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From the Department of Pathology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Israel.
| Correspondence to: Nurit Rosenzweig-Bublil, Department of Pathology, Sackler School of Medicine, Tel-Aviv University 69978, Ramat-Aviv, Israel (e-mail: rosenswe{at}post.tau.ac.il). |
| Received for publication March 25, 2005; accepted for publication September 27, 2005. |
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Abstract |
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Key words: Phenylephrine, prostatic intraepithelial neoplastic, neoangiogenesis, collagen, prostate
The close relationship between epithelium and stroma enables the transition of many growth factors, neurotransmitters, neuromodulators, cytokines, hormones, and the like, producing paracrine or autocrine effects that lead to a mutual crosstalk (Chung, 1995; Chung and Davies, 1996). As can be expected, epithelial-stromal interaction plays an important role not only in the normal development of the prostate but also in its tumorogenicity (Cunha, 1994; Chung and Davies, 1996; Wong and Wang, 2000).
Human prostatic stroma smooth muscle cells and blood vessels express various subtypes of adrenergic receptors (Lepor et al, 1993; McVary et al, 1998). We have previously shown that daily administration for 1 month of phenylephrine (PE), an alpha-1 adrenergic analog, induces atypical prostate hyperplasia in rats (Golomb et al, 1998) resembling those of prostate intraepithelial neoplasia (PIN) in men (Bostwick et al, 1993; Epstein, 1995). These lesions were also accompanied by mild fibrosis and a few inflammatory cells (Golomb et al, 1998). Chronic inflammation of longstanding duration has been linked to the development of prostatic carcinoma and proliferative inflammatory atrophy of the human prostate (De Marzo et al, 1999). Recently we defined the evolutionary progression of the PIN lesions, as induced by PE in rats, from 8 hours postinjection until 14 days of treatment (Rosenzweig et al, 2004). It was found that PE induced a biphasic reaction: a subacute inflammatory exudate that took place in the prostate stroma but that was partially resolved later on. The presence of this inflammatory exudate preceded the epithelial changes that evolve into PIN lesions. In addition, resident macrophages and mast cells, known for their capacity to secrete various growth factors and cytokines, were also found in elevated numbers in the rat prostate (Rosenzweig et al, 2004).
Most of the histopathological studies on pharmacological induction of benign prostatic hyperplasia (BPH) or PIN in rats have so far focused on the epithelial proliferative lesions rather than on the stromal ones. To the best of our knowledge, the behavior of various stromal elements has not been reported as yet in the experimental induction of neoplastic events in the rat prostate. However, studies on connective tissue constituents (fibrocytes, myofibroblasts, collagen, and elastic fibers) in human prostate stroma were reported in both normal and carcinoma patients, in whom the stromal mass is predominant and much denser (Chung and Davies, 1996; Chagas et al, 2002; Zhang et al, 2003) than in rodents (Scolnik et al, 1994; Nemeth and Lee, 1996).
To better understand the microenvironmental role of prostate stroma in the initiation and promotion of aberrant epithelial growth, we have investigated the pathogenetic evolution of major connective tissue fibrillar and cellular component changes by quantitative histochemical and immunohistochemical (IHC) methods following PE-induced PIN lesions and have compared these within control rats.
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Materials and Methods |
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The animals were randomly divided into 3 groups, comprising 1 experimental (9 rats) and 2 control (4 rats each) groups. The experimental group was treated daily with PE (Sigma Chemical Co, Rehovot, Israel) dissolved in saline (10 mg/mL), subcutaneously injected at a dose of 10 mg/kg body weight (BW)/d for a period of 30 days. The control groups consisted of untreated rats and those that received an equivalent volume of saline for the same period of time. Since no histological differences were encountered between these 2 groups, they were unified into 1 control group.
For the assessment of the cellular proliferation rate, a single dose of 5'-bromo-2'-deoxyuridine (BrdU; 50 mg/kg BW) (Sigma) was injected IP 48 hours prior to the end of the experimental period (Yu et al, 1992).
Histopathology Processing
The rats were euthanized with an overdose of pentobarbital (IP). The
ventral prostate was excised and fixed overnight in Stieve solution at 4°C
(Lillie, 1965). Afterward, the
samples were histologically processed, and paraffin-embedded sections were
stained by Harris hematoxylin-eosin for pathological diagnosis. In addition,
toluidine blue staining for identification of the mastocyte metachromasia was
used (Sheehan and Hrapchak,
1980). For a general examination of the prostatic stroma, the
Gomori trichrome stain was used (Lillie,
1965). To obtain evidence of fine elastic fibrils, the Pinkus
method was used (Pinkus and Hunter,
1960). The quantitative analysis of the collagen fibers was
performed using picro-sirius red (PSR) stain visualized under polarized light
(Junqueira et al, 1979), using
cross-Nicol prisms (Olympus, Tokyo, Japan). A spectra of colors ranging from
red-orange to yellow-green micropatches
(Junqueira et al, 1979;
Whittaker et al, 1994) was
noted. For the quantitative analysis, the frequency of each color spot was
established (see "Statistical Analysis" section).
IHC Staining
The stromal cells were visualized by IHC methods as follows: deparaffinized
and rehydrated tissue sections were blocked for endogenous peroxidase using a
mixture of methanol 3% H2O2 solution. Antigen retrieval
was achieved by heating in a programmable microwave (Amana, Newton, Iowa). The
sections were placed in 0.1 M citric acid buffer solution (pH 6.5) and
microwaved twice (5 minutes each time) in a pressure cooker (Nordic Ware,
Minneapolis, Minn).
For the characterization of the stromal cells, the following antibodies at their respective dilutions were used: for fibrocytes and myofibroblasts, both mouse monoclonal anti-vimentin (1: 100; Novocastra, Newcastle, United Kingdom) and goat polyclonal anti–actin isoforms of broad range (1:75; Santa Cruz, Calif); for monocyte-derived macrophages, mouse monoclonal anti-ED2 (1:50; IQ Products, Groningen, The Netherlands); for the identification of S-phased proliferating cells, monoclonal anti-BrdU (1:200; Dako, Glostrup, Denmark). For the immunoreaction, biotinylated secondary antibodies (rabbit anti-goat, goat anti-mouse; Sigma) and the HRP-avidin system (Zymed, Calif) were used. The color reaction was detected by DAB-H2O2 substrate (Zymed), according to manufacturer protocols. For each immunoreaction, negative controls of adjacent sections were performed by omitting either the primary antibody or both primary and secondary antibodies.
Statistical Analysis
The comparative quantitative assessment for positive immunoreactive cells
was performed for each histological section on 4 different, randomly chosen
fields. Thereafter the individual values were regrouped and their mean values
were calculated. For cell examination, a 200x final magnification was
used. Cell identification was conducted according to their positive
immunoreactivity, and morphological patterns and their number per unit surface
area (1 mm2) were assessed.
Similarly, the color spectra from polarized PSR-stained sections were taken by Olympus DP-50 digital camera using a 10x objective. Color separation of the red, orange, and yellow collagen fibers was performed using the Adobe Photoshop 7.0 ME. For the quantitative study, the selected colors were copied to another file to generate a new gray-scale picture (Tiff format), which was examined using NIH image 1.63 software. In order to avoid interference with the broken cells or fibrils and, eventually, the microscopic dust particles, a minimal reliable unit of 15 pixels per particle size was chosen. The sum of pixels of a given color was calculated for each section, and the group means (± standard deviation) was statistically compared between the treated and untreated rats.
The statistical significance of the means was assessed by multiple pairwise Student's t test (P < .05).
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Results |
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The increase in mesenchymal cell numbers after PE treatment was substantiated by their high proliferative rate (Figure 3A through C). The control prostates showed isolated BrdU-positive cells in both epithelial and stromal compartments (Figure 3A). After the PE treatment, their frequency in absolute numbers was significantly increased (P < .05 and .025 respectively) (Figure 3B and C). However, when their relatively increased ratio was considered, the stromal cells presented a higher proliferative rate (P < .05) compared to the epithelial cells (Figure 3C, insert).
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Picro-sirius red selectively stained the collagen fibrils. Analyzed under the polarized light, they appear as a well-structured network in both interacinar and interlobular spaces, being more conspicuous among the treated than the control animals (Figure 4E and F). The collagenous material is disposed as thin lamellae presenting various diffraction colors ranging from red to orange and yellow patches. The computerized digital analysis of these colored patches (Figure 4G) reveals an increase in yellow and orange patches in the treated groups. This increase is indicative of a de novo synthesis induced by PE in the prostate stroma.
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Discussion |
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The increase in stromal cell population can be attributed to the sympathomimetic stimulation of PE. A genetic excessive-evoked sympathetic activation is a major feature in the development of high blood pressure among the spontaneously hypertensive rats (SHR) (Magee and Schofield, 1994; Julius, 1996). When fibroblasts and vascular smooth muscle cells originating from the SHR strain are grown in vitro, they show an increased proliferative rate (Paquet et al, 1989; Guicheney et al, 1991) as well as an increase in the number of their prostatic epithelium cells (Matityahou et al, 2003). Moreover, we have previously reported that SHR develop BPH spontaneously (Golomb et al, 2000). When mesenchymal stromal cells of human BPH origin are treated in vitro with norepinephrine (Smith et al, 1999) or PE (Boesch et al, 1999), they show an increased expression of smooth muscle cytoskeleton proteins such as actin and myosin, as expressed by their differentiation into smooth muscle phenotype (Magee et al, 2000). This effect was inhibited by doxazosin, an alpha-1 adrenergic antagonist (Boesch et al, 1999; Smith et al, 1999; Magee et al, 2000). The alpha-1 adrenergic blockade down-regulates the myosin heavy-chain mRNA expression in human BPH (Lin et al, 2001). According to Kanagawa et al (2003), norepinephrine increases the DNA synthesis and, subsequently, cell proliferation of the human prostate stromal cells by activating the MAP kinase (MAPK) signaling pathway. However, this stimulatory effect was less effective on the epithelial cells (Kanagawa et al, 2003).
It is noteworthy that steroid sex hormones do not influence the expression of the contractile filaments and therefore neither enhanced nor inhibited the norepinephrine and doxazosin effect (Magee et al, 2000). It seems that the prostatic smooth muscle cells are less responsive to hormonal alteration than the fibroblasts (Zhao et al, 1992).
Alpha smooth muscle actin expression is considered a marker of choice for myofibrillar modulation and is also transiently expressed by myofibroblasts during experimental wound healing (Gabbiani et al, 2003). It was suggested (De Wever and Mareel, 2003) that the myofibroblasts might play a causal role in the transition from the noninvasive toward the invasive tumor phenotype, as has been observed in various tumors including human prostatic carcinoma (PCa) (Rowley 1998–99; Tuxhorn et al, 2002). Nowadays, the fibroblasts are considered to trans-differentiate into myofibroblasts by expressing both alpha actin as well as vimentin micro filaments (De Wever and Mareel, 2003). In such a case, myofibroblasts and their precursor fibrocytes may well be involved in the synthesis of stromal fibrillar constituents. The overgrowth of these cells could obviously lead to prostatic hyperplasia directly and/or through local mesenchymal epithelial interaction (Cunha, 1994; Thomson et al, 2002).
It is well accepted that ECM serves as a microenvironmental reservoir for the various growth factors necessary for the growth and remodulation of the tissue, thus offering the anchoring scaffolding for the surrounding ducto-acinar complex. This can explain the inability of primary human prostatic epithelial cells to proliferate in vitro in the absence of stromal cells (Hall et al, 2002). On the other hand, the paucity of stromal elements that characterizes different strains of rat prostates might be attributed to their innate refractivity to develop hyperplastic lesions spontaneously with advancing age (Scolnik et al, 1994).
We have shown that continuous PE administration by itself favors the maintenance of the inflammatory exudate over the acute reactive phase in the rat prostate (Rosenzweig et al, 2004). In fact, a persistent chronic inflammatory exudate promotes fibroblast proliferation and angiogenesis, ultimately leading to scar organization and fibrosis. The repair process begins as early as 24 hours postinjury (Kumar et al, 2005).
The increase in the monocyte-derived macrophages and, to a lesser extent, the mast cells may well explain their role in fibrotic scar formation (Frangogiannis et al, 2002), as these cells are known to produce cytokines and growth factors. Fibrotic changes were observed in human BPH and PCa (Chagas et al, 2002; Zhang et al, 2003). The smooth muscle cells that were found subadjacent to the hyperplastic acini and also in well-differentiated PCa are totally absent in the poorly differentiated carcinoma and metastases (Deering et al, 1995). Various conditions have been reported to accompany prostatic fibrosis in men, such as chronic ischemia (Kozlowski et al, 2001), proliferative inflammatory atrophy (De Marzo et al, 1999; Putzi and De Marzo, 2000), as well as in cases of chronic administration of testosterone in baboons (Karr et al, 1984) and estradiol in rats (Zhao et al, 1992). A detailed review of the literature regarding the functional resemblance between wound repair and reactive stroma in many cancers, including prostatic cancer, was offered by Rowley (1998–99).
The increase in the microvascular density, as observed among PE-treated rats, is indicative of the existence of neoangiogenesis, which occurs during the healing stage observed following the early phase of PE effect (Rosenzweig et al, 2004). Tumors are known to alter their own microenvironment by creating new blood vessels to hasten their growth (Guiot et al, 2003). According to Bostwick and Iczkowski (1998), the microvascular density is a good criterion for the assessment of the degree of angiogenesis and is therefore considered an independent prognostic factor for patients with PCa. Blood vessel density in human PCa was found to be twice as high as in normal prostate (Deering et al, 1995). Angiogenesis could be accelerated both in BPH and PCa via epidermal growth factor induction of vascular endothelial growth factor gene expression. In an IHC study it was found that vascular endothelial growth factor was absent in the normal human prostate, and only the epithelia from BPH and, to a lesser extent, in PCa cell lines exhibited strong immunoreactivity (Ravindranath et al, 2001). A recent review of tumor angiogenesis is offered by Verheul et al (2004).
It is known that collagen is the most ubiquitous protein in animals and the key molecule of the ECM organization. The trichrome stain is a classical routine technique to identify the presence of connective tissue as a whole, but it cannot afford the identification of fine fibrillar collagen. Moreover, it is known that the normal rat prostatic stroma contains a negligible amount of connective tissue elements (Lin et al, 2001). Therefore, we approached a more adequate technique, such as polarized-light picrosirius red staining, in order to obtain a better resolution for the collagen fibrils. The color spectra of the collagen fibers depend on their size (Junqueira et al, 1979; Modis, 1991) or their packing alignment and orientation in the ECM (Nimni, 1980; Pickering and Boughner, 1991). The presence of yellow fibers is considered to reflect decreased packing density or size, therefore corresponding to thin and young fibers, whereas the orange-red ones that are thicker do represent polymerized, old fibers (Dayan et al, 1989). These data seems to reflect the evolution of collagenetic process as encountered in our model. The significant increased amounts of the yellow and orange fibers found among PE-treated rats correspond to the synthesis of new, young, relatively unorganized collagen fibers. At the beginning the fibrils are short (1–3 µm), but later on, as a result of a fusion process, larger fibrils are generated, creating a shaft network (Kadler et al, 2000). The continuous deposition of new collagen fibers creates thickened fibers, thus leading to denser ECM material. Such increases in the collagen content corroborate with the increase in the fibrocytes and in the myofibroblasts number, since these cells are known to be involved in collagen synthesis and secretion (Bosman and Stamenkovic, 2003). In human BPH, this collagen network is much increased and appears denser (Chagas et al, 2002), but it is relatively reduced in PCa, in which some disintegration of the fibers was reported (Morrison et al, 2000). The latter was correlated to the Gleason score and is regarded as a predictor of patient survival (Burns-Cox et al, 2001).
Little information is as yet available with regard to the amount and location of the elastic fibers in the prostate gland, since the fibers are known to intermingle among the collagen fibers (Scott and Vesely, 1995). This may explain their similar location patterns, which are similar to the collagen fibers. Although the presence of fine elastic fibrils appears in scar tissues (Roten et al, 1996) as well as in normal human prostate and PCa (Zhang et al, 2003), the time course of their appearance has not been elucidated. However, a study of human skin scar formation revealed that focal appearances of thin elastic filaments do appear relatively late in the third month after the initiation of the lesion (Roten et al, 1996).
In conclusion, chronic PE administration was found to induce definite stromal changes, such as inflammatory leukocytic exudate, as well as a significant increase in both cellular and fibrillar elements, including neoangiogenesis. According to Mareel and Leroy (2003), the combination of all these lesions is considered as a desmoplastic process frequently encountered in various neoplastic conditions. The findings presented here raise the question as to whether other xenobiotics capable of inducing prostatic hyperplasia might also promote similar stromal lesions in rats. Understanding the factors capable of stimulating or monitoring stromal growth may open new therapeutic strategies in the prevention of the fibrotic process, with a view to attenuating the pathogenesis and evolution of prostatic disorders.
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Acknowledgments |
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Footnotes |
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