RESUMO
Neuroprotection is among the potential treatment options for glaucoma and other retinal pathologies characterized by the loss of retinal ganglion cells (RGCs). Here, we examined the impact of a neural stem (NS) cell-based intravitreal co-administration of two neuroprotective factors on the survival of axotomized RGCs. To this aim we used lentiviral vectors to establish clonal NS cell lines ectopically expressing either glial cell line-derived neurotrophic factor (GDNF) or ciliary neurotrophic factor (CNTF). The modified NS cell lines were intravitreally injected either separately or as a 1:1 mixture into adult mice one day after an optic nerve lesion, and the number of surviving RGCs was determined in retinal flat-mounts two, four and eight weeks after the lesion. For the transplantation experiments, we selected a GDNF- and a CNTF-expressing NS cell line that promoted the survival of axotomized RGCs with a similar efficacy. Eight weeks after the lesion, GDNF-treated retinas contained 3.8- and CNTF-treated retinas 3.7-fold more RGCs than control retinas. Of note, the number of surviving RGCs was markedly increased when both factors were administered simultaneously, with 14.3-fold more RGCs than in control retinas eight weeks after the lesion. GDNF and CNTF thus potently and synergistically rescued RGCs from axotomy-induced cell death, indicating that combinatorial neuroprotective approaches represent a promising strategy to effectively promote the survival of RGCs under pathological conditions.
Assuntos
Fator Neurotrófico Ciliar/administração & dosagem , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Células-Tronco Neurais/transplante , Fármacos Neuroprotetores/administração & dosagem , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Axotomia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fator Neurotrófico Ciliar/metabolismo , Sinergismo Farmacológico , Vetores Genéticos , Lentivirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Compressão Nervosa , Células-Tronco Neurais/metabolismo , Fármacos Neuroprotetores/metabolismo , Traumatismos do Nervo ÓpticoRESUMO
Engulfment and cell motility 1/dedicator of cytokinesis 180 (Elmo1/Dock180) is a bipartite guanine nucleotide exchange factor for the monomeric GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1). Elmo1/Dock180 regulates Rac1 activity in a specific spatiotemporal manner in endothelial cells (ECs) during zebrafish development and acts downstream of the Netrin-1/Unc5-homolog B (Unc5B) signaling cascade. However, mechanistic details on the pathways by which Elmo1/Dock180 regulates endothelial function and vascular development remained elusive. In this study, we aimed to analyze the vascular function of Elmo1 and Dock180 in human ECs and during vascular development in zebrafish embryos. In vitro overexpression of Elmo1 and Dock180 in ECs reduced caspase-3/7 activity and annexin V-positive cell number upon induction of apoptosis. This protective effect of Elmo1 and Dock180 is mediated by activation of Rac1, p21-activated kinase (PAK) and AKT/protein kinase B (AKT) signaling. In zebrafish, Elmo1 and Dock180 overexpression reduced the total apoptotic cell and apoptotic EC number and promoted the formation of blood vessels during embryogenesis. In conclusion, Elmo1 and Dock180 protect ECs from apoptosis by the activation of the Rac1/PAK/AKT signaling cascade in vitro and in vivo. Thus, Elmo1 and Dock180 facilitate blood vessel formation by stabilization of the endothelium during angiogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Vasos Sanguíneos/metabolismo , Neovascularização Fisiológica , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/genética , Vasos Sanguíneos/crescimento & desenvolvimento , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas rac de Ligação ao GTP/biossíntese , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
RATIONALE: The transcription factor HOXC9 belongs to the homeobox gene family acting as developmental morphogen in several species. HOXC9 is EXPRESSED in different vascular beds in vivo. Yet vascular functions of HOXC9 have not been studied. OBJECTIVE: This study was aimed at characterizing HOXC9 functions in human vascular endothelial cells and in zebrafish vascular development. METHODS AND RESULTS: HOXC9 was abundantly expressed in resting human umbilical vein endothelial cells and was downregulated by hypoxia. Overexpression of HOXC9 inhibited endothelial cell proliferation, migration, and tube formation in vitro. Expression profiling and chromatin immunoprecipitation experiments in human umbilical vein endothelial cells identified interleukin 8 as the major HOXC9 target and demonstrated the direct binding of HOXC9 to the interleukin 8 promotor. HOXC9 overexpression led to reduced endothelial interleukin 8 production, whereas HOXC9 silencing increased interleukin 8. The antimigratory and antiangiogenic effect of HOXC9 overexpression could be rescued by external interleukin 8 administration. Corresponding to the cellular experiments, endothelial-specific overexpression of HOXC9 and morpholino-based interleukin 8 loss-of-function experiments inhibited zebrafish vascular development. CONCLUSION: The data identify HOXC9 as an endothelial cell active transcriptional repressor promoting the resting, antiangiogenic endothelial cell phenotype in an interleukin 8-dependent manner.
Assuntos
Células Endoteliais/fisiologia , Proteínas de Homeodomínio/genética , Hipóxia/fisiopatologia , Interleucina-8/fisiologia , Neovascularização Fisiológica/fisiologia , Proteínas de Peixe-Zebra/genética , Animais , Capilares/citologia , Capilares/fisiologia , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Regulação para Baixo/fisiologia , Células Endoteliais/citologia , Proteínas de Homeodomínio/metabolismo , Humanos , Veias Umbilicais/citologia , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Biohybrid hydrogels combining electrically neutral synthetic polymers and highly anionic glycosaminoglycans (GAGs) offer exciting options for regenerative therapies as they allow for the electrostatic conjugation of various growth factors. Unraveling details of ionization and structure within such networks defines an important analytical challenge that requires the extension of current methodologies. Here, we present a mean-field approach to quantify the density of ionizable groups, GAG concentration, and cross-linking degree of such hydrogels based on experimental data from microslit electrokinetics and ellipsometry. An exemplary poly(ethylene glycol)-heparin system was analyzed to demonstrate how electrostatic fingerprints of hydrogels obtained by the introduced strategy can sensitively display composition and structure of the polymer networks.
Assuntos
Hidrogéis/química , Eletricidade Estática , Glicosaminoglicanos/química , Heparina/química , Cinética , Polietilenoglicóis/químicaRESUMO
Retinal ganglion cells (RGCs) comprise a heterogenous group of projection neurons that transmit visual information from the retina to the brain. Progressive degeneration of these cells, as it occurs in inflammatory, ischemic, traumatic or glaucomatous optic neuropathies, results in visual deterioration and is among the leading causes of irreversible blindness. Treatment options for these diseases are limited. Neuroprotective approaches aim to slow down and eventually halt the loss of ganglion cells in these disorders. In this review, we have summarized preclinical studies that have evaluated the efficacy of cell-based neuroprotective treatment strategies to rescue retinal ganglion cells from cell death. Intraocular transplantations of diverse genetically nonmodified cell types or cells engineered to overexpress neurotrophic factors have been demonstrated to result in significant attenuation of ganglion cell loss in animal models of different optic neuropathies. Cell-based combinatorial neuroprotective approaches represent a potential strategy to further increase the survival rates of retinal ganglion cells. However, data about the long-term impact of the different cell-based treatment strategies on retinal ganglion cell survival and detailed analyses of potential adverse effects of a sustained intraocular delivery of neurotrophic factors on retina structure and function are limited, making it difficult to assess their therapeutic potential.
RESUMO
BACKGROUND: Neuronal ceroid lipofuscinosis (NCL) is a group of rare and fatal neurodegenerative lysosomal storage diseases. Progressive retinal degeneration and loss of vision are among the characteristic symptoms of affected patients. A brain-directed enzyme replacement therapy has been shown to significantly attenuate the neurological symptoms in CLN2 patients and is currently the only approved therapy for NCL; however, there is presently no treatment option for retinal dystrophy in NCL. OBJECTIVE: This short review aims to give an overview of preclinical studies that have developed and evaluated therapeutic strategies for the treatment of retinal dystrophy in animal models of different NCL forms. MATERIAL AND METHODS: The key findings of preclinical studies that have achieved positive therapeutic effects on retinal structure and/or function using different treatment strategies are summarized and discussed. RESULTS AND CONCLUSION: The published data on preclinical studies demonstrate the efficacy of different therapeutic strategies to attenuate retinal degeneration and vision loss in animal models for different NCL forms. It remains to be seen whether these promising results can be confirmed in future clinical studies.
Assuntos
Lipofuscinoses Ceroides Neuronais , Distrofias Retinianas , Animais , Modelos Animais de Doenças , Terapia de Reposição de Enzimas , Humanos , Lipofuscinoses Ceroides Neuronais/complicações , Lipofuscinoses Ceroides Neuronais/diagnóstico , Lipofuscinoses Ceroides Neuronais/terapia , Retina , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/terapia , Tripeptidil-Peptidase 1RESUMO
We have recently demonstrated that neural stem cell-based intravitreal co-administration of glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) confers profound protection to injured retinal ganglion cells (RGCs) in a mouse optic nerve crush model, resulting in the survival of ~38% RGCs two months after the nerve lesion. Here, we analyzed whether this neuroprotective effect is long-lasting and studied the impact of the pronounced RGC rescue on axonal regeneration. To this aim, we co-injected a GDNF- and a CNTF-overexpressing neural stem cell line into the vitreous cavity of adult mice one day after an optic nerve crush and determined the number of surviving RGCs 4, 6 and 8 months after the lesion. Remarkably, we found no significant decrease in the number of surviving RGCs between the successive analysis time points, indicating that the combined administration of GDNF and CNTF conferred lifelong protection to injured RGCs. While the simultaneous administration of GDNF and CNTF stimulated pronounced intraretinal axon growth when compared to retinas treated with either factor alone, numbers of regenerating axons in the distal optic nerve stumps were similar in animals co-treated with both factors and animals treated with CNTF only.
Assuntos
Morte Celular/efeitos dos fármacos , Fator Neurotrófico Ciliar/uso terapêutico , Fator Neurotrófico Derivado de Linhagem de Célula Glial/uso terapêutico , Células Ganglionares da Retina/metabolismo , Animais , Modelos Animais de Doenças , Injeções Intravítreas , CamundongosRESUMO
Neuronal ceroid lipofuscinosis (NCL) type 1 (CLN1) is a neurodegenerative storage disorder caused by mutations in the gene encoding the lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1). CLN1 patients suffer from brain atrophy, mental and motor retardation, seizures, and retinal degeneration ultimately resulting in blindness. Here, we performed an in-depth analysis of the retinal phenotype of a PPT1-deficient mouse, an animal model of this condition. Reactive astrogliosis and microgliosis were evident in mutant retinas prior to the onset of retinal cell loss. Progressive accumulation of storage material, a pronounced dysregulation of various lysosomal proteins, and accumulation of sequestosome/p62-positive aggregates in the inner nuclear layer also preceded retinal degeneration. At advanced stages of the disease, the mutant retina was characterized by a significant loss of ganglion cells, rod and cone photoreceptor cells, and rod and cone bipolar cells. Results demonstrate that PPT1 dysfunction results in early-onset pathological alterations in the mutant retina, followed by a progressive degeneration of various retinal cell types at relatively late stages of the disease. Data will serve as a reference for future work aimed at developing therapeutic strategies for the treatment of retinal degeneration in CLN1 disease.
Assuntos
Fenótipo , Degeneração Retiniana/genética , Tioléster Hidrolases/genética , Animais , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Células Bipolares da Retina/metabolismo , Células Bipolares da Retina/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Tioléster Hidrolases/deficiência , Tioléster Hidrolases/metabolismoRESUMO
To gain insight into the morphogenetic functions of the recognition molecule tenascin in the central nervous system, we have studied its localization in the developing and lesioned adult mouse optic nerve using light and electron microscopic immunocytochemistry. Since tenascin is a secreted molecule, we have analysed the tenascin-synthesizing cells in tissue sections of retinae and optic nerves by in situ hybridization. A weak and homogeneous tenascin immunoreactivity was detectable in the developing retinal nerve fibre layer and optic nerve of 14-day-old mouse embryos, the earliest developmental age investigated. In the optic nerve of neonatal and 1-week-old animals, a high number of tenascin messenger RNA (mRNA)-containing cells were present, and antibodies to tenascin labelled the surfaces of astrocytes and unmyelinated retinal ganglion cell axons. With increasing age, expression of tenascin in the optic nerve was down-regulated at the mRNA and protein levels. At the fourth postnatal week, blood vessels in the optic nerve and collagen fibrils in the vicinity of meningeal fibroblast-like cells still showed significant immunoreactivity, but the optic nerve tissue proper no longer did so. In adult animals, tenascin was no longer detectable in association with blood vessels located in the myelinated part of the optic nerve, and meninges were only weakly immunoreactive. Also, tenascin mRNA-containing cells were no longer detectable in the myelinated part of the adult mouse optic nerve and few labelled cells were found in the meninges. In the retina, ganglion cells contained no detectable levels of tenascin mRNA at any of the developmental ages analysed. No significant up-regulation of tenascin expression was seen in the nerve tissue proper of transected proximal (i.e. retinal) and distal (i.e. cranial) optic nerve stumps of adult mice during the first 4 weeks after lesioning, the time period studied. However, collagen fibrils associated with meningeal fibroblast-like cells and located near the lesion site became strongly tenascin-immunoreactive 2 days after lesioning. Also, some blood vessels at the lesion site became immunoreactive. We conclude that tenascin in the optic nerve is synthesized by glial cells and not by retinal ganglion cells. The detectability of tenascin at embryonic ages suggests that it may mediate neurite growth in vivo. The absence of a strong, lesion-induced up-regulation of tenascin expression in the regeneration-prohibitive mouse optic nerve contrasts with the lesion-induced pronounced up-regulation in the regeneration-permissive peripheral nervous system, and may indicate a functional involvement of tenascin in regenerative processes. The high tenascin positivity of collagen fibrils at early postnatal ages and after lesioning suggests that tenascin expression may be correlated with mitotic activity of the associated meningeal fibroblast-like cells. Finally, tenascin may be involved in the process of vascularization, since the molecule is associated with blood vessels in developing and adult lesioned, but not intact adult, optic nerves.
RESUMO
HOXC9 belongs to the family of homeobox transcription factors, which are regulators of body patterning and development. HOXC9 acts as a negative regulator on blood endothelial cells but its function on lymphatic vessel development has not been studied. The hyaluronan receptor homologs stabilin 1 and stabilin 2 are expressed in endothelial cells but their role in vascular development is poorly understood. This study was aimed at investigating the function of HOXC9, stabilin 2 and stabilin 1 in lymphatic vessel development in zebrafish and in endothelial cells. Morpholino-based expression silencing of HOXC9 repressed parachordal lymphangioblast assembly and thoracic duct formation in zebrafish. HOXC9 positively regulated stabilin 2 expression in zebrafish and in HUVECs and expression silencing of stabilin 2 phenocopied the HOXC9 morphant vascular phenotype. This effect could be compensated by HOXC9 mRNA injection in stabilin 2 morphant zebrafish embryos. Stabilin 1 also regulated parachordal lymphangioblast and thoracic duct formation in zebrafish but acts independently of HOXC9. On a cellular level stabilin 1 and stabilin 2 regulated endothelial cell migration and in-gel sprouting angiogenesis in endothelial cells. HOXC9 was identified as novel transcriptional regulator of parachordal lymphangioblast assembly and thoracic duct formation in zebrafish that acts via stabilin 2. Stabilin 1, which acts independently of HOXC9, has a similar function in zebrafish and both receptors control important cellular processes in endothelial cells.