Telocytes are major constituents of the angiogenic apparatus

The current study investigated role of telocytes (TCs) in angiogenesis during embryonic development of quail using immunohistochemistry (IHC), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The angiogenic apparatus consisted of TCs, endothelial cells, and macrophages. TCs were identified morphologically by their telopodes and podoms using TEM and SEM and immunohistochemically using CD34, and vascular endothelial growth factor (VEGF). TCs also expressed CD68. TCs formed a three-dimensional network and established direct contact with blood vessels, sprouting endothelial cells, and active macrophages, while exerting their effect through paracrine signaling. VEGF was also expressed by endothelial cells and macrophages. Matrix metalloproteinase–9 (MMP-9) was expressed by TCs, endothelial cells, and macrophages. In conclusion, the expression of VEGF by TCs, endothelial cells, and macrophages is required for the proliferation and migration of endothelial cells and vascular growth. The expression of MMP-9 by TCs, endothelial cells, and macrophages is essential for the degradation of extracellular matrix (ECM) components during neoangiogenesis. Macrophages may facilitate phagocytosis and elimination of the degraded ECM components.

Immunohistochemical procedures of VEGF. Two-step immunohistochemical staining procedures using the DAKO EN Vision System and HRP peroxidase were applied 16 , Paraffin sections 5-µm thick were cut using the Richert Leica RM 2125 microtome. The sections were dewaxed, rehydrated, and washed by PBS (pH 7.4) three times for 5 min. Blocking of the endogenous peroxidase was carried out using drops of 3% hydrogen peroxide in methanol for 20 min at room temperature. The section was thoroughly rinsed using running tap water for 10 min. Antigen retrieval was performed by applying 10-mm sodium citrate buffer (pH 6.0; Table 2). The sodium citrate buffer was heated in a water bath for 20 min to 95-98 °C followed by cooling for 20 min at room temperature. Sections were rinsed for 5 min by PBS (pH 7.4) three times. Blocking nonspecific background staining was carried out using drops of blocking serum (DAKO) to cover the sections for 5 min at room temperature. www.nature.com/scientificreports/ The primary antibody was incubated with the sections. The antibodies were successfully used in avian species 17 . Table 3 lists the identity, sources, and the working dilutions of the antibodies used in the immunohistochemical technique. The slides were washed in by PBS (pH 7.4) three times for 5 min and incubated with secondary antibody at room temperature for 30 min. The slides were washed again with PBS (pH 7.4) three times for 5 min and incubated for 5-10 min at room temperature with DAB and substrate chromogen, which revealed a brown color at the antigen site. The counterstain, Harris hematoxylin, was applied for 30 s. The sections were dehydrated by ethanol alcohol 90% and 100% II, cleared in xylene, and covered using DPX. Immunohistochemical stained sections were examined using the Leitz Dialux20 microscope provided with the Canon PowerShot A95 digital camera. Negative controls were performed using the same procedures, except using the primary antibodies.   19 . The fixative was prepared as follows: 10 mL of 25% paraformaldehyde, 10 mL of 50% glutaraldehyde, 50 mL phosphate buffer, and 30 mL distilled water. Five samples were used for each age (i.e., days 5 and 8 of incubation). The neck skin was carefully excised and trimmed to a small-sized length of 2.0 to 3.0 mm. Karnovsky fixative ( Table 2) was used at 4 °C overnight. The samples underwent postfixation by osmium tetroxide, dehydration, impregnation in a mixture of alcohol/resin and pure resin, resin embedding, and crystallization in an oven at 60 °C. Semithin sections were taken at 1 μm using an ultramicrotome Ultracut E (Reichert-Leica, Germany) and stained with toluidine blue 20,21 and Periodic acid-Schiff (PAS) 22 . Staining of semithin sections was performed after dissolving the resin using a saturated alcoholic solution of sodium hydroxide. The stained sections were examined by a Leitz Dialux 20 microscope and a Canon digital camera (Canon PowerShot A95).
TEM. Ultra-thin sections (60 nm) of resin-embedded samples at embryonic days 5 and 8 were cut using the Reichert ultra-microtome. The sections were stained using uranyl acetate followed by lead citrate for 15 min for each stain. The stained grids were examined using a JEOL100CX II transmission electron microscope at the central laboratory of South Valley University, Egypt.

SEM.
The samples were fixed in Karnovsky's fixative and washed. Sample washing was performed using the Na-phosphate buffer (pH 7.3) for four times at 15 min. Samples were postfixed in 1% osmic acid in 0.1 M Naphosphate buffer for an additional 2 h at room temperature and washed in Na-phosphate buffer. The samples were dehydrated by ascending grades of alcohol (50%, 70%, and 90%) for 30 min at each concentration and 100% for 2 days with many changes. Samples were treated with isoamyl acetate for 2 days and dried using the critical-point drying method with the Critical Point Drying Procedure Polaron E3000 CPD apparatus (Germany). Sample coating with gold was performed using the JEOL 1100 E-ion sputtering device (Japan) and examined with a JEOL SEM (JSM 5500 LV) at 10 kV, at the central laboratory of South Valley University, Egypt.

Coloring of TEM and SEM images.
To distinguish different types of cells and structures, coloring of TEM and SEM images was performed using the Photo Filter 6.3.2 program. The black and white image was colored using adjust then color balance to change the color to a desirable colors and degrees. Coloring transfer was performed by the stamp tool located at the right panel. Changing color was performed again using adjust then color balance to color another cell or structure. The methods used were previously described by many authors 2,4,23-31 .

Ethical approval. The National Ethics Committee of South Valley University and veterinary authorities in
South Valley University Province, Egypt, approved the method of this study. ' All procedures were performed in accordance with the relevant guidelines and regulations 32 . Arrival guidelines the study was carried out in compliance with the ARRIVE guidelines 33 .

Discussion
The current study investigated the role of TCs during the early stages of angiogenesis in embryonic quail. Using TEM, SEM, and IHC, typical TCs were identified forming a 3D network in the neck skin. TCs had strong immunoaffinity for CD34 CD34 is a member protein of the transmembrane sialomucin protein family and is used for the identification of hematopoietic stem cells and non-hematopoietic progenitors, including vascular endothelial progenitors and embryonic fibroblasts, multipotent mesenchymal stromal cells, interstitial dendritic cells, and epithelial progenitors. Functional implications of CD34 are linked to cell adhesion 34 , proliferation, and inhibition of differentiation of stem or progenitor cells 35 .
Perivascular TCs formed a heterocontact with the blood vessels and release of secretory vesicles for paracrine singling. TCs established contact with sprouting ECs, which indicated neovascularization. They also expressed VEGF, which is essential for the development and growth of the circulatory system and for the regulation of proliferation and migration of vascular ECs 36 . The findings of the current study suggest the role of TCs in angiogenesis, which is supported by previous studies. They secrete VEGF and endothelial growth factor, which regulate www.nature.com/scientificreports/ angiogenesis and the proliferation of ECs 6 . The angiogenic role of TCs is also noted during myocardial infarction. TCs produce angiogenic microRNAs, such as let-7e, 10a, 21, 27b, 100, 126-3p, 130a, 143, 155, and 503 37 . TCs communicated through direct contact and paracrine signaling with active macrophages. Avian macrophage identified by their rounded profile and contained large phagosomes with materials of different stages of digestion 38 . The relation between TCs and macrophages may reveal that TCs involved in the phagocytic activities via indirect pathway. The effect of TCs on the activation of macrophages has been studied using an in vitro coculture of peritoneal macrophages (pMACs). The authors showed that communication between pMACs and TCs occurs through heterocellular junctions and the paracrine mode, and they suggest that activation of pMACs occurs via the mitochondrial signaling pathway 39 .
MMP, or matrixins, are a type of endopeptidases. They are specialized to degrade the extracellular matrix components and other proteins and contribute to tissue remodeling 40 . Based on the biochemical properties, MMP subtypes are categorized as collagenases, gelatinases, stromelysins, and membrane-type MMPs (MT-MMPs) 41 . Gelatinases are gelatinase A (MMP-2) and gelatinase B (MMP-9) 42 . MMP-9 plays a critical role in angiogenesis, immune cell migration, activation of cytokines and chemokines, and progression and metastasis of cancer cells [43][44][45] . MMP-9 degrades collagen types IV, V, XIk´, XIVl´, elastin, aggrecan, link protein, decorinr, lamininn, entactin, SPARCq, myelin basic proteinm, ∞2Mn, ∞1Pli, IL-1βj, and proTNF-∞k 46,47 . In the current study, TCs exhibited proteolytic activities that had a strong immunoaffinity for MMP-9. MMP-9 plays an essential role in the steps of angiogenesis and degrades the basement membrane of capillaries. MMP-9 also promotes EC migration 43 . Vascular pruning requires post hypoxia activation of MMP-9 48 . CD68 is a sialomucin belongs to class D scavenger receptor. CD68 protein is common in late endosomes and lysosomes. Thus, CD68 protein is found in the granules of macrophages, and other cells of the mononuclear phagocyte system such as Kupffer in the liver, microglial cells in the brain, Hofbauer cell in the placenta, osteoclast in bone 49 . CD68 is also expressed by other immune cells including dendritic cells, neutrophils, basophils, and mast cells, activated T-cells and some proportion of mature B-cells as well as epithelium of renal tubules 50 . Increased CD68 expression in macrophage is associated with high vascularity 51 . In the current study, TCs expressed CD68 as well as macrophages. Stromal cells/telocytes express CD68 in the human adult trigeminal ganglion 52 . Cd68 is one of the complement Receptors that expressed by antigen presenting cells. Complement Receptors are implicated in in cell migration and phagocytosis and immune regulation. Complement regulatory proteins that expressed by antigen presenting cells have an essential role in limiting cell activation. Complement receptors cooperate with different receptors to regulate myeloid cell responses 53 .    www.nature.com/scientificreports/ In the current study, TCs formed 3D network at the site of neovascularization. Nehls and his colleagues noticed that the initial endothelial sprouting may not depend on pericyte. Despite of pericyte is one of the initiative types of cells that invade the nascent vascular tissue and distribute at the tip of the growing endothelial sprouts 54 . Pericyte has an essential role during angiogenesis. Development of the endothelial tube is associated with pericytes, that the endothelial sprouting act as a migration sign. pericytes are derived from the differentiation of resident mesenchymal precursors or migration from the adventitia of the adjacent vessel 55 . This process is known as pericyte-driven angiogenic process in which endothelial cells are preceded and guided by migrating pericytes 56 . pericytes may inhibit endothelial growth and migration 57,58 . Previous studies revealed a remarkable correlation between pericyte contribution and microvessel stabilization 59,60 . pericyte investment is linked to maintenance of capillary integrity in vivo 61 .Formation of vascular basal lamina and investment of the pericytes occurs at the end of the proliferative stage and the onset of the mature or quiescent stage of capillary activity 62 . endothelial cell-pericyte interactions stimulate upregulation of basal lamina-encoding genes and proteins, including fibronectin laminin, and integrins 63 . On the other hand, pericytes invasion may establish tubes and enhance the subsequent penetration of endothelial cells 64 . NG2 expressing pericyte progenitors located in close proximity   www.nature.com/scientificreports/ to blood vessels located closed to blood vessels 65 . Thus, the reciprocal interaction between endothelial cells and pericytes has a fundamental role in angiogenic process.
In conclusion, TCs facilitate angiogenesis. They express VEGF, which promotes endothelial proliferation and migration. They also release MMP-9, which plays a critical role in the degradation of the capillary basement membrane, EC migration, and vascular pruning.

Data availability
All data generated or analysed during this study are available on the author.