Prostatic hyperplasia: Vascularization, hemodynamic and hormonal analysis of dogs treated with finasteride or orchiectomy.

As a consequence of a hormonal imbalance, Prostatic Hyperplasia (PH) is characterized by increased prostate volume, along with higher local angiogenesis and vascularization. Orchiectomy is the common treatment for dogs, however it is not an option for breeding animals. Thus, finasteride arises as the drug of choice for stud dogs. Therefore, the aim of this study was to evaluate the effects of orchiectomy or finasteride therapies on hormonal and vascular dynamics of PH dogs. Fifteen dogs, aged 6-13 years were assigned to: Untreated Group (dogs diagnosed with PH-n = 5), Finasteride treated group (PH dogs treated with finasteride-n = 5) and Orchiectomy treated group (PH dogs submitted to orchiectomy-n = 5). Evaluations were performed in a monthly interval (first day of treatment; after 30 and 60 days). Doppler ultrasonography was performed to measure prostatic volume, vascularization and hemodynamic profile of prostatic artery. Dihydrotestosterone, estrogen and testosterone concentrations were measured. At day 60, prostatic biopsy was performed for histological, immunohistochemical and qPCR analysis for VEGF-A expression. At day 60, vascularization score was higher in untreated compared to treated groups (finasteride and orchiectomy). Furthermore, VEGF-A expression was lower in the Orchiectomy Treated Group, but VEGF-A was immunohistochemically lower in both treated groups (finasteride and orchiectomy) compared to the Untreated Group. The efficiency of finasteride treatment in reducing clinical signs, prostate volume and vascularization appears to be similar to orchiectomy. In conclusion, both PH medical and surgical therapy lead to reduction in prostate dimension and VEGF-A expression and, consequently, lower local vascularization. However, orchiectomy promotes marked hormonal changes, which ultimately lead to prostate atrophy.

1. Introduction

Prostatic Hyperplasia (PH) is the most common male disease of canine senescence [1], with incidence of 95% in non-castrated dogs aged 9 years and 50% and 16%, respectively, in those aged 5 and 2 years [2]. The most common observed clinical signs of PH in dogs are tenesmus, dysuria, hematuria and hematospermia [3]. However, symptomatic PH occurs when the increase in prostate volume compresses urinary and intestinal segments [3]. Canine PH has high similarity with the disease in elderly men, featuring the dog as a good animal model for the study on humans [4].

PH is triggered by a specific hormonal imbalance of aged dogs and men, characterized by 40% less testosterone and 60% more estradiol concentration compared to young individuals [5]. In addition, there is increase in prostatic conversion of testosterone into dihydrotestosterone (DHT), catalyzed by the 5-alpha reductase enzyme [6]. High DHT concentration leads to increased production and expression of prostate growth factors (e.g. vascular endothelial growth factor [VEGF]), giving rise to hyperplasia of the prostatic gland [6].

The definitive treatment for PH in dogs is bilateral orchiectomy, which aims to cease testosterone production and, ultimately, reduce its conversion into DHT [7]. Such endocrine change leads to lower expression of prostate VEGF, resulting in reduced blood flow to the prostatic parenchyma [8]. However, gonadectomy is not an option for stud dogs of high genetic value [7] or PH-affected men [9]. Although no medical therapy for PH is effective such as orchiectomy in reducing clinical signs and prostatic size [10], conservative treatment should to be considered for special-case patients. Estrogen therapy has been initially used for such purpose, aiming to block luteinizing hormone release and, consequently, testosterone synthesis by Leydig cells [11]. However, estrogen treatment is not considered fully safe due to its toxic potential and side effects in males [12]. Thus, GnRH analogs or antagonists have been considered choices for PH treatment, while indirectly reducing testosterone concentrations [13]. Although effective in decreasing prostate size, blood flow and hormone levels, GnRH treatment interferes with testicular sperm production and male fertility, which is not an advantage comparing to gonadectomy in dogs [14]. In this context, PH therapy with finasteride is an alternative for decreasing DHT concentration by 5α-reductase inhibition, reducing prostate PH volume and clinical signs without altering the seminal quality of dogs [15,16]. In addition, finasteride is the drug of choice for PH treatment in men [17]. It has been previously shown that finasteride was capable of reducing prostate volume and local vascularization after 60 days of treatment in dogs [18]. However, the effect of finasteride on tissue expression of vascular factors has not been deeply explored both in humans and dogs [19].

Therefore, this study aimed to characterize the expression of prostatic VEGF, hemodynamic and blood flow changes of the prostatic artery and hormonal PH levels of dogs submitted to finasteride orchiectomy treatment.

2. Materials and methods

2.1. Animals and experimental study

The Bioethics Committee of the School of Veterinary Medicine and Animal Science—University of São Paulo (protocol number 7122171213) approved the current study. Privately owned dogs of several breeds were used in this study, and the animals were included only after owner consent.

Fifteen dogs of several breeds, with body weights ranging from 10–30 kg and aged 6 to 13 years were presumptively diagnosed with PH through clinical signs of hematospermia, tenesmus, dysuria and hematuria, as well as prostatic biometry by B-mode ultrasonography [18,20,21]. Dogs were randomly assigned to 3 experimental groups, according to the treatment mode:

Untreated Group (n = 5);

Finasteride treated Group (n = 5): dogs treated with 5 mg/per animal/day of finasteride (Finasterida®/Medley®) [22];

Orchiectomy treated Group (n = 5).

To assure the appropriate sample size, an analysis was conducted with the SAS Power and Sample Size 12 (SAS Institute Inc., Cary, NC, USA). A retrospective analysis of the variables indicated there was a power of 0.99, which is considered an acceptable statistical power (at least 0.8). Hence, 5 males per group were sufficient to demonstrate significant differences in the data.

Doppler ultrasonography and hormonal evaluations were performed in a monthly interval during 60 days, considering the first day of the diagnosis, medical treatment or gonadectomy. A close clinical follow-up was performed with special attention to possible adverse effects and remission of clinical signs. After 2 months of treatment or diagnosis, dogs were subjected to prostatic biopsy in order to obtain prostate fragments for histological analysis, immunohistochemical and qPCR VEGF-A expression. Under general anesthesia, dogs were placed in dorsal recumbence and a parapenile small incision was performed. An 18-gauge semi-automatic Tru-Cut needle was inserted through the incision and, by means of an ultrasound-guide, prostatic tissue samples were collected through at least two punctures of one of the prostatic lobes [23,24].

The fragments were submitted to real-time PCR processing or immediately washed in 0.9% saline solution, fixed in 10% buffered formaldehyde phosphate solution for 24 hours and posteriorly stored in 70% alcohol until inclusion in paraffin.

2.2. B-mode and doppler prostate ultrasonography

Prostatic ultrasonographic assessment was performed transabdominally with a linear 5.5 MHz transducer (M5 Mindray®, Shenzhen, China). Dogs were placed in dorsal recumbency and caudal abdominal region was sheaved before transabdominal scanning.

Prostatic volume (PV) was evaluated by B-mode ultrasonography using the bladder as a window, measuring height and length in the sagittal plane and width in the axial plane. PV was calculated using the formula: PV (cm3) = 0.487 × L × W × (DL+DT):2 + 6.38 (L = length; DL = depth on longitudinal section; DT = depth on transverse section; W = width) [25]. The expected prostate volume (EPV) was used to estimate the prostatic volume according to the dog weight, i.e., a prostatic volume control for each dog bodyweight, considering the following formula: EPV = 8.48 + (0.238 × Kg body weight) [20,25].

Prostatic tissue perfusion and blood flow velocity of the prostatic artery were evaluated by Doppler ultrasonography. Prostatic artery was scanned at the hypogastric abdominal region and located cranio-dorsal to the prostate gland [20,26]. Color flow Doppler was used to map the vessel and subsequently pulsed-wave Doppler was used to characterize the waveform. Blood sample volume was positioned at the artery center and all measurements were obtained with an angle of ≤60°, making proper angle correction whenever necessary. A total of 9 stable waves of the prostatic artery were obtained to calculate the average of each variable. The following blood flow velocity parameters were automatically calculated by the Doppler machine software, using mathematical formulas or Pourcelot index: peak systolic velocity (PSV), end diastolic velocity (EDV), resistance index [RI = (PSV - EDV) / PSV], pulsatility index [PI = (PSV - EDV) / mean velocity], time average maximum velocity (TAMAX) and peak systolic: diastolic velocity [S/D = (PSV / EDV)].

For the qualitative evaluation of the prostate tissue perfusion, prostate scanning was preferably carried out in longitudinal section by B-mode ultrasonography, followed by color flow Doppler. Prostate vascularization was graded on an arbitrary scale of 1–3, being 1 –the minimum degree of prostate vasculature; 2 –the intermediary degree of vascularization and 3 –the maximum degree of prostate vasculature. Analysis was performed always by only one analyzer.

2.3. Hormonal profile of testosterone, estrogen and dihydrotestosterone

Blood samples (3 mL) from all dogs were collected by puncture of the right or left cephalic or jugular veins into vacuum tubes without an anticoagulant at monthly intervals during the morning period and allowed to clot at room temperature. The tubes were then centrifuged for 10 minutes at 1500 × g, and serum was separated in aliquots of 0.5 mL and stored at −20°C until hormone analysis.

Testosterone and estrogen concentration was measured using a commercial radiomunoassay kit (BECKMAN Coulter Company IM-1841 / IM-1119-Immunotech, Prague, Czech Republic and BECKMAN Coulter Company IM-1841 / DSL4800-Immunotech, Prague, Czech Republic, respectively), both previously validated for dogs [13,27] For dihydrotestosterone, competitive ELISA kit (BECKMAN, Active® DSL9600i-Immunotech-, Prague, Czech Republic) was used [28]. The limit detection for the testosterone assay was 0.01 ng/mL and the intra-assay coefficient of variation was 4.52%. For estrogen, the limit detection test was 1.12 pg/mL and the coefficient of variation ranged from 7.15% to 3.45%. For dihydrotestosterone assay, the limit detection was 6.58% and the coefficient of variation ranged from 9.86% to 3.03%.

2.4. Histological evaluation of prostatic fragments

For the evaluation of the histo-morphological appearance of the prostate tissue, 5 μm histological sections were dewaxed and stained with hematoxylin-eosin (HE) for analysis under light microscopy (Nikon, Eclipse E200, Japan). PH was considered to occur according to the classification of Palmieri, et al. [29]. In other words, PH consisted of normal prostate architecture, whilst an increased amount of secretory epithelium. The alveoli region was expected to be larger and presenting papillary projections, as well as dilated and cystic, lined by columnar to small cuboidal cells with absence of secretory activity. The atrophic areas had mild to moderate increased stroma.

2.5. Immunohistochemical reaction for vascular endothelial growth factor (VEGF-A)

For the immunohistochemical analysis of VEGF-A, 3 μm prostate sections were used. Antigen retrieval was performed in Tris/EDTA buffer at pH 9 with the primary antibody and subsequently with the secondary antibody. A mouse monoclonal antiserum specific for VEGF-A (VG-1, AB1316, ABCAM, UK; diluted 1 in 100) was used in an overnight system. Subsequently, an incubation with antirabbit IgG secondary antibody (STAR124P, Bio-Rad Laboratories, USA; diluted 1:1000) was performed, followed by the addition of 3,3’-diaminobenzidine substrate (during 5 minutes) and haematoxylin counterstain [30].

All immunohistochemical reactions were accompanied by a negative control as previously described [31, 32], consisted of prostate tissue from the Untreated Group lacking application of primary antibody. For the positive control, we used canine mammary gland tumor, previously attested by others [33-35] and also as a guarantee of the cross-reactivity of the primary antibody with canine tissue. Evaluations were performed by microscopy (Leitz, Dialux 20, Germany) at 400x magnification by three operators in a single-blind manner (results were calculated as the mean value of three evaluations). Only the cytoplasm of the prostate glandular epithelium within ten fields was analyzed, by using the relationship between the percentage of stained area (0–0 to 5%, 1–5 to 33%; 2–33 to 66%; 3—above 66%) and intensity (0—negative, 1—low, 2—moderate, 3—high). Thus, a score was defined by the following calculation: Score = Percentage of stained area x staining intensity [36].

2.6. Evaluation of prostatic vascular endothelial growth factor (VEGF-A) by quantitative PCR

After biopsy, prostate fragments were transferred to cryotubes, immersed in liquid nitrogen and stored in -80°C for subsequent RNA extraction. Samples were macerated and RNA extraction was performed using the RNAspin mini Illustrative extraction kit (GE Healthcare Life Sciences, Piscataway, NJ). After extraction, the RNA was quantified by NanoDrop spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA), using 1 μL of the sample. Subsequently, 1 μg of total RNA was used for cDNA synthesis, through the commercial Superscript VILO cDNA Synthesis kit (Life Technologies). Then, synthesized cDNA was quantified using the Qubit 2.0 Fluorometer (Qubit dsDNA BR, Life Techonologies) and samples were stored at -20°C.

To determine the gene expression of vascular endothelial growth factor (VEGF-A), the primer was selected according to Giantin, et al. [30] (Forward primer: 5’- CGTGCCCACTGAGGAGTT-3’/Reverse primer: 5’- GCCTTGATGAGGTTTGATCC-3’). The primers of the endogenous genes B2M (Forward primer: 5’-TCCTCATCCTCCTCGCT-3’/Reverse primer: 5’-TTCTCTGCTGGGTGTCG-3’) and GAPDH (Forward primer: 5’-GTAGTGAAGCAGGCATCGGA-3’/Reverse primer: 5’-GTCGAAGGTGGAAGAGTGGG-3’) were designed using online primer-Blast software (http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome, NCBI, NIH, USA). The standardization of the primers was performed through a standard serial dilution curve of samples 1:10 with 7 points.

Reactions were performed in triplicates for each sample in Realplex2 Mastercycler (Eppendorf, Hamburg, Germany). For each reaction, the SYBR Greener kit qPCR Supermix Universal (Life Technologies) and 2 μL of the cDNA were used. The qPCR program consisted of 2 minutes at 50°C, 10 minutes at 95°C and 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. The concentration of the primers was defined as 200:200 for VEGF-A and GAPDH and 300:300 for B2M.

The efficiency of the reaction was calculated by the PCR software Realplex, with ideal efficiency considered between 0.9 and 1.1. The amplification of each gene in each sample was evaluated to verify if Ct values were contained in the linear standard curve. The qRT-PCR data were analyzed using the ΔΔCt method [37]. The geometric mean of housekeeping genes GAPDH and B2M were used to normalize the relative quantification of target genes. The Ct value of this sample was then subtracted from the other samples to generate the value of ΔΔCt and consequently the Fold Change of gene expression.

2.7. Statistical analysis

Data were evaluated using SAS System for Windows (SAS Institute Inc., Cary, NC, USA). Effects of experimental group, time of evaluation, and interaction between these factors were estimated by repeated measures ANOVA (SAS MIXED procedure) for the ultrasonographic parameters and hormonal profile. Differences between treatments were analyzed by means of parametric and non-parametric tests, according to the residual normality (Gaussian distribution) and homogeneity of variance. Whenever one of these assumptions has not been complied, the data was transformed. If transformations were not successful, non-parametric tests were used. Testosterone concentration, estrogen concentration, dihydrotestosterone concentration, pulsatility index, peak systolic:diastolic velocity and vascularization score were log transformed. In the absence of significant interactions, the effect of groups were analyzed by merging all the evaluation moments, and conversely, evaluation times were compared by combining all groups, taking into account the Bonferroni correction (PROC GLIMMIX). Otherwise, comparisons were made taking into account the main effects.

Differences between groups were analyzed by an orthogonal contrast. Orthogonal comparisons were performed to determine the two main effects: effect of treatment (Untreated Group vs. Finasteride Treated Group + Orchiectomy Treated Group) and the effect of both treatments (Finasteride Treated Group vs. Orchiectomy Treated Group). The Least Significant Difference (LSD) test was used to compare moments of evaluation within the experimental period.

For prostatic volume (PV) and expected prostatic volume (EPV), results were analyzed comparing all three experimental groups (Untreated, Finasteride Treated and Orchiectomy Treated groups) using LSD test in each moment of evaluation (Day 0, 30 and 60). Moreover, in order to compare PV and EPV, Student t test (parametric variables) and Wilcoxon test (nonparametric variables) were used.

The relative qPCR data were analyzed by the MIXED procedure, as described by Steibel, et al. [38]. The response variables were also submitted to Spearman correlation analysis. Results are reported as untransformed means ± S.E.M. Level of significance was P<0.05.

3. Results

Significant interactions (P<0.05) between groups (Untreated vs. Finasteride Treated vs. Orchiectomy Treated groups) and moments (Days 0 vs. 30 vs. 60) were observed for prostate volume, vascularization score, peak systolic: diastolic velocity (S/D), resistance index (RI) and testosterone and dihydrotestosterone concentrations (Table 1).

Table 1

Probability values for the interaction between the main effects of treatment (Untreated vs. Finasteride Treated vs. Orchiectomy Treated) and time (0 vs. 30 vs. 60 days) on ultrasonographic analysis and hormonal concentrations.

	Treatment	Time	Treatment x Time
PVS (cm/s)	0.44	0.42	0.47
EDV (cm/s)	0.13	0.16	0.33
TAMAX (cm/s)	0.32	0.33	0.56
S/D	0.03	0.02	0.01
Resistance index (RI)	0.08	0.15	0.02
Pulsatility index (PI)	0.43	0.98	0.97
Prostatic volume (cm3)	0.20	<0.01	<0.01
Vascularization score (1–3)	<0.01	<0.01	0.03
Estrogen (pg/mL)	0.30	0.02	0.72
Testosterone (ng/mL)	<0.01	<0.01	<0.01
Dihydrotestosterone (pg/mL)	<0.01	<0.01	0.01

PSV: Peak systolic velocity; EDV: End diastolic velocity; TAMAX: Time average maximum velocity; S/D: Peak systolic: diastolic velocity.

After 30 days of the onset of the experiment, all dogs of the Finasteride Treated and the Orchiectomy Treated groups no longer presented the initial clinical signs (hematospermia, tenesmus, dysuria or hematuria). Moreover, dogs of the Untreated Group remained clinically stable, allowing the continuity of the experimental design.

3.1. B-mode and doppler ultrasonographic analysis

Prostate volume was higher than EPV on day 0 and day 30 in all experimental groups (Fig 1). However, no difference between PV and EPV occurred in day 60 for the Finasteride Treated Group (PV: 42.5±12.3 cm3; EPV: 14.3±0.6 cm3) and the Orchiectomy Treated Group (PV: 16.2±2.4 cm3; EPV: 15.13±0.3 cm3) (Fig 1). After 60 days, prostate volume of the Orchiectomy Treated Group was lower than the other groups (Fig 1). Moreover, a prostate volume reduction of 67.4±6.1% was observed in orchiectomized dogs, different from those treated with finasteride (35.2±5.4%) and untreated (15.4±7.9% increase in prostate volume).

Fig 1

Prostate Volume (PV–cm3) and Expected Prostate Volume (EPV–cm3) in the Untreated, Finasteride Treated and Orchiectomy Treated groups at day 0 (A), 30 (B) and 60 (C). *indicate significant differences between PV and EPV and a,b indicate significant differences between groups (P<0.05).

The Orchiectomy Treated Group had significant increase in peak systolic: diastolic velocity (S/D) of the prostatic artery at 60 days, compared to the Finasteride Treated Group (Fig 2A). However, at this time-point, the Untreated Group had lower values of S/D compared to both treatment groups. In addition, S/D was lower in the Untreated Group on day 30, compared to the other groups (Fig 2A). Significant increase in resistance index of the prostatic artery was observed over time for the Orchiectomy Treated Group (Fig 2B). Moreover, on day 60, RI was lower in the Untreated Group compared to both treatment groups (Fig 2B). In the Finasteride and Orchiectomy Treated groups, there was a significant reduction in vascularization score over time (Fig 2C). On days 30 and 60, the Untreated Group had the highest vascularization score compared to both the Finasteride and Orchiectomy Treated groups (Fig 2C). Furthermore, on day 30 and 60, the Orchiectomy Treated Group had lower prostate vascularization score compared to the Finasteride Treated Group (Fig 2C).

Fig 2

Peak systolic: diastolic velocity (A) and resistance index (B) of the prostatic artery and prostate vascularization score (C) in the Untreated, Finasteride Treated and Orchiectomy Treated groups throughout the experimental period. *indicate: Contrast 1 –comparison between Untreated vs. Treatments (Finasteride and Orchiectomy) at P<0.0001. †indicate: Contrast 2 –comparison between Finasteride vs. Orchiectomy Treated groups at P<0.05.

The Untreated Group had higher end diastolic velocity (EDV) compared to both treatment groups. In addition, EDV of the Finasteride Treated Group was higher than Orchiectomy Treated Group (Table 2). Time average maximum velocity (TAMAX) was lower in the Orchiectomy Treated Group compared to the Finasteride Treated Group (Table 2).

Table 2

Mean ± SE of prostate ultrasonographic parameters and estrogen concentrations of Untreated, Finasteride Treated and Orchiectomy Treated groups and Probability values (P) of statistical orthogonal contrast analysis.

	Groups			Contrast
	Untreated	Finasteride Treated	Orchiectomy Treated	C1	C2
PSV (cm/s)	71.2±2.5	73.3±4.1	64.3±4	0.6	0.08
EDV(cm/s)	20.3±0.78	18.8±1.5	14.9±1.3	0.03	0.03
TAMAX (cm/s)	26±1.90	28.7±2.1	22.4±1.9	0.8	0.03
Pulsatility index	2.1±0.2	1.9±0.1	2.5±0.2	0.5	0.08
Estrogen (pg/mL)	6.74±2.1	3.62±0.7	2.99±0.5	0.1	0.6

Contrast C1 –Comparison between Untreated and Treated Groups (Finasteride Treated Group + Orchiectomy Treated Group); Contrast C2 –Comparison between Finasteride Treated Group and Orchiectomy Treated Group; PSV: Peak systolic velocity; EDV: End diastolic velocity; TAMAX: Time average maximum velocity; S/D: Peak systolic: diastolic velocity.

3.2. Hormonal profile

No significant difference between groups or time-points was verified for estrogen concentrations (Table 2). On the other hand, a significant reduction in testosterone concentrations was observed in the Orchiectomy Treated Group throughout the experiment (Fig 3A). The Untreated Group had higher testosterone concentrations compared to both treatment groups (Fig 3A), however, the Orchiectomy Treated Group had lower testosterone concentrations than the Finasteride Treated Group on days 30 and 60 (Fig 3A).

Fig 3

Testosterone (A) and dihydrotestosterone (B) concentrations in the Untreated, Finasteride Treated and Orchiectomy Treated groups throughout the experimental period. *indicate: Contrast 1 –comparison between Untreated vs. Treatments (Finasteride and Orchiectomy) at P<0.01. †indicate: Contrast 2 –comparison between Finasteride vs. Orchiectomy Treated groups at P<0.0001.

After 30 and 60 days, dihydrotestosterone concentrations decreased significantly in the Orchiectomy Treated Group, compared to the Finasteride treated Group, although the latest group had gradual decrease in DHT concentrations (Fig 3B). The Untreated Group had higher DHT concentrations on day 60 compared to both treatment groups (Fig 3B).

3.3. Histological analysis

The Untreated and Finasteride Treated groups had similar histological pattern, with hypertrophy of the prostate glandular epithelium and increased space between glands, glandular proliferation, papillary projection and mild stromal fibrosis. Several alveoli were dilated and cystic, presenting stasis of secretory activity. Inflammatory cells were observed in both groups.

On the other hand, the Orchiectomy Treated Group presented massive reduction of the glandular epithelium, remaining only connective tissue, characterizing an atrophic prostate gland.

3.4. Vascular endothelial growth factor analysis by immunohistochemical and qPCR

Regarding prostatic immunostaining score for VEGF-A, the Untreated Group had higher VEGF-A expression compared to both treatment groups (Fig 4). The Finasteride Treated Group was superior than the Orchiectomy Treated Group (Fig 4). In addition, the Orchiectomy Treated Group had lower expression of VEGF-A, compared to the Untreated Group (P<0.004 –Fig 5) and the Finasteride Treated Group (P<0.01 –Fig 5).

Fig 4

(I) Cytoplasmatic and glandular epithelium immunostaining for VEGF-A in the Untreated Group (arrow, A), Finasteride Treated Group (arrow, B) and Orchiectomy Treated Group (arrow, C). 200x. (II) Mean and standard error of the immunostaining score for VEGF. *indicate: Contrast 1 –comparison between Untreated vs. Treatments (Finasteride and Orchiectomy) at P<0.0001. †indicate: Contrast 2 –comparison between Finasteride vs. Orchiectomy Treated groups at P<0.02.

Fig 5

Relative quantification of prostatic VEGF-A gene expression.

Values are expressed in log2 (FC, Fold Change). *indicate P<0.01. **indicate P<0.05. Legend: UNT–Untreated Group; FIN–Finasteride Treated Group; ORC–Orchiectomy Treated Group.

3.5. Correlation analysis

Positive correlations were observed in the Finasteride Treated Group between prostate volume and vascularization score (r = 0.69; p = 0.0039). On the other hand, resistance index of the prostatic artery correlated negatively with prostate volume (r = -0.76; p = 0.0008) in the Orchiectomy Treated Group. In addition, there was a positive correlation between dihydrotestosterone concentrations and vascularization score (r = 0.85; p = 0.0002) in the Orchiectomy Treated Group.

4. Discussion

The present study aimed to evaluate possible changes in prostate vascularization and hemodynamic, as well as the hormonal profile of dogs submitted to medical or surgical treatment (finasteride or orchiectomy, respectively) for prostatic hyperplasia.

In PH dogs, an imbalance between testosterone and estrogen is observed [5], ultimately increasing dihydrotestosterone (DHT) concentrations [6]. In fact, in the present experiment, all animals presumptively diagnosed with PH (day 0) had characteristic hormonal profile (estrogen, DHT and testosterone) [39]. However, only orchiectomized dogs had significant decrease in DHT and testosterone concentrations. Surprisingly, the hormonal profile of the Finasteride Treated Group remained unchanged. However, it is noteworthy that men treated with finasteride present reduction of serum DHT concentrations only after 10 weeks of treatment [40]. Therefore, it was hypothesize that significant reductions in DHT concentrations may occur only after a long period of finasteride administration, since significant prostatic changes (atrophy of the prostate gland) were observed only after 9 weeks in dogs [41].

Finasteride treatment promotes reduction in prostatic size, reaching the expected prostatic volume (EPV) after 60 days of treatment, similarly to previous studies [18, 42,43]. Moreover, according to Iguer-Ouada; Verstegen [44], in our study, finasteride was effective in reducing PH clinical signs. On the other hand, dogs submitted to orchiectomy had remarkable prostate volume reduction, which can be attributed to the significant decrease in testosterone and DHT concentrations soon after gonadectomy. In fact, surgical treatment can reduce 50% the prostatic volume by 3 weeks after orchiectomy [45], ultimately resulting in prostate atrophy [46]. In our experiment, orchiectomy led to prostatic atrophy 60 days after surgical procedure, reducing 67.4% the prostatic volume, as verified by the connective tissue content of the prostate glandular epithelium on histological analysis.

Untreated and finasteride-treated dogs had hypertrophy of the glandular tissue in a centrifugal manner, which is a peculiar histological finding of PH [29,47]. Among factors that stimulate mitosis of the prostatic parenchyma (e.g. epidermal factor, keratinocyte growth factor and insulin-like growth factors), VEGF-A is considered the main factor responsible for local angiogenesis [6,48]. Our data show that untreated PH dogs had the highest vascularization score throughout the experimental period, as well as the highest immunostaining score for VEGF-A in the prostate epithelium, similarly to previous data on men [49,50] and dogs [35,51,52]. Moreover, positive correlation between prostate volume and local blood flow was observed for untreated and finasteride-treated dogs, suggesting that increased prostatic size is followed by a concomitant increase in local vascularization in PH dogs [18,20,31].

Although we have not performed a tissue VEGF-A evaluation before treatment, a decrease in prostate vascularization score throughout the experimental period was observed in the Finasteride Treated and Orchiectomy Treated groups. This was attested by lower prostate immunostaining for VEGF-A and VEGF-A expression after 60 days of treatment, compared to untreated dogs. The most important modulator of the VEGF-A expression in the prostate tissue is the dihydrotestosterone concentrations [50]. Thus, reduction of DHT concentrations leads to lower VEGF-A expression, ultimately inhibiting neovascularization. In fact, a positive correlation between DHT concentrations and prostate vascularization score in the Orchiectomy Treated Group was verified, suggesting that the prostatic blood flow is under the control of the hormonal profile. The Orchiectomy Treated Group showed remarkable decrease in DHT and testosterone concentrations throughout the experimental period, and also significant reduction in prostate VEGF-A expression after surgery. Based on these results, it was suggested that severe androgen deprivation alters protein synthesis in a post-translational manner before the transcription of mRNA in prostatic tissues of dogs. Therefore, our data can be considered for further studies on prostate cancer and oncological treatments [51].

Despite the decrease in prostatic VEGF-A expression, local vascularization and prostate volume (35.2% reduction) in the Finasteride Treated Group, similarly to observed in men [52], hormonal change during the experimental period was not verified. Simultaneously to 5α-reductase blockage, finasteride also acts modulating the expression of prostatic androgen receptors [15,53]. Thus, it was hypothesized that the negative finasteride regulation of androgen receptors is the main causative factor for the reduction of prostatic volume during the initial period of treatment (while DHT concentrations have not yet changed), consequently, reducing tissue expression of VEGF-A and angiogenesis stimulus [53,54]. However, such assumption should be investigated in further studies regarding the local androgen receptor gene expression following long-term finasteride treatment. It could also be inferred that finasteride may also inhibit prostatic DHT receptors, thus blocking DHT hypertrophic action on prostate parenchyma. On the other hand, surgical treatment (orchiectomy) promotes intense prostate atrophy, leading to morpho-functional changes such as reduction of growth factors gene expression.

Gonadectomy is considered an important surgical treatment for the reduction of prostate volume, local vascularization and blood flow [53]. In our experiment, orchietomized dogs had lower tissue perfusion, as observed in ultrasonographic analysis. Thus, it was concluded that the sharp decrease in androgen profile (testosterone and DHT) after gonadectomy led to significant reduction in prostate blood flow. In fact, prostatic hemodynamics is primarily regulated by androgen hormones [55-60]. Thus, the expected reduction in prostate blood supply after testicle removal is an indirect signal of treatment effectiveness [52]. In our experiment, negative correlation between prostate volume and resistance index of the prostatic artery in the Orchiectomy Treated Group was observed, suggesting that the increase in prostatic intravascular resistance can be used as a marker of prostatic size reduction and severe atrophy.

In conclusion, both PH medical and surgical therapy (finasteride and orchiectomy) led to reduction in prostate dimension and immunostaining for VEGF-A, consequently, lower local vascularization. However, orchiectomy promoted more marked androgenic decrease and severe prostate atrophy, in turn altering prostatic artery hemodynamic and even the VEGF-A expression. These results show a favorable prognosis for PH treated dogs and suggest further studies focused on the diagnosis and treatment of PH in dogs.

(XLSX)

Click here for additional data file.

28 Apr 2020

PONE-D-20-09715

Benign Prostatic Hyperplasia: vascularization, hemodynamic and hormonal analysis of dogs treated with finasteride or orchiectomy

PLOS ONE

Dear Vannucchi

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit and interest but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

You will carefully address all the comments of the two reviewers. Especially, you will have to demonstrate the specificity of VEGF-A immunolabelling illustrated in Figure 5, by testing different anti-VEGF-A antibodies and  showing control labelling with the secondary antibodies used. You will pay a special attention to the terminology recommended by both reviewers and improve the writing of the manuscript.

We would appreciate receiving your revised manuscript by Jun 12 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Jean-Léon Thomas

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide additional details regarding the dogs used in your study and ensure you have described the source. For more information regarding PLOS' policy on materials sharing and reporting, see " ext-link-type="uri" xlink:type="simple">https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-materials."

3. In your Methods section, please provide additional details regarding participant consent from the owners of the animals. In the ethics statement in the Methods and online submission information, please ensure that you have specified (a) whether consent was informed and (b) what type you obtained (for instance, written or verbal). If the need for consent was waived by the ethics committee, please include this information.

4. Please amend either the title on the online submission form (via Edit Submission) or the title in the manuscript so that they are identical.

5. Please upload a copy of Figure 5, to which you refer in your text on line 315. If the figure is no longer to be included as part of the submission please remove all reference to it within the text.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript describes the morphological, ultrasonographic appearance of the canine prostate gland, associated with VEGF protein and gene expression in dogs with prostatic hyperplasia treated with orchiectomy or finasteride. It is an interesting manuscript; however, this reviewer has several concerns regarding the study design and results presentation. Please, see my major and minor comments below.

Major concerns

1. This is one of the major concerns regarding this manuscript. Authors show in the figure cytoplasmic VEGF expression in the prostatic epithelial cells and this finding it is not expected. VEGF-A expression should not be present in epithelial prostatic cells. As authors can confirm in Palmieri et. Al. (2014) (https://doi.org/10.1177/0300985814549951), VEGF-A express only internal blood vessel and not epithelial cells. For human, is the same (https://www.proteinatlas.org/ENSG00000112715-VEGFA/tissue). Unfortunately, in this reviewer opinion, all VEGF-A results are just unspecific binding. Please, check the previous literature to confirm that the VEGF-A expression was misinterpreted in this manuscript.

2. It is widely accepted that canine prostatic hyperplasia (HP) is the most common prostatic disease in intact dogs and authors highlighted this point in introduction section. Thus, using only 15-affected dogs (5 in each group) it is a lower number of subjects. Have authors performed a power analysis? In this reviewer opinion, since PH is a common and the procedure performed are not invasive, authors should include more subjects in each group.

3. Benign prostatic hyperplasia is a redundant term in pathology and in both humans and dogs this term is gradually replaced by Prostatic Hyperplasia. Recently, a group of international veterinary pathologists and clinicians covered by the Oncology Pathology Working Group, have proposed the current Histopathological Terminology Standards for the Reporting of Prostatic Epithelial Lesions in Dogs (Palmieri et al. 2019, doi: 10.1016/j.jcpa.2019.07.005). Thus, it is strongly suggested to use the standardized terminology.

4. A very similar research performed by this own research group (Angrimani et al. 2018, doi: 10.1016/j.theriogenology.2018.03.031) was previously published in Theriogenology. The authors had similar groups. In the previously published paper, the authors included a group of heath dogs without PH clinical signs. Why authors omitted their own similar research work? Are the same subjects used in both studies?

5. This study is lacking a group of Heath dogs. It will be interesting to include a group of heath dogs and a power analysis to confirm that five dogs in each group is statistically significant.

6. The tissue biopsy was performed at the diagnosis ang 60 days treatment? It is unclear how PH was diagnosed. Was biopsy performed or only ultrasound examination?

7. Histological PH analysis. It will be interesting to morphologically compare tissue samples at the diagnosis and 60 days after treatment. Besides that, considerer changing the reference Palmieri, et al. [29] for Palmieri et al. 2019 (doi: 10.1016/j.jcpa.2019.07.005).

8. There is no explanation about the tissue biopsy procedure. Please, clarify how biopsy was performed including, procedures and anatomical site of the tissue biopsy.

9. Immunohistochemistry – the positive and negative controls provided are not a real control. Omitting primary antibody is not a negative control. Why mammary gland was used a positive control do VEGF-A? Since mammary gland is a epithelia tissue, is not expected to express VEGF-A. How about the cross-reactivity of the primary antibody with canine tissue.

10. Results, lines 309-313 – Histology, how authors can confirm homogeneity during biopsy procedure? The results section is lacking correlation among the results.

11. The discussion section is poorly presented with focus on hormonal and ultrasonographic finding and authors perform confrontation of their results with the previously literature seems more literature review than a discussion section. Authors should explain their results, providing convincing hypotheses for the results, instead just confront their results with the previous literature.

Minor point.

1. Standardize description of VEGF-A gene and protein. Usually, gene are writing using italic letters. Abstract, lines 37-38 – “VEGF qPCR 37 expression was”, is not VEGF-A qPCR expression is VEGF-A gene expression or just VEGF-A expression

2. Results section should be organized using subheadings.

3. In conclusion section, the conclusions are based mainly in VEGF-A expression. However, in the whole manuscript, the VEGF-A expression is poorly presented.

4. The English should be revised for spelling and grammar errors.

5. The figure captions are poorly described. The Fig 4 present immunohistochemical images without proper caption. The Fig 4 caption says “Figure 4. Testosterone (A) and dihydrotestosterone (B) concentration in Untreated, Finasteride treated, and Orchiectomy treated groups during the experimental period. * indicate: Contrast – Comparison between Untreated vs. Treatments (Finasteride and 663 Orchiectomy). † indicate: Contrast 2 – Comparison between Finasteride vs. Orchiectomy treated groups. * indicate p”. However, the figure shows Fig 4, A, B, C and D. i.e. the figure 4c seems only tissue stroma (poorly representative biopsy). However, there is no caption and it is uncertain the arrow in this image.

Reviewer #2: Canine prostatic hyperplasia is considered since decades a good animal model for the study of the human counterpart. Several papers focusing BPH are present in literature and the manuscript PONE-D-20-09715 “Benign Prostatic Hyperplasia: vascularization, hemodynamic and hormonal analysis of dogs treated with finasteride or orchiectomy” is in the vein.

The study is interesting however, major revision is required before reconsidering for publication.

A revision of the English will contribute to improve the whole manuscript

A general issue: finasteride. No description of finasteride in the abstract nor in the introduction. Finasteride is not considered among keywords. Finasteride is maybe the key of the study and its composition, action, brief story are totally omitted. It has been already employed in dogs? Yes, but it is not highlighted in the introduction.

Hyperplasia or hypertrophy? An old issue: both. However, let use hyperplasia – BPH.

ABSTRACT is long but not incisive.

Page 2 line 22 – 24. Delete “is related…..prostatic cells”

Page 2 line 24. spend a couple of word on finasteride or put in brackets (anti-androgen)

Page 2 line 30. Delete “prostatic”at the beginning of the sentence and add it between “measure” and “volume”

Page to lines 34-35. “Was similar to expect prostatic volume” – unclear

Page 2 lines 43-44. “more marked androgenic decerase and secvere prostate atrophy” – improve English.

Tables. OK

KEYWORDS. delete "carnivore reproduction" and add finasteride

INTRODUCTION

Insert at least couple of sentences on finasteride

Page 3 line 52. …change the last part of the sentence with “the dog as a good animal model for the study of the human counterpart”.

Page 4 line 71. Since “they” reduce

MATERIAL METHODS reports that study was approved by the Bioethics Committee of the School, but it is not known if the dogs enrolled in the study were from kennel or were owned dogs. In this latter case, owner consensus was required anyway. Please clarify and specify in the text – kennel or owned dogs?

Histological evaluation of prostatic fragments must be improved.

Page 7 line 163. Delete “submitted to deparaffinization” and simply write “section were dewaxed”

Page 8 lines 174-176. Source of the secondary antibody employed in immunohistochemistry.

RESULTS

Page 13 line 303. After 30 days from

Page 13 lines 309-313. Histologic description is poor. Please improve. Did you see cystic acini? Stasis of glandular secretion? Epithelial layer/s of the acini: one, more? Papillary growth in the lumen? Histologic differences between untreated and finasteride groups?

Did you observe inflammatory cells in untreated group?

DISCUSSION is long: shortening is required - - half a page at least.

Issue: nobody knows if untreated and finasteride prostates were different at the beginning of the study, because there is not a time 0 biopsy. It is sure in my opinion that finasteride had a positive effect, however I suggest to remark that you had no idea of the entity of hyperplasia in single cases before tratment.

Page 15 line 349. In corroboration – According to….

TABLES FIGURES. OK

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

16 May 2020

Jean-Léon Thomas

Editor-in-Chief

PLOS-ONE

March 15, 2020

Dear Dr. Jean-Léon Thomas,

We want to thank the opportunity of submitting our manuscript to this prestigious journal and greatly appreciate all the efforts spent on reviewing our work. We are certain that your contributions greatly improved the quality of this manuscript. We hope our modifications make it suitable for acceptance.

We have accepted all the reviewers’s suggestions. Some interesting points were raised, which certainly make the text clearer and easier to understand.

Ms. N. PONE-D-20-09715- "Benign Prostatic Hyperplasia: vascularization, hemodynamic and hormonal analysis of dogs treated with finasteride or orchiectomy"

Reviewer #1: The suggestions of this reviewer have been addressed and written in BLUE in the revised manuscript.

Authors show in the figure cytoplasmic VEGF expression in the prostatic epithelial cells and this finding it is not expected. VEGF-A expression should not be present in epithelial prostatic cells. As authors can confirm in Palmieri et. al. (2014) (https://doi.org/10.1177/0300985814549951), VEGF-A express only internal blood vessel and not epithelial cells. For human, is the same (https://www.proteinatlas.org/ENSG00000112715-VEGFA/tissue). Unfortunately, in this reviewer opinion, all VEGF-A results are just unspecific binding. Please, check the previous literature to confirm that the VEGF-A expression was misinterpreted in this manuscript.

We agree with the reviewer on the observation of VEGF immunolocalization. In fact, Palmieri et al. (2014) described the lack of VEGF expression in epithelial cells. However, Shidaifat et al., 2005 (doi: 10.1507/endocrj.k07-009) and Chevalier et al., 2002 (doi: 10.1016/s0303-7207(01)00728-6) also showed VEGF expression in stromal and epithelial compartments. Furthermore, Walsh et al., 2002 (doi: 10.1038/sj.pcan.4500575) observed that 50% of PH cases had positive score for VEGF in epithelial cells. Thus, in order to clear such information, the discussion section was modified accordingly in Lines 400-401.

It is widely accepted that canine prostatic hyperplasia (HP) is the most common prostatic disease in intact dogs and authors highlighted this point in introduction section. Thus, using only 15-affected dogs (5 in each group) it is a lower number of subjects. Have authors performed a power analysis? In this reviewer opinion, since PH is a common and the procedure performed are not invasive, authors should include more subjects in each group.

In fact, the number of subjects was our concern and we fully agree with the reviewer. Thus, before finalizing the experiment, we performed a power analysis by SAS Power and Sample Size 12 (SAS Institute Inc., Cary, NC, USA), in order to assure the appropriate sample size. We obtained a power of 0.99, which is considered an acceptable statistical power (at least 0.8). More information on the experimental design and sample size was provided in Lines 103-107.

Benign prostatic hyperplasia is a redundant term in pathology and in both humans and dogs this term is gradually replaced by Prostatic Hyperplasia. Recently, a group of international veterinary pathologists and clinicians covered by the Oncology Pathology Working Group, have proposed the current Histopathological Terminology Standards for the Reporting of Prostatic Epithelial Lesions in Dogs (Palmieri et al. 2019, doi: 10.1016/j.jcpa.2019.07.005). Thus, it is strongly suggested to use the standardized terminology.

We agree with the reviewer and the terminology was modified throughout.

A very similar research performed by this own research group (Angrimani et al. 2018, doi: 10.1016/j.theriogenology.2018.03.031) was previously published in Theriogenology. The authors had similar groups. In the previously published paper, the authors included a group of heath dogs without PH clinical signs. Why authors omitted their own similar research work? Are the same subjects used in both studies?

We have been researching PH over the years and several distinct data were obtained. The manuscript referred by the reviewer (Angrimani et al. 2018, doi: 10.1016/j.theriogenology.2018.03.031) had the main goal of analyzing the effect of PH itself (and also finasteride) on prostate vascularization, therefore a non-PH group was necessary. On the other hand, the present manuscript has the objective of comparing two modalities of PH treatment, which does not require a non-PH group in the experimental design. In addition, the subjects of both experiments were different. However, we agree with the reviewer and used such information in Lines 78-79.

This study is lacking a group of Heath dogs. It will be interesting to include a group of heath dogs and a power analysis to confirm that five dogs in each group is statistically significant.

The present manuscript has the main objective of comparing two modalities of PH treatment, thus, we believe that the experimental design does not require a non-PH group. Additionally, we were ethically concerned of performing a needless prostatic biopsy in healthy dogs, under the risk of internal hemorrhage and urethra lesions. However, we partially agree with the reviewer and with this in mind we opt to analyze our data as an orthogonal contrast, in which a combination of groups increases the number of subjects per group and allow for obtaining estimates of main, nested and interaction effects. Such explanation was included in Lines 265-269.

The tissue biopsy was performed at the diagnosis ang 60 days treatment? It is unclear how PH was diagnosed. Was biopsy performed or only ultrasound examination?

We agree with the reviewer for the lack of methodological information. Therefore, we modified and included more information in Lines 95-97 (presumptive PH diagnosis) and Lines 111-118 (prostate biopsy timing and technique).

Histological PH analysis. It will be interesting to morphologically compare tissue samples at the diagnosis and 60 days after treatment. Besides that, considerer changing the reference Palmieri, et al. [29] for Palmieri et al. 2019 (doi: 10.1016/j.jcpa.2019.07.005).

We agree with the reviewer. However, we opt to perform a single prostate biopsy after 60 days of treatment for ethical reasons (two procedures within a two-month interval) of owned dogs. The indicated reference was included accordingly in Line 180.

There is no explanation about the tissue biopsy procedure. Please, clarify how biopsy was performed including, procedures and anatomical site of the tissue biopsy.

We apologize for the lack of information. Thus, more information on this subject was included in Lines 111-118.

Immunohistochemistry – the positive and negative controls provided are not a real control. Omitting primary antibody is not a negative control. Why mammary gland was used a positive control do VEGF-A? Since mammary gland is a epithelia tissue, is not expected to express VEGF-A. How about the cross-reactivity of the primary antibody with canine tissue.

We apologize for the lack of information. Thus, more information on this subject was included in Lines 197-201.

Results, lines 309-313 – Histology, how authors can confirm homogeneity during biopsy procedure? The results section is lacking correlation among the results.

We apologize for the lack of information. Thus, more information on this subject was included in Lines 180-184 and 344-346.

The discussion section is poorly presented with focus on hormonal and ultrasonographic finding and authors perform confrontation of their results with the previously literature seems more literature review than a discussion section. Authors should explain their results, providing convincing hypotheses for the results, instead just confront their results with the previous literature.

Discussion was fully revised and modified.

Standardize description of VEGF-A gene and protein. Usually, gene are writing using italic letters. Abstract, lines 37-38 – “VEGF qPCR expression was”, is not VEGF-A qPCR expression is VEGF-A gene expression or just VEGF-A expression.

It was modified accordingly throughout.

Results section should be organized using subheadings.

It was modified accordingly.

Minor Concerns: In conclusion section, the conclusions are based mainly in VEGF-A expression. However, in the whole manuscript, the VEGF-A expression is poorly presented.

We agree with the reviewer and the Conclusion was modified accordingly in Lines 449-453.

Minor Concerns: The English should be revised for spelling and grammar errors.

The manuscript was submitted to the correction of a native English speaker and all the grammar and spelling errors were corrected throughout. We are sending attached a translation certificate.

The figure captions are poorly described. The Fig 4 present immunohistochemical images without proper caption. However, the figure shows Fig 4, A, B, C and D. i.e. the figure 4c seems only tissue stroma (poorly representative biopsy). However, there is no caption and it is uncertain the arrow in this image.

We apologize for our mistake and, in fact, figure captions were exchanged. All figure captions were reviewed and modified accordingly.

We have to clarify that only tissue stroma was observed in our biopsy analysis of the Orchiectomy Treated Group. Thus, in order to clear such query, we added this information in Lines 344-346.

Reviewer #2: The suggestions of this reviewer have been addressed and written in RED in the revised manuscript.

Major Concerns: A revision of the English will contribute to improve the whole manuscript

The manuscript was submitted to the correction of a native English speaker and all the grammar and spelling errors were corrected throughout. We are sending attached a translation certificate.

A general issue: finasteride. No description of finasteride in the abstract nor in the introduction. Finasteride is not considered among keywords. Finasteride is maybe the key of the study and its composition, action, brief story are totally omitted. It has been already employed in dogs? Yes, but it is not highlighted in the introduction.

We agree with the reviewer and, in fact, this information was missing. Thus, we modified the Abstract and the Introduction section accordingly in Lines 21-24 and Lines 77-81, respectively.

Hyperplasia or hypertrophy? An old issue: both. However, let use hyperplasia – BPH.

We agree with the reviewer that both tissue alterations occur in BPH. However, according to reviewer 1, the novel nomenclature of BPH is Prostatic Hyperplasia, which we adopt throughout.

Abstract Concerns: ABSTRACT is long but not incisive.

We modified the abstract accordingly.

Introduction Concerns: Insert at least couple of sentences on finasteride

In fact, information on finasteride was missing. We included more information in Lines 77-81.

MATERIAL METHODS reports that study was approved by the Bioethics Committee of the School, but it is not known if the dogs enrolled in the study were from kennel or were owned dogs. In this latter case, owner consensus was required anyway. Please clarify and specify in the text – kennel or owned dogs?

We apologize for the lack of information, which was provided in Lines 92-93.

Material Methods Concerns: Histological evaluation of prostatic fragments must be improved.

More information was included in Lines 180-184.

Material Methods Concerns: Page 8 lines 174-176. Source of the secondary antibody employed in immunohistochemistry.

More information was included in Lines 193-196.

Results Concerns: Page 13 lines 309-313. Histologic description is poor. Please improve. Did you see cystic acini? Stasis of glandular secretion? Epithelial layer/s of the acini: one, more? Papillary growth in the lumen? Histologic differences between untreated and finasteride groups? Did you observe inflammatory cells in untreated group?

More information was included in Lines 339-343.

DISCUSSION is long: shortening is required - - half a page at least.

We agree with the reviewer, therefore the discussion was revised.

Issue: nobody knows if untreated and finasteride prostates were different at the beginning of the study, because there is not a time 0 biopsy. It is sure in my opinion that finasteride had a positive effect, however I suggest to remark that you had no idea of the entity of hyperplasia in single cases before tratment.

We agree with the reviewer. However, we opt to perform a single prostate biopsy after 60 days of treatment for ethical reasons (two procedures within a two-month interval) of owned dogs. More information was included accordingly in Lines 405-409.

We are submitting the revised manuscript to be revaluated for publication.

Thank you for your consideration.

Sincerely,

Camila Infantosi Vannucchi, PhD

Professor, Department of Animal Reproduction - University of São Paulo

2 Jun 2020

Prostatic Hyperplasia: vascularization, hemodynamic and hormonal analysis of dogs treated with finasteride or orchiectomy

PONE-D-20-09715R1

Dear Dr. Vannucchi,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Jean-Léon Thomas

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors provided a revised manuscript with all concerns properly addressed. After reviewing the revised version of this manuscript, I have no further comments and I recommend the revised version of this manuscript for publication in PLos One.

Stay safe.

Reviewer #2: In the second version of the manuscript all suggestions given have been considered. In particular, Finasteride was better described, hyperplasia instead of hypertrophy was used, an improvement of Materials and Methods was performed. Discussion was also revised.

The manuscript, in my opinion, is now suitable for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

16 Jun 2020

PONE-D-20-09715R1

Prostatic Hyperplasia: vascularization, hemodynamic and hormonal analysis of dogs treated with finasteride or orchiectomy

Dear Dr. Vannucchi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Jean-Léon Thomas

Academic Editor

PLOS ONE