DISTRIBUTION OF PDGFR+ CELLS AND INTERSTITIAL CELLS OF CAJAL IN THE HUMAN FETAL GUT

Two types of interstitial cells, interstitial cells of Cajal (ICC) and "fibroblast-like" cells, recently named platelet-derived growth factors receptor positive (PDGFR+) cells, are present within the muscular layer of gastrointestinal (GI) tract. ICC and PDGFR+ cells represent different classes of cells with unique ultrastructure, molecular phenotype and function, and they occupy the same anatomical niches in the GI tract. It is considered that PDGFR+ cells such as ICC, mediate enteric inhibitory neurotransmission. Platelet-derived growth factors (PDGFs) are major mitogens for many cell types of mesenchymal origin, like fibroblasts and smooth muscle cells, and during embryogenesis, PDGF signaling is important in organogenesis. In the available literature, there is no data on the presence and distribution of PDGFR immunoreactive cells in the human intestine during fetal period. The aim of this study was to identify PDGFR immunoreactive cells in the gut of human fetuses, as well as to determine their distribution in relation to smooth muscle cells, ICC and enteric nerve structures. The material consisted of 12 Human Fetuses, gestational age from 10 to 12 weeks. The immunohistochemical test was carried out with the PDGFR- antibody, ICC were identified using the C-kit antibody, while the muscle structures were demonstrated by the Desmin antibody. During the development of the human intestine, at week 11, PDGFR immunoreactive cells are present within the circular muscle layer, while they are absent in the myenteric plexus region and in the parts below the serosa. Unlike them, ICC are present only around the inception of the myenteric plexus ganglia. In the fetal period of the human development, PDGFR immunoreactive cells are present in all parts of the intestines, they are localized within the circular muscle layer and do not coincide with the ICC. Acta Medica Medianae 2020;59(1):51-59.


Introduction
The muscular layer of the gastrointestinal (GI) tract is a complex tissue that, in addition to smooth muscle cells, contains a variety of cellular phenotypes. Within the muscular layer of GI tract, there are at least two types of interstitial cells, interstitial cells of Cajal (ICC) and cells called "fibroblast-like" or "ICC-like cells" (1,2). These interstitial cells of mesenchymal origin form gap junctions with each other and with surrounding smooth muscles thus conducting electrical signal and regulating muscle excitability (3). Interstitial cells of Cajal express c-kit (4), so immunohistochemical labeling of the Kit receptor has enabled the reliable identification and study of ICC function and pathophysiology (5) on human and animal material.
The studies of ICC development suggest that some types of ICC and smooth muscle cells have a common precursor that expresses the c-kit (6). Kit signaling is thought to be necessary for the separation of mesenchymal precursors during differentiatition toward ICC or smooth muscle cells, for maintaining the ICC phenotype, and for normal postnatal development (7). The pacemaker role of the ICC around the myenteric plexus (ICC-MY), which spontaneously generate slow waves necessary for peristaltic contraction, has recently been confirmed (3,8). The ICC within the muscle layer (intramuscular ICC -ICC-IM) have a role as mediators of 52 cholinergic and nitrinergic neurotransmission (9)(10)(11), participate in afferent signaling and integration of sensory-motor function as an element of the afferent branch of the gastrointestinal reflex (12). ICC also have a sensory role in the transduction of mechanical stimuli, that is, they function as stretch receptors (13). Loss and dysfunction of ICC have been demonstrated in numerous motility disorders (14)(15)(16)(17)(18). Contrary to the studies of ICC, the study of "fibroblast-like" cells has only recently been made possible by immunohistochemical labeling of platelet derived growth factor receptor A (PDGFR) (19). These cells are different from ICC, but they occupy the same anatomical niches in the GI tract in mice, primates, and humans (19)(20)(21)(22)(23)(24). ICC and PDGFR+ cells represent different classes of cells with unique ultrastructure, molecular phenotype and function.
Smooth muscle cells are electrically connected to ICC and PDGFR+ cells via gap junctions and form an integrated unit -smooth muscle cell, ICC and PDGFR+ cells (SIP) syncytium (3). SIP cells express different receptors and ion channels, and changes in conductivity in any type of SIP cell affect the excitability and reactions of the syncytium. Interstitial cells are also found in various other smooth muscle organs; however, in most cases the physiological and pathophysiological role of these cells is not clearly defined (25,26). PDGFR+ cells are also closely related to the motor neurons varices and are intertwined with ICC around neural processes (19,27). Immunohistochemical studies have shown that PDGFRα+ cells in the musculature of the GI tract express small conductance Ca 2+ -activated K + (SK3) channels, and mediate enteric inhibitory responses to purines in GI muscles (28)(29)(30). This observation, together with the findings that PDGFR+ cells are very closely related to enteric motor neurons and electrically paired with smooth muscle cells (31), suggests that they, such as ICC, may mediate enteric inhibitory neurotransmission. Major mitogens for many cell types of mesenchymal origin, like fibroblasts and smooth muscle cells, are platelet-derived growth factors (PDGFs) (32). During embryogenesis, PDGF signaling is important in organogenesis (33), while adult cells of multiple organs expressing PDGF ligands and receptors often play an important role in the pathophysiology of various disorders, including GI dysmotility (34).
In the available literature, there is no data on the presence and distribution of PDGFR immunoreactive cells in the human intestine during fetal period.

Aim
The aim of this study was to identify PDGFR immunoreactive cells in the gut of human fetuses, as well as to determine their distribution in relation to smooth muscle cells, ICC and enteric nerve structures.

Material
The material consisted of 12 human fetuses, in the gestational age from 10 to 12 weeks. The tissue material was obtained from the Institute of Pathology, Clinical Center Niš, after legal abortions and premature births due to prepartal deaths. Gestational ages were estimated by anatomical criteria according to the crown-rump length, biparietal diameter, and foot length, as well as from the anamnestic data on pregnancy age. There were no gastrointestinal disorders in the specimens, and both sexes were represented in the sample. A macroscopic examination was performed in detail and only specimens that did not undergo post-mortem changes were selected. The study was approved by the Ethics Committee of the University of Niš Faculty of Medicine, and was performed within the internal project no. 22 of the University of Niš Faculty of Medicine.

Tissue preparation
Gut specimens were isolated and fixed in formaldehyde (10%), paraffin embedded, sequentially sectioned at 4 μm, and routinely H&E stained due to histological examination.

Immunohistochemistry
The specimens were exposed to PDGFR antibodies, anti-c-kit antibodies to investigate ICC, and smooth muscle cells were immunohistochemically labeled with anti-desmin (DES) antibodies. Section deparaffinization was performed in xylol and descending series of alcohol rinses (less than 1 min each) followed by rehydration in distilled water. The tissue sections were incubated after blocking endogenous peroxidase (3% H2O2 for 10 min at room temperature) with the primary antibody in a humidified chamber at room temperature for one hour, followed by rinses in a phosphate-buffered solution (0.1 M PBS, pH 7.4). The primary antibodies were dissolved in Dako antibody diluent (EnVisionTM FLEX DM830 Code: К8006, Dako, Denmark). After secondary antibody administration (EnVisionTM FLEX SМ802, Code: К8000, Dako, Denmark) for 45 min at room temperature, immune complexes were visualized by the Daco REAL EnVisionTM Detection System, Code: k5007 (Dako, Denmark). Mayer's haematoxylin was used for counterstaining of all immunolabeled sections, and immunoreactivity was absent in negative controls in which the primary antibody was omitted. The primary antibodies used in the research and their respective dilutions are listed in Table 1.

Results
During the development of the human intestine, at week 11, immunoreactivity to desmin is present in all parts of the gut tissue. Desmin immunoreactivity is present in all parts of the intestine in the form of a concentric band of cells (Figure 1), which by their localization correspond to the circular muscle layer, while the longitudinal layer is not yet differentiated. Only in the terminal portions of the foregut and initial portions of the midgut are individual desmin immunopositive cells localized immediately below the serosa, representing the origin of the longitudinal muscle layer. PDGFR immunoreactivity is present in all parts of the human primitive gut (Figure 2) at week 11. PDGFR immunoreactive cells are present within the circular muscle layer, while they are not present in the myenteric plexus (MP) region, or in the parts below the serosa. PDGFR+ cells are elongated, spindle-shaped cells, oriented in parallel to the longitudinal axis of the smooth muscle cells within the circular gut muscle layer. In the submucosal region, there are two PDGFR low immunoreactive cells (Figure 2 arrowhead).
In week 11 of human development, c-kit immunoreactive cells are present in all parts of the gut in the MP region, as continuous rows and nets of cells present around the MP ganglia, at the outer border of the circular muscle layer (Figure 3). C-kit immunoreactive cells lie at the edges of the inception of the MP ganglia but they are not present within them. They are also absent within the circular muscle layer, as well as in the region below the serosa. In submucosa, in the area where submucosal ganglions develop, c-kit immunoreactivity is also absent. C-kit immunoreactive cells are multipolar with large round or oval nuclei, a small body, and numerous thin processes. Their processes form a network around the MP ganglia. In addition to ICC, a large number of c-kit immunoreactive mast cells are present, but they are easily distinguished from ICC on the basis of their shape and granular content.  The comparison of desmin, C-kit and PDGFRα immunoreactivity in the distal midgut clearly shows that the localization of C-kit and PDGFR immunoreactive cells is significantly different (Figure 4).

Discussion
Based on the desmin immunohistochemistry results of our research, we can observe that in all parts of the fetal intestine, the circular muscular layer develops first, and only later the longitudinal layer, following the principle of proximal-distal gradient. These results are consistent with the previous studies (35,36). During 11 th and 12 th week of development, C-kit immunopositive cells, which correspond to ICC, are present in all parts of the human fetal intestine in the MP region and are surrounding the inception of the MP ganglia, but they are absent within the circular muscle layer. This finding is also consistent with the results of previous studies, which indicate that intramuscular ICC develop only in the late fetal period, while certain ICC subtypes develop after birth (37)(38)(39).
Platelet derived growth factor (PDGF) is a major mitogen for many cells of mesenchymal origin, including fibroblasts and smooth muscle cells, which is why PDGF signaling is especially important during embryogenesis (34,40). PDGF receptor A (PDGFR) is a receptor present on the surface of a large number of cell types, which binds one of the PDGF isoforms, causing cellular growth and differentiation during organ development and is responsible for the normal functioning of tissues and organs (41,42). Previously considered mainly a developmental growth factor receptor in the GI tract, the discovery of receptor tyrosine kinase PDGFR expression in "fibroblast-like" cells within tunica muscularis (19) has opened the door to new trials and definitions of the role of these cells (23,27). The most significant finding of our study is that PDGFR immunoreactive cells are present in all parts of the human intestine in the early fetal development period. These cells are localized within the circular muscle layer, on the contrary, no PDGFR+ cells have been observed in the MP region as well as in parts just below the serosa. An identical localization of PDGFR immunoreactive cells also exists in the gut of adults, with the difference that they are also present in the MP region in adults, where they form three-dimensional networks. This position of PDGFR immunoreactive cells is consistent with the fact about their possible role in neurotransmission of signaling from ICC to smooth muscle cells (23,43). It has been previously reported that PDGFR+ cells have important roles in the morphogenesis of small intestinal mucosa villus of the mouse (44,45). PDGFR+ cells were also found in the subepithelial layer of the adult guinea pig GI tract (46). However, the details of the distribution and functions of PDGFR+ cells in fetus GI tract have not been reported.
Kurahashi et al. have described a specific type of PDGFR+ cells in the lamina propria of the human GI tract (47), and suggested that subepithelial PDGFR+ cells have a role in sensory and secretomotor signaling, proliferation, differentiation, and apoptosis of epithelial cells, and in epithelial cellular pathology, including inflammatory responses and tumorigenesis. Subepithelial PDGFR+ cells in adults form a sheath just beneath the epithelium and cover the crypts from their base to the luminal surface of the epithelium. It is suggested that these cells may have modulatory functions in immune and sensory responses and in the maintenance of mucosal homeostasis, but the roles of these cells in physiological and pathophysiological processes are still unknown. In our research, there was a low PDGFR submucosal immunoreactivity during the early period of fetal development, and dominant PDGFR were present within the circular muscle layer. As already mentioned, due to their close contact with enteric nerve endings and smooth muscle cells within the circular muscle layer, it is assumed that these cells primarily play a role in the neuromodulation of peristalsis.
Another important result of our study is that PDGFR cells differ from ICC and that they are functionally close but still different cell types of interstitial cells. In adults, PDGFR cells are widely distributed within the MP region and in circular and longitudinal muscle layers throughout the human colon (23). Blair et al. (21) have shown relationships between enteric neurons and interstitial cells in primates. They have shown that PDGFR+ cells are closely associated with ICC and occupy the same anatomical niches as ICC-MY and ICC-IM. However, in contrast to the distribution of intestinal cells in adults, we showed in our study that, in the fetal period, PDGFR activity was not observed in the ICC-MY domains around the ganglia. This finding indicates that PDGFR cells develop later than the ICC within GI tract. Further, our results show that unlike the region of MP, PDGF cells are present in the circular muscle in the fetal period and there are still no differentiated ICC-IM.
Ultrastructural studies show that enteric neurons do not effectuate any direct contact with smooth muscle cells or synaptic specializations; on the other hand, ICC make close contacts with both cholinergic and nitrinergic neurons, forming synapse-like connections at one end and gap junctions with smooth muscle cells (3,48). Calcium activated chloride channels (Ano1) are highly expressed and exclusively by ICC throughout the GI tract (21,49), so the major excitatory neurotransmitter -acetylcholine induces depolarization by the activation of Ano1 currents (50). ICC-IM also respond to inhibitory neurotransmitters between neurons and smooth muscles (51). PDGFR+ are very similar to ICC-IM in adults, and also form gap junctions with surrounding smooth muscle cells (3,20,21). PDGFR+ cells express guanylate cyclase, purinergic P2Y1 receptors and small conductance Ca 2+ -activated K + (SK3K + ) channels (23,27,52) which indicates that PDGFR+ cells mediate enteric inhibitory neurotransmision. It has been confirmed that PDGFR+ cells, like ICC, generate inhibitory post junctional responses in GI muscles (27). Gap junctions provide electrical coupling between cells, such that induction of a K + current in PDGFR+ cells results in hyperpolarization, first of these cells and immediately after that in net hyperpolarization of the SIP syncytium and reduce muscle contraction.
In contrast to our results, PDGFR+ cells in adults form a network adjacent to ICC around the myenteric ganglia (23). ICC-MY have a pacemaker role to generate spontaneous electrical activity, while the function of myenteric PDGFR+ is still unknown. Since they are interconnected with ICC-MP, they may have a role in the propagation and modulation of the electrical peristaltic waves.
At present, little is known about the involvement of PDGFR+ cells in GI motor dysfunction. It is certain that the changes in purinergic neural inputs could have effects on colonic motility. Enhanced activation of PDGFR in mice, contribute to the development of GI fibrosis and sarcoma (53). In a recent study (54), it has been concluded that colonic transit disorder may be due to the downregulation of the Kit and Ano1 channels and the upregulation of SK3 channels in PDGFR+ cells, suggesting that the imbalance between ICC and PDGFR distribution might be a possible reason for gut dysmotility. Furthermore, some stromal tumors (GIST) are positive for PDGFR (55,56), so it is possible that these cells, like ICC, can be malignantly transformed.

Conclusion
In the fetal period of human development, PDGFR immunoreactive cells are present in all parts of the intestine, localized within the circular muscle layer, and do not coincide with ICC.