Mesenchymal Stromal Cells Facilitate Tip Cell Fusion Downstream of BMP-Mediated Venous Angiogenesis-Brief Report.

BACKGROUND: The goal of this study was to identify and characterize cell-cell interactions that facilitate endothelial tip cell fusion downstream of BMP (bone morphogenic protein)-mediated venous plexus formation. METHODS: High resolution and time-lapse imaging of transgenic reporter lines and loss-of-function studies were carried out to study the involvement of mesenchymal stromal cells during venous angiogenesis. RESULTS: BMP-responsive stromal cells facilitate timely and precise fusion of venous tip cells during developmental angiogenesis. CONCLUSIONS: Stromal cells are required for anastomosis of venous tip cells in the embryonic caudal hematopoietic tissue.

Ocular proteomic and transcriptomic changes with aging in a rabbit model of lensectomy with intraocular lens insertion.

Children that undergo intraocular surgery have an exaggerated postoperative response compared to adults that can result in significant postoperative challenges and reduced post-operative visual acuity. Rabbits were used as an animal model for investigating aging differences, treatment options, and surgical techniques for anterior chamber surgical interventions due to similarities in anterior chamber size and decreasing postoperative response with age. In our study, juvenile and adult rabbits underwent lensectomy with intraocular lens (IOL) insertion to determine how ocular RNA transcripts and proteins change with age. Rabbits underwent lensectomy with IOL insertion, and aqueous humor (AH) was collected immediately prior to surgery and at the peak of the postoperative response on post-operative day 3. Proteins related to coagulation and inflammation were assessed using targeted mass spectrometry. In addition, the cornea and iris/ciliary body tissues were dissected, and transcripts analyzed using RNA sequencing. While clinically, juvenile rabbits have greater fibrin formation following intraocular surgery compared to older rabbits, this change does not appear to be related to relative abundance levels of coagulation and inflammatory proteins in the AH. Gene transcript levels from a variety of immune response and inflammatory pathways reflected significant increases when comparing operated to unoperated ocular tissues, indicating the significant impact that surgery has on each ocular structure. This work further advances our understanding of how the rabbit eye proteomic and transcriptomic changes in response to surgery with aging, as we seek to ultimately identify the mechanisms for the exaggerated postoperative responses after pediatric intraocular surgery.

Ablation of mpeg+ Macrophages Exacerbates mfrp-Related Hyperopia.

Purpose: Proper refractive development of the eye, termed emmetropization, is critical for focused vision and is impacted by both genetic determinants and several visual environment factors. Improper emmetropization caused by genetic variants can lead to congenital hyperopia, which is characterized by small eyes and relatively short ocular axial length. To date, variants in only four genes have been firmly associated with human hyperopia, one of which is MFRP. Zebrafish mfrp mutants also have hyperopia and, similar to reports in mice, exhibit increased macrophage recruitment to the retina. The goal of this research was to examine the effects of macrophage ablation on emmetropization and mfrp-related hyperopia. Methods: We utilized a chemically inducible, cell-specific ablation system to deplete macrophages in both wild-type and mfrp mutant zebrafish. Spectral-domain optical coherence tomography was then used to measure components of the eye and determine relative refractive state. Histology, immunohistochemistry, and transmission electron microscopy were used to further study the eyes. Results: Although macrophage ablation does not cause significant changes to the relative refractive state of wild-type zebrafish, macrophage ablation in mfrp mutants significantly exacerbates their hyperopic phenotype, resulting in a relative refractive error 1.3 times higher than that of non-ablated mfrp siblings. Conclusions: Genetic inactivation of mfrp leads to hyperopia, as well as abnormal accumulation of macrophages in the retina. Ablation of the mpeg1-positive macrophage population exacerbates the hyperopia, suggesting that macrophages may be recruited in an effort help preserve emmetropization and ameliorate hyperopia.

Brain-Derived Neurotrophic Factor as a Treatment Option for Retinal Degeneration.

This review discusses the therapeutic potential of brain-derived neurotrophic factor (BDNF) for retinal degeneration. BDNF, nerve growth factor (NGF), neurotrophin 3 (NT-3) and NT-4/NT-5 belong to the neurotrophin family. These neuronal modulators activate a common receptor and a specific tropomyosin-related kinase (Trk) receptor. BDNF was identified as a photoreceptor protectant in models of retinal degeneration as early as 1992. However, development of effective therapeutics that exploit this pathway has been difficult due to challenges in sustaining therapeutic levels in the retina.

Loss of Zebrafish Mfrp Causes Nanophthalmia, Hyperopia, and Accumulation of Subretinal Macrophages.

Purpose: Mutations in membrane frizzled-related protein (MFRP) are associated with nanophthalmia, hyperopia, foveoschisis, irregular patches of RPE atrophy, and optic disc drusen in humans. Mouse mfrp mutants show retinal degeneration but no change in eye size or refractive state. The goal of this work was to generate zebrafish mutants to investigate the loss of Mfrp on eye size and refractive state, and to characterize other phenotypes observed. Methods: Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 methods were used to generate multiple frameshift mutations in zebrafish mfrp causing premature translational stops in Mfrp. Spectral-domain optical coherence tomography (SD-OCT) was used to measure eye metrics and refractive state, and immunohistochemistry was used to study adult eyes. Gene expression levels were measured using quantitative PCR. Results: Zebrafish Mfrp was shown to localize to apical and basal regions of RPE cells, as well as the ciliary marginal zone. Loss of Mfrp in mutant zebrafish was verified histologically. Zebrafish eyes that were mfrp mutant showed reduced axial length causing hyperopia, RPE folding, and macrophages were observed subretinally. Visual acuity was reduced in mfrp mutant animals. Conclusions: Mutation of zebrafish mfrp results in hyperopia with subretinal macrophage infiltration, phenocopying aspects of human and mouse Mfrp deficiency. These mutant zebrafish will be useful in studying the onset and progression of Mfrp-related nanophthalmia, the cues that initiate the recruitment of macrophages, and the mechanisms of Mfrp function.

Dynamic smad-mediated BMP signaling revealed through transgenic zebrafish.

Bone morphogenic protein (BMP) signaling is fundamental to development, injury response, and homeostasis. We have developed transgenic zebrafish that report Smad-mediated BMP signaling in embryos and adults. These lines express either enhanced green fluorescent protein (eGFP), destabilized eGFP, or destabilized Kusabira Orange 2 (KO2) under the well-characterized BMP Response Element (BRE). These fluorescent proteins were found to be expressed dynamically in regions of known BMP signaling including the developing tail bud, hematopoietic lineage, dorsal eye, brain structures, heart, jaw, fins, and somites, as well as other tissues. Responsiveness to changes in BMP signaling was confirmed by observing fluorescence after activation in an hsp70:bmp2b transgenic background or by inhibition in an hsp70:nog3 background. We further demonstrated faithful reportage by the BRE transgenic lines following chemical repression of BMP signaling using an inhibitor of BMP receptor activity, dorsomorphin. Overall, these lines will serve as valuable tools to explore the mechanisms and regulation of BMP signal during embryogenesis, in tissue maintenance, and during disease.