Diabetes induces stromal remodelling and increase in chondroitin sulphate proteoglycans of the rat ventral prostate

Abstract

Extracellular matrix (ECM) remodelling is an important process involved in prostate cancer progression. Alterations in ECM caused by diabetes in different tissues such as kidney is well described; however, it is poorly investigated in prostate. The aim of this study was to evaluate changes in ECM of rat prostate showing gland atrophy caused by diabetes and their implications in development of malignant lesions. Diabetes was induced in Wistar rats using alloxan (45 mg/kg bw). After 90 days of diabetes onset, animals were killed and ventral prostate was removed and prepared for light microscopy following immunoreaction for fibronectin, chondroitin sulphate and Picrossirius staining for collagen fibres. Proteoglycans (PG) were identified at transmission electron microscopy after fixation with Cuprolinic Blue. Diabetes led to a thickening of 25% in the acinar basement membrane accompanied by increase and disorganization of its proteoglycans (P1). Three additional populations of prostatic stromal PGs were identified: collagen fibril linked (P2) and interstitial (P3) and (P4) PGs. Diabetes increased P3 and mainly P4 which had higher dimension and accumulated around the smooth muscle cells. In addition, an increase in chondrotin sulphate (33%, mainly in sites where P4 were noted) and collagen (44%) was noted in diabetic rats, whereas fibronectin did not change. Atrophic changes observed in rat ventral prostate after diabetes are accompanied by stromal remodelation related to increase in collagen and chondroitin sulphate proteoglycans. Thus, diabetes can promote a stromal microenvironment rich in elements that could favour cell migration, proliferation and pathological process.

Figure 1

Light and transmission electron microscopy of the rat ventral prostate from control (a–c) and diabetic group (d–f). (a and b) Overview of acini in the ventral prostate and detail of the secretory epithelium (e) and interstitial stroma (s). (c) Secretory epithelium of control animals show typical columnar cells (e) with elongated nucleus (n) and very prominent area endoplasmic reticulum (er). Stromal region below the BM (lb) has a typical organization with smooth muscle cells (smc) and fibroblasts (f) in layers. (d and e) General features of the ventral prostate of diabetic animals, showing reduced gland portions and atrophied epithelium (e). Stroma (s). (f) Cubic epithelium (e) consisted of flat cells with reduced nuclei (n) and cytoplasmic organelles, but the secretory activity is evident in the cell apex (arrow). In the stroma (s), fibrils and cellular disruption is clear, showing breakdown of the fibromuscular layer. Smooth muscle cells (smc) and fibroblast extensions (arrowhead). Scale bars: (a, d) 400 μm; (b, e) 20 μm; (c) 2.01 μm; (f) 2.54 μm.

Figure 5

Evaluation of the extracellular matrix in the ventral prostate of control (a, c, e) and diabetic (b, d, f, g, h) rats after Picrosirius-Hematoxylin staining (a and b) and immunohistochemistry for fibronectin (c and d) and chondroitin sulphate (e–h). Control animals display large amount of collagen (a), fibronectin (c) and chondroitin sulphate (e), all of them are located predominantly in the subepithelial region and blood vessels (v). Epithelium (e). Chronic diabetes promotes an increase in the elements of the prostatic extracellular matrix. Collagen (b) presents denser fibres dispersed throughout the stroma indicating desmoplasia. Fibronectin (d) does not show significant changes, but presents points of interrupted expression under the acini (arrow). Chondroitin sulphate (f) shows clear increase in the immunoreaction and stromal distribution. There are also points of intense positivity for CS (g and h), indicating a greater concentration of these molecules around blood vessels (v), smooth muscle cells (arrows) and at points of BM (arrowhead). Scale bars: (a–f) 25 μm; (g) 50 μm; (h) 46 μm.

Figure 2

Rat ventral prostate from control (a, c) and diabetic (b, d) group after ultrastructural cytochemistry with Cuprolin Blue. Diabetic animals show marked thickening of the BM (lb) mainly in the lamina densa region (arrows). Collagen (co) associated with BM also presents a clear increase of fibril diameter when compared to control group. Smooth muscle cell (smc), fibroblast (f). (c) P1s occurring at regular intervals in the internal and external faces of the lamina densa of epithelial MB (arrow head). (d) In diabetic animals, these P1s appear disorganized and larger (arrow head). Scale bars: (a, b) 1.56 μm; (c, d) 0.44 μm.

Figure 4

P4 characterization in the ventral prostate of control (a, b) and diabetic (c–i) rats after ultrastructural cytochemistry with Cuprolinic Blue. In control animals P4 appear as long filaments (a), (arrows), normally associated with each other and separated by an amorphous material (b). In the prostate of the diabetic group, such proteoglycans occur with the same morphological characteristic and association with the amorphous material (c). Moreover, filaments are thicker and associate themselves end-to-end forming long structures of high electron density (d and e). These P4s occur throughout the stromal region (arrow) including interstitial stroma (f), but they form continuous layers mainly in the region of the acinar BM (g) and between smooth muscle cells (h and i). Smooth muscle cell (smc), epithelium (e). Scale bars: (a, f, g, i) 0.56 μm; (b, c, e) 0.171 μm; (d) 0.2 μm; (h) 0.6 μm.

Figure 3

Rat ventral prostate of control (a and c) and diabetic (b and d) animals after ultrastructural cytochemistry with Cuprolinic Blue showing P2 and P3. (a and b) P2s appear associated with collagen fibrils at regular intervals of 46 nm, usually in dense bands, in both controls and diabetic group. Diabetes does not change the distribution of these proteoglycans. (c) P3s are found near the smooth muscle cells (smc) or free in the interstitial stroma. P4 (arrowhead). (d) In diabetic animals, the distribution of such interstitial proteoglycans is more abundant and diffuse. Fibroblast (f). Scale bars: (a, b) 0.35 μm; (c, d) 0.28 μm.