Angiogenesis, the formation of new blood vessels from pre-existing vessels, is not only a critical step in embryogenesis and wound healing, but also contributes to the pathogenesis of tumor growth and chronic inflammation [8, 10, 21, 23, 24]. It is a complex process involving extensive interaction between the cells, soluble factors and extracellular matrix (ECM) components. Angiogenesis is initiated by vasodilatation and increased permeability. The formation of a vascular network requires different sequential steps including; the release of proteases from activated endothelial cells, degradation of the basement membrane surrounding the existing vessels, differentiation and migration of the endothelial cells into the interstitial space, endothelial cell proliferation forming tubular structures, lumen formation, generation of new basement membrane with the recruitment of pericytes and finally of fusion of newly formed vessels and initiation of blood flow [18, 34–39].
As it is well known, hypoxia results in both angiogenesis and inflammation , and thus the production of growth factors increases [28–31]. Angiogenesis provides oxygen and nutrients for the metabolic needs of the cells that are present at inflammatory sites . Therefore, we considered that the expanded avascular mesangial matrix regions of the glomeruli could induce the angiogenesis by creating a hypoxic state.
In our microscopic examinations, we observed an increased mesangial matrix with inflammatory areas including macrophages, and various steps of angiogenesis including vasodilatation and endothelial proliferation in pre-existing capillaries, as well as, angiogenic cell islands without basal lamina and lumen formation in immature capillaries containing basal lamina in glomeruli of acute and chronic PAN nephrotic rats. During angiogenesis, the transport of plasma proteins such as fibrinogen and plasminogen from the blood stream into the surrounding tissue is excessively increased. These plasma proteins provide a convenient environment for the migrating angiogenic cells . In our light microscopic studies, we observed angiogenic cell islands and immature capillaries surrounded by dense mesangial matrix areas. We considered that this situation could be correlated with highly concentrated plasma proteins leaked out the pre-existing dilated capillaries around this area.
The angiogenic cell islands can be easily distinguished from the surrounding cells by their big nuclei with peripherally located heterochromatin. These cell islands had no basal lamina yet. When the lumen formation of angiogenic cell islands were completed after the polarization stage, the basal lumina has appeared . As evidence to this argument, in our microscopic and ultrastructural images, we observed more basophilic cytoplasm of immature capillaries and prominent increase in the granular endoplasmic reticulum cisterns which is responsible for the formation of the basal lamina components. Moreover, some researchers have argued that endothelial apoptosis is effective on the primitive lumen formation stage of the angiogenic cell islands . In our study, we observed apoptotic endothelial cells with advanced lumen formation of immature capillaries in our ultrastructural images as a support this argument.
In our microscopic and ultrastructural images, macrophages were the most abundant cell type in the angiogenic regions of the increased avascular matrix. Various cell types and cell products induce or modulate angiogenesis. The major of these cells are macrophages. In all steps of angiogenesis, macrophages take place by their secretary activity. It was reported that they had angiogenic characteristics when exposed to low oxygen supply . Macrophages are among the main sources of metalloproteases (e.g. collagenases) and serine proteases (e.g. elastase and plasminogen activator). These enzymes can degrade ECM molecules, modulate the mechanical framework, and lead to the released ECM-bound growth factors. In addition to proteases, macrophages produce several factors that induce migration of endothelial cells. Most of them also support other stages of the angiogenic process such as proliferation or differentiation of endothelial cells. Their inducing effects on migration seem to be sufficient for the initial neovascularization as migrating endothelial cells can form sprouts without proliferation. Thus, macrophages release several factors that do not directly induce angiogenesis but act indirectly by attracting or activating angiogenic cells. This activity occurs in all phase of the angiogenic process [8, 18, 41]. Hence we considered that the macrophages in this increased avascular matrix were activated due to the presence of insufficient oxygen and induced the angiogenesis.
We also showed the existence of the foam cells in the expanded avascular mesangial matrix of glomeruli in PAN rats. In some diseases, e.g. focal and segmental glomerulosclerosis and hyalinosis, mesangial cells (MCs) exhibit foam cell like morphology. These lipid-laden MCs have impaired phagocytic capacity and disrupted cytoskeletons. Studies have demonstrated that IGF-1 (insulin-like growth factor-1) induces MC to transform into foam cell by phagocytosing lipids [42, 43]. We considered that these cells frequently observed around the angiogenic regions could be also responsible from the development of angiogenesis like the other macrophages.
In scarred kidneys, it was reported that PD-ECGF expression is elevated and its level of expression is correlated with pathological angiogenesis [17, 20, 22–24]. In this study, we detected occasional angiogenic regions in expanded avascular mesangial matrix of the glomeruli in acute and chronic PAN induced rats. We showed elevated PD-ECGF immunostaining in the glomeruli of acute and chronic PAN induced rats. When we compared control and experimental groups, number of total angiogenic stages in experimental groups increased in parallel with PD-ECGF immuno-reactivity results (Table 4, 5). These results supported our ultrastructural findings observation related to angiogenesis development. Thus these findings are consistent with the results of recent studies that have indicated that PD-ECGF expression level is correlated with the number of microvessels in various pathological conditions and may contribute to neovascularization [17, 18, 20, 22–24]. Therefore, as many researchers, we expect that PD-ECGF may become one of the major targets of angiogenesis therapy in future .