These results suggest the importance of swapping of the lipid partners of different headgroups in the reaction step. This study shows that Arg324 plays a role in the reaction cycle via complex intra-protein and protein-lipid interactions.
Assuntos
Arginina/metabolismo , Fosfolipídeos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Cálcio/metabolismo , Cinética , Fosforilação , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Eletricidade EstáticaRESUMO
BACKGROUND: Sprouting angiogenesis is an important mechanism for morphogenetic phenomena, including organ development, wound healing, and tissue regeneration. In regenerative medicine, therapeutic angiogenesis is a clinical solution for recovery from ischemic diseases. Mesenchymal stem cells (MSCs) have been clinically used given their pro-angiogenic effects. MSCs are reported to promote angiogenesis by differentiating into pericytes or other vascular cells or through cell-cell communication using multiple protein-protein interactions. However, how MSCs physically contact and move around ECs to keep the sprouting angiogenesis active remains unknown. METHODS: We proposed a novel framework of EC-MSC crosstalk analysis using human umbilical vein endothelial cells (HUVECs) and MSCs obtained from mice subcutaneous adipose tissue on a 3D in vitro model, microvessel-on-a-chip, which allows cell-to-tissue level study. The microvessels were fabricated and cultured for 10 days in a collagen matrix where MSCs were embedded. RESULTS: Immunofluorescence imaging using a confocal laser microscope showed that MSCs smoothed the surface of the microvessel and elongated the angiogenic sprouts by binding to the microvessel's specific microstructures. Additionally, three-dimensional modeling of HUVEC-MSC intersections revealed that MSCs were selectively located around protrusions or roots of angiogenic sprouts, whose surface curvature was excessively low or high, respectively. CONCLUSIONS: The combination of our microvessel-on-a-chip system for 3D co-culture and image-based crosstalk analysis demonstrated that MSCs are selectively localized to concave-convex surfaces on scaffold structures and that they are responsible for the activation and stabilization of capillary vessels.
Assuntos
Células-Tronco Mesenquimais , Neovascularização Fisiológica , Camundongos , Humanos , Animais , Comunicação Celular , Células-Tronco Mesenquimais/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Dispositivos Lab-On-A-ChipRESUMO
The formation of mature vasculature through angiogenesis is essential for adequate wound healing, such that blood-borne cells, nutrients, and oxygen can be delivered to the remodeling skin area. Neovessel maturation is highly dependent on the coordinated functions of vascular endothelial cells and perivascular cells, namely pericytes (PCs). However, the underlying mechanism for vascular maturation has not been completely elucidated, and its role in wound healing remains unclear. In this study, we investigated the role of Ninjurin-1 (Ninj1), a new molecule mediating vascular maturation, in wound healing using an inducible PC-specific Ninj1 deletion mouse model. Ninj1 expression increased temporarily in NG2-positive PCs in response to skin injury. When tamoxifen treatment induced a decreased Ninj1 expression in PCs, the neovessels in the regenerating wound margins were structurally and functionally immature, but the total number of microvessels was unaltered. This phenotypic change is associated with a reduction in PC-associated microvessels. Wound healing was significantly delayed in the NG2-specific Ninj1 deletion mouse model. Finally, we showed that Ninj1 is a crucial molecule that mediates vascular maturation in injured skin tissue through the interaction of vascular endothelial cells and PCs, thereby inducing adequate and prompt wound healing.
RESUMO
OBJECTIVE: Endothelial progenitor cells (EPCs) play an important role in the self-healing of a vascular injury by participating in the reendothelialization that limits vascular remodeling. We evaluated whether prostaglandin I(2) plays a role in the regulation of the function of EPCs to limit vascular remodeling. METHODS AND RESULTS: EPCs (Lin(-)cKit(+)Flk-1(+) cells) were isolated from the bone marrow (BM) of wild-type (WT) mice or mice lacking the prostaglandin I(2) receptor IP (IP(-/-) mice). Reverse transcription-polymerase chain reaction analysis showed that EPCs among BM cells specifically express IP. The cellular properties of EPCs, adhesion, migration, and proliferation on fibronectin were significantly attenuated in IP-deficient EPCs compared with WT EPCs. In contrast, IP agonists facilitated these functions in WT EPCs, but not in IP-deficient EPCs. The specific deletion of IP in BM cells, which was performed by transplanting BM cells of IP(-/-) mice to WT mice, accelerated wire injury-mediated neointimal hyperplasia in the femoral artery. Notably, transfused WT EPCs, but not IP-deficient EPCs, were recruited to the injured vessels, participated in reendothelialization, and efficiently rescued the accelerated vascular remodeling. CONCLUSIONS: These findings clearly indicate that the prostaglandin I(2)-IP system is essential for EPCs to accomplish their function and plays a critical role in the regulation of vascular remodeling.
Assuntos
Endotélio Vascular/metabolismo , Epoprostenol/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica/fisiologia , Animais , Adesão Celular , Movimento Celular , Proliferação de Células , Endotélio Vascular/patologia , Fibronectinas/metabolismo , Hiperplasia/metabolismo , Hiperplasia/patologia , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Knockout , Modelos Animais , Receptores de Epoprostenol/genética , Receptores de Epoprostenol/metabolismo , Transdução de Sinais/fisiologia , Túnica Íntima/lesões , Túnica Íntima/metabolismo , Túnica Íntima/patologiaRESUMO
Fabry disease is an X-linked lysosomal storage disorder caused by mutations of the α-galactosidase A gene (GLA), and the disease is a relatively prevalent cause of left ventricular hypertrophy mimicking idiopathic hypertrophic cardiomyopathy. We assessed clinically 5 patients of a three-generation family and also searched for GLA mutations in 10 family members. The proband had left ventricular hypertrophy with localized thinning in the basal posterior wall and late gadolinium enhancement (LGE) in the near-circumferential wall in cardiovascular magnetic resonance images and her sister had vasospastic angina pectoris without organic stenosis of the coronary arteries. LGE notably appeared in parallel with decreased α-galactosidase A activity and increased NT-pro BNP in our patients. We detected a new GLA missense mutation (G195V) in exon 4, resulting in a glycine-to-valine substitution. Of the 10 family members, 5 family members each were positive and negative for this mutation. These new data extend our clinical and molecular knowledge of GLA gene mutations and confirm that a novel missense mutation in the GLA gene is important not only for a precise diagnosis of heterozygous status, but also for confirming relatives who are negative for this mutation.
Assuntos
Doença de Fabry/diagnóstico , Doença de Fabry/genética , Hipertrofia Ventricular Esquerda/diagnóstico , Hipertrofia Ventricular Esquerda/genética , Mutação de Sentido Incorreto/genética , alfa-Galactosidase/genética , Adulto , Substituição de Aminoácidos/genética , Bloqueio de Ramo/diagnóstico , Bloqueio de Ramo/genética , Bloqueio de Ramo/patologia , Angiografia Coronária , Vasoespasmo Coronário/diagnóstico , Vasoespasmo Coronário/genética , Vasoespasmo Coronário/patologia , Análise Mutacional de DNA , Ecocardiografia , Eletrocardiografia , Éxons/genética , Doença de Fabry/patologia , Feminino , Genótipo , Glicina/genética , Humanos , Hipertrofia Ventricular Esquerda/patologia , Japão , Imageamento por Ressonância Magnética , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Miocárdio/patologia , Peptídeo Natriurético Encefálico/sangue , Linhagem , Fragmentos de Peptídeos/sangue , Processamento de Sinais Assistido por Computador , Valina/genética , Adulto JovemRESUMO
Sarcopenia is a pathophysiological malfunction induced by skeletal muscle atrophy. Several studies reported an association between sarcopenia-induced cardiac cachexia and poor prognosis in heart disease. However, due to lack of an established animal models, the underlying mechanism of disturbed cardiac repair accompanied with sarcopenia remains poorly understood. Here, we developed a novel sarcopenia-induced cardiac repair disturbance mouse model induced by tail suspension (TS) after cardiac ischemia and reperfusion (I/R). Importantly, we identified a specific exosomal-microRNA marker, miR-16-5p, in the circulating exosomes of I/R-TS mice. Of note, sarcopenia after I/R disturbed cardiac repair and raised the level of circulating-exosomal-miR-16-5p secreting from both the atrophic limbs and heart of TS mice. Likewise, miR-16-5p mimic plasmid disturbed cardiac repair in I/R mice directly. Additionally, in neonatal rat ventricular myocytes (NRVMs) cultured in vitro under hypoxic conditions in the presence of a miR-16-5p mimic, we observed increased apoptosis through p53 and Caspase3 upregulation, and also clarified that autophagosomes were decreased in NRVMs via SESN1 transcript interference-mediated mTOR activation. In conclusion, we show the pro-apoptotic effect of sarcopenia-derived miR-16-5p, which may be behind the exacerbation of myocardial infarction. Therefore, miR-16-5p can be a novel therapeutic target in the context of cardiac repair disturbances in sarcopenia-cachexia.