Angiogenesis contribution to the pathophysiology of nasal polyposis is still controversial. The pathognomonic tissueremodeling of this clinical condition is frequently attributed to the underlying chronic inflammatory process. However, the combination of the chronic inflammatory tissueremodeling with the associatededema is required to produce the extensive structural impairment that characterizes CRSwNP. The role of endothelial cells in the production and evolution of CRSwNP was explored in this work based on the tissue growth hypothesis in driving edema. The poor fluid compartment control was assumed to be the result of functional effects on the vascular wall, as polyp tissue required three times higher transudation volume to reach the same interstitial hydrostatic pressure of the healthy nasal mucosa during inflammation.
Inflammatory mediators that produce tissue remodelling and modulate angiogenesis induction in chronic processes can indeed produce early effects on the differentiation of endothelial cells, inducing an increase in capillary permeability, tip cell migration, and vascular budding. Late functional changes and vessel stabilization are probably myofibroblasts and pericytes dependent, but the effects of early soluble mediators and extracellular matrix-derived molecules upon endothelial cell morphology were previously detected and could be studied even in endothelial cell cultures. The Gallus domesticus allows simultaneous measurements of edema changes and effects upon tissue development with high reproducibility without causing animal suffering[14, 25]. Implants of cells or tissue fragments do not result in rejection by the egg, which provides a nutrient-rich environment capable of maintaining tissue viability for a few hours and, in some situations, for several days[26, 27].
Healthy mucosa implants reduced or halted embryo development and CAM vasculature growth. The measured vascular areas were smaller in the eggs from the healthy mucosa group, and the branching indexes were lower than those observed in the eggs of the control and polyp groups. The healthy mucosa effects were attributed to the presence of interrupted vessels, and a smaller and less anastomosed network. Quantitative results were a priori independent on the CAM size, as the measurements considered size standardized ROI areas. Differences would have been described as being even greater if the membrane size was considered and the whole vessel areas were measured. Underlying pathophysiological events were interpreted in terms of the failure of the healthy mucosa fragments to contribute timely to their perfusion through pro-angiogenic biochemical signaling. The qualitative evidence of directional growth of capillary vessels was absent in the periphery of healthy mucosa implants, while it was abundant in the periphery of polyp implants. Also, the mucosa implants presented no signs of effective perfusion at the end of the experiment, while the polyp implants looked like the original tissue that was implanted and adhered to the CAMs’ surfaces.
Experimental results support the previous inference about the role of induction of angiogenesis in CRSwNP in the disease relapses after surgical removal of polyps, abehavior known to be dependent on its clinical endotype.
The sustained implant viability in the CAM model relies on the development of a neovascular assembly for the effective tissue perfusion that occurs through coordinated angiogenesis processes.This possibility has been previously explored for studies of tumor and artificial tissues implants[16, 21].While the healthy mucosa implants produced only regressive effects throughout the CAM area, the biochemical signaling of polyp fragments synergized the embryo's innate inflammatory mechanisms and the growing vascular network to ease liquid and cell percolation in the implanted regions.
The outcome of the polyp implants’ interactions in the model was well-perfused CAM membranes with atypical vessel tree topologies. The interstitial hydrostatic pressure and fluid compartment control by the implants were probably relevant to this peculiar behaviour of polyp implants. Indeed, nasal polyp interstitial hydrostatic pressure was reported to be very low, when compared to healthy nasal mucosa.On the other hand, the highly positive interstitial hydrostatic pressure in the stroma of malignant tissues is associated with perfusion impairment, making inadequate delivery of therapeutic agents. Loss of complexity and atypical symmetry could be easily suggested by the qualitative aspects shown in Fig. 3 for both experimental groups. Quantitative protocols for analysis of angiogenesis were recently reviewed and several techniques can be of help in future studies[30–32].Present results show that both the measured vascular area and the branching index measured in the healthy mucosa implanted eggs were lower than expected for the controls. It could be concluded that both types of tissue implants interacted and modified the CAM vasculature as the healthy mucosa produced negative effects and the polyp as modified growth patterns positively.
Regarding morphogenesis and tissue regeneration, it also should be argued if the slightly vascularized polyp tissue could modify the CAM vasculature structure without affecting the embryo development and growth because polyp and control group embryos reached the equivalent size, weight, and development stage. At least for the interval E6-E8, the alternative possibility is that the polyp implants actively stimulate host cell populations. As mucosal growth abnormalities are common clinical findings in CRSwNP, this implies biochemical signaling for tissue growth and remodeling produced by the polyp tissue cells[33, 34].
Inflammatory cells and also stem cells present in the implants were considered the main cell populations able to interact with the developing egg tissues to produce the observed effects. Inflammatory cells are expected to either produce regressive changes or inflammation in the egg. Human mesenchymal stem cells were previously reported to remain alive in the egg environment, to be able to migrate to embryo tissues and to interfere with vasculogenesis and tissue growth[26, 35]. It was previously demonstrated that the nasal polyp-derived stem cells differentially modulated the angiogenesis-related genes expression, VEGF, and FGF10.VEGF and FGF10 proteins were reported to participate in the development of embryo limbs, which was an embryo differentiation process taking place in the same stage the assays were conducted[36, 37].Inflammatory cells from CRSwNP, however, were recently reported to produce increased immune stimulation through the Receptor for the Activator of Nuclear factor Kappa-B (RANK) pathway[38, 39].The inflammatory response stimulation by RANK and RANK-ligand coupling, besides the induction of a Th2 type response, cooperates to undermine the barrier function of the growing vasculature, producing edema-prone neovessels.It was assumed that the mesenchymal stem cells present in the implanted polyp fragments together with the polyp inflammatory cells, while preserving to some extent, could signal for the extended survival of both the implant cell populations and host tissues, and reduce inflammatory rejection of the tissue implants. Not only are the production of proangiogenic growth factors important, but also the migratory capacity and the possibility of neovasculature stabilization may be determined by the vascularisation behaviour of structures implanted on the CAM, as reported in the interaction between glioblastoma and epithelial stem cells.The proposition that the results should be attributed to angiogenesis signalling by polyp mesenchymal stem cells in such an inflammatory environment was favoured by setting the forty-eight hours period between the tissue implantation and data harvesting. During this interval, cell migration and proliferation were less likely to occur extensively and labile inflammatory cytokines co-stimulatory effects would still be observed as well.
Further studies should confirm the polyp and mucosa-derived mesenchymal stem cells' immunoregulatory and pro-angiogenic interactions. The application of undifferentiated mesenchymal cells derived from the human placenta in cell therapy is defended because the anti-inflammatory and immunomodulatory properties of these cells were demonstrated to stimulate tissue regeneration and repair.