Prostatic Fibrosis is Associated with Lower Urinary Tract Symptoms

Jinjin Ma, Mehrnaz Gharaee-Kermani, Lakshmi Kunju, John M. Hollingsworth,Jeremy Adler, Ellen M. Arruda and Jill A. Macoska*From the Departments of Mechanical Engineering (JM, EMA), Urology (MGK, JMH, JAM), Pathology (LK), Pediatrics and CommunicableDiseases (JA) and Biomedical Engineering (EMA) and Program in Macromolecular Science and Engineering (EMA), University of Michigan,Ann Arbor, Michigan

Purpose: Current therapies for male lower urinary tract symptoms secondary toprostate enlargement prevent hormonal effects on prostate growth and inhibitsmooth muscle contraction to ease bladder neck and urethral pressure. However,lower urinary tract symptoms can be refractory to these therapies, suggestingthat additional biological processes not addressed by them may also contribute tolower urinary tract symptoms. Aging associated fibrotic changes in tissue archi-tecture contribute to dysfunction in multiple organ systems. Thus, we testedwhether such changes potentially have a role in impaired urethral function andperhaps in male lower urinary tract symptoms.Materials and Methods: Periurethral tissues were obtained from a whole pros-tate ex vivo and from 28 consecutive men treated with radical prostatectomy.Lower urinary tract symptoms were assessed using the American UrologicalAssociation symptom index. Prostate tissues were subjected to mechanical test-ing to assess rigidity and stiffness. Fixed sections of these tissues were evaluatedfor collagen and elastin content, and glandularity to assess fibrosis. Statisticalanalysis included the Student t test and calculation of Pearson correlation coef-ficients to compare groups.Results: Periurethral prostate tissues demonstrated nonlinear viscoelastic me-chanical behavior. Tissue from men with lower urinary tract symptoms wassignificantly stiffer (p � 0.0016) with significantly higher collagen content (p � 0.0038)and lower glandularity than that from men without lower urinary tract symptoms(American Urological Association symptom index 8 or greater vs 7 or less).Conclusions: Findings show that extracellular matrix deposition and fibrosischaracterize the periurethral prostate tissue of some men with lower urinarytract symptoms. They point to fibrosis as a factor contributing to lower urinarytract symptom etiology.

Key Words: prostate, prostatic hyperplasia, fibrosis, collagen, lower urinary

Abbreviations

and Acronyms

AUASI � American UrologicalAssociation symptom index

LUTS � lower urinary tractsymptoms

Submitted for publication January 26, 2012.Study received institutional review board ap-

proval.Supported by Grant P20DK090770 (JAM).Supplementary material for this article can be

obtained at http://jurology.com.* Correspondence: Department of Urology,

University of Michigan, 6217 Cancer Center, 1500East Medical Center Dr., Ann Arbor, Michigan48109-0944 (telephone: 734-647-8121; FAX: 734-647-9271; e-mail: jcoska@umich.edu).

tract symptoms

LOWER urinary tract symptoms second-ary to prostate enlargement manifest asurinary frequency and urgency, de-creased force of stream, incomplete blad-der emptying, hesitancy, intermittencyand nocturia.1 If left untreated, LUTScan progress to bladder dysfunction, uri-

nary retention and renal impairment.1–5

0022-5347/12/1884-1375/0THE JOURNAL OF UROLOGY®

© 2012 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RES

In men medical treatment for LUTSincludes 5�-reductase inhibitors and/or�1-adrenergic receptor antagonists. Al-though these therapies ameliorate symp-toms, they can produce adverse effectsthat require termination of the thera-peutic regimen and LUTS can become

refractory to these approaches.1–5 Thus,

http://dx.doi.org/10.1016/j.juro.2012.06.007Vol. 188, 1375-1381, October 2012

EARCH, INC. Printed in U.S.A.www.jurology.com 1375

PROSTATIC FIBROSIS IS ASSOCIATED WITH LOWER URINARY TRACT SYMPTOMS1376

there is a need to explore other therapeutic targets toprovide LUTS relief with improved tolerability andduration.

A potential target is prostatic fibrosis, which toour knowledge has been unexplored. Fibrosis is anaberrant version of the normal wound healing pro-cess that is characterized by myofibroblast accumu-lation, collagen deposition, extracellular matrix re-modeling and tissue rigidization.6–8 Numerous studiesshow that aging and inflammation associated fibroticchanges in tissue architecture contribute to dysfunc-tion and disease in multiple organ systems, includingpancreatic dysfunction in type 2 diabetes,9,10 chronicobstructive pulmonary disease,11,12 cirrhotic nonalco-holic fatty acid liver disease13,14 and Crohn disease,which is part of the spectrum disorder termed inflam-matory bowel disease.15,16 If the prostate, like othersoft tissues, is susceptible to fibrotic changes associ-ated with inflammation and aging, such changes couldproduce stiffer tissue architecture and a consequentnegative impact on urethral function, promoting uri-nary obstructive symptoms.

To investigate prostatic fibrosis as a potentialcontributing factor to LUTS we established param-eters to measure prostatic tissue stiffness and deter-mined whether these measures correlated with his-topathology and collagen content indicating fibrosisin a series of patients with or without evidence ofLUTS. Results revealed that extracellular matrixdeposition characterizes the periurethral prostatetissue of some men with LUTS and point to fibrosisas a previously unrecognized factor contributing toLUTS etiology.

MATERIALS AND METHODS

Tissue Procurement and PreparationAn entire prostate was resected at autopsy within 24hours after death from a 66-year-old male who died ofchronic lymphocytic leukemia. Information on LUTS orprostate cancer was not available in the medical record forthis patient. Periurethral tissue was also procured at sur-gery from 28 consecutive men undergoing radical prosta-tectomy, of whom 21 had completed the AUASI within 30days before surgery. All prostate tissues, and annotatedclinical and pathology information were obtained withinstitutional review board approval. All tissues were re-ceived in 10% RPMI medium and processed immediatelyfor mechanical and histological studies.

Mechanical TestingThe postmortem prostate was processed within 48 hoursafter death. Nine tissue slices were taken parallel and 9were taken perpendicular to the urethra. Slices weretrimmed to a mean � SD of 12.3 � 1.7 � 3.5 � 0.4 � 3.3 �0.4 mm (fig. 1, A). Prostatectomy tissues were processed

within 24 hours of procurement and trimmed to a mean of

9.5 � 2.2 � 1.7 � 0.5 � 2.0 � 0.6 mm. All trimmed tissueswere subjected to uniaxial load-unload mechanical testingusing a constant strain rate of 0.01 per second to attain amaximum strain of 0.3 with a custom-built tensile tester(fig. 1, B). Synchronized force and image recordings werecompiled using LabVIEW™.17 The terminal slope of thenominal stress vs nominal strain response is the tan-gent modulus or passive stiffness of prostate tissue inkPa, expressed as force per unit area, with higher tan-gent modulus values corresponding to greater tissuestiffness.

Histological EvaluationThe 18 tissue slices from the postmortem prostate andtissues adjacent to those subjected to mechanical testingfrom the prostatectomy specimens were fixed in 10% phos-phate buffered formalin, embedded in paraffin, sectionedand stained with hematoxylin and eosin. We determinedthe percent of glandular epithelium and stroma, and theextent of inflammatory infiltrate, which was assessed asabsent, minimal, mild or severe. Elastin content was as-sessed by Verhoeff staining. Collagen content was as-sessed by Masson trichrome staining using a previouslydescribed methodology.18 Briefly, sections were digitallyimaged with a PathScan Enabler IV (Advanced imagingConcepts, Princeton, New Jersey) and color segmentedusing a subprogram in MATLAB (R2010a, MathWorks®)that separates and quantifies color elements from trichromeimages, permitting quantitation of blue stained areas cor-responding to extracellular collagen. The extracellular col-lagen I portion of the tissue section (numerator) was di-vided by the area of the tissue section (denominator) andmultiplied by 100 to provide the percent of collagen in thattissue section.

Statistical AnalysisThe Student t test with p �0.05 considered statisticallysignificant and Pearson correlation coefficients were cal-culated to compare groups.

RESULTS

Periurethral Prostate Tissues Demonstrate

Nonlinear Viscoelastic Mechanical Behavior

Nodular hyperplasia was evident upon opening thepostmortem prostate along the urethra (fig. 1, A).Stress-strain curves generated from uniaxial load-unload mechanical testing of the 9 tissue sectionsparallel and 9 perpendicular to the urethra weresimilar between sections and nodules, and also non-linear and time dependent (fig. 1, C). This suggestedthat, like other soft tissues, periurethral prostatetissues showed viscoelastic mechanical behav-ior.19–22 In the 9 tissue sections taken parallel vs the9 taken perpendicular to the urethra average tan-gent modulus was 450 � 77 vs 560 � 180 kPa,average stromal content was 78% � 12% vs 88% �12% and average collagen content was 59% � 9% vs66% � 10% (fig. 1, D). Thus, despite some localized

differences in tensile mechanics, and glandular and

ff elast

PROSTATIC FIBROSIS IS ASSOCIATED WITH LOWER URINARY TRACT SYMPTOMS 1377

collagen content these periurethral tissues werelargely homogeneous for these properties.

Histologically Homogenous Periurethral

Prostate Tissues are Mechanically Isotropic

Postmortem prostate tissues were relatively aglan-dular with a 5% to 35% glandular content and 65%to 95% stromal content (fig. 1, D). All tissue sectionsshowed minimal or mild inflammatory infiltrate andthe extent of inflammation evaluated for all sec-tions was similar. Masson trichrome and Verhoeffstaining revealed an average collagen content of49% to 67% and minimal elastin content, respec-tively (fig. 1, E). The average tangent modulus, stro-mal content and collagen content of the 9 tissuesections parallel or perpendicular to the urethrawere similar and not significantly different within orbetween groups (fig. 1, D). This suggested that theperiurethral tissues examined were largely homoge-neous for these properties and mechanically isotro-

Figure 1. Postmortem prostate tissue mechanical testing and hopened along urethra. Periurethral tissue showed some hyperplB, tensile tester had dual actuators driven by stepper motors andat actuator ends. Specimen was gripped by micro artery clampSamples were loaded to maximum strain and unloaded at concompiled. C, typical stress-strain uniaxial load-unload curve gendicular to urethra. Response curve was consistent with nonlineamodulus, and collagen and stromal content of 9 tissue secphotomicrographs show parallel inner tissue sections. Deep breduced from �50. Same section (ii). Verhoeff elastin stain, redcollagen was assessed at about 80% collagen (iii). Masson trichrnonspecific staining. Black areas indicate elastin fibers. Verhoe

pic.

Periurethral Tissue Collagen Content

and Mechanical Stiffness/Rigidity

are Positively Correlated with LUTS

After validating the approaches used to mechanicallytest postmortem prostate tissue we used these meth-ods to similarly test periurethral tissue from 28 pros-tatectomy specimens. One piece of tissue was availablefor testing from each of 10 patients and 2 or more wereavailable for each of the remaining 18. The tangentmodulus for these tissues was 9 to 2,390 kPa (me-dian 502). The median collagen content of thesetissues was 32%. The average tangent modulus oftissues with a collagen content of greater than 32%was significantly higher than that of tissues with acollagen content of less than 32%. This indicatedthat greater tissue stiffness directly correlated withhigher collagen content due to fibrosis associated ex-tracellular matrix deposition (r � 0.60, p � 0.0038,table 1 and fig. 2, A).

The median glandular content of these tissues was

ical assessment. A, prostate was trimmed of excess tissue andine sections were taken parallel and 9 perpendicular to urethra.ted on crossed roller slides with load cell on 1 actuator and gripser grips. Grips hung in trough to submerge specimen in saline.rue strain rate. Synchronized force and image recordings were

from mechanical testing of tissue sections parallel or perpen-time dependent or viscoelastic mechanical behavior. D, tangentwere each averaged and SD (error bars) was calculated. E,as indicate high collagen content (i). Masson trichrome stain,

from �50. Human cirrhotic liver serving as positive control fortain, reduced from �50. Human aorta (iv). Brown areas indicatein stain, reduced from �50.

istologasia. Nmounin largstant teratedr andtionslue areucedome s

40% (range 0% to 70%). The average tangent modulus

)

PROSTATIC FIBROSIS IS ASSOCIATED WITH LOWER URINARY TRACT SYMPTOMS1378

of tissues with less than 40% glandular contents washigher than that of tissues with greater than 40%glandular contents. Although these differences werenot statistically significant, they indicated a trendtoward a correlation of greater tissue stiffness withlower tissue glandular content (p � 0.080, fig. 2, B).

The average tangent modulus of tissues of the 12patients with an AUASI score of 7 or less was lessthan that of the 9 with a score of 8 or greater. This

Table 1. Tangent modulus

No. Pts Median/Cutoff (rang

Content: 28Collagen 32% (1.6–58.3Glandular 40% (0–70

AUASI 21 7 (0–31Transrectal ultrasound prostate vol 19 60, 40 cc (23.4–196Age at surgery 28 58 yrs (43–70

Figure 2. Mechanical testing and histological assessment of pecollagen content and tangent modulus of tissues from 28 pattangent modulus of tissues from 28 patients. Arrows indicate meD and E, representative photomicrographs show high glandularpatient 090611a. E, patient 061411. F and G, photomicrogra

associated with high mechanical stiffness. F, patient 090611b. G, patie

was a highly statistically significant difference, in-dicating that greater tissue stiffness directly corre-lated with moderate/severe LUTS in these patients(r � 0.82, p � 0.0016).

Figure 2, D and E shows examples of trichromestained tissues representative of periurethral pros-tate tissues with high glandular and low collagencontent associated with low mechanical stiffness.Figure 2, F and G show those with low glandular

Mean � SD Tangent Modulus (kPa)

p ValueMedian or Less Greater Than Median

477 � 340 1,211 � 794 0.00381,113 � 862 654 � 431 0.08

390 � 215 1,184 � 758 0.0016829 � 770, 910 � 890 930 � 713, 857 � 652 0.77, 0.88842 � 683 846 � 760 0.99

ral prostatectomy tissue obtained at prostatectomy. A, percentArrows indicate median. B, percent glandular epithelium and, AUASI and tangent modulus of tissues from 21 of 28 patients.

w collagen content associated with low mechanical stiffness. D,ow representative low glandular and high collagen content

e)

))))

riurethients.dian. Cand lophs sh

nt 092010. D to F, Masson trichrome stain, reduced from �50.

PROSTATIC FIBROSIS IS ASSOCIATED WITH LOWER URINARY TRACT SYMPTOMS 1379

and high collagen content associated with high me-chanical stiffness.

Mild/moderate histological inflammation was ob-served in 5 of 13 (38%) vs 9 of 15 patient tissues(60%) with a tangent modulus below vs above themedian of 502 kPa. These differences were not sta-tistically significant but they suggested a trend to-ward higher inflammation levels associated withgreater tissue stiffness. Elastin staining was evalu-able for periurethral tissues from 24 of the 28 pa-tients but it was largely negative with staining evi-dent in tissue from only 8 of 24 patients (33%).

AUASI scores, including 0 to 7—absent/mild, 8 to19—moderate and 20 to 35—severe, were availablefor 21 of the 28 study patients. Of the men 12, 8 and1 described symptoms that were absent/mild, mod-erate and severe, respectively. The average tangentmodulus of tissues from the 9 patients describing amoderate/severe AUASI score was significantly higherthan that of tissues from the 12 with a score in theabsent/mild range (p � 0.0016). This indicated thatgreater tissue stiffness directly correlated with mod-erate/severe LUTS (r � 0.82, table 1). Men withmoderate/severe symptoms scored significantlyworse in several categories, particularly intermit-tent stream (table 2).

Periurethral Tissue Stiffness was

Independent of Prostate Volume and Patient Age

Transrectal ultrasound measurements, available for19 of the 28 prostates subjected to mechanical testing,were used to calculate prostate volume. The averagetangent modulus for periurethral tissues from pros-

Table 2. AUASI score components

Pt No.IncompleteEmptying

FrequentUrination

InterSt

AUASI 7 or less:1 0 02 0 03 0 14 0 15 0 26 0 17 1 18 2 2

Totals 3 8AUASI 8 or greater:

1 0 42 0 03 3 14 2 25 1 26 5 47 3 38 0 5

Totals 14 21

p Value 0.069 0.026 0.003

tates smaller or larger than the median volume of 60 ccwas not statistically significant. Since median prostatevolume was skewed toward higher volume prostates,data were reevaluated using 40 cc as a cutoff. Theaverage tangent modulus for tissues from prostatessmaller or larger than 40 cc was again not statisticallysignificant (table 1). Together these results suggestthat periurethral tissue stiffness was independent ofprostate volume.

Median age at surgery of these 28 patients was 58years (range 43 to 73). The average tangent modulusfor periurethral tissues from men younger or olderthan 58 years was not statistically significant (table 1).Notably average age at surgery was 59 � 6 yearsand in 20 of the 28 men (70%) ages clustered be-tween 53 and 64 years.

DISCUSSION

We propose what is to our knowledge a previouslyunexplored mechanism that might promote LUTS inmen, namely prostate tissue stiffening consequentto fibrosis. Results demonstrate the feasibility ofmeasuring prostate tissue stiffness, the associationof high levels of tissue stiffness with collagen con-tent and fibrosis, and the further association of thesemeasures with LUTS. Such periurethral tissue fi-brosis could decrease urethral flexibility and compro-mise the ability of the prostatic urethra to expand toaccommodate urinary flow during micturition, whichcould manifest as obstructive symptoms.

A limitation of the current study was the inabilityto collect and examine transurethral resection of the

UrgencyWeak

Stream Straining Nocturia

0 0 0 01 0 1 11 0 0 10 1 0 10 1 0 10 2 0 10 4 0 00 1 0 2

2 9 1 7

2 0 0 20 5 3 20 2 0 22 2 1 11 5 3 11 3 0 12 5 1 35 4 0 3

13 26 8 15

mittentream

00011100

3

02224522

19

0.035 0.020 0.089 0.018

PROSTATIC FIBROSIS IS ASSOCIATED WITH LOWER URINARY TRACT SYMPTOMS1380

prostate tissues procured after treatment for benignprostatic hyperplasia and prostatic enlargement.This inability was due to the current standard ofcare at our institution, which is based on laser ab-lation of prostate tissue. Although it is effective, thismethod does not permit tissue collection for researchstudies. Another limitation is the lack of informationon LUTS in study patients other than preoperativeAUASI scores. Since most patients are referred toour institution for prostatectomy after the prostatecancer diagnosis, information on a history of LUTSis not available. Also, it is unclear whether periure-thral fibrosis reflects fibrosis only of the transitionalzone of the prostate or whether it may indicate morewidespread fibrosis including the peripheral zoneand lower urinary tract. It is also unclear whethersuch more extensive fibrosis would impact urethralfunction. Furthermore, in 20 of the 28 men (70%)treated with prostatectomy ages clustered between53 and 64 years. Thus, we could not adequately testthe role of aging in the development of fibrotic changes inprostate tissue architecture. Despite these limitations webelieve that our study clearly associates periurethral fi-brosis with LUTS and provides the rationale for furtherstudy and perhaps therapeutic targeting of the mecha-nisms contributing to prostatic fibrosis.

Although to our knowledge it is novel in the set-ting of LUTS, aging associated fibrotic changes intissue architecture contribute to dysfunction and dis-ease in multiple organ systems.6 Fibrosis can gener-ally be considered an errant wound healing process inresponse to chronic inflammation. Chronic inflamma-tion has been noted in the prostate in the context ofprostatitis and histological inflammatory infiltrate.6,8

Prostatitis is a common condition that accounts foralmost 2 million ambulatory care visits annually in theUnited States.23 Epidemiological data reveal an asso-ciation between chronic prostatic inflammation (ieprostatitis) and subsequent development of LUTS.24,25

Also, chronic inflammation has been noted in severalhistological studies of the prostate.25–29 Togetherthese studies suggest that, as in other organ sys-tems, inflammatory changes in the prostate conse-

quent to aging or infection may promote progressive

REFERENCES

of life in a racially and ethnically diverse random toms, overactive bladder,

fibrosis and changes in tissue architecture, contrib-uting to urinary obstructive symptoms.

Multiple cell types can differentiate or dedifferen-tiate into myofibroblasts (fig. 3, A).30 Many if not allof these cell types comprise the prostatic tissue mi-croenvironment, suggesting that dynamic changesin tissue composition may develop in the prostatedue to aging and inflammatory processes. Our datasuggest that fibrosis is one of these processes and itis associated with LUTS. Tissue fibrosis along withcellular proliferation/prostatic enlargement and smoothmuscle hypercontractility may act independently orin combination to promote male LUTS (fig. 3, B).Moreover, these 3 pathological processes likely over-lap, in that the cell types involved in these processescan accumulate and transdifferentiate. If so, ther-apy targeting these 3 pathological processes may beneeded to adequately alleviate the symptoms andpathobiology contributing to male LUTS.

CONCLUSIONS

To our knowledge we report the first study showingthat fibrotic changes affecting periurethral prostatictissue are associated with increased mechanicalstiffness and LUTS. Thus, some patients with LUTSmay benefit from antifibrotic treatment alone orcombined with the currently prescribed regimen of5�-reductase inhibitors and �1-adrenergic receptor

Figure 3. Prostate fibrotic development and contribution tomale LUTS. A, multiple cell types that can differentiate intomyofibroblastic cells. B, 3 major pathobiological processes thatcan act alone or in combination to promote male LUTS.

antagonists.22

1. Laborde EE and McVary KT: Medical manage-ment of lower urinary tract symptoms. Rev Urol,suppl., 2009; 11: S19.

2. Wei JT, Calhoun E and Jacobsen SJ: Urologicdiseases in America project: benign prostatichyperplasia. J Urol 2005; 173: 1256.

3. Kupelian V, Wei JT, O’Leary MP et al: Prevalence oflower urinary tract symptoms and effect on quality

sample: the Boston Area Community Health (BACH)Survey. Arch Intern Med 2006; 166: 2381.

4. Parsons JK, Bergstrom J, Silberstein J et al:Prevalence and characteristics of lower urinarytract symptoms in men aged � or � 80 years.Urology 2008; 72: 318.

5. Irwin DE, Kopp ZS, Agatep B et al: Worldwideprevalence estimates of lower urinary tract symp-

urinary incontinence

and bladder outlet obstruction. BJU Int 2011;108: 1132.

6. Pohlers D, Brenmoehl J, Loffler I et al: TGF-betaand fibrosis in different organs—molecular path-way imprints. Biochim Biophys Acta 2009; 1792:746.

7. Hinz B: Formation and function of the myofibro-blast during tissue repair. J Invest Dermatol

2007; 127: 526.

PROSTATIC FIBROSIS IS ASSOCIATED WITH LOWER URINARY TRACT SYMPTOMS 1381

8. Wynn TA: Cellular and molecular mechanisms offibrosis. J Pathol 2008; 214: 199.

9. Detlefsen S, Sipos B, Feyerabend B et al: Pan-creatic fibrosis associated with age and ductalpapillary hyperplasia. Virchows Arch 2005; 447:800.

10. Donath MY, Schumann DM, Faulenbach M et al:Islet inflammation in type 2 diabetes: from met-abolic stress to therapy. Diabetes Care, suppl.,2008; 31: S161.

11. Scotton CJ and Chambers RC: Molecular targetsin pulmonary fibrosis: the myofibroblast in focus.Chest 2007; 132: 1311.

12. Gharaee-Kermani M, Hu B, Phan SH et al: Recentadvances in molecular targets and treatment ofidiopathic pulmonary fibrosis: focus on TGFbetasignaling and the myofibroblast. Curr Med Chem2009; 16: 1400.

13. Frith J, Day CP, Henderson E et al: Non-alcoholicfatty liver disease in older people. Gerontology2009; 55: 607.

14. Novo E, di Bonzo LV, Cannito S et al: Hepaticmyofibroblasts: a heterogeneous population ofmultifunctional cells in liver fibrogenesis. IntJ Biochem Cell Biol 2009; 41: 2089.

15. Rieder F and Fiocchi C: Intestinal fibrosis inIBD—a dynamic, multifactorial process. Nat Rev

Gastroenterol Hepatol 2009; 6: 228.

16. Goldacre MJ: Demography of aging and the ep-idemiology of gastrointestinal disorders in theelderly. Best Pract Res Clin Gastroenterol 2009;23: 793.

17. Larkin LM, Calve S, Kostrominova TY et al: Struc-ture and functional evaluation of tendon-skeletalmuscle constructs engineered in vitro. Tissue Eng2006; 12: 3149.

18. Adler J, Swanson SD, Schmiedlin-Ren P et al:Magnetization transfer helps detect intestinal fi-brosis in an animal model of Crohn disease.Radiology 2011; 259: 127.

19. Clark RE: Stress-strain characteristics of freshand frozen human aortic and mitral leaflets andchordae tendineae. Implications for clinical use.J Thorac Cardiovasc Surg 1973; 66: 202.

20. Ghista DN and Rao AP: Mitral-valve mechanics—stress-strain characteristics of excised leaflets,analysis of its functional mechanics and its med-ical application. Med Biol Eng 1973; 11: 691.

21. Lanir Y and Fung YC: Two-dimensional mechani-cal properties of rabbit skin. I. Experimental re-sults. J Biomech 1974; 7: 29.

22. Lanir Y and Fung YC: Two-dimensional mechani-cal properties of rabbit skin. II. Experimental re-sults. J Biomech 1974; 7: 171.

23. Habermacher GM, Chason JT and Schaeffer AJ:Prostatitis/chronic pelvic pain syndrome. Annu

Rev Med 2006; 57: 195.

24. St Sauver JL, Jacobson DJ, McGree ME et al:Longitudinal association between prostatitis anddevelopment of benign prostatic hyperplasia.Urology 2008; 71: 475.

25. Krieger JN, Lee SW, Jeon J et al: Epidemiologyof prostatitis. Int J Antimicrob Agents, suppl.,2008; 31: S85.

26. Roehrborn CG, Kaplan SA, Noble WD et al: Theimpact of acute or chronic inflammation in base-line biopsy on the risk of clinical progression ofBPH: results from the MTOPS study. J Urol,suppl., 2005; 173: 346, abstract 1277.

27. Robert G, Descazeaud A, Nicolaiew N et al: Inflam-mation in benign prostatic hyperplasia: a 282 patients’immunohistochemical analysis. Prostate 2009; 69:1774.

28. Delongchamps NB, de la Roza G, Chandan V et al:Evaluation of prostatitis in autopsied prostates—ischronic inflammation more associated with benignprostatic hyperplasia or cancer? J Urol 2008; 179:1736.

29. Bierhoff E, Vogel J, Benz M et al: Stromal nod-ules in benign prostatic hyperplasia. Eur Urol1996; 29: 345.

30. Hinz B, Phan SH, Thannickal VJ et al: The myo-fibroblast: one function, multiple origins. Am J

Pathol 2007; 170: 1807.