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Investigative Urology: Regional Concentration of Basic Fibroblast Growth Factor in Normal and Benign Hyperplastic Human Prostates
0022-5347/95/1533-0839$03.00/0 OF UROI.<)GY Copyright 0 1995 by AMERICAN UI~OLOCICAIL ASSOCIATION, INC.
THEJOURNAL
Vol. 153,839-843,March 1995 Printed in U.S.A.
REGIONAL CONCENTRATION OF BASIC FIBROBLAST GROWTH FACTOR IN NORMAL AND BENIGN HYPERPLASTIC HUMAN PROSTATES FRANK P. BEGUN,* MICHAEL T. STORY, KATHLEEN A. HOPP, ELLEN SHAPIRO AND
RUSSELL K. LAWSON From the Departments of Urology and Biochemistry, The Medical College of Wisconsin, and the Department of Veterans Affairs, Clement J . Zablocki Medical Center, Milwaukee, Wisconsin
ABSTRACT
Basic fibroblast grown factor (bFGF) is a potent mitogen for mesenchymal cells, including fibroblasts cultured from prostate, and has been postulated to play a role in t h e development of benign prostatic hyperplasia (BPH). If this is the case, it might be expected that bFGF levels would be elevated in the adenomas of BPH and in the periurethral region of the prostate where BPH is believed to arise. This study was undertaken to test this hypothesis. The concentration of bFGF was evaluated in 31 prostates, 13 normal glands and 18 with BPH. A method for quantitating bFGF by radioimmunoassay was developed that enabled growth factor levels to be correlated to t h e geographic region of the prostate and t h e histopathology of t h e specimen. A 2- to 3-fold higher concentration of bFGF (ng./g. of tissue) was noted in the benign hyperplastic prostates when compared with t h e adult normal glands. Pubertal specimens demonstrated low growth factor levels comparable to those observed in t h e normal adult group. Two prepubertal prostates analyzed had high levels similar to those measured in the hyperplastic glands. While t h e levels of bFGF i n the normal adult prostates were highest in the periurethral region, statistical analysis failed to demonstrate a s i g d i c a n t difference. Similarly, quantitative morphometric evaluation failed to demonstrate any significant differences in bFGF concentration related to t h e proportion of stromal, epithelial, or lumenal elements in the tissue sections. KEY WORDS: growth substances, prostatic hypertrophy Benign prostatic hyperplasia (BPH) is the most common nonmalignant neoplasm in men. This is evidencedby the fact that more than 350,000 surgical procedures are performed each year to treat this condition. The result is a total yearly expenditure in the United States that exceeds 1 billion dollars.' Although this disorder is an almost universal phenomenon, its pathogenesis is poorly understood. There is general agreement that BPH begins in the inner regions of the prostate. McNeal has localized the early changes of BPH to an area termed the transition zone, located adjacent to the urethra in the proximal region of the gland between the verumontanum and the bladder neck.' The first lesions appear in the third and fourth decades of life and precede the clinical symptoms by 10 to 30 year^.^*^ Benign prostatic hyperplasia is believed to begin with stromal alternations that stimulate the growth and differentiation of epithelial cells.4-" This suggests that stromal- epithelial interactions are critical in the development of BPH. Our laboratory has isolated a member of the heparin-binding growth factor family from the human p r ~ s t a t e . ' ~This .~~ growth factor, basic fibroblast growth factor (bFGF), has been postulated to be responsible for the stromal and/or epithelial hyperplasia that occurs in BPH.I4 If bFGF is a mediator of these interactions, it might be expected that levels of the growth factor would be elevated in glands demonstrating hyperplasia compared with normal prostates. It might also occur in greater concentrations in the periurethral regions of prostates with early hyperplastic changes as well
as in the adenomas of more advanced BPH. The current study was undertaken to test this hypothesis. METHODS
Tissue specimens. Whole prostates were obtained from adult men undergoing open prostatectomy for treatment of obstructive uropathy. Prostates were also available from from young men, adolescents and children who were cadaver renal transplant donors. Specimens were determined to be normal or BPH by histological examination by a pathologist. Appropriate consent was obtained prior to removal of the prostate gland in all instances. Sectioning method. The whole prostate gland was placed in a sterile container, packed in ice and transported to the laboratory. The gland was weighed and a 0.75- to 1-cm. thick slice was taken, midway between the bladder neck and the verumontanum, in the transaxial plane. The section was laid flat on a Petri dish and immediately frozen at -2OC. The remainder of the prostate was sent for histopathological analysis. The frozen specimen was further divided in half in the same transaxial plane. In this manner, 2 adjacent 0.4- to 0.5-cm. cross-sectionalslices were obtained. The freezing process prevented degradation of the bFGF by proteolytic enzymes and facilitated sectioning and handling of the tissue. Subsequent procedures were carried out at OC. The two adjacent slices were kept in their normal orientation and again laid flat in the Petri dish. Using a razor blade, horizontal and vertical cuts, 3-4 mm. apart, were made through the upper slice and carried part way down into the lower section. In this fashion, the upper slice was completely divided into a numAccepted for publication September 9, 1994. * Requests for reprints: Department of Urology, Froedtert Memo- ber of cubes that weighed 50 to 200 mg. The underlying rial Lutheran Hosuital. . ,9200 West Wisconsin Ave.. Milwaukee, Wis- specimen was marked with corresponding grooves for orienConsin 53226. This work was supported by P.H.S. grant DK31063.and by the tation. A map was made of the cut surface of the prostate, and each cube was assigned a number. The cubes were placed Department of Veterans AfFaus Medical Research Semce. 839
840
REGIONAL CONCENTRATION OF bFGF IN HUMAN PROSTATES
in a preweighed 50 ml. conical centrifuge tube and weighed. aspect of the gland) and capsule. The transition zone was not Five ml. of 50 mM. Tris, 1.55 M. NaC1, pH 7.6, containing 1readily identifiable as a distinct regional entity in many of EDTA (10 mM.), phenylmethanesulfonyl fluoride (1 mM.), the normal prostates. Therefore, sections through the tranGl-(tosylamide)-2-phenylethyl chloromethyl ketone (0.3 sition zone were included in either the glandular or periuremM.), N-ethylmaleimide (0.05 mM.), soybean trypsin inhib- thral region depending on their geographic location. Color-assisted quantitative image analysis. The stained tisitor (10 mg.ll.), leupeptin (1mg./l.) and pepstatin A (4 pM.) was added. All protease inhibitors were from Sigma (St. sue sections were viewed under a Wild-Leitz Microscope usLouis, Missouri). The specimens were stored at -2OC. The ing the 6.3 Planafluotar objective and a Variolum adapter lower tissue slice was fixed in formalin and embedded in under 250 x magnification (Ernst Leitz, Wetzlar, Germany). paraffin, and the whole mount section was stained with he- A high resolution JVC TK-870U color video camera (Victor matoxylin and eosin. Using this method, the geographic lo- Co., Tokyo, Japan) was used to capture the image of the cation of each specimen that was processed for bFGF levels histologic section that was displayed on a 12-inch color monitor. The image was simultaneously digitized using the AT could be ascertained. Tissue homogenization. The tissue was thawed and homog- Vista inside a Bell System 220 personal computer (Bell Comenized 3 times with a Polytron (Brinkmann Instruments, puter Co., Austin, Texas). The automated analysis was perInc., Westbury, New York) at a setting of 5 for 10 seconds Formed by the Bioquant Image Analysis System (BQ MEG with a 30-second cooling interval between homogenizations. N-Vista: R & M Biometrics, Nashville, Tennessee). The BQ The homogenate was centrifuged at 23,000 x g for 30 min- MEG IV-Vista is a color system image analysis that discrimutes using a Beckman Model 52-21 centrifuge (Beckman inates color differences of stained tissue sections. The threshInstruments, Inc., Palo Alto, California). The supenatant was olds were set to identify the various tissue components of the filtered (0.2 pm. Acrodisc membrane; Gelman Sciences, Inc., prostate. The area densities corresponding to each of these Ann Arbor, Michigan) and stored frozen at -2OC until deter- tissue components were calculated for each full screen of the color monitor. The mean percentage of stromal tissue ( S ) , mination of bFGF content by radioimmunoassay (RIA). Radioimmunoassay. Basic FGF was iodinated using the glandular epithelium (E) and glandular lumina (L) were deiodogen method described by Salacinski et al.15 The RIA was termined from the tissue sections obtained. Statistical analysis. The mean bFGF concentration [bFGF] performed using recombinant human bFGF (Amgen Biologicals, Thousand Oaks, California) as ligand and rabbit anti- was calculated for each whole prostate gland. When possible, serum raised against bovine serum albumin conjugated (1- the regional concentrations of bFGF were analyzed. Two 24) bFGF provided by Andrew Baird (Whittier Institute for normal adult prostates and 4 BPH prostates were randomly Diabetes and Endocrinology, San Diego, California). The chosen for quantitative image analysis. Three to 5 areas with epitope of the protein bFGF (1-146) recognized by the anti- the highest and a similar number with the lowest [bFGFl for serum is the sequence 11-15.16 The antiserum reacts with each gland were selected. The percentage of S, E and L for the the amino-terminal extended form of bFGF (1-154) isolated corresponding tissue sections was calculated. An analysis of from human p r 0 ~ t a t e . It l ~has not been reported whether the variance (ANOVA) of the S%, E% and L% by low versus high antiserum recognizes 24,23, and 22 kD forms of the protein [bFGF] for each patient was performed. In addition, the data that appear to be localized in the cell n u ~ l e u s . ' The ~ anti- from all patients were combined and rank ordered according serum does not cross-react with acidic FGF and keratinocyte to [bFGF]. An analysis of variance of S%, E% and L% by low growth factor (M.T.S., unpublished data), that are structur- versus high [bFGF] ranking was also carried out. Whole ally similar to bFGF and identified in pro~tate.'~.'' The RIA gland and regional mean values were compared for the varwas performed as previously described." The bFGF content ious groups by T-test. of the samples was derived from the bFGF standard curve using an Apple IIE and a curve fitting software program RESULTS (Interactive Microware Inc., State College, Pennsylvania). Evaluation of the bFGF content of normal and BPH prosData from the dose-response of prostate homogenates was tates. A total of 31 prostates, 13 normal and 18 BPH, was plotted on logit-log paper. Parallel dose response curves were analyzed for [bFGF]. The mean [bFGF], determined by RIA, indicative of equimolar competition of the ligand and sample was expressed as ng. bFGF/g. tissue. The mean [bFGFl valfor antibody. Basic FGF values were expressed as ng./g. ues for the normal and BPH prostates are summarized in tissue. The intra- and interassay coefficients of variation tables 1 and 2. The mean [bFGF] for the entire normal were 9.5% and 12.1%, respectively. prostate group was 100.1 2 12.8 compared with 156.1 2 7.3 Definition of prostatic anatomy. The various regions of the for the BPH group. This difference was significantly differprostate were defined so that meaningful statistical analysis of the data could be carried out. The convention used was similar to that set forth by M~Neal.'.~Specimens obtained TABLE1. Whole gland mean BFGFI for normal prostates from the transplant donors provided a complete cross-section of the gland. However, prostates obtained from patients unGland Number Age (yrs.) Weight ( m s . ) @FGFI* dergoing open prostatectomy would be expected to contain Prepubertal predominantly adenoma and periurethral elements. The pos143.7 2 7.8 (13) 771 0.3 2.4 776 4 2 234.2 t 22.5 (11) terior prostate and capsule were rarely included. Therefore, Pubertal the whole prostates were divided into the following regions: 9 83.4 f 3.9 (14) 707 14 periurethral (those sections containing or immediately con14 99.1 2 8.2 (20) 729 16 tiguous to the urethral mucosa, including the transition 9.7 84.3 2 6.3 (17) 912 17 zone), glandular (those sections in the mid-portion of the 749 18 20 114.1 2 5.2 (43) Adult prostate gland adjacent, lateral and posterior to the urethra, 867 21 15.2 79.7 2 3.6 (23) including the posterior prostate but not the capsule), anterior 772 23 22 103.3 f 7.4 (39) (glandular elements anterior to the urethra) and capsule. 703 23 40.5 80.9 f 16.7 (30) The open prostatectomy specimens were divided into the 866 23 ND 61.3 2 3.7 (29) 69 1 30 21.5 62.6 t 3.5 (33) following regions: periurethral (those sections containing or 913 42 15.9 90.2 5 9.6 (19) immediately contiguous to the urethral mucosa), glandular 713 43 22 64.6 ? 2.6 (24) (adenomatous elements adjacent, lateral and posterior to the * Mean tbFGF1 (ng./g.) f the standard error of the mean. The number Of periurethral r e ~ o n )anterior , (glandular elements anterior to soecimens analvzed is indicated in Darentheses. the urethra), posterior (true prostatic tissue at the posterior ' N D = Not determined.
841
REGIONAL CONCENTRATION O F bFGF I N HUMAN PROSTATES
-TABLE2.
Gland Number
Whole gland mean lbFGFl for BPH glands Aae
Weight ( e m s . )
bFGF Content as a Function of Age
[bFGFl*
36 ND 142.0 f 18 (37) 688 44 96.5t 6.3 (67) 21.5 762 77 53 152.6 t 4.2 (74) 733 58 ND 219.0t 12.4 (19) 674 88 169.3 2 6.7 (33) 60 885 96 61 167.9 2 3.7 (109) 754 62 151.1t 6.4 (50) ND 728 63 36.5 164.2 2 6.0 (41) 752 63 48 196.2t 5.8 (19) 785 65 29 205.4 t 9.7 (40) 774 89 145.22 8.5 (66) 66 702 148.1t 6.7 (75) 116 750 68 70 ND 125.0 ? 10.5 (34) 689 115.22 5.2 (52) 725 73 110 167.6 2 5.0 (72) 74 75 798 112.8t 4.6 (86) 75 858 70 156.2 2 6.3 (51) 77 886 285 732 79 85 176.1 t 4.6 (102) * Mean [bFGFI ( n d g . ) f the standard error of the mean. The number of specimens analyzed is indicated in parentheses ND = Not determined.
I
0
0
I*
20
0
I
BPH
40
80
60
Age (years) ent; however, the data were more informative when the normal prostate group was subdivided into prepubertal, pubertal and adult specimens. The mean [bFGF] value for the adult prostates was 77.5 2 3.4 compared with 189.0 ? 32.0 for the prepubertal and 95.2 ? 6.3 for the pubertal group (table 3). The mean [bFGFl values for the BPH prostates were significantly higher than those of the adult normal glands (p = .001). The mean [bFGF] of the 2 prepubertal specimens was similar to that of the BPH group, while the mean [bFGF] of the pubertal glands was comparable to that of the adult normal group and significantly lower than the BPH specimens (p = .002). Thus, the [bFGFl of the BPH prostates was twice that of the adult normal glands. These data are graphically illustrated in the figure which depicts the mean [bFGF] values plotted as a function of age and pathology. Regional LbFGF] analysis. The data from the analysis of tbFGF1 in various regions of the prostates are summarized in table 4. There were some specimens in both the adult normal prostate group and the BPH group that did not contain samples from all of the represented regions of the gland. This was particularly the case for the BPH group since the posterior and capsular regions are rarely excised. The mean [bFGF] for the glandular regions of the adult normal prostates were as follows: 80.8 f 7.9 (glandular), 92.1 ? 13.6 (anterior) and 60.8 (posterior). The periurethral region had a somewhat higher mean [bFGF] of 108.5 ? 13.7. However, this was not significantly different when compared with the glandular areas. The capsular [bFGFl of the adult normal glands was 59.8 2 10.6. This was lower than the periurethral and glandular regions but also not statistically significant. The mean RFGF] for the adenomatous portions of the BPH prostates was fairly uniform, with values of 148.2 ? 8.9 for the glandular, 148.1 2 21.4 for the anterior and 161.8 2 63.8 for the posterior regions. The periurethral areas had a mean concentration of 168.8 t 16.1 that was not significantly different from the surrounding glandular elements. However, the bFGF content of the capsular regions
bFGF concentration as function of age TABLE 4. Reeional IbFGFl in adult normal and BPH prostates heion
Adult Normal
108.5 -t 13.7(6) Periurethral 80.8 ‘t 7.9 (5) Glandular 92.1 f 13.6(6) Anterior 60.8(1) Posterior 59.8 2 10.6(2) Capsule * Mean bFGF1 (ngJg.) ‘t the standard error of the prostates analyzed is indicated in parentheses.
BPH 168.8 ‘t 16.1 (11) 148.2 2 8.9 (12) 148.1 2 21.4 (6) 161.8 2 63.8 (2) 72.6 2 5.5 (4) mean. The number of
was 72.6 2 5.5. The difference between this value and those of the periurethral and glandular regions was statistically significant (p = .003, p = .001, respectively). Quantitative image analysis. Data from the quantitative image analysis evaluation of 6 individual prostates are presented in table 5. The low and high [bFGF] ranges are indicated along with the corresponding mean %S, %E, %L for each gland. Five of the 6 prostates failed to demonstrate significant differences in the percentages of the various tissue components in the regions with the highest and lowest [bFGFl. Prostate 762 had a mean %S of 56.12 and 70.30 for the regions with the lowest and highest [bFGFl, respectively (F(1,9) = 9.85,p 5.01). Correspondingmean %E values were 28.5 and 14.2 for the same areas (F(1,9) = 18.55, p 5 .003). Samples from all 6 prostates were combined and rank ordered according to [bFGF]. The bottom and top 50%represented the ”low” and “high”bFGF groups, respectively. Analyses of variance were computed, using these groups, on the %E, %S and %L. The mean %S values of 65.35 for the low [bFGF] regions and 68.45 for the high bFGF1 regions did not differ significantly (F(1,47) = .74, p = .39). The mean %E of 17.71 and 16.00 for the low and high [bFGF] areas also failed to show statistical significance (F(1,47) = .56, p = .46). A similar analysis for %L resulted in mean values of 16.94 and 15.55 for the low and high [bFGF] regions (F(1,47) = .50, p = .48).
TABLE3. ComDarison of whole eland mean BFGFI for the different age groups ~~
~~~
Mean* [bFGFl
Nonadult Normal
Prepubertal
Pubertal
X 126.5 2 21.4 X X Nonadult Normal (6) X 189.0 2 32.0 X X Prepubertal (2) X X p = ,035 95.2t 6.3 Pubertal (4) NS p = .04 p = ,002 77.5 2 3.4 Adult Normal (7) p = ,002 NS NS 156.1 t 7.3 Adult BPH (18) * Mean fiFGFl (ngJg.) 2 the standard error of the mean. The number of prostates analyzed is indicated in parentheses. NS = Not significant.
Adult Normal
X X
X X p = ,001
REGIONAL CONCENTRATION OF bFGF IN HUMAN PROSTATES
842
TABLE5. Analvsis of variance o f Dement tissue components b y low us. high BFGFI for each prostate
-
~~
8 Stroma
Prostate Number
Mean 885 (BPH)
% Epithelum
8 Lumen
Growth Factor Range Low
=
40-93
(N = 3)
ANOVA
Mean
High = 201-212 (N = 3) Low=53-66 ( N = 5 )
69.5 56.12
732 (BPH)
High = 147335 (N = 5) Low = 88-98 (N = 5)
70.30 58.46
886 (BPH)
High = 247359 (N = 5) Low = 8&91 (N = 3)
61.76 62.93
867 (Normal Adult)
High = 204-281 (N = 5) Low=5459 ( N = 3 )
80.94 72.78
866 (Normal Adult)
High = 102-115 (N = 3) hw=20-31 (N=4)
68.43 64.80
High = 78-98
65.82
F(1,5) = .02
F(1,9) = 18.55**
F(1,9) = ,001 15.46 18.20
F(1.9) = 1.35
F(1,9) = .39 20.18 19.30
18.06 17.80 F(1.7) = 3.20
F(1,7) = 2.87 8.56 13.87
F(1,7) = 2.01 10.50 13.43
F(1,5) = .72
F(1,5) = .25 19.10 13.63
F(1,5) = .07 12.47 21.60
F(1,7) = .48
F(1,7) = .04 15.45
-
14.67 15.38
14.2 23.34 F(1,9) = ,204
ANOVA
13.47
15.80 28.5 F(1,9) = 9.85*
(N = 4)
Mean
F(1.5) = 1.25
F(1,5) = .29 762 (BPH)
ANOVA
9.83
76.7
F(1,7) = .56 18.70
* p 5 .01 ** p 5 ,003 DISCUSSION
We reasoned that if bFGF plays a pivotal role in the etiology of BPH it might be anticipated that higher levels of the growth factor would be present in the region of the prostate immediately adjacent to the urethra, where BPH is believed to arise.'s3 Elevated bFGF levels might also be expected in younger men who are undergoing early prostatic changes recognized as microscopic BPH.'l Similarly, elevated levels of bFGF might be anticipated in older men in regions of the prostate that are developing macroscopic BPH.'* A method for quantitating bFGF was developed that enabled growth factor levels to be correlated with the geographic region of the prostate and the histopathology of the specimen. This method overcame the shortcomings in quantitation of immunohistochemistry and in situ hybridization. However, because of the heterogeneity of prostate tissue and the need to use at least 50 mg. of tissue for bFGF quantitation, the possibility existed that the tissue used to determine [bFGF] could be different from the adjacent tissue used for histopathology evaluation. In addition, the procedure that was used to analyze bFGF does not distinguish between extracellular and intracellular multiple molecular weight forms of bFGF. It is believed that 24, 23 and 22 kD forms of the protein, initiated from CUG codons, are exclusively located in the cell nucleus, while the 18 kD form, initiated from the AUG codon, is preferentially exported to the cell surface.17 A number of investigators have subsequently identified bFGF in the nucleus, suggesting that the growth factor may act through intracellular pathways. The physiological significance of these findings to the etiology of BPH remains to be determined. Our studies indicated that BPH tissue had a 2- to 3-fold higher [bFGFJ than adult prostates without hyperplastic changes (table 3). These findings are in agreement with the bFGF-like biological activity, 4-fold higher in BPH than in normal prostate, reported by Nishi and associates and the BPH-associated increase in bFGF mRNA reported by Mori et al.23 The mean age of the 7 adult normal prostate donors in our study was 29 t 9 years versus 64 ? 11years for the 18 BPH specimens. The age of the donors of the 3 normal prostates analyzed in Mori's study was 49 ? 1years versus 71 ? 7 years for the 12 BPH specimens. The age of donors of normal prostate ranged from 37 to 44 years, but the age of BPH donors was not reported in Nishi's paper. In our study and the study by Mori, the age of donors of normal prostate was significantly lower than BPH donors. Therefore, it is conceivable that the increase in [bFGFl in the BPH group is age-related, rather than BPH-related. Further studies are needed to address this possibility.
''
The regional bFGF concentration of normal adult prostates was found to be less uniform than that of the hyperplastic glands (table 4). The periurethral regions had the highest mean [bFGF] values and the anterior and glandular regions had somewhat lower values, while the posterior and capsular regions had the lowest [bFGFl. The data, although not statistically significantly, were consistent with our hypothesis that elevated bFGF levels might be expected in the region of the prostate where BPH is known to arise.3 However, only 3 normal prostates from men 30 years or older were studied (table 1).It will be important to expand the data base of normal prostates from men in the third and fourth decades of life, when microscopic BPH is first observed," to support or refute our preliminary data. The adenomas of BPH begin periurethrally and grow outward, displacing and compressing the surrounding elements of the true prostate. Enucleation of the prostate during open surgery for BPH removes only the hyperplastic region of the gland. The data illustrate that bFGF levels in these glands were uniformly elevated over those of normal prostate in all regions except the capsule (table 4). This distribution of bFGF in BPH supports our contention that the growth factor plays an important role in the hyperplastic process. Adult normal prostates had [bFGFl that were similar to those concentrations found in the capsular regions of BPH glands. This may represent a "background" level of bFGF that is present in many tissues.'* A higher concentration of bFGF was found in 2 prepubertal specimens as compared with pubertal and normal adult prostates (table 1).The bFGF levels in the adolescent glands were low and statistically comparable to those of the normal adult group. These glands are undergoing growth as a result of androgen stimulation, and the bFGF levels might be expected to be elevated. Unfortunately, the small number of specimens available for evaluation precluded the formation of any conclusions. Interestingly, when analyzed histologically, prepubertal specimens contained a much higher proportion of stroma than did the normal adult prostate^.'^ It has been shown that fibroblasts cultured from human prostate synthesize bFGF.'" It is possible that the high levels of bFGF observed in the prepubertal prostates was a function of the stromal content of the gland. Immunohistochemical staining of the prostate for bFGF has demonstrated that the stroma is a rich source of the growth factor.26 Our unpublished observations indicated that, while the stroma stained intensely, the epithelial cells also contained bFGF. The hyperplastic regions of individual prostates often demonstrate wide variations between adjacent tissue specimens in IbFGFl. The increased [bFGF] in these regions or in BPH,
REGIONAL CONCENTRATION OF bFGF IN HUMAN PROSTATES when compared with normal adult prostate, may be related to a n increase in the proportion of stromal element^.^^^^^ To test t h e hypothesis that prostatic bFGF levels are a function of the stromal content of t h e tissue, quantitative morphometry was performed on selected prostate specimens. Individual sections demonstrating t h e highest and lowest [bFGF] were selected for analysis. Our findings did not support a relationship between [bFGFl and t h e proportion of stroma present in the prostatic tissue. This suggests that bFGF might be distributed uniformly in t h e stroma, epithelium and luminal compartments. However, it is unlikely that the growth factor is present i n the lumen of secretory ducts since it lacks a secretory signal ~ e q u e n c e . ~An ’ alternative explanation is related to the procedure used to evaluate tissue histology and quantitate bFGF. Those sections that were analyzed for bFGF and those used for quantitative morphometry were adjacent but not t h e same. Because of t h e heterogeneity of the prostate, morphometric evaluation may have been performed on tissue that differed histologically from that used for [bFGFl determination. Although in situ hybridization is not quantitative, t h e technique can be expected to provide information as t o the cellular compartments responsible for [bFGFl production in the prostate. These studies a r e in progress i n our laboratory. Acknowledgement. We are grateful to Dr. Andrew Baird for antiserum to bFGF. We t h a n k Mary Molter and Bonnie Livingston for technical assistance and Kathy Kropidlowski for manuscript preparation. REFERENCES
1. Holtgrewe, H. L., Mebust, W. K., Dowd, J. B., Cockett, A. T. K., Peters, P. C. and Proctor, C.: Transurethral prostatectomy: practice aspects of the dominant operation in American urology. J. Urol., 141: 248, 1989. 2. McNeal, J. E.: Origin and evolution of benign prostatic enlargement. Invest. Urol., 15:340, 1978. 3. McNeal, J . E.: Anatomy of the prostate and morphogenesis of BPH. In: New Approaches to the Study of Benign Prostatic Hyperplasia. Edited by F. A. Kimball, A. E. Buhl and D. B. Carter. New York: Alan R. Liss, Inc., pp 27-53, 1984. 4. Reischauer, F.: Die Entstehung der sogenannten prostatahypertrophie. Virchows Arch. Path. Anat., 256.357, 1925. 5. Moor, R. A.: Benign hypertrophy of the prostate: a morphological study. J. Urol., 5 0 680, 1943. 6. Deming, C. L. and Neumann, C.: Early phases of prostatic hyperplasia. Surg. Gynecol. Obstet., 68:155, 1939. 7. Ahluwalia, M. S. and Tandon, H. D.: Nodular hyperplasia of the prostate in North India: an autopsy study. J. Urol., 9 3 94, 1965. 8. Pradhan, B. K. and Chandra, K.: Morphogenesis of nodular hyperplasia-prostate. J. Urol., 113: 210, 1975. 9. Cunha, G. R.: Androgenic effects upon prostatic epithelium are mediated via trophic influences from stroma. In New Approaches to the Study of Benign Prostatic Hyperplasia. Edited bv F. A. Kimball. A. E. Buhl and B. D. Carter. New York Alan d. Liss, pp. 81-102, 1984. 10. Chung, L. W. K., Matsuura, J.. Rocco. A. K., Thompson, T. C., Miller, G. J . and Runner, M. C.: A new mouse model for prostatic hyperplasia: induction of adult prostatic overgrowth by fetal urogenital sinus implants. In New Approaches to the Study of Benign Prostatic Hyperplasia. Edited by F. A. Kimball, A. E. Buhl and D. B. Carter. New York: Alan R. Liss, pp. 291-306, 1984. 11. LeDuc, I. E.: The anatomy of the prostate and the pathology of early benign hypertrophy. J. Urol., 4 2 1217, 1939. 12. Story, M. T., Sasse, J., Jacobs, S. C. and Lawson, R. K.: Prostatic
a43
growth factor: purification and structural relationship to basic fibroblast growth factor. Biochemistry, 2 6 3843, 1987. 13. Story, M. T., Esch, F., Shimasaki, S., Sasse, J., Jacobs, S. C. and Lawson, R. K.: Amino-terminal sequence of a large form of basic fibroblast growth factor isolated from human benign prostatic hyperplastic tissue. Biochem. Biophys. Res. Commun., 142:702, 1987. 14. Lawson, R. K.: The natural history of benign prostatic hyperplasia. AUA Update Series 5(19), 1986. 15. Salacinski, P., Mclean, C., Sykes, J., Clement-Jones, V. and Lowry, P. J.: Iodination of proteins, glycoproteins and peptides using a solid-phaseoxidizing agent, 1,3,4,6-tetrachloro-3a,6adiphenyl glycoluril (Iodogen).Anal. Biochem., 117:136, 1981. 16. Baird, A. and Ling, N.: Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in vitro: implications for a role of heparinase-like enzymes in the neovascular response. Biochem. Biophys. Res. Commun., 142 428, 1987. 17. Florkiewicz,R. Z., Baird, A. and Gonzalez,A.-M.: Multiple forms of bFGF: differential nuclear and cell surface localization. Growth Factors, 4: 265, 1991. 18. Mannson, P.-E., Adams, P., Kan, M. and McKeehan, W. L.: Heparin-binding growth factor gene expression and receptor characteristics in normal rat prostate and two transplantable rat prostate tumors. Cancer Res., 4 9 2485, 1989. 19. Yan, G., Fukabori, Y., Nikolaropoulost, S., Wang, F. and McKeehan, W. L.: Heparin-binding keratinocyte growth factor is a candidate stromal to epithelial cell andromedin. Mol. Endocrinol., 6 2123, 1992. 20. Story, M. T., Livingston, B., Baeten, L., Swartz, S. J., Jacobs, S. J., Begun, F. P. and Lawson, R. K.: Cultured human prostate-derived fibroblasts produce a factor that stimulates their growth with properties indistinguishable from basic fibroblast growth factor. Prostate, 1 5 355, 1989. 21. Isaacs, J. T. and Coffey, D. S.: Etiology and disease process of benign prostatic hyperplasia. Prostate, suppl., 2 33, 1989. 22. Nishi, N., Matuo, Y., Kunitomi, K, Takenaka, I., Usami, M., Kotake, T. and Wada, F.: Comparative analysis of growth factors in normal and pathologic human prostates. Prostate, 1 3 39, 1988. 23. Mori, H., Maki, M., Oishi, R., Jaye, M., Igarashi, K, Yoshida, 0. and Hatanaka, M.: Increased expression of genes for basic fibroblast growth factor type 2 in human benign prostatic hyperplasia. Prostate, 16 71, 1990. 24. Gospodarowicz, D., Neufeld, G. and Schweigerer, L.: Fibroblast growth factor: structural and biological properties. J . Cell. Physiol. suppl., 5: 15, 1987. 25. Shapiro, E., Perlman, E. and Lepor, H.: Morphogenesis of the prostate. American Academy of Pediatrics, 60th Annual Meeting, p. 93, 1991. 26. Sherwood. E. R.. Fone. C.-Y.. Lee. C. and Kozlowski. J. M.: Basic fibroblast growth -factor:’ a potential mediator of stromal growth in the human prostate. Endocrinology, 130 2955, 1992. 27. Bartsch, G., Brungger, A., Schweikert, U., Hintner, H., Stanzl, U., Marth, C. L., Daxenbichler, G. and Rohr, H. P.: The importance of stromal tissue in benign prostatic hyperplasia: morphological, immunofluorescence, and endocrinological investigations. In New Approaches to the Study of Benign Prostatic Hyperplasia. Edited by F. A. Kimball, A. E. Buhl and D. B. Carter. New York: Alan R. Liss, pp. 159-179, 1984. 28. Shapiro, E., Becich, M. J., Hartanto, V. and Lepor, H.: The relative proportion of stromal and epithelial hyperplasia is related to the development of symptomatic benign prostate hyperplasia. J . Urol., 147: 1293, 1992. 29. Abraham, J. A., Whang, J., Tumolo, A., Mergia, A., Friedman, J., Gospodarowicz, D. and Fiddes, J.: Human basic fibroblast growth factor: nucleotide sequence and genomic organization. E.M.B.O.J., 5 2523, 1986.
THEJOURNAL
Vol. 153,839-843,March 1995 Printed in U.S.A.
REGIONAL CONCENTRATION OF BASIC FIBROBLAST GROWTH FACTOR IN NORMAL AND BENIGN HYPERPLASTIC HUMAN PROSTATES FRANK P. BEGUN,* MICHAEL T. STORY, KATHLEEN A. HOPP, ELLEN SHAPIRO AND
RUSSELL K. LAWSON From the Departments of Urology and Biochemistry, The Medical College of Wisconsin, and the Department of Veterans Affairs, Clement J . Zablocki Medical Center, Milwaukee, Wisconsin
ABSTRACT
Basic fibroblast grown factor (bFGF) is a potent mitogen for mesenchymal cells, including fibroblasts cultured from prostate, and has been postulated to play a role in t h e development of benign prostatic hyperplasia (BPH). If this is the case, it might be expected that bFGF levels would be elevated in the adenomas of BPH and in the periurethral region of the prostate where BPH is believed to arise. This study was undertaken to test this hypothesis. The concentration of bFGF was evaluated in 31 prostates, 13 normal glands and 18 with BPH. A method for quantitating bFGF by radioimmunoassay was developed that enabled growth factor levels to be correlated to t h e geographic region of the prostate and t h e histopathology of t h e specimen. A 2- to 3-fold higher concentration of bFGF (ng./g. of tissue) was noted in the benign hyperplastic prostates when compared with t h e adult normal glands. Pubertal specimens demonstrated low growth factor levels comparable to those observed in t h e normal adult group. Two prepubertal prostates analyzed had high levels similar to those measured in the hyperplastic glands. While t h e levels of bFGF i n the normal adult prostates were highest in the periurethral region, statistical analysis failed to demonstrate a s i g d i c a n t difference. Similarly, quantitative morphometric evaluation failed to demonstrate any significant differences in bFGF concentration related to t h e proportion of stromal, epithelial, or lumenal elements in the tissue sections. KEY WORDS: growth substances, prostatic hypertrophy Benign prostatic hyperplasia (BPH) is the most common nonmalignant neoplasm in men. This is evidencedby the fact that more than 350,000 surgical procedures are performed each year to treat this condition. The result is a total yearly expenditure in the United States that exceeds 1 billion dollars.' Although this disorder is an almost universal phenomenon, its pathogenesis is poorly understood. There is general agreement that BPH begins in the inner regions of the prostate. McNeal has localized the early changes of BPH to an area termed the transition zone, located adjacent to the urethra in the proximal region of the gland between the verumontanum and the bladder neck.' The first lesions appear in the third and fourth decades of life and precede the clinical symptoms by 10 to 30 year^.^*^ Benign prostatic hyperplasia is believed to begin with stromal alternations that stimulate the growth and differentiation of epithelial cells.4-" This suggests that stromal- epithelial interactions are critical in the development of BPH. Our laboratory has isolated a member of the heparin-binding growth factor family from the human p r ~ s t a t e . ' ~This .~~ growth factor, basic fibroblast growth factor (bFGF), has been postulated to be responsible for the stromal and/or epithelial hyperplasia that occurs in BPH.I4 If bFGF is a mediator of these interactions, it might be expected that levels of the growth factor would be elevated in glands demonstrating hyperplasia compared with normal prostates. It might also occur in greater concentrations in the periurethral regions of prostates with early hyperplastic changes as well
as in the adenomas of more advanced BPH. The current study was undertaken to test this hypothesis. METHODS
Tissue specimens. Whole prostates were obtained from adult men undergoing open prostatectomy for treatment of obstructive uropathy. Prostates were also available from from young men, adolescents and children who were cadaver renal transplant donors. Specimens were determined to be normal or BPH by histological examination by a pathologist. Appropriate consent was obtained prior to removal of the prostate gland in all instances. Sectioning method. The whole prostate gland was placed in a sterile container, packed in ice and transported to the laboratory. The gland was weighed and a 0.75- to 1-cm. thick slice was taken, midway between the bladder neck and the verumontanum, in the transaxial plane. The section was laid flat on a Petri dish and immediately frozen at -2OC. The remainder of the prostate was sent for histopathological analysis. The frozen specimen was further divided in half in the same transaxial plane. In this manner, 2 adjacent 0.4- to 0.5-cm. cross-sectionalslices were obtained. The freezing process prevented degradation of the bFGF by proteolytic enzymes and facilitated sectioning and handling of the tissue. Subsequent procedures were carried out at OC. The two adjacent slices were kept in their normal orientation and again laid flat in the Petri dish. Using a razor blade, horizontal and vertical cuts, 3-4 mm. apart, were made through the upper slice and carried part way down into the lower section. In this fashion, the upper slice was completely divided into a numAccepted for publication September 9, 1994. * Requests for reprints: Department of Urology, Froedtert Memo- ber of cubes that weighed 50 to 200 mg. The underlying rial Lutheran Hosuital. . ,9200 West Wisconsin Ave.. Milwaukee, Wis- specimen was marked with corresponding grooves for orienConsin 53226. This work was supported by P.H.S. grant DK31063.and by the tation. A map was made of the cut surface of the prostate, and each cube was assigned a number. The cubes were placed Department of Veterans AfFaus Medical Research Semce. 839
840
REGIONAL CONCENTRATION OF bFGF IN HUMAN PROSTATES
in a preweighed 50 ml. conical centrifuge tube and weighed. aspect of the gland) and capsule. The transition zone was not Five ml. of 50 mM. Tris, 1.55 M. NaC1, pH 7.6, containing 1readily identifiable as a distinct regional entity in many of EDTA (10 mM.), phenylmethanesulfonyl fluoride (1 mM.), the normal prostates. Therefore, sections through the tranGl-(tosylamide)-2-phenylethyl chloromethyl ketone (0.3 sition zone were included in either the glandular or periuremM.), N-ethylmaleimide (0.05 mM.), soybean trypsin inhib- thral region depending on their geographic location. Color-assisted quantitative image analysis. The stained tisitor (10 mg.ll.), leupeptin (1mg./l.) and pepstatin A (4 pM.) was added. All protease inhibitors were from Sigma (St. sue sections were viewed under a Wild-Leitz Microscope usLouis, Missouri). The specimens were stored at -2OC. The ing the 6.3 Planafluotar objective and a Variolum adapter lower tissue slice was fixed in formalin and embedded in under 250 x magnification (Ernst Leitz, Wetzlar, Germany). paraffin, and the whole mount section was stained with he- A high resolution JVC TK-870U color video camera (Victor matoxylin and eosin. Using this method, the geographic lo- Co., Tokyo, Japan) was used to capture the image of the cation of each specimen that was processed for bFGF levels histologic section that was displayed on a 12-inch color monitor. The image was simultaneously digitized using the AT could be ascertained. Tissue homogenization. The tissue was thawed and homog- Vista inside a Bell System 220 personal computer (Bell Comenized 3 times with a Polytron (Brinkmann Instruments, puter Co., Austin, Texas). The automated analysis was perInc., Westbury, New York) at a setting of 5 for 10 seconds Formed by the Bioquant Image Analysis System (BQ MEG with a 30-second cooling interval between homogenizations. N-Vista: R & M Biometrics, Nashville, Tennessee). The BQ The homogenate was centrifuged at 23,000 x g for 30 min- MEG IV-Vista is a color system image analysis that discrimutes using a Beckman Model 52-21 centrifuge (Beckman inates color differences of stained tissue sections. The threshInstruments, Inc., Palo Alto, California). The supenatant was olds were set to identify the various tissue components of the filtered (0.2 pm. Acrodisc membrane; Gelman Sciences, Inc., prostate. The area densities corresponding to each of these Ann Arbor, Michigan) and stored frozen at -2OC until deter- tissue components were calculated for each full screen of the color monitor. The mean percentage of stromal tissue ( S ) , mination of bFGF content by radioimmunoassay (RIA). Radioimmunoassay. Basic FGF was iodinated using the glandular epithelium (E) and glandular lumina (L) were deiodogen method described by Salacinski et al.15 The RIA was termined from the tissue sections obtained. Statistical analysis. The mean bFGF concentration [bFGF] performed using recombinant human bFGF (Amgen Biologicals, Thousand Oaks, California) as ligand and rabbit anti- was calculated for each whole prostate gland. When possible, serum raised against bovine serum albumin conjugated (1- the regional concentrations of bFGF were analyzed. Two 24) bFGF provided by Andrew Baird (Whittier Institute for normal adult prostates and 4 BPH prostates were randomly Diabetes and Endocrinology, San Diego, California). The chosen for quantitative image analysis. Three to 5 areas with epitope of the protein bFGF (1-146) recognized by the anti- the highest and a similar number with the lowest [bFGFl for serum is the sequence 11-15.16 The antiserum reacts with each gland were selected. The percentage of S, E and L for the the amino-terminal extended form of bFGF (1-154) isolated corresponding tissue sections was calculated. An analysis of from human p r 0 ~ t a t e . It l ~has not been reported whether the variance (ANOVA) of the S%, E% and L% by low versus high antiserum recognizes 24,23, and 22 kD forms of the protein [bFGF] for each patient was performed. In addition, the data that appear to be localized in the cell n u ~ l e u s . ' The ~ anti- from all patients were combined and rank ordered according serum does not cross-react with acidic FGF and keratinocyte to [bFGF]. An analysis of variance of S%, E% and L% by low growth factor (M.T.S., unpublished data), that are structur- versus high [bFGF] ranking was also carried out. Whole ally similar to bFGF and identified in pro~tate.'~.'' The RIA gland and regional mean values were compared for the varwas performed as previously described." The bFGF content ious groups by T-test. of the samples was derived from the bFGF standard curve using an Apple IIE and a curve fitting software program RESULTS (Interactive Microware Inc., State College, Pennsylvania). Evaluation of the bFGF content of normal and BPH prosData from the dose-response of prostate homogenates was tates. A total of 31 prostates, 13 normal and 18 BPH, was plotted on logit-log paper. Parallel dose response curves were analyzed for [bFGF]. The mean [bFGF], determined by RIA, indicative of equimolar competition of the ligand and sample was expressed as ng. bFGF/g. tissue. The mean [bFGFl valfor antibody. Basic FGF values were expressed as ng./g. ues for the normal and BPH prostates are summarized in tissue. The intra- and interassay coefficients of variation tables 1 and 2. The mean [bFGF] for the entire normal were 9.5% and 12.1%, respectively. prostate group was 100.1 2 12.8 compared with 156.1 2 7.3 Definition of prostatic anatomy. The various regions of the for the BPH group. This difference was significantly differprostate were defined so that meaningful statistical analysis of the data could be carried out. The convention used was similar to that set forth by M~Neal.'.~Specimens obtained TABLE1. Whole gland mean BFGFI for normal prostates from the transplant donors provided a complete cross-section of the gland. However, prostates obtained from patients unGland Number Age (yrs.) Weight ( m s . ) @FGFI* dergoing open prostatectomy would be expected to contain Prepubertal predominantly adenoma and periurethral elements. The pos143.7 2 7.8 (13) 771 0.3 2.4 776 4 2 234.2 t 22.5 (11) terior prostate and capsule were rarely included. Therefore, Pubertal the whole prostates were divided into the following regions: 9 83.4 f 3.9 (14) 707 14 periurethral (those sections containing or immediately con14 99.1 2 8.2 (20) 729 16 tiguous to the urethral mucosa, including the transition 9.7 84.3 2 6.3 (17) 912 17 zone), glandular (those sections in the mid-portion of the 749 18 20 114.1 2 5.2 (43) Adult prostate gland adjacent, lateral and posterior to the urethra, 867 21 15.2 79.7 2 3.6 (23) including the posterior prostate but not the capsule), anterior 772 23 22 103.3 f 7.4 (39) (glandular elements anterior to the urethra) and capsule. 703 23 40.5 80.9 f 16.7 (30) The open prostatectomy specimens were divided into the 866 23 ND 61.3 2 3.7 (29) 69 1 30 21.5 62.6 t 3.5 (33) following regions: periurethral (those sections containing or 913 42 15.9 90.2 5 9.6 (19) immediately contiguous to the urethral mucosa), glandular 713 43 22 64.6 ? 2.6 (24) (adenomatous elements adjacent, lateral and posterior to the * Mean tbFGF1 (ng./g.) f the standard error of the mean. The number Of periurethral r e ~ o n )anterior , (glandular elements anterior to soecimens analvzed is indicated in Darentheses. the urethra), posterior (true prostatic tissue at the posterior ' N D = Not determined.
841
REGIONAL CONCENTRATION O F bFGF I N HUMAN PROSTATES
-TABLE2.
Gland Number
Whole gland mean lbFGFl for BPH glands Aae
Weight ( e m s . )
bFGF Content as a Function of Age
[bFGFl*
36 ND 142.0 f 18 (37) 688 44 96.5t 6.3 (67) 21.5 762 77 53 152.6 t 4.2 (74) 733 58 ND 219.0t 12.4 (19) 674 88 169.3 2 6.7 (33) 60 885 96 61 167.9 2 3.7 (109) 754 62 151.1t 6.4 (50) ND 728 63 36.5 164.2 2 6.0 (41) 752 63 48 196.2t 5.8 (19) 785 65 29 205.4 t 9.7 (40) 774 89 145.22 8.5 (66) 66 702 148.1t 6.7 (75) 116 750 68 70 ND 125.0 ? 10.5 (34) 689 115.22 5.2 (52) 725 73 110 167.6 2 5.0 (72) 74 75 798 112.8t 4.6 (86) 75 858 70 156.2 2 6.3 (51) 77 886 285 732 79 85 176.1 t 4.6 (102) * Mean [bFGFI ( n d g . ) f the standard error of the mean. The number of specimens analyzed is indicated in parentheses ND = Not determined.
I
0
0
I*
20
0
I
BPH
40
80
60
Age (years) ent; however, the data were more informative when the normal prostate group was subdivided into prepubertal, pubertal and adult specimens. The mean [bFGF] value for the adult prostates was 77.5 2 3.4 compared with 189.0 ? 32.0 for the prepubertal and 95.2 ? 6.3 for the pubertal group (table 3). The mean [bFGFl values for the BPH prostates were significantly higher than those of the adult normal glands (p = .001). The mean [bFGF] of the 2 prepubertal specimens was similar to that of the BPH group, while the mean [bFGF] of the pubertal glands was comparable to that of the adult normal group and significantly lower than the BPH specimens (p = .002). Thus, the [bFGFl of the BPH prostates was twice that of the adult normal glands. These data are graphically illustrated in the figure which depicts the mean [bFGF] values plotted as a function of age and pathology. Regional LbFGF] analysis. The data from the analysis of tbFGF1 in various regions of the prostates are summarized in table 4. There were some specimens in both the adult normal prostate group and the BPH group that did not contain samples from all of the represented regions of the gland. This was particularly the case for the BPH group since the posterior and capsular regions are rarely excised. The mean [bFGF] for the glandular regions of the adult normal prostates were as follows: 80.8 f 7.9 (glandular), 92.1 ? 13.6 (anterior) and 60.8 (posterior). The periurethral region had a somewhat higher mean [bFGF] of 108.5 ? 13.7. However, this was not significantly different when compared with the glandular areas. The capsular [bFGFl of the adult normal glands was 59.8 2 10.6. This was lower than the periurethral and glandular regions but also not statistically significant. The mean RFGF] for the adenomatous portions of the BPH prostates was fairly uniform, with values of 148.2 ? 8.9 for the glandular, 148.1 2 21.4 for the anterior and 161.8 2 63.8 for the posterior regions. The periurethral areas had a mean concentration of 168.8 t 16.1 that was not significantly different from the surrounding glandular elements. However, the bFGF content of the capsular regions
bFGF concentration as function of age TABLE 4. Reeional IbFGFl in adult normal and BPH prostates heion
Adult Normal
108.5 -t 13.7(6) Periurethral 80.8 ‘t 7.9 (5) Glandular 92.1 f 13.6(6) Anterior 60.8(1) Posterior 59.8 2 10.6(2) Capsule * Mean bFGF1 (ngJg.) ‘t the standard error of the prostates analyzed is indicated in parentheses.
BPH 168.8 ‘t 16.1 (11) 148.2 2 8.9 (12) 148.1 2 21.4 (6) 161.8 2 63.8 (2) 72.6 2 5.5 (4) mean. The number of
was 72.6 2 5.5. The difference between this value and those of the periurethral and glandular regions was statistically significant (p = .003, p = .001, respectively). Quantitative image analysis. Data from the quantitative image analysis evaluation of 6 individual prostates are presented in table 5. The low and high [bFGF] ranges are indicated along with the corresponding mean %S, %E, %L for each gland. Five of the 6 prostates failed to demonstrate significant differences in the percentages of the various tissue components in the regions with the highest and lowest [bFGFl. Prostate 762 had a mean %S of 56.12 and 70.30 for the regions with the lowest and highest [bFGFl, respectively (F(1,9) = 9.85,p 5.01). Correspondingmean %E values were 28.5 and 14.2 for the same areas (F(1,9) = 18.55, p 5 .003). Samples from all 6 prostates were combined and rank ordered according to [bFGF]. The bottom and top 50%represented the ”low” and “high”bFGF groups, respectively. Analyses of variance were computed, using these groups, on the %E, %S and %L. The mean %S values of 65.35 for the low [bFGF] regions and 68.45 for the high bFGF1 regions did not differ significantly (F(1,47) = .74, p = .39). The mean %E of 17.71 and 16.00 for the low and high [bFGF] areas also failed to show statistical significance (F(1,47) = .56, p = .46). A similar analysis for %L resulted in mean values of 16.94 and 15.55 for the low and high [bFGF] regions (F(1,47) = .50, p = .48).
TABLE3. ComDarison of whole eland mean BFGFI for the different age groups ~~
~~~
Mean* [bFGFl
Nonadult Normal
Prepubertal
Pubertal
X 126.5 2 21.4 X X Nonadult Normal (6) X 189.0 2 32.0 X X Prepubertal (2) X X p = ,035 95.2t 6.3 Pubertal (4) NS p = .04 p = ,002 77.5 2 3.4 Adult Normal (7) p = ,002 NS NS 156.1 t 7.3 Adult BPH (18) * Mean fiFGFl (ngJg.) 2 the standard error of the mean. The number of prostates analyzed is indicated in parentheses. NS = Not significant.
Adult Normal
X X
X X p = ,001
REGIONAL CONCENTRATION OF bFGF IN HUMAN PROSTATES
842
TABLE5. Analvsis of variance o f Dement tissue components b y low us. high BFGFI for each prostate
-
~~
8 Stroma
Prostate Number
Mean 885 (BPH)
% Epithelum
8 Lumen
Growth Factor Range Low
=
40-93
(N = 3)
ANOVA
Mean
High = 201-212 (N = 3) Low=53-66 ( N = 5 )
69.5 56.12
732 (BPH)
High = 147335 (N = 5) Low = 88-98 (N = 5)
70.30 58.46
886 (BPH)
High = 247359 (N = 5) Low = 8&91 (N = 3)
61.76 62.93
867 (Normal Adult)
High = 204-281 (N = 5) Low=5459 ( N = 3 )
80.94 72.78
866 (Normal Adult)
High = 102-115 (N = 3) hw=20-31 (N=4)
68.43 64.80
High = 78-98
65.82
F(1,5) = .02
F(1,9) = 18.55**
F(1,9) = ,001 15.46 18.20
F(1.9) = 1.35
F(1,9) = .39 20.18 19.30
18.06 17.80 F(1.7) = 3.20
F(1,7) = 2.87 8.56 13.87
F(1,7) = 2.01 10.50 13.43
F(1,5) = .72
F(1,5) = .25 19.10 13.63
F(1,5) = .07 12.47 21.60
F(1,7) = .48
F(1,7) = .04 15.45
-
14.67 15.38
14.2 23.34 F(1,9) = ,204
ANOVA
13.47
15.80 28.5 F(1,9) = 9.85*
(N = 4)
Mean
F(1.5) = 1.25
F(1,5) = .29 762 (BPH)
ANOVA
9.83
76.7
F(1,7) = .56 18.70
* p 5 .01 ** p 5 ,003 DISCUSSION
We reasoned that if bFGF plays a pivotal role in the etiology of BPH it might be anticipated that higher levels of the growth factor would be present in the region of the prostate immediately adjacent to the urethra, where BPH is believed to arise.'s3 Elevated bFGF levels might also be expected in younger men who are undergoing early prostatic changes recognized as microscopic BPH.'l Similarly, elevated levels of bFGF might be anticipated in older men in regions of the prostate that are developing macroscopic BPH.'* A method for quantitating bFGF was developed that enabled growth factor levels to be correlated with the geographic region of the prostate and the histopathology of the specimen. This method overcame the shortcomings in quantitation of immunohistochemistry and in situ hybridization. However, because of the heterogeneity of prostate tissue and the need to use at least 50 mg. of tissue for bFGF quantitation, the possibility existed that the tissue used to determine [bFGF] could be different from the adjacent tissue used for histopathology evaluation. In addition, the procedure that was used to analyze bFGF does not distinguish between extracellular and intracellular multiple molecular weight forms of bFGF. It is believed that 24, 23 and 22 kD forms of the protein, initiated from CUG codons, are exclusively located in the cell nucleus, while the 18 kD form, initiated from the AUG codon, is preferentially exported to the cell surface.17 A number of investigators have subsequently identified bFGF in the nucleus, suggesting that the growth factor may act through intracellular pathways. The physiological significance of these findings to the etiology of BPH remains to be determined. Our studies indicated that BPH tissue had a 2- to 3-fold higher [bFGFJ than adult prostates without hyperplastic changes (table 3). These findings are in agreement with the bFGF-like biological activity, 4-fold higher in BPH than in normal prostate, reported by Nishi and associates and the BPH-associated increase in bFGF mRNA reported by Mori et al.23 The mean age of the 7 adult normal prostate donors in our study was 29 t 9 years versus 64 ? 11years for the 18 BPH specimens. The age of the donors of the 3 normal prostates analyzed in Mori's study was 49 ? 1years versus 71 ? 7 years for the 12 BPH specimens. The age of donors of normal prostate ranged from 37 to 44 years, but the age of BPH donors was not reported in Nishi's paper. In our study and the study by Mori, the age of donors of normal prostate was significantly lower than BPH donors. Therefore, it is conceivable that the increase in [bFGFl in the BPH group is age-related, rather than BPH-related. Further studies are needed to address this possibility.
''
The regional bFGF concentration of normal adult prostates was found to be less uniform than that of the hyperplastic glands (table 4). The periurethral regions had the highest mean [bFGF] values and the anterior and glandular regions had somewhat lower values, while the posterior and capsular regions had the lowest [bFGFl. The data, although not statistically significantly, were consistent with our hypothesis that elevated bFGF levels might be expected in the region of the prostate where BPH is known to arise.3 However, only 3 normal prostates from men 30 years or older were studied (table 1).It will be important to expand the data base of normal prostates from men in the third and fourth decades of life, when microscopic BPH is first observed," to support or refute our preliminary data. The adenomas of BPH begin periurethrally and grow outward, displacing and compressing the surrounding elements of the true prostate. Enucleation of the prostate during open surgery for BPH removes only the hyperplastic region of the gland. The data illustrate that bFGF levels in these glands were uniformly elevated over those of normal prostate in all regions except the capsule (table 4). This distribution of bFGF in BPH supports our contention that the growth factor plays an important role in the hyperplastic process. Adult normal prostates had [bFGFl that were similar to those concentrations found in the capsular regions of BPH glands. This may represent a "background" level of bFGF that is present in many tissues.'* A higher concentration of bFGF was found in 2 prepubertal specimens as compared with pubertal and normal adult prostates (table 1).The bFGF levels in the adolescent glands were low and statistically comparable to those of the normal adult group. These glands are undergoing growth as a result of androgen stimulation, and the bFGF levels might be expected to be elevated. Unfortunately, the small number of specimens available for evaluation precluded the formation of any conclusions. Interestingly, when analyzed histologically, prepubertal specimens contained a much higher proportion of stroma than did the normal adult prostate^.'^ It has been shown that fibroblasts cultured from human prostate synthesize bFGF.'" It is possible that the high levels of bFGF observed in the prepubertal prostates was a function of the stromal content of the gland. Immunohistochemical staining of the prostate for bFGF has demonstrated that the stroma is a rich source of the growth factor.26 Our unpublished observations indicated that, while the stroma stained intensely, the epithelial cells also contained bFGF. The hyperplastic regions of individual prostates often demonstrate wide variations between adjacent tissue specimens in IbFGFl. The increased [bFGF] in these regions or in BPH,
REGIONAL CONCENTRATION OF bFGF IN HUMAN PROSTATES when compared with normal adult prostate, may be related to a n increase in the proportion of stromal element^.^^^^^ To test t h e hypothesis that prostatic bFGF levels are a function of the stromal content of t h e tissue, quantitative morphometry was performed on selected prostate specimens. Individual sections demonstrating t h e highest and lowest [bFGF] were selected for analysis. Our findings did not support a relationship between [bFGFl and t h e proportion of stroma present in the prostatic tissue. This suggests that bFGF might be distributed uniformly in t h e stroma, epithelium and luminal compartments. However, it is unlikely that the growth factor is present i n the lumen of secretory ducts since it lacks a secretory signal ~ e q u e n c e . ~An ’ alternative explanation is related to the procedure used to evaluate tissue histology and quantitate bFGF. Those sections that were analyzed for bFGF and those used for quantitative morphometry were adjacent but not t h e same. Because of t h e heterogeneity of the prostate, morphometric evaluation may have been performed on tissue that differed histologically from that used for [bFGFl determination. Although in situ hybridization is not quantitative, t h e technique can be expected to provide information as t o the cellular compartments responsible for [bFGFl production in the prostate. These studies a r e in progress i n our laboratory. Acknowledgement. We are grateful to Dr. Andrew Baird for antiserum to bFGF. We t h a n k Mary Molter and Bonnie Livingston for technical assistance and Kathy Kropidlowski for manuscript preparation. REFERENCES
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