Angiogenesis in the Gonadal Capillary Network of the Chick Embryo

Seventy-one chick embryos of both sexes at the 35 Hamburger and Hamilton (H-H) developmental stage were processed for scanning electron microscopy of vascular corrosion casts and of critical point dried specimens, as well as transmission electron- and light microscopy, in order to study the angiogenic structures. The gonadal subepithelial capillary network was located at the level of the tunica albuginea under the covering epithelium. The casts showed a densely-meshed capillary network and numerous sprouting (nodular protrusions or capillary sprouts) and non-sprouting (enlarged vessels and angiogenic holes) angiogenic structures that were randomly distributed and mixed. Four types of angio-genic holes were encountered in the casts: primary (diameter \u3c 2.5 μm), secondary (diameter \u3e 2.5 μm), tertiary (variable diameter and circular narrowings on one side), and open angiogenic holes. We suggest that the different morphologies reflect evolution of these holes. Furthermore, the open angiogenic hole would probably either form nodular protrusions at its open ends, which tend to join with other nodular protrusions and neighboring capillaries and form new vessels; or there would be fusion with two or more neighboring open holes. Correlative critical point dried sections showed fenestrations in the capillary walls and transcapillary pillars that corresponded to the angiogenic holes found in the casts. Ultrathin sections of the vessels presented typical characteristics of growing endothelium: large nuclei with loosely textured chromatin, abundant cytoplasm rich in cell organelles and intraluminal endothelial processes

Technical Improvements in Corrosion Casting of Small Specimens: A Study on Mesonephric Tubules and Vessels of Chicken Embryos

The injection technique for corrosion casting of small, embryonic material can be improved by using a chemical ligature (cyanocrylate). With this simple method, leakage of the resin at the injection site is prevented and the mechanical stability of the cannula-vessel coupling is improved. The blood vascular system of chicken embryos as small as Hamburger-Hamilton stage 24 (approximately 4.5 days of incubation) has successfully been injected using this procedure. Corrosion casts of the mesonephric tubular system have been made in a similar manner. Additionally, a simple way for secure transport of the fragile casts by immersion in 10% gelatin is suggested

Reply by A. Carretero et al.

Dear Editor, The study of vascular casts in the embryo by means of the scanning electron microscope represents the main subject of our research. We fully agree with DeRuiter and Gittenberger-de Groot in that corrosion casting is a valuable tool to study the early angiogenesis in embryos

Vascular Architecture of the Lactating and Non-Lactating Teat of the Bitch: A Scanning Electron- and Light Microscope Study

Tissues from fourteen mammary glands of eight bitches were prepared for scanning electron microscopy of vascular corrosion casts and for histology to study the vasculature of the lactating and non-lactating teats. The densely-meshed mammary dermal capillary network formed ridges and troughs. The teat ducts were vascularized by a relatively densely-meshed capillary network which drained into veins longitudinally oriented to the ducts. Between eight and fifteen teat duct openings were seen on the tip of the teat, that were sometimes divided by a septum. The inner vascularization of the teat showed that the main papillary arteries divided into undulating secondary papillary arteries which presented numerous semi-constrictions and loops. Their structure may help during erection of the teat. Arteriovenous anastomoses found at different points may participate in blood flow maintenance during suckling, heat regulation and teat erection. Veins freely anastomosed and ran longitudinally to the axis of the teat. They exhibited numerous bicuspid valves. In non-lactating teats, vessels showed the same main architecture and characteristics mentioned above, although these were considerably less marked. The structure of the vascular elements in the teat of the bitch could favor blood flow during suckling and suggest that vessels adapt to the physiological situation

Programmed cell senescence during mammalian embryonic development

Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locations, including the mesonephros and the endolymphatic sac of the inner ear, which we have analyzed in detail. Mechanistically, senescence in both structures is strictly dependent on p21, but independent of DNA damage, p53, or other cell-cycle inhibitors, and it is regulated by the TGF-beta/SMAD and PI3K/FOXO pathways. Developmentally programmed senescence is followed by macrophage infiltration, clearance of senescent cells, and tissue remodeling. Loss of senescence due to the absence of p21 is partially compensated by apoptosis but still results in detectable developmental abnormalities. Importantly, the mesonephros and endolymphatic sac of human embryos also show evidence of senescence. We conclude that the role of developmentally programmed senescence is to promote tissue remodeling and propose that this is the evolutionary origin of damage-induced senescence

Vascular interstitial cells in retinal arteriolar annuli are altered during hypertension

The authors thank Veronica Melgarejo, Lorena Noya, and Angel Vazquez for technical assistance. Supported by grants from Instituto de Salud Carlos III (PI12/00605, PI16/00719, SAF2014-59945-R, and Red de Investigación Renal REDinREN 12/0021/0013), Spain; Fundação para a Ciência e a Tecnologia (SFRH/BPD/102573/2014, SFRH/BD/95330/2013), Ministerio da Educação e Ciência, Portugal; and Fondo Europeo de Desarrollo Regional (FEDER).Supported by grants from Instituto de Salud Carlos III (PI12/00605, PI16/00719, SAF2014-59945-R, and Red de Investigacion Renal REDinREN 12/0021/0013), Spain; Fundacao para a Ciencia e a Tecnologia (SFRH/BPD/102573/2014, SFRH/BD/95330/2013), Ministerio da Educacao e Ciencia, Portugal; and Fondo Europeo de Desarrollo Regional (FEDER).PURPOSE. It has been suggested that arteriolar annuli localized in retinal arterioles regulate retinal blood flow acting as sphincters. Here, the morphology and protein expression profile of arteriolar annuli have been analyzed under physiologic conditions in the retina of wildtype, β-actin-Egfp, and Nestin-gfp transgenic mice. Additionally, to study the effect of hypertension, the KAP transgenic mouse has been used. METHODS. Cellular architecture has been studied using digested whole mount retinas and transmission electron microscopy. The profile of protein expression has been analyzed on paraffin sections and whole mount retinas by immunofluorescence and histochemistry. RESULTS. The ultrastructural analysis of arteriolar annuli showed a different cell population found between endothelial and muscle cells that matched most of the morphologic criteria established to define interstitial Cajal cells. The profile of protein expression of these vascular interstitial cells (VICs) was similar to that of interstitial Cajal cells and different from the endothelial and smooth muscle cells, because they expressed b-actin, nestin, and CD44, but they did not express CD31 and a-SMA or scarcely express F-actin. Furthermore, VICs share with pericytes the expression of NG2 and platelet-derived growth factor receptor beta (PDGFR-β). The high expression of Ano1 and high activity of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase observed in VICs was diminished during hypertensive retinopathy suggesting that these cells might play a role on the motility of arteriolar annuli and that this function is altered during hypertension. CONCLUSIONS. A novel type of VICs has been described in the arteriolar annuli of mouse retina. Remarkably, these cells undergo important molecular modifications during hypertensive retinopathy and might thus be a therapeutic target against this disease

Novel use of plga microspheres to create an animal model of glaucoma with progressive neuroretinal degeneration

Progressive degeneration of neuroretinal tissue with maintained elevated intraocular pressure (IOP) to simulate chronic glaucoma was produced by intracameral injections of poly (lactic-co-glycolic) acid (PLGA) microspheres (Ms) in rat eyes. The right eye of 39 rats received different sizes of PLGA-Ms (2 µL suspension; 10% w/v): 14 with 38–20 µm Ms (Ms38/20 model) and 25 with 20–10 µm particles (Ms20/10 model). This novel glaucoma animal model was compared to the episcleral vein sclerosis (EPI) model (25 eyes). Injections were performed at baseline, two, four and six weeks. Clinical signs, IOP, retina and optic nerve thicknesses (using in vivo optical coherence tomography; OCT), and histological studies were performed. An IOP increment was observed in all three groups, however, the values obtained from the PLGA-Ms injection resulted lower with a better preservation of the ocular surface. In fact, the injection of Ms20/10 created a gentler, more progressive, and more sustained increase in IOP. This IOP alteration was correlated with a significant decrease in most OCT parameters and in histological ganglion-cell count for the three conditions throughout the eight-week follow-up. In all cases, progressive degeneration of the retina, retinal ganglion cells and optic nerve, simulating chronic glaucoma, was detected by OCT and corroborated by histological study. Results showed an alternative glaucoma model to the well-known episcleral vein model, which was simpler to perform, more reproducible and easier to monitor in vivo

Chronic glaucoma using biodegradable microspheres to induce intraocular pressure elevation. Six-month follow-up

Background: To compare two prolonged animal models of glaucoma over 24 weeks of follow-up. A novel pre-trabecular model of chronic glaucoma was achieved by injection of biodegradable poly lactic-co-glycolic acid (PLGA) microspheres (10–20 µm) (Ms20/10) into the ocular anterior chamber to progressively increase ocular hypertension (OHT). Methods: Rat right eyes were injected to induce OHT: 50% received a suspension of Ms20/10 in the anterior chamber at 0, 2, 4, 8, 12, 16 and 20 weeks, and the other 50% received a sclerosing episcleral vein injection biweekly (EPIm). Ophthalmological clinical signs, intraocular pressure (IOP), neuroretinal functionality measured by electroretinography (ERG), and structural analysis of the retina, retinal nerve fiber layer (RNFL) and ganglion cell layer (GCL) protocols using optical coherence tomography (OCT) and histological exams were performed. Results: Both models showed progressive neuroretinal degeneration (p < 0.05), and contralateral eye affectation. The Ms20/10 model showed a more progressive increase in IOP and better preservation of ocular surface. Although no statistical differences were found between models, the EPIm showed a tendency to produce thicker retinal and thinner GCL thicknesses, slower latency and smaller amplitude as measured using ERG, and more aggressive disturbances in retinal histology. In both models, while the GCL showed the greatest percentage loss of thickness, the RNFL showed the greatest and earliest rate of thickness loss. Conclusions: The intracameral model with biodegradable microspheres resulted more like the conditions observed in humans. It was obtained by a less-aggressive mechanism, which allows for adequate study of the pathology over longer periods. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

The development and growth of tissues derived from cranial neural crest and primitive mesoderm is dependent on the ligation status of retinoic acid receptor γ:evidence that retinoic acid receptor γ functions to maintain stem/progenitor cells in the absence of retinoic acid

Retinoic acid (RA) signaling is important to normal development. However, the function of the different RA receptors (RARs)-RARα, RARβ, and RARγ-is as yet unclear. We have used wild-type and transgenic zebrafish to examine the role of RARγ. Treatment of zebrafish embryos with an RARγ-specific agonist reduced somite formation and axial length, which was associated with a loss of hoxb13a expression and less-clear alterations in hoxc11a or myoD expression. Treatment with the RARγ agonist also disrupted formation of tissues arising from cranial neural crest, including cranial bones and anterior neural ganglia. There was a loss of Sox 9-immunopositive neural crest stem/progenitor cells in the same anterior regions. Pectoral fin outgrowth was blocked by RARγ agonist treatment. However, there was no loss of Tbx-5-immunopositive lateral plate mesodermal stem/progenitor cells and the block was reversed by agonist washout or by cotreatment with an RARγ antagonist. Regeneration of the caudal fin was also blocked by RARγ agonist treatment, which was associated with a loss of canonical Wnt signaling. This regenerative response was restored by agonist washout or cotreatment with the RARγ antagonist. These findings suggest that RARγ plays an essential role in maintaining stem/progenitor cells during embryonic development and tissue regeneration when the receptor is in its nonligated state

Long-term retinal PEDF overexpression prevents neovascularization in a murine adult model of retinopathy

Neovascularization associated with diabetic retinopathy (DR) and other ocular disorders is a leading cause of visual impairment and adult-onset blindness. Currently available treatments are merely palliative and offer temporary solutions. Here, we tested the efficacy of antiangiogenic gene transfer in an animal model that mimics the chronic progression of human DR. Adeno-associated viral (AAV) vectors of serotype 2 coding for antiangiogenic Pigment Epithelium Derived Factor (PEDF) were injected in the vitreous of a 1.5 month-old transgenic model of retinopathy that develops progressive neovascularization. A single intravitreal injection led to long-term production of PEDF and to a striking inhibition of intravitreal neovascularization, normalization of retinal capillary density, and prevention of retinal detachment. This was parallel to a reduction in the intraocular levels of Vascular Endothelial Growth Factor (VEGF). Normalization of VEGF was consistent with a downregulation of downstream effectors of angiogenesis, such as the activity of Matrix Metalloproteinases (MMP) 2 and 9 and the content of Connective Tissue Growth Factor (CTGF). These results demonstrate long-term efficacy of AAV-mediated PEDF overexpression in counteracting retinal neovascularization in a relevant animal model, and provides evidence towards the use of this strategy to treat angiogenesis in DR and other chronic proliferative retinal disorders