Activation of adult mammalian retinal stem cells in vivo via antagonism of BMP and sFRP2

Background The adult mammalian retina does not have the capacity to regenerate cells lost due to damage or disease. Therefore, retinal injuries and blinding diseases result in irreversible vision loss. However, retinal stem cells (RSCs), which participate in retinogenesis during development, persist in a quiescent state in the ciliary epithelium (CE) of the adult mammalian eye. Moreover, RSCs retain the ability to generate all retinal cell types when cultured in vitro, including photoreceptors. Therefore, it may be possible to activate endogenous RSCs to induce retinal neurogenesis in vivo and restore vision in the adult mammalian eye. Methods To investigate if endogenous RSCs can be activated, we performed combinatorial intravitreal injections of antagonists to BMP and sFRP2 proteins (two proposed mediators of RSC quiescence in vivo), with or without growth factors FGF and Insulin. We also investigated the effects of chemically-induced N-methyl-N-Nitrosourea (MNU) retinal degeneration on RSC activation, both alone and in combination withthe injected factors. Further, we employed inducible Msx1-CreERT2 genetic lineage labeling of the CE followed by stimulation paradigms to determine if activated endogenous RSCs could migrate into the retina and differentiate into retinal neurons. Results We found that in vivo antagonism of BMP and sFRP2 proteins induced CE cells in the RSC niche to proliferate and expanded the RSC population. BMP and sFRP2 antagonism also enhanced CE cell proliferation in response to exogenous growth factor stimulation and MNU-induced retinal degeneration. Furthermore, Msx1-CreERT2 genetic lineage tracing revealed that CE cells migrated into the retina following stimulation and/or injury, where they expressed markers of mature photoreceptors and retinal ganglion cells. Conclusions Together, these results indicate that endogenous adult mammalian RSCs may have latent regenerative potential that can be activated by modulating the RSC niche and hold promise as a means for endogenous retinal cell therapy to repair the retina and improve vision.

Non-Pigmented Ciliary Epithelium-Derived Extracellular Vesicles Loaded with SMAD7 siRNA Attenuate Wnt Signaling in Trabecular Meshwork Cells In Vitro

Primary open-angle glaucoma is established by the disruption of trabecular meshwork (TM) function. The disruption leads to increased resistance to the aqueous humor (AH), generated by the non-pigmented ciliary epithelium (NPCE). Extracellular vesicles (EVs) participate in the communication between the NPCE and the TM tissue in the ocular drainage system. The potential use of NPCE-derived EVs to deliver siRNA to TM cells has scarcely been explored. NPCE-derived EVs were isolated and loaded with anti-fibrotic (SMAD7) siRNA. EV’s structural integrity and siRNA loading efficiency were estimated via electron microscopy and fluorescence. Engineered EVs were added to pre-cultured TM cells and qRT-PCR was used to verify the transfer of selected siRNA to the cells. Western blot analysis was used to evaluate the qualitative effects on Wnt-TGFβ2 proteins’ expression. EVs loaded with exogenous siRNA achieved a 53% mRNA knockdown of SMAD7 in TM cells, resulting in a significant elevation in the levels of β-Catenin, pGSK3β, N-Cadherin, K-Cadherin, and TGFβ2 proteins in TM cells. NPCE-derived EVs can be used for efficient siRNA molecule delivery into TM cells, which may prove to be beneficial as a therapeutic target to lower intraocular pressure (IOP).

Ultrastructure of the gills ciliary epithelium of Limnoperna fortunei (Dunker 1857), the invasive golden mussel

Background Limnoperna fortunei is a bivalve mollusk originally from southern Asia that invaded South America in the 1990's. Its high efficiency in pumping and filtering water and the capacity to promote strong adhesion to different substrates allowed the adaptation of this invasive species, associated with several environmental and economic damages. A deep understanding of the biology and ecology aspects of L. fortunei is necessary to outline effective strategies to manage its invasion. Mollusk gills are important structures responsible for several biological functions including breathing and feeding. In this work, we characterized the ultrastructure of L. fortunei gills and its ciliary epithelium using transmission and scanning electron microscopy. This is the first report of the L. fortunei gills ciliary epithelial cells visualized with high resolution and detailed morphology. Results The analysis showed a highly organized and large amount of ciliary structures (frontal cilia, laterofrontal cilia, and lateral cilia) on the entire length of the branchial epithelium. Mitochondria, smooth endoplasmic reticulum and glycogen granules were abundantly found in the epithelial cells of the gills, suggesting that all this energetic apparatus could be related to the morpho-functional structure of the cilia. Vesicles possibly containing mucus could also be observed in these cells, suggesting that they might be related to L. fortunei mechanism of selection and/or rejection of captured particles suspended in water. Conclusions Our data suggest the mechanism used by this mollusk for particles capture and selection, which could contribute to a better understanding of important aspects of invasion and decide on more efficient and economic strategies of population control.

Glucocorticoid agonists enhance retinal stem cell self-renewal and proliferation

Background Adult mammalian retinal stem cells (RSCs) readily proliferate, self-renew, and generate progeny that differentiate into all retinal cell types in vitro. RSC-derived progeny can be induced to differentiate into photoreceptors, making them a potential source for retinal cell transplant therapies. Despite their proliferative propensity in vitro, RSCs in the adult mammalian eye do not proliferate and do not have a regenerative response to injury. Thus, identifying and modulating the mechanisms that regulate RSC proliferation may enhance the capacity to produce RSC-derived progeny in vitro and enable RSC activation in vivo. Methods Here, we used medium-throughput screening to identify small molecules that can expand the number of RSCs and their progeny in culture. In vitro differentiation assays were used to assess the effects of synthetic glucocorticoid agonist dexamethasone on RSC-derived progenitor cell fate. Intravitreal injections of dexamethasone into adult mouse eyes were used to investigate the effects on endogenous RSCs. Results We discovered that high-affinity synthetic glucocorticoid agonists increase RSC self-renewal and increase retinal progenitor proliferation up to 6-fold without influencing their differentiation in vitro. Intravitreal injection of synthetic glucocorticoid agonist dexamethasone induced in vivo proliferation in the ciliary epithelium—the niche in which adult RSCs reside. Conclusions Together, our results identify glucocorticoids as novel regulators of retinal stem and progenitor cell proliferation in culture and provide evidence that GCs may activate endogenous RSCs.

A rare case of surgical treatment of a patient with gastric cancer with Cartagener syndrome

Kartagener Syndrome (CS) is a rare autosomal recessive disease that is a part of primary ciliary dyskinesia (PCD), and is characterized by a triad of syndrome including bronchiectasis, polysinusitis and a complete mirror arrangement of the internal organs of the chest and abdominal cavities (situs inversus). In most patients, bronchiectasis in the lungs develops from the first years of life and progresses in time. The tardy diagnostic leads to a poor prognosis of the disease and adduce the development of persistent violations of the function of external respiration. Most often, patients with CS are observed by physicians and pulmonologists from early childhood, since respiratory infections often require annual hospitalization in specialized medical institutions due to abnormalities of the ciliary epithelium of the respiratory tract. However, to date, there are no generally accepted recommendations for the management and treatment of patients in this category. This article describes the case of a newly diagnosed CS in an older patient undergoing surgical treatment for gastric cancer. Despite the pronounced concomitant pathology on the part of the bronchopulmonary system, the perioperative period in the patient proceeded relatively satisfactorily and did not entail an increase in the length of hospitalization.