RESUMO
Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in the U.S. The vision-threatening processes of neuroglial and vascular dysfunction in DR occur in concert, driven by hyperglycemia and propelled by a pathway of inflammation, ischemia, vasodegeneration, and breakdown of the blood retinal barrier. Currently, no therapies exist for normalizing the vasculature in DR. Here, we show that a single intravitreal dose of adeno-associated virus serotype 2 encoding a more stable, soluble, and potent form of angiopoietin 1 (AAV2.COMP-Ang1) can ameliorate the structural and functional hallmarks of DR in Ins2Akita mice, with sustained effects observed through six months. In early DR, AAV2.COMP-Ang1 restored leukocyte-endothelial interaction, retinal oxygenation, vascular density, vascular marker expression, vessel permeability, retinal thickness, inner retinal cellularity, and retinal neurophysiological response to levels comparable with nondiabetic controls. In late DR, AAV2.COMP-Ang1 enhanced the therapeutic benefit of intravitreally delivered endothelial colony-forming cells by promoting their integration into the vasculature and thereby stemming further visual decline. AAV2.COMP-Ang1 single-dose gene therapy can prevent neurovascular pathology, support vascular regeneration, and stabilize vision in DR.
Assuntos
Angiopoietina-1/uso terapêutico , Proteína de Matriz Oligomérica de Cartilagem/uso terapêutico , Diabetes Mellitus Tipo 1/complicações , Retinopatia Diabética/terapia , Modelos Animais de Doenças , Terapia Genética , Retina/patologia , Angiopoietina-1/química , Angiopoietina-1/genética , Angiopoietina-1/metabolismo , Animais , Proteína de Matriz Oligomérica de Cartilagem/química , Proteína de Matriz Oligomérica de Cartilagem/genética , Proteína de Matriz Oligomérica de Cartilagem/metabolismo , Células Cultivadas , Terapia Combinada/efeitos adversos , Cruzamentos Genéticos , Retinopatia Diabética/imunologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/transplante , Terapia Genética/efeitos adversos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Injeções Intravítreas , Leucócitos/citologia , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/patologia , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Estabilidade Proteica , Distribuição Aleatória , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Retina/imunologia , Retina/metabolismo , SolubilidadeRESUMO
The origin and developmental mechanisms underlying coronary vessels are not fully elucidated. Here we show that myocardium-derived angiopoietin-1 (Ang1) is essential for coronary vein formation in the developing heart. Cardiomyocyte-specific Ang1 deletion results in defective formation of the subepicardial coronary veins, but had no significant effect on the formation of intramyocardial coronary arteries. The endothelial cells (ECs) of the sinus venosus (SV) are heterogeneous population, composed of APJ-positive and APJ-negative ECs. Among these, the APJ-negative ECs migrate from the SV into the atrial and ventricular myocardium in Ang1-dependent manner. In addition, Ang1 may positively regulate venous differentiation of the subepicardial APJ-negative ECs in the heart. Consistently, in vitro experiments show that Ang1 indeed promotes venous differentiation of the immature ECs. Collectively, our results indicate that myocardial Ang1 positively regulates coronary vein formation presumably by promoting the proliferation, migration and differentiation of immature ECs derived from the SV.
Assuntos
Angiopoietina-1/metabolismo , Vasos Coronários/embriologia , Células-Tronco Embrionárias/fisiologia , Coração/embriologia , Miocárdio/metabolismo , Angiopoietina-1/genética , Animais , Diferenciação Celular/fisiologia , Quimera , Primers do DNA/genética , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Our previous studies demonstrated that enhanced epithelial cell proliferation is important for healing of experimental esophageal ulcers. However, the roles of angiogenesis, its major mediator, vascular endothelial growth factor (VEGF), and the mechanism(s) regulating VEGF expression during esophageal ulcer healing remain unknown. Esophageal ulcers were induced in rats by focal application of acetic acid. We studied expressions of hypoxia-inducible transcription factor-1 alpha (HIF-1 alpha), an activator of the VEGF gene, and VEGF by reverse transcriptase-polymerase chain reaction, Western blotting, and immunostaining. To determine the efficacy of VEGF gene therapy in esophageal ulcer healing, we studied whether a single local injection of plasmid cDNA encoding recombinant human VEGF(165) affects ulcer healing and angiogenesis. Esophageal ulceration induced HIF-1 alpha protein expression and VEGF gene activation reflected by increased VEGF mRNA (240%) and VEGF protein (310%) levels. HIF-1 alpha protein was expressed in microvessels bordering necrosis where it co-localized with VEGF. Injection of cDNA encoding VEGF(165) significantly enhanced angiogenesis and accelerated esophageal ulcer healing. These results: 1) suggest that HIF-1 alpha may mediate esophageal ulceration-triggered VEGF gene activation, 2) indicate an essential role of VEGF and angiogenesis in esophageal ulcer healing, and 3) demonstrate the feasibility of gene therapy for the treatment of esophageal ulcers.