A Dual 5α-Reductase Inhibitor Dutasteride Caused Reductions in Vascular Density and Area in Benign Prostatic Hyperplasia

Objectives. Dutasteride, a dual 5α-reductase inhibitor, is used to treat benign prostatic hyperplasia. Nevertheless, its histopathological effects on the morphometrics of blood vessels and glands are still controversial. The aim here was to assess the histopathological effects of dutasteride in cases of benign prostatic hyperplasia in a retrospective study. Methods. Patients with benign prostatic hyperplasia more than 40 cm3 in prostatic volume were administered 0.5 mg of dutasteride daily or left untreated prior to receiving a transurethral resection of the prostate. Images of sections stained with hematoxylin/eosin and with anti-CD31 antibody were analyzed. Results. In the dutasteride-treated group, the duration of administration was 16.3 ± 8.1 weeks. Artery/arteriole density and vein/venule density in benign prostatic tissue were both lower in the dutasteride-treated group than in the control group. The vein/venule area as a percentage of the whole area was also lower in the dutasteride-treated group, while the artery/arteriole area did not show a significant difference. Glandular/CD31-expressing vessel densities as well as glandular/CD31-expressing vessel areas were comparable between the two groups. Conclusions. Dutasteride reduced the artery/arteriole and vein/venule densities and the proportion of vein/venule area in the tissue of patients with benign prostatic hyperplasia.


Introduction
Benign prostatic hyperplasia (BPH) is a histologic diagnosis that refers to the proliferation of smooth muscle and epithelial cells within the prostatic transition zone, for which dihydrotestosterone (DHT) is the primary androgen responsible in elderly men [1]. As the initial treatment for symptomatic BPH, pharmacotherapy with 1 -adrenergic blockers, 5reductase inhibitors, antimuscarinic agents, or a combination thereof is oen used [2]. Type 1 and type 2 isoenzymes of 5 -reductase are present throughout the body [3], and dutasteride, a dual 5 -reductase inhibitor, acts competitively and speci�cally on type 1 and type 2 isoenzymes to inhibit the conversion of testosterone to the more potent DHT [4][5][6]. In comparison with �nasteride, a 5 -reductase inhibitor selectively acting on type 2 isoenzyme [7], dutasteride is a 45fold greater inhibitor of type 1 5 -reductase, and a 2.5-fold greater inhibitor of type 2 5 -reductase [8][9][10].
Patients with BPH who do not respond suitably to pharmacotherapy oen undergo transurethral resection of the prostate (TUR-P), the gold standard for the surgical treatment of BPH, and microwave thermotherapy [11] in order to improve symptoms of bladder outlet obstruction. Several studies assessing clinical and histopathological perioperative effects of dutasteride have been reported [12][13][14], in which staining of CD31 or CD34 in endothelium was mainly used to detect "microvessels. " Data delineating histopathological effects of long-term dutasteride administration on BPH tissue are still lacking. erefore, we conducted a retrospective comparative study to analyze histopathological changes in BPH tissue between patients given dutasteride for a relatively long time and controls without preoperative medication.
Blood vessels are composed of aorta, arteries, arterioles, capillaries, venules, veins, and cava. In the prostatic tissue, arteries and arterioles are identi�able with light microscopy as blood vessels with thick walls of obvious muscular media.
ere are no speci�c histologic features that accurately distinguish small arteries from larger arterioles. For convenience, arterioles are said to have a diameter of less than 100 m [15]. On the contrary, veins and venules are identi�ed as blood vessels with thin muscular wall and the latter is also supposed to be less than 100 m in diameter. Capillaries have neither a muscular media nor an elastic lamella, while a single but complete layer of endothelial cells lies on a basement membrane [15]. Capillaries are generally difficult to distinguish from lymphatic vessels by light-microscopic morphology. Additionally, it was reported that the vascular endothelial markers CD31 and CD34 are occasionally expressed even in lymphatic endothelial cells [16,17], which may make it more difficult to de�nitely distinguish capillaries and lymphatic vessels. e de�nition of microvessels does not appear in textbooks of pathology [15], but seems to be explained as blood vessels of the microcirculatory system, that is, capillaries, arterioles, and venules [18].
In the present study, we assessed parameters concerning arteries and arterioles as well as veins and venules in resected prostate tissue, as they have solid vascular walls and are �rmly identi�ed by examining sections stained with hematoxylin and eosin using light microscopy. Additionally, we counted the number of vascular vessels in the tissue by staining endothelial cells with anti-CD31 antibody. Arteries/arterioles and veins/venules are much larger than capillaries, and it is reasonable to hypothesize that they contribute more to the operative blood loss in the perioperative period. e aim of this study was to assess the histopathological effects of dutasteride administration in the tissue of patients with benign prostatic hyperplasia in a retrospective study. Dutasteride reduced artery/arteriole and vein/venule densities and vein/venule area.

Patients and Methods
2.1. Ethics Statement. is study was conducted in accordance with the Helsinki declaration aer approval by the Ethics Committee of Kanto Rosai Hospital. e committee approved the use of oral consent documented in the electronic chart for each patient, as the study was retrospective and nonrandomized.

Clinical
Design. is is a retrospective study to investigate effects of dutasteride administered before transurethral resection of the prostate in Japanese BPH patients. Twentyseven BPH patients whose prostate volume determined by transabdominal ultrasonography was more than 40 cm 3 were enrolled between February 2010 and April 2011. eir serum PSA levels were less than 4.0 ng/mL, otherwise transrectal prostate biopsy had already shown no cancer in the prostate. ey had not received LHRH agonists, antiandrogens, estrogens, and 5 -reductase inhibitors and had not experienced any type of prostatic surgery or radiation therapy to the pelvis before. Patients with severe cardiovascular disease, liver disease, renal failure, or bleeding tendency were excluded, as they were not suitable for TUR-P. Patients were administered 0.5 mg of dutasteride once daily (GlaxoSmithKline, UK) for some period ( 5 , or else they underwent a transurethral resection of the prostate (TUR-P) without dutasteride treatment ( . A daily 0.5 mg tablet is the therapeutic dose approved for treating symptomatic BPH in Japan. Age, body height, body weight, body mass index, and duration of administration of dutasteride were recorded preoperatively. e transurethral resection was carried out by three surgeons with irrigation in 3% D-sorbitol (Uromatic S, Baxter, USA). Patients were not transfused with autologous or allogeneic blood of any type in the perioperative period. Operation time for TUR-P, amount of intravenous crystalloid infusion during TUR-P, and weight of resected prostate tissue were recorded as perioperative data. Pre-and postoperative blood hemoglobin (Hb), hematocrit (Hct), and serum sodium (Na) levels were also determined.

Morphometric Analyses of BPH Tissue.
As histopathological data, resected prostate tissue was assessed for artery/arteriole density (AD), vein/venule density (VD), and glandular density (GD). Additionally, artery/arteriole area (AA), vein/venule area (VA), and glandular area (GA) were also evaluated as a proportion of the whole area. Resected prostate tissue was �xed in 10% formalin. As a preliminary experiment, some sections were subjected to Elastica Van Gieson staining in order to distinguish between elastic �bers, collagen �bers, and muscle �bers. All sections were stained with hematoxylin and eosin. Additionally, all sections were immunohistochemically stained with anti-CD31 monoclonal antibody (JC70A, DAKO, Denmark) following the manufacturer's recommendations. en, a section of one chip at the center of the tissue slide as well as an additional four sections of chips around the �rst section were scanned with a microscopy (B�51, Olympus, Japan) at a magni�cation of 40. e histology of each section was captured into a Windows computer with a digital camera (DS-Fi1, Nikon Instruments, Japan) using image-analyzing soware (NIS-Elements D, Nikon Instruments, Japan). A scale bar of 1000 m was placed on each image for calibration in the histological analysis.
ereaer, images of sections were analyzed with imageanalyzing soware (Image J, http://rsbweb.nih.gov/ij/) in order to count numbers of arteries/arterioles, veins/venules, and glands in sections stained with hematoxylin and eosin. Outer lines of whole sections were traced with a freehandselections tool of Image J in order to measure the traced areas. Outlines of the lumens of arteries/arterioles and veins/venules were also traced in a similar manner to measure their intraluminal areas. Intraglandular areas were thresholded and measured also using Image J. e sums of the number of arteries/arterioles or veins/venules in �ve sections from a patient divided by the sum of the whole areas of �ve sections were regarded as AD and VD, respectively. GD was similarly calculated. e sums of all artery/arteriole areas or of all vein/venule areas in �ve sections divided by the sum of the whole areas of �ve sections were regarded as AA and VA, respectively. GA was similarly calculated. Supposing that vascular lumens are completely round, the diameter ( ) of a lumen was calculated by the formula; 4 , where is the measured area of the lumen. Density and area of CD31expressing vessels were similarly calculated with sections stained with anti-CD31 antibody. More precisely, outlines of the lumens of CD31-expressing vessels were traced to measure their intraluminal areas. e sums of the number of CD31-expressing vessels in �ve sections from a patient divided by the sum of the whole areas of �ve sections were regarded as CD31D. e sums of all areas of CD31-expressing vessels in �ve sections divided by the sum of the whole areas of �ve sections were regarded as CD31A.

Statistical
Analyses. e statistical analysis was performed using the Mann-Whitney U test for intergroup comparisons. Spearman's rank correlation test and the linear regression analysis were performed to analyze correlations between AD/VD and the duration of administration of dutasteride and to draw least squares regression lines. Values were expressed as the mean ± SD.
was regarded as statistically signi�cant.

Clinical Parameters in the Perioperative Period.
Characteristics of patients are listed in Table 1. ere were no signi�cant differences in age, height, body weight, body mass index, and prostate volume at the �rst visit between the control and dutasteride-treated groups. In the dutasteridetreated group, the period of dutasteride administration was 16 3±8 1 weeks and reduction in prostate volume was 28 2%± 3 2%. Perioperative parameters of TUR-P are also listed in Table 1. ere were no signi�cant differences in operation time, amount of intravenous crystalloid infusion during TUR-P, weight of resected prostate tissue, and perioperative changes in Hb, Hct, and serum Na.

Morphometric Effects of Dutasteride Administration on BPH Tissue.
Prostatic cancer was not detected in any of the resected prostatic samples. e numbers of vascular vessels detected in all 27 cases either with hematoxylin/eosin staining or with CD31 staining were 724 and 3390 in total, that is, 26.8 and 125.6/case, respectively. e difference in the number of vessels detected either with hematoxylin/eosin staining or with CD31 staining was more in vessels whose diameters were less than 100 m compared with larger vessels, as shown in Figure 1. Figure 2 shows the typical histology of BPH with hematoxylin and eosin staining and with Elastica Van Gieson staining. Arteries/arterioles and veins/venules were able to be distinguished by the thick muscular media. As the identi�cation of arteries/arterioles and veins/venules was similar between Elastica Van Gieson staining and staining with hematoxylin and eosin, the histological data for arteries/arterioles and veins/venules were basically obtained using sections stained with hematoxylin and eosin.
With hematoxylin/eosin staining, the numbers of arteries/arterioles and veins/venules were 182 and 542 in all 27 cases, that is, 6.7 and 20.1/case, respectively, while vessels without media such as capillaries were not counted. Arteries/arterioles, veins/venules, and capillaries were essentially indistinguishable with CD31 staining. Figure 3 shows the Horizontal line: diameters of lumens, blue bars: CD31-expressing vessels, and red bars: artery/arteriole and vein/venule. e difference in the number of vessels detected either with hematoxylin/eosin staining or with CD31 staining was more in vessels whose diameters were less than 100 m compared with larger vessels.
typical histology of BPH with CD31 staining. Histological data for the resected BPH tissues are listed in Table 2. AD, VD, and VA were signi�cantly lower in the dutasteride-treated group than the control group. AA tended to be lower in the dutasteride-treated group. GD and GA were comparable between the groups. CD31D and CD31A were also similar between the two groups. As shown in Figure 4 (lower panels), VD and VA were negatively correlated with the duration of administration of dutasteride ( 2 and 0.003, resp.). Least squares regression lines were drawn. Neither AD nor AA showed a signi�cant correlation with the duration of dutasteride treatment ( and 0.089, resp., Figure 3 (upper panels)). CD31D and CD31A were not correlated with the duration of administration either (data not shown).

Discussion
Blood vessels allow haematopoietic cells to eliminate the organism for immune surveillance, supply oxygen/nutrients, and dispose of waste leading to bene�cial effects for tissue growth and regeneration. Angioblasts differentiate into endothelial cells and then assemble to form a vascular vessel (vasculogenesis) followed by subsequent sprouting expansion of the vascular network (angiogenesis) [19]. Human and rat prostate endothelial cells play an important role in androgen responses in the prostate, as they express functional androgen receptor and are androgen sensitive [19,20].
Hahn et al. [12] showed that the perioperative administration of dutasteride had no effects on microvessel density (MVD) in prostatic tissue of BPH patients given preoperative dutasteride for 2 or 4 weeks compared with placebo controls in a prospective, randomized, and multicenter study,  [14] also showed that dutasteride had no effect on prostatic MVD in BPH patients given pre-operative dutasteride for 2 to 4 weeks compared with controls given no drug, where MVD was calculated as a mean of counts of CD34-positive microvessels in 10 consecutive high-power �elds. e CD31D, density of CD31positive vessels, is much lower in the present study compared with the densities of microvessels in other studies. e distribution of CD31-positive vessels is not even throughout the resected prostatic chips with some portion containing few vessels. In the present study, all CD31-positive vessels in �ve chips were counted, then the CD31D was calculated by dividing the number of the vessels by the whole area of those chips. us, the calculated densities were not very high. Concerning preoperative �nasteride administration and MVD in resected prostatic tissue, Donohue et al. reported that inhibition of expression of vascular endothelial growth factor and reduction of MVD were observed aer two weeks administration of 5 mg daily of �nasteride, where  MVD was assessed by counting CD31-positive microvessels in 10 nonoverlapping, consecutive high-power �elds [21]. Furthermore, Lekas et al. [22] reported in a prospective, randomized study that �nasteride administration for 25.3 weeks on average to BPH patients resulted in the suppression of MVD as well as vascular endothelial growth factor and hypoxia-inducible factor-1 expression in a time-dependent manner, where MVD was assessed by counting CD34positive microvessels in the three most vascularized highpower �elds. e vessels with media identi�ed with hematoxylin/eosin staining in the present study seem mainly to be arterioles and venules of relatively smaller diameter, as the peak of the distribution of calculated diameters of those vessels was 50-100 m. e duration of dutasteride administration of 16.3 weeks in the present study was relatively long compared with that in previous studies, up to 4 weeks. is may be why dutasteride administration before TUR-P in this study caused reductions of AD, VD, and VA in the prostatic tissue partly in a time-dependent manner. Otherwise, the counting of numerous capillaries in other reports might have concealed more important in�uences of dutasteride on larger blood vessels. In other words, although larger blood vessels tend to be in�uenced by dutasteride more than capillaries, the much more numerousness of the latter contributes to the computed values of the vessel densities and gets changes of the larger vessels indiscernible. Actually, androgens are reported to limit the number of new vessels developed while they contribute to the presence of larger vessels [23] and androgen deprivation therapy induces changes in relatively large vessels [24]. Measurements of luminal areas of blood vessels in the prostate were not made in the preceding reports describing effects of 5 -reductase inhibitors, which can be a useful tool for evaluating effects of drugs on blood vessels. As they showed clearer effects of dutasteride including time dependency, veins/venules might be more susceptible to dutasteride than arteries/arterioles. CD31D and CD31A were comparable in the control and dutasteride-treated groups. is may be because the numbers of capillaries in the BPH tissues outweigh those of larger vessels with media and capillaries may not be in�uenced by dutasteride. Additionally, CD31 might have picked up lymphatic vessels as well. en, it may be worth evaluating larger vascular vessels with media by morphologically and morphometrically investigating specimens under direct vision and not only relying on staining with endothelial markers such as CD31 and CD34.
In the rat prostate, levels of angiogenetic factors such as hypoxia-inducible factor-1 and vascular endothelial growth factor (VEGF) were lower in the dutasteride-treated groups than in the control group [14]. In human BPH tissues, VEGF expression was signi�cantly associated with androgen receptor and type 2 5 -reductase expression, but not with type 1 5 -reductase expression [25]. ese result could support the present �nding of decreased vascularity in BPH tissues with dutasteride administration possibly through the suppression of androgen signaling pathways.
Andriole et al. reported that prostate cancer patients administered 10 mg of dutasteride daily for 1 week followed by 5 mg daily for more than 45 days in total showed a decrease ISRN Urology 7 in MVD by 45% in cancer tissue in addition to a trend toward increased apoptosis of cancer cells when compared with controls without dutasteride administration [26]. In this paper, the dose of dutasteride was much higher than that used for BPH patients, and so the signi�cant decrease in MVD in prostatic tissue might have been demonstrated aer a shorter period of dutasteride administration than in the present paper. Another possibility is that the interstitial microvessels in malignant prostatic tissue differ from those in benign tissue in quality. Andriole et al. also reported in a more recent article that the administration of 0.5 mg of dutasteride daily over a 4-year study period (REDUCE study) reduced the risk of incident prostate cancer and acute urinary retention by 22.8% and 77.3%, respectively, compared with the control [27].
As the administration of �nasteride for as little as two weeks reduced prostatic MVD [15], the actions of �nasteride on prostatic microvessels may be faster and more profound than those of dutasteride. Additional inhibition of the type 1 5 -reductase by a dual 5 -reductase inhibitor may antagonize the microvessel suppressive activity of type 2-speci�c 5 -reductase inhibitors.
In benign prostatic tissue, we did not detect differences in glandular density and the proportion of intraglandular area regardless of dutasteride administration. Such morphometrics seem rarely reported, probably because glands in benign prostatic tissue have complicated folds. We could calculate values by thresholding intraglandular spaces with the thresholding function of Image J. In the report by Andriole et al. described above [17], dutasteride caused a decrease of 18% in the width of benign prostatic epithelial cells in patients with prostate cancer. Considering that densities and areas of arteries/arterioles and veins/venules, but not glands in benign prostatic tissue decreased with dutasteride as reported in this study, the main target of dutasteride in reducing BPH size could be the interstitium including blood vessels, not glands.
Perioperative 4-6 weeks' administration of dutasteride had no effect in reducing blood loss during or aer TUR-P [12,13], while two patients in the control group in the latter article required a postoperative blood transfusion compared to no patient in the dutasteride-treated group. Similarly in the present study, blood loss was comparable between the two groups as estimated from the perioperative changes in blood hemoglobin and hematocrit. us, the reduction in the density and area of blood vessels in BPH did not directly lead to decreased blood loss in the perioperative period. In conclusion, dutasteride administration for 16.3 ± 8.1 weeks before TUR-P caused reductions of AD, VD, and VA in BPH tissue partly in a time-dependent manner. Blood loss estimated by the perioperative changes in blood Hb and Hct was comparable between the control and dutasteride-treated groups.