RESUMO
Single-cell RNA sequencing (scRNA-seq) has enabled the identification of novel gene signatures and cell heterogeneity in numerous tissues and diseases. Here we review the use of this technology for Fuchs' Endothelial Corneal Dystrophy (FECD). FECD is the most common indication for corneal endothelial transplantation worldwide. FECD is challenging to manage because it is genetically heterogenous, can be autosomal dominant or sporadic, and progress at different rates. Single-cell RNA sequencing has enabled the discovery of several FECD subtypes, each with associated gene signatures, and cell heterogeneity. Current FECD treatments are mainly surgical, with various Rho kinase (ROCK) inhibitors used to promote endothelial cell metabolism and proliferation following surgery. A range of emerging therapies for FECD including cell therapies, gene therapies, tissue engineered scaffolds, and pharmaceuticals are in preclinical and clinical trials. Unlike conventional disease management methods based on clinical presentations and family history, targeting FECD using scRNA-seq based precision-medicine has the potential to pinpoint the disease subtypes, mechanisms, stages, severities, and help clinicians in making the best decision for surgeries and the applications of therapeutics. In this review, we first discuss the feasibility and potential of using scRNA-seq in clinical diagnostics for FECD, highlight advances from the latest clinical treatments and emerging therapies for FECD, integrate scRNA-seq results and clinical notes from our FECD patients and discuss the potential of applying alternative therapies to manage these cases clinically.
RESUMO
Corneal diseases are one of the leading causes of moderate-to-severe visual impairment and blindness worldwide, after glaucoma, cataract, and retinal disease in overall importance. Given its tendency to affect people at a younger age than other blinding conditions such as cataract and glaucoma, corneal scarring poses a huge burden both on the individuals and society. Furthermore, corneal scarring and fibrosis disproportionately affects people in poorer and remote areas, making it a significant ophthalmic public health problem. Traditional medical strategies, such as topical corticosteroids, are not effective in preventing fibrosis or scars. Corneal transplantation, the only effective sight-restoring treatment for corneal scars, is curbed by challenges including a severe shortage of tissue, graft rejection, secondary conditions, cultural barriers, the lack of well-trained surgeons, operating rooms, and well-equipped infrastructures. Thanks to tremendous research efforts, emerging therapeutic options including gene therapy, protein therapy, cell therapy and novel molecules are in development to prevent the progression of corneal scarring and compliment the surgical options currently available for treating established corneal scars in clinics. In this article, we summarise the most relevant preclinical and clinical studies on emerging therapies for corneal scarring in recent years, showing how these approaches may prevent scarring in its early development.
Assuntos
Catarata , Doenças da Córnea , Lesões da Córnea , Glaucoma , Humanos , Cicatriz/terapia , Cicatriz/complicações , Lesões da Córnea/terapia , Lesões da Córnea/complicações , Doenças da Córnea/tratamento farmacológico , Doenças da Córnea/complicações , Glaucoma/complicações , Catarata/complicaçõesRESUMO
Retinal neovascularization, or pathological angiogenesis in the retina, is a leading cause of blindness in developed countries. Transforming growth factor-ß-activated kinase 1 (TAK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) activated by TGF-ß1 and other proinflammatory cytokines. TAK1 is also a key mediator of proinflammatory signals and plays an important role in maintaining vascular integrity upon proinflammatory cytokine stimulation such as TNFα. However, its role in pathological angiogenesis, particularly in retinal neovascularization, remains unclear. Here, we investigate the regulatory role of TAK1 in human endothelial cells responding to inflammatory stimuli and in a rat model of oxygen-induced retinopathy (OIR) featured retinal neovascularization. Using TAK1 knockout human endothelial cells that subjected to inflammatory stimuli, transcriptome analysis revealed that TAK1 is required for activation of NFκB signaling and mediates its downstream gene expression related to endothelial activation and angiogenesis. Moreover, pharmacological inhibition of TAK1 by 5Z-7-oxozeaenol attenuated angiogenic activities of endothelial cells. Transcriptome analysis also revealed enrichment of TAK1-mediated NFκB signaling pathway in the retina of OIR rats and retinal neovascular membrane from patients with proliferative diabetic retinopathy. Intravitreal injection of 5Z-7-oxozeaenol significantly reduced hypoxia-induced inflammation and microglial activation, thus attenuating aberrant retinal angiogenesis in OIR rats. Our data suggest that inhibition of TAK1 may have therapeutic potential for the treatment of retinal neovascular pathologies.
Assuntos
Doenças Retinianas , Neovascularização Retiniana , Animais , Humanos , Camundongos , Ratos , Citocinas/uso terapêutico , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Lactonas/uso terapêutico , Camundongos Endogâmicos C57BL , Neovascularização Patológica/patologia , NF-kappa B , Oxigênio , Doenças Retinianas/patologia , Neovascularização Retiniana/metabolismoRESUMO
Retinal neovascularization is a severe complication of proliferative diabetic retinopathy (PDR). MicroRNAs (miRNAs) are master regulators of gene expression that play an important role in retinal neovascularization. In this study, we show that miR-143-3p is significantly downregulated in the retina of a rat model of oxygen-induced retinopathy (OIR) by miRNA-sequencing. Intravitreal injection of synthetic miR-143 mimics significantly ameliorate retinal neovascularization in OIR rats. miR-143 is identified to be highly expressed in the neural retina particularly in the ganglion cell layer and retinal vasculature. In miR-143 treated cells, the functional evaluation showed a decrease in cell migration and delayed endothelial vessel-like tube remodeling. The multiomics analysis suggests that miR-143 negatively impacts endothelial cell activity through regulating cell-matrix adhesion and mediating hypoxia-inducible factor-1 signaling. We predict hub genes regulated by miR-143 that may be involved in mediating endothelial cell function by cytoHubba. We also demonstrate that the retinal neovascular membranes in patients with PDR principally consist of endothelial cells by CIBERSORTx. We then identify 2 hub genes, thrombospondin 1 and plasminogen activator inhibitor, direct targets of miR-143, that significantly altered in the PDR patients. These findings suggest that miR-143 appears to be essential for limiting endothelial cell-matrix adhesion, thus suppressing retinal neovascularization.
Assuntos
MicroRNAs , Neovascularização Retiniana , Animais , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Oxigênio/efeitos adversos , Ratos , Retina/metabolismo , Neovascularização Retiniana/terapiaRESUMO
Angiogenesis plays a critical role in both physiological responses and disease pathogenesis. Excessive angiogenesis can promote neoplastic diseases and retinopathies, while inadequate angiogenesis can lead to aberrant perfusion and impaired wound healing. Transforming growth factor ß activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, is a key modulator involved in a range of cellular functions including the immune responses, cell survival and death. TAK1 is activated in response to various stimuli such as proinflammatory cytokines, hypoxia, and oxidative stress. Emerging evidence has recently suggested that TAK1 is intimately involved in angiogenesis and mediates pathogenic processes related to angiogenesis. Several detailed mechanisms by which TAK1 regulates pathological angiogenesis have been clarified, and potential therapeutics targeting TAK1 have emerged. In this review, we summarize recent studies of TAK1 in angiogenesis and discuss the crosstalk between TAK1 and signaling pathways involved in pathological angiogenesis. We also discuss the approaches for selectively targeting TAK1 and highlight the rationales of therapeutic strategies based on TAK1 inhibition for the treatment of pathological angiogenesis.
Assuntos
Sistemas de Liberação de Medicamentos , MAP Quinase Quinase Quinases/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Transdução de Sinais , Animais , Humanos , Neovascularização Patológica/patologiaRESUMO
Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.
Assuntos
Terapia Genética , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Neovascularização Retiniana/genética , Neovascularização Retiniana/terapia , Animais , Hipóxia Celular , Dependovirus/metabolismo , Modelos Animais de Doenças , Feminino , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Injeções Intravítreas , Domínios Proteicos , Ratos Sprague-Dawley , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Transgenes , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Purpose: Corneal injury that occurs after burning with alkali initiates wound-healing processes, including inflammation, neovascularization, and fibrosis. Excessive reactions to injury can reduce corneal transparency and thereby compromise vision. The NADPH oxidase (Nox) enzyme complex is known to be involved in cell signaling for wound-healing angiogenesis, but its role in corneal neovascularization has been little studied. Methods: The center corneas of wild-type and Nox4 knockout (KO) mice were injured with 3 µL 1 M NaOH, while the contralateral corneas remained untouched. On day 7, mRNA expression levels of NADPH oxidase isoforms, the proangiogenic factors VEGF-A and TGFß1, and proinflammatory genes ICAM-1 and VCAM-1 were determined. Corneal neovascularization and fibrosis were visualized using PECAM-1 antibody and picrosirius red staining, respectively, on the same day. Results: Expressions of both Nox2 and Nox4 gene isoforms as well as the above genes were markedly increased in the injured corneas at 7 days. Injured corneas showed neovascularization and fibrosis as well as an increase in clinical opacity score. All responses stimulated by alkali burn were abrogated in Nox4 KO mice. Conclusions: Nox4 could be a new target to treat pathologic corneal wound-healing responses and such targeting might prevent blindness caused by burn injuries.
Assuntos
Queimaduras Químicas/enzimologia , Lesões da Córnea/enzimologia , Queimaduras Oculares/induzido quimicamente , NADPH Oxidase 4/metabolismo , Cicatrização/fisiologia , Animais , Regulação Enzimológica da Expressão Gênica/fisiologia , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/genética , Reação em Cadeia da Polimerase em Tempo Real , Hidróxido de Sódio , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Background: Cardiomyocytes derived from pluripotent stem cells are immature. Maturation of cardiomyocytes is a multifactorial dynamic process that involves various factors in vivo that cannot be fully recapitulated in vitro. Here, we report a novel tissue engineering chamber with an integrated electrical stimulator and electrodes that will allow wireless electrical stimulation of cardiac tissue in vivo. Materials and Methods: Immunocompromised rats were implanted with tissue engineering chambers containing the stimulator and electrodes, and control chambers (chambers with electrical stimulator but without the electrodes) in the contralateral limb. Each chamber contained cardiomyocytes derived from human induced pluripotent stem cells (iPSCs). After 7 days of chamber implantation, the electrical stimulators were activated for 4 h per day, for 21 consecutive days. Results: At 4 weeks postimplantation, cardiomyocytes derived from human iPSCs survived, were assembled into compact cardiac tissue, and were perfused and vascularized by the host neovessels. Conclusion: This proof-of-principle study demonstrates the biocompatibility of the tissue engineering chamber with integrated electrical stimulator and electrodes. This could be utilized to study the influence of continuous electrical stimulation on vascularized cardiac or other tissues in vivo.
RESUMO
Dry eye disease (DED) has become common on a global scale in recent years. There is a wide prevalence of DED in different countries based on various ethnicities and environment. DED is a multifactorial ocular disorder. In addition to advanced age and gender, such factors as living at high altitude, smoking, pterygium, prolonged use of consumer electronics or overingesting of caffeine or multivitamins are considered to be the major risk factors of DED. We report the DED epidemiology in Taiwan firstly in this article. According to the pathophysiological factors and changes inthe composition of the tear film in DED, it can be categorized into several subtypes, including lipid anomaly dry eye, aqueous tear deficiency, allergic and toxic dry eye among others. Each subtype has its own cause and disease management; therefore, it is important for ophthalmologists to identify the type through literature review and investigation. The management of DED, relies not only on traditional medications such as artificial tears, gels and ointments, but also newer treatment options such as acupuncture, SYL1001, and nanomedicine therapy. We also conducted a comprehensive literature review including common subtypes and treatment of DED. Clearly, more clinical trials are needed to assess the efficacy and safety of the various treatments and common subtypes of DED.
RESUMO
Ocular neovascularization is a common pathological feature in diabetic retinopathy and neovascular age-related macular degeneration that can lead to severe vision loss. We evaluated the therapeutic efficacy of a novel endogenous inhibitor of angiogenesis, the calreticulin anti-angiogenic domain (CAD180), and its functional 112-residue fragment, CAD-like peptide 112 (CAD112), delivered using a self-complementary adeno-associated virus serotype 2 (scAAV2) in rodent models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. The expression of CAD180 and CAD112 was elevated in human umbilical vein endothelial cells transduced with scAAV2-CAD180 or scAAV2-CAD112, respectively, and both inhibited angiogenic activity in vitro. Intravitreal gene delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly inhibited ischemia-induced retinal neovascularization in rat eyes (CAD180: 52.7% reduction; CAD112: 49.2% reduction) compared to scAAV2-mCherry, as measured in retinal flatmounts stained with isolectin B4. Moreover, the retinal structure and function were unaffected by scAAV2-CAD180 or scAAV2-CAD112, as measured by optical coherence tomography and electroretinography. Moreover, subretinal delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly attenuated laser-induced choroidal neovascularization in mouse eyes compared to scAAV2-mCherry, as measured by fundus fluorescein angiography (CAD180: 62.4% reduction; CAD112: 57.5% reduction) and choroidal flatmounts (CAD180: 40.21% reduction; CAD112: 43.03% reduction). Gene delivery using scAAV2-CAD180 or scAAV2-CAD112 has significant potential as a therapeutic option for the management of ocular neovascularization.
Assuntos
Inibidores da Angiogênese/biossíntese , Calreticulina , Dependovirus , Retinopatia Diabética , Neovascularização Retiniana , Transdução Genética , Inibidores da Angiogênese/genética , Angiografia , Animais , Calreticulina/biossíntese , Calreticulina/genética , Retinopatia Diabética/diagnóstico por imagem , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Retinopatia Diabética/fisiopatologia , Eletrorretinografia , Feminino , Vetores Genéticos , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Degeneração Macular/diagnóstico por imagem , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/fisiopatologia , Camundongos , Ratos , Ratos Sprague-Dawley , Neovascularização Retiniana/diagnóstico por imagem , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/fisiopatologia , Tomografia de Coerência Óptica , Proteína Vermelha FluorescenteRESUMO
The benefits of adult stem cells for repair of the heart have been attributed to the repertoire of salutary paracrine activities they appear to exert. We previously isolated human W8B2+ cardiac stem cells (CSCs) and found they powerfully influence cardiomyocytes and endothelial cells to collectively promote cardiac repair and regeneration. Here, the complexity of the W8B2+ CSC secretomes was characterised and examined in more detail. Using ion exchange chromatography to separate soluble proteins based on their net surface charge, the secreted factors responsible for the pro-survival activity of W8B2+ CSCs were found within the low and medium cation fractions. In addition to the soluble proteins, extracellular vesicles generated from W8B2+ CSCs not only exhibited pro-survival and pro-angiogenic activities, but also promoted proliferation of neonatal cardiomyocytes. These extracellular vesicles contain a cargo of proteins, mRNA and primary microRNA precursors that are enriched in exosomes and are capable of modulating collectively many of the cellular pathways involved in protein metabolism, cell growth, as well as cellular responses to stress and organisation of the extracellular matrix. Thus the W8B2+ CSC secretome contains a multitude of bioactive paracrine factors we have now characterised, that might well be harnessed for therapeutic application for cardiac repair and regeneration.
Assuntos
Células-Tronco Adultas/metabolismo , Fatores Biológicos/metabolismo , Vesículas Extracelulares/química , MicroRNAs/metabolismo , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia por Troca Iônica , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , RatosRESUMO
Diabetic cardiomyopathy is a major contributor to the increasing burden of heart failure globally. Effective therapies remain elusive, in part due to the incomplete understanding of the mechanisms underlying diabetes-induced myocardial injury. The objective of this study was to assess the direct impact of insulin replacement on left ventricle structure and function in a rat model of diabetes. Male Sprague-Dawley rats were administered streptozotocin (55 mg/kg i.v.) or citrate vehicle and were followed for 8 weeks. A subset of diabetic rats were allocated to insulin replacement (6 IU/day insulin s.c.) for the final 4 weeks of the 8-week time period. Diabetes induced the characteristic systemic complications of diabetes (hyperglycaemia, polyuria, kidney hypertrophy) and was accompanied by marked left ventricle remodelling (cardiomyocyte hypertrophy, left ventricle collagen content) and diastolic dysfunction (transmitral E/A, left ventricle-dP/dt). Importantly, these systemic and cardiac impairments were ameliorated markedly following insulin replacement, and moreover, markers of the diabetic cardiomyopathy phenotype were significantly correlated with the extent of hyperglycaemia. In summary, these data suggest that poor glucose control directly contributes towards the underlying features of experimental diabetic cardiomyopathy, at least in the early stages, and that adequate replacement ameliorates this.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Cardiomiopatias Diabéticas/prevenção & controle , Ventrículos do Coração/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Estreptozocina , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Biomarcadores/sangue , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/induzido quimicamente , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/patologia , Cardiomiopatias Diabéticas/fisiopatologia , Progressão da Doença , Fibrose , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Masculino , Miocardite/patologia , Miocardite/fisiopatologia , Miocardite/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Fatores de Tempo , Remodelação Ventricular/efeitos dos fármacosRESUMO
Choroidal neovascularization (CNV) is a common pathological feature in neovascular age-related macular degeneration, which is the leading cause of vision loss among elderly populations in developed countries. This study evaluated the effect of a novel endogenous inhibitor of angiogenesis, calreticulin anti-angiogenic domain (CAD), subconjunctivally delivered by an adenoviral vector (Ad-CAD) in a rat model of laser-induced CNV. CAD was expressed in Ad-CAD-infected cells and inhibited the angiogenic activity in human umbilical vein endothelial cells in vitro. CAD expression was also found in various ocular tissues after in vivo subconjunctival Ad-CAD injection. Via bioluminescence imaging it is shown that a single subconjunctival injection of Ad-luciferase induced the expression of the transgene in the injected eyes within 24 h, which lasted for at least 112 days. Forty-two days after subconjunctival injection of Ad-CAD, retinal structure and function were unaffected, as measured using optical coherence tomography and electroretinography, respectively. After laser injury, subconjunctival Ad-CAD gene delivery significantly inhibited CNV lesions as measured via choroid flat-mounts (51% reduction at 21 days; p < 0.001), as well as by fundus fluorescein angiography (19.3%, 28.2%, 31%, and 27.5% reductions at days 21, 28, 35, and 42, respectively; p < 0.05) in rats. The data suggest that subconjunctival Ad-CAD gene therapy could effectively inhibit laser-induced CNV and might be an attractive therapeutic approach for the management of choroidal neovascularization.
Assuntos
Calreticulina/genética , Neovascularização de Coroide/terapia , Técnicas de Transferência de Genes , Degeneração Macular Exsudativa/terapia , Animais , Calreticulina/uso terapêutico , Neovascularização de Coroide/genética , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos/uso terapêutico , Células Endoteliais da Veia Umbilical Humana , Humanos , Ratos , Retina/efeitos dos fármacos , Retina/patologia , Degeneração Macular Exsudativa/genética , Degeneração Macular Exsudativa/patologiaRESUMO
Diabetic retinopathy (DR), a chronic and progressive complication of diabetes mellitus, is a sight-threatening disease characterized in the early stages by neuronal and vascular dysfunction in the retina, and later by neovascularization that further damages vision. A major contributor to the pathology is excess production of vascular endothelial growth factor (VEGF), a growth factor that induces formation of new blood vessels and increases permeability of existing vessels. Despite the recent availability of effective treatments for the disease, including laser photocoagulation and therapeutic VEGF antibodies, DR remains a significant cause of vision loss worldwide. Existing anti-VEGF agents, though generally effective, are limited by their short therapeutic half-lives, necessitating frequent intravitreal injections and the risk of attendant adverse events. Management of DR with gene therapies has been proposed for several years, and pre-clinical studies have yielded enticing findings. Gene therapy holds several advantages over conventional treatments for DR, such as a longer duration of therapeutic effect, simpler administration, the ability to intervene at an earlier stage of the disease, and potentially fewer side-effects. In this review, we summarize the current understanding of the pathophysiology of DR and provide an overview of research into DR gene therapies. We also examine current barriers to the clinical application of gene therapy for DR and evaluate future prospects for this approach.
Assuntos
Retinopatia Diabética/terapia , Terapia Genética/métodos , Neovascularização Patológica/terapia , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Retinopatia Diabética/genética , Retinopatia Diabética/fisiopatologia , Epigênese Genética , Terapia Genética/efeitos adversos , Humanos , Neovascularização Patológica/genética , Neovascularização Patológica/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Mitochondria are morphologically dynamic organelles constantly undergoing processes of fission and fusion that maintain integrity and bioenergetics of the organelle: these processes are vital for cell survival. Disruption in the balance of mitochondrial fusion and fission is thought to play a role in several pathological conditions including ischemic heart disease. Proteins involved in regulating the processes of mitochondrial fusion and fission are therefore potential targets for pharmacological therapies. Mdivi-1 is a small molecule inhibitor of the mitochondrial fission protein Drp1. Inhibiting mitochondrial fission with Mdivi-1 has proven cytoprotective benefits in several cell types involved in a wide array of cardiovascular injury models. On the other hand, Mdivi-1 can also exert antiproliferative and cytotoxic effects, particularly in hyperproliferative cells. In this review, we discuss these divergent effects of Mdivi-1 on cell survival, as well as the potential and limitations of Mdivi-1 as a therapeutic agent.
RESUMO
Histone deacetylase (HDAC) inhibitors are known to suppress abnormal development of blood vessels. Angiogenic activity in endothelial cells depends upon NADPH oxidase 4 (Nox4)-dependent redox signalling. We set out to study whether the HDAC inhibitor trichostatin A (TSA) affects Nox4 expression and angiogenesis. Nox4 expression was measured by real time PCR and Western blot analysis in endothelial cells. Hydrogen peroxide (H2 O2 ) was measured by amplex(®) red assay in endothelial cells. Nox4 was knocked down by Nox4 shRNA. In vitro angiogenic activities such migration and tubulogenesis were assessed using wound healing and Matrigel assays, respectively. In vivo angiogenic activity was assessed using subcutaneous sponge assay in C57Bl/6 and Nox4-deficient mice. Trichostatin A reduced Nox4 expression in a time- and concentration-dependent manner. Both TSA and Nox4 silencing decreased Nox4 protein and H2 O2 . Mechanistically, TSA reduced expression of Nox4 via ubiquitination of p300- histone acetyltransferase (p300-HAT). Thus, blocking of the ubiquitination pathway using an inhibitor of ubiquitin-activating enzyme E1 (PYR-41) prevented TSA inhibition of Nox4 expression. Trichostatin A also reduced migration and tube formation, and these effects were not observed in Nox4-deficient endothelial cells. Finally, transforming growth factor beta1 (TGFß1) enhanced angiogenesis in sponge model in C57BL/6 mice. This response to TGFß1 was substantially reduced in Nox4-deficient mice. Similarly intraperitoneal infusion of TSA (1 mg/kg) also suppressed TGFß1-induced angiogenesis in C57BL/6 mice. Trichostatin A reduces Nox4 expression and angiogenesis via inhibition of the p300-HAT-dependent pathway. This mechanism might be exploited to prevent aberrant angiogenesis in diabetic retinopathy, complicated vascular tumours and malformations.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , NADPH Oxidases/antagonistas & inibidores , Neovascularização Fisiológica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Capilares/efeitos dos fármacos , Capilares/metabolismo , Movimento Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Camundongos , Modelos Biológicos , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , Oxirredução/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Ubiquitinação/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Adult stem cells continue to promise opportunities to repair damaged cardiac tissue. However, precisely how adult stem cells accomplish cardiac repair, especially after ischemic damage, remains controversial. It has been postulated that the clinical benefit of adult stem cells for cardiovascular disease results from the release of cytokines and growth factors by the transplanted cells. Studies in animal models of myocardial infarction have reported that such paracrine factors released from transplanted adult stem cells contribute to improved cardiac function by several processes. These include promoting neovascularization of damaged tissue, reducing inflammation, reducing fibrosis and scar formation, as well as protecting cardiomyocytes from apoptosis. In addition, these factors might also stimulate endogenous repair by activating cardiac stem cells. Interestingly, stem cells discovered to be resident in the heart appear to be functionally superior to extra-cardiac adult stem cells when transplanted for cardiac repair and regeneration. In this review, we discuss the therapeutic potential of cardiac stem cells and how the proteins secreted from these cells might be harnessed to promote repair and regeneration of damaged cardiac tissue. We also highlight how recent controversies about the efficacy of adult stem cells in clinical trials of ischemic heart disease have not dampened enthusiasm for the application of cardiac stem cells and their paracrine factors for cardiac repair: the latter have proved superior to the mesenchymal stem cells used in most clinical trials in the past, some of which appear to have been conducted with sub-optimal rigor.
Assuntos
Células-Tronco Adultas/transplante , Isquemia Miocárdica/terapia , Miócitos Cardíacos/citologia , Neovascularização Fisiológica , Comunicação Parácrina/fisiologia , Ensaios Clínicos como Assunto , Humanos , Transplante de Células-Tronco , Resultado do TratamentoRESUMO
Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 µm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 µL in collagen scaffold vs 22.5±2.3 µL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 µL in collagen scaffold with human ASCs vs 25.7±1.9 µL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo.