VEGF-B, a homolog of VEGF discovered a long time ago, has not been considered an important target in antiangiogenic therapy. Instead, it has received little attention from the field. In this study, using different animal models and multiple types of vascular cells, we revealed that although VEGF-B is dispensable for blood vessel growth, it is critical for their survival. Importantly, the survival effect of VEGF-B is not only on vascular endothelial cells, but also on pericytes, smooth muscle cells, and vascular stem/progenitor cells. In vivo, VEGF-B targeting inhibited both choroidal and retinal neovascularization. Mechanistically, we found that the vascular survival effect of VEGF-B is achieved by regulating the expression of many vascular prosurvival genes via both NP-1 and VEGFR-1. Our work thus indicates that the function of VEGF-B in the vascular system is to act as a ''survival,'' rather than an ''angiogenic'' factor and that VEGF-B inhibition may offer new therapeutic opportunities to treat neovascular diseases.apoptosis ͉ vascular survival ͉ ocular neovascularization
MicroRNA (miRNA) expression in fetal human retinal pigment epithelium (hfRPE), retina, and choroid were pairwise compared to determine those miRNAs that are enriched by 10-fold or more in each tissue compared with both of its neighbors. miRs-184, 187, 200a/200b, 204/211, and 221/222 are enriched in hfRPE by 10- to 754-fold compared with neuroretina or choroid (P<0.05). Five of these miRNAs are enriched in RPE compared with 20 tissues throughout the body and are 10- to 20,000-fold more highly expressed (P<0.005). miR-204 and 211 are the most highly expressed in the RPE. In addition, expression of miR-204/211 is significantly lower in the NCI60 tumor cell line panel compared with that in 13 normal tissues, suggesting the progressive disruption of epithelial barriers and increased proliferation. We demonstrated that TGF-beta receptor 2 (TGF-betaR2) and SNAIL2 are direct targets of miR-204 and that a reduction in miR-204 expression leads to reduced expression of claudins 10, 16, and 19 (message/protein) consistent with our observation that anti-miR-204/211 decreased transepithelial resistance by 80% and reduced cell membrane voltage and conductance. The anti-miR-204-induced decrease in Kir7.1 protein levels suggests a signaling pathway that connects TGF-betaR2 and maintenance of potassium homeostasis. Overall, these data indicate a critical role for miR-204/211 in maintaining epithelial barrier function and cell physiology.
In retinitis pigmentosa, loss of cone photoreceptors leads to blindness, and preservation of cone function is a major therapeutic goal. However, cone loss is thought to occur as a secondary event resulting from degeneration of rod photoreceptors. Here we report a genome editing approach in which adeno-associated virus (AAV)-mediated CRISPR/Cas9 delivery to postmitotic photoreceptors is used to target the Nrl gene, encoding for Neural retina-specific leucine zipper protein, a rod fate determinant during photoreceptor development. Following Nrl disruption, rods gain partial features of cones and present with improved survival in the presence of mutations in rod-specific genes, consequently preventing secondary cone degeneration. In three different mouse models of retinal degeneration, the treatment substantially improves rod survival and preserves cone function. Our data suggest that CRISPR/Cas9-mediated NRL disruption in rods may be a promising treatment option for patients with retinitis pigmentosa.
In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/βA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy. We demonstrated that CRYBA1 coimmunoprecipitates with the ATP6V0A1/V0-ATPase a1 subunit. Interestingly, in mice when Cryba1 (the gene encoding both the βA3- and βA1-crystallin forms) is knocked out specifically in RPE, V-ATPase activity is decreased and lysosomal pH is elevated, while cathepsin D (CTSD) activity is decreased. Fundus photographs of these Cryba1 conditional knockout (cKO) mice showed scattered lesions by 4 months of age that increased in older mice, with accumulation of lipid-droplets as determined by immunohistochemistry. Transmission electron microscopy (TEM) of cryba1 cKO mice revealed vacuole-like structures with partially degraded cellular organelles, undigested photoreceptor outer segments and accumulation of autophagosomes. Further, following autophagy induction both in vivo and in vitro, phospho-AKT and phospho-RPTOR/Raptor decrease, while pMTOR increases in RPE cells, inhibiting autophagy and AKT-MTORC1 signaling. Impaired lysosomal clearance in the RPE of the cryba1 cKO mice also resulted in abnormalities in retinal function that increased with age, as demonstrated by electroretinography. Our findings suggest that loss of CRYBA1 causes lysosomal dysregulation leading to the impairment of both autophagy and phagocytosis.
Distinct mutations in the centrosomal-cilia protein CEP290 lead to diverse clinical findings in syndromic ciliopathies. We show that CEP290 localizes to the transition zone in ciliated cells, precisely to the region of Y-linkers between central microtubules and plasma membrane. To create models of CEP290-associated ciliopathy syndromes, we generated Cep290(ko/ko) and Cep290(gt/gt) mice that produce no or a truncated CEP290 protein, respectively. Cep290(ko/ko) mice exhibit early vision loss and die from hydrocephalus. Retinal photoreceptors in Cep290(ko/ko) mice lack connecting cilia, and ciliated ventricular ependyma fails to mature. The minority of Cep290(ko/ko) mice that escape hydrocephalus demonstrate progressive kidney pathology. Cep290(gt/gt) mice die at mid-gestation, and the occasional Cep290(gt/gt) mouse that survives shows hydrocephalus and severely cystic kidneys. Partial loss of CEP290-interacting ciliopathy protein MKKS mitigates lethality and renal pathology in Cep290(gt/gt) mice. Our studies demonstrate domain-specific functions of CEP290 and provide novel therapeutic paradigms for ciliopathies.
SUMMARY:Entactin-1 (nidogen-1) is an ubiquitous component of basement membranes. From in vitro experiments, entactin-1 was assigned a role in maintaining the structural integrity of the basement membrane because of its binding affinity to other components, such as type IV collagen and laminin. Entactin-1 also interacts with integrin receptors on the cell surface to mediate cell adhesion, spreading, and motility. Targeted disruption of the entactin-1 gene in the mouse presented in this study revealed a duplication of the entacin-1 locus. Homozygous mutants for the functional locus lacked entactin-1 mRNA and protein and often displayed seizure-like symptoms and loss of muscle control in the hind legs. The behavior patterns suggested the presence of neurologic deficits in the central nervous system, thus providing genetic evidence linking entactin-1 to proper functions of the neuromuscular system. In homozygous mutants, structural alterations in the basement membranes were found only in selected locations including brain capillaries and the lens capsule. The morphology of the basement membranes in other tissues examined superficially appeared to be normal. These observations suggest that the lost functions of entactin-1 result in pathologic changes that are highly tissue specific. (Lab Invest 2002, 82:1617-1630.
Color, an important visual cue for survival, is encoded by comparing signals from photoreceptors with different spectral sensitivities. The mouse retina expresses a short wavelength-sensitive and a middle/long wavelength-sensitive opsin (S- and M-opsin), forming opposing, overlapping gradients along the dorsal-ventral axis. Here, we analyzed the distribution of all cone types across the entire retina for two commonly used mouse strains. We found, unexpectedly, that ‘true S-cones’ (S-opsin only) are highly concentrated (up to 30% of cones) in ventral retina. Moreover, S-cone bipolar cells (SCBCs) are also skewed towards ventral retina, with wiring patterns matching the distribution of true S-cones. In addition, true S-cones in the ventral retina form clusters, which may augment synaptic input to SCBCs. Such a unique true S-cone and SCBC connecting pattern forms a basis for mouse color vision, likely reflecting evolutionary adaptation to enhance color coding for the upper visual field suitable for mice’s habitat and behavior.
Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell.
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