Int Urogynecol J (2009) 20:985–990 DOI 10.1007/s00192-009-0876-z

ORIGINAL ARTICLE

Caldesmon expression is decreased in women with anterior vaginal wall prolapse: a pilot study Peter Takacs & Marc Gualtieri & Mehdi Nassiri & Keith Candiotti & Alessia Fornoni & Carlos A. Medina

Received: 20 August 2008 / Accepted: 19 March 2009 / Published online: 3 April 2009 # The International Urogynecological Association 2009

Abstract Introduction and hypothesis The purpose of this study is to compare vaginal caldesmon expression in women with and without anterior vaginal wall prolapse. Methods Vaginal tissues were sampled in women with (n= 11) or without (n=11) vaginal wall prolapse. Caldesmon messenger RNA (mRNA) expression was assessed by quantitative real-time polymerase chain reaction. Immunohistochemistry and digital image analysis were used to determine caldesmon protein expression in the histologic sections. Results There were no significant differences in demographic data between the two groups. Caldesmon mRNA expression was significantly decreased in the vaginal tissue from women with anterior vaginal wall prolapse compared to women without prolapse [(caldesmon mean ± SD mRNA P. Takacs (*) : M. Gualtieri : C. A. Medina Department of Obstetrics and Gynecology (D-50), University of Miami, Miller School of Medicine, Jackson Memorial Hospital, P.O. Box 016960, Miami, FL 33010, USA e-mail: [email protected] M. Nassiri Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, USA K. Candiotti Department of Anesthesiology, University of Miami, Miller School of Medicine, Miami, USA A. Fornoni Diabetes Research Institute, University of Miami, Miller School of Medicine, Miami, USA

expression in relative units) 0.03±0.03 vs 0.17±0.17, P= 0.02]. The fractional area of nonvascular caldesmon staining in the vagina of women with anterior vaginal wall prolapse was significantly decreased compared to women without prolapse [mean ± SD (0.09±0.04 vs 0.16±0.09, P= 0.03)]. Conclusions Vaginal caldesmon expression is significantly decreased in women with anterior vaginal wall prolapse compared to normal subjects. Keywords Caldesmon . Prolapse . Smooth muscle cell . Vagina

Introduction Pelvic organ prolapse (POP) is a distressing problem for many women, leading to surgery for 6% of all women in the US [1]. The etiology of POP is not clearly understood, however, several factors thought to play a role in the development of POP include abnormal degradation/synthesis of collagen in the connective tissue, site-specific fascial defects, and denervation of the pelvic floor [2–4]. Recently, Boreham et al. and others have proposed that alterations in the smooth muscle content and function may contribute to the development of POP [5–9]. Several studies of the vagina have shown a discernible difference in the morphological features of smooth muscle cells in women with POP compared to women without [5– 9]. However, the cellular and molecular mechanism for this dysfunction is not completely understood. Myosin phosphorylation and calcium-calmodulin binding are the two key regulatory factors in smooth muscle cell contraction [10, 11]. Appropriate myosin phosphorylation requires a fine tuning mechanism via thin filament

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regulation. A major part of this regulatory mechanism is the actin binding protein caldesmon heavy isoform (hcaldesmon). This isoform is found almost entirely in smooth muscle cells and inhibits actomyosin cross-bridge cycling by inhibiting actin-activated myosin adenosine triphosphatase (ATPase) activity [12]. It is also well established that the activity of the thin filaments toward myosin is independently regulated by calcium because native thin filaments isolated from smooth muscles confer a calcium-dependent regulation on unregulated myosin from skeletal or smooth muscle [13]. There is substantial evidence that caldesmon-based regulation is involved in modifying smooth muscle calcium sensitivity and relaxation [13, 14]. Based on these previous reports, we hypothesized that vaginal caldesmon expression is decreased in POP. Diminished vaginal expression of caldesmon in the prolapsed vagina may increase the contractility of the vagina, compensating for the loss of smooth muscle cells commonly found in POP. To test our hypothesis, we have studied full thickness vaginal wall samples from women with or without anterior vaginal wall prolapse to determine the caldesmon RNA and protein expression.

Material and methods Tissue samples of the anterior vaginal wall were obtained from women undergoing abdominal or vaginal hysterectomy for benign reasons (cases with endometriosis were excluded) at the University of Miami, Miller School of Medicine, Jackson Memorial Hospital, between December 1, 2005 and October 31, 2006. Institutional review board approval was obtained prior to the start of the study, and every patient signed an informed consent form prior to surgery, allowing the excision of tissue samples and their use for research purposes. For the morphometric, histological measurements, 11 patients with and 11 patients without POP were enrolled. All women in the POP group had stage 2 or greater anterior vaginal wall relaxation. All patients with POP sampled had a central vaginal wall defect as demonstrated by preoperative examination. The site of tissue collection was standardized due to the fact that the fraction of smooth muscle in the vaginal muscularis may vary throughout the vagina. After removal of the uterus, full thickness samples of the anterior vaginal wall were obtained from the vaginal cuff at the anterior midline portion of the vaginal wall with Metzenbaum scissors. Removal of the sample was performed carefully to avoid any crush injury to the samples. All patients underwent an assessment of POP stages based on the international POP quantitative system [15]. Demographic and pertinent

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clinical information was recorded prospectively and stored in a dedicated database. Tissue preparation Anterior vaginal wall samples were fixed in Tissue-Tek® Xpress™ molecular fixative, (Sakura Finetek Torrance, CA, USA) and then processed by a recently described automated microwave-based rapid tissue-processing instrument (Tissue-Tek® Xpress™, Sakura Finetek, Torrance, CA, USA). Immunohistochemistry Four-micrometer paraffin sections were melted overnight at 37°C, cleaned in xylene, and hydrated in decreasing grades of ethanol. After blockage of endogenous peroxidase activity with a solution of hydrogen peroxide and methanol, slides were sequentially treated with the primary mouse antibody, biotinylated anti-mouse immunoglobulin, and streptavidin-biotin-peroxidase complex (LSAB™+/HRP kit, Dako, Carpinteria, CA, USA). Diaminobenzidine was used as chromogen in the presence of hydrogen peroxide. Slides were then counterstained with hematoxylin. All reactions were carried out at room temperature (22°C). To identify the smooth muscle cells, anti-smooth muscle actin antibody was used (monoclonal mouse, 1:250, 30 min incubation, clone 1A4, catalog #0851 Dako). Caldesmon expression was studied with a monoclonal mouse antibody, 1:100, 30 min incubation (clone h-CD catalog #M3557 Dako). An antigen retrieval step was used for caldesmon using citrate buffer and a steamer for 30 min. For a negative control, normal mouse serum was substituted for the antibody. Image analysis: determination of nonvascular smooth muscle fractional area Smooth muscle cells were identified by specific staining with antibodies to α-actin. Stained sections were analyzed with a Nikon Eclipse 80i microscope and an image analysis system (ImageJ, NIH, Bethesda, MD, USA). The selection technique of similar features on digitized immunohistochemical images has been described previously [6, 7, 16]. The vaginal muscularis (excluding vascular smooth muscle) was outlined manually on each cross-section, and α-actin staining was identified in each slide. Each adjacent slide was stained for h-caldesmon. Thereafter, the area of hcaldesmon and α-actin staining within the nonvascular muscularis was quantified by computer software (ImageJ). The fraction of smooth muscle in the area of interest was determined by computation of the area of α-actin staining that was relative to the total area of nonvascular muscularis.

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Table 1 Clinical characteristics of women with and without anterior vaginal wall prolapse Age (mean ± SD) Parity (median, range) Vaginal parity (median, range) Smoking (n,%) Menopause (n,%) Body mass index (mean ± SD) Abdominal hysterectomy (n,%) Vaginal hysterectomy (n,%) POP stage (median, range)

The proportion of muscularis immunoreactivity with smooth muscle α-actin was expressed as a fraction of the total muscularis area. The same method was applied to the h-caldesmon stained slides. The examiner was blinded to the clinical history. RNA extraction Total RNA extraction was performed by addition of Trizol reagent (GibcoBRL, Gaithersburg, MD, USA) and subsequent homogenization of 50-μm thick sections of paraffin blocks with a tissue tearor (Biospec Product Inc., Bartlesville, OK, USA). From the homogenized tissue, RNA was extracted with chloroform, followed by isopropyl precipitation on ice. The RNA pellets were resuspended in 300μl of diethylpyrocarbonate-treated water. RNA concentrations were measured on ND-1000 Spectrophotometer V.3.2.1. (NanoDrop Technologies, Wilmington, DE, USA). Real-time polymerase chain reaction Quantitative real-time polymerase chain reaction (PCR) using TaqMan chemistry was performed. Primers and probes for caldesmon (CALD1), smooth muscle myosin heavy chain 11 (MYH11), and control genes were obtained from Applied Biosystems (TaqMan gene expression assay, Foster City, CA, USA). The expression levels of the two endogenous control genes, glyceraldehyde-3-phosphate dehydrogenase and peptidylprolyl isomerase A (cyclophilin A), were measured by real-time quantitative PCR. All

POP group (n=11)

Non-POP group (n=11)

P value

52±6 1 (1–4) 1 (1–4) 0 7 (63) 28±3 5 (45) 6 (55) 3 (2–4)

48±5 1 (1–5) 1 (1–5) 0 5 (45) 29±5 7 (64) 4 (36) 0 (0)

NS NS NS NS NS NS NS NS <0.01

probes were labeled with FAM dye and MGB. First-strand complementary DNA (cDNA) was synthesized using highcapacity cDNA archive kit according to the manufacturer’s instructions (Applied Biosystems) from 500 ng total RNA. PCR was performed using 1 μl of cDNA template in a 20-μl reaction volume, with TaqMan universal PCR master mix, on the iCycler thermal cycler (Bio-Rad, Hercules, CA, USA). PCR conditions had an initial AmpliTaq gold DNA polymerase activation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s, and annealing and extension at 60°C for 1 min. The threshold cycle (Ct) of the target gene was then normalized to the geometric mean of the control genes or myosin. Statistical methods Continuous data were compared using Student’s t test, if the distribution of samples was normal, or the Mann– Whitney U test, if the sample distribution was asymmetrical. Differences were considered significant when P value was less than 0.05. All statistical calculations were performed using the SigmaStat software (SPSS Inc, Chicago, IL, USA).

Results Demographic characteristics of the women with and without anterior vaginal wall prolapse are described in Table 1. There were no significant differences in age, parity,

Table 2 Vaginal caldesmon expression in women with and without anterior vaginal wall prolapse

Caldesmon mRNA expression (mean ± SD, relative units) Myosin mRNA expression (mean ± SD, relative units) Caldesmon mRNA expression normalized to myosin (mean ± SD, relative units) Nonvascular caldesmon staining area (mean ± SD, percentage)

POP group (n=11)

Non-POP group (n=11)

P value

0.03±0.03 0.94±1.64 1.22±0.16 0.09±0.04

0.17±0.17 2.54±2.81 3.08±1.98 0.16±0.09

0.02 0.02 0.01 0.01

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Fig. 1 Immunohistochemical analysis of smooth muscle actin in the anterior vaginal wall (×100). Anterior vaginal wall biopsy from a patient without anterior vaginal wall prolapse was immunostained with antibody to actin

Fig. 3 Immunohistochemical analysis of caldesmon in the anterior vaginal wall (×100). Anterior vaginal wall biopsy from a patient without anterior vaginal wall prolapse was immunostained with antibody to caldesmon

menopausal status, or hormone replacement therapy between the two groups (Table 1). Additionally, the stage of the anterior vaginal wall prolapse was higher in the POP group compared to the non-POP group {median, range [3 (2–4) vs 0 (0)], P<0.01}. Caldesmon messenger RNA (mRNA) expression was significantly decreased in the vaginal tissue from women with anterior vaginal wall prolapse compared to women without prolapse [(caldesmon mean ± SD mRNA expression in relative units) 0.03±0.03 vs 0.17±0.17, P=0.02, Table 2]. Similarly, smooth muscle myosin heavy chain mRNA expression was significantly decreased in the vaginal tissue from women with anterior vaginal wall prolapse compared to women without prolapse [(myosin

mean ± SD mRNA expression in relative units) 0.94±1.64 vs 2.54±2.81, P=0.02, Table 2]. In addition, the expression level of caldesmon mRNA normalized to myosin was also significantly decreased in the women with anterior vaginal wall prolapsed compared to women without POP [(caldesmon mRNA expression normalized to myosin, mean ± SD, relative units) 1.22±0.16 vs 3.08±1.98, P= 0.01, Table 2]. Immunostaining revealed a well-defined, uniform distribution of smooth muscle cells in patients without POP (Fig. 1). In the POP patients, smooth muscle distribution was irregular and haphazard (Fig. 2). Quantitative morphometric analysis revealed that the ratio of the fractional area of nonvascular smooth muscle in the vaginal wall biopsies

Fig. 2 Immunohistochemical analysis of smooth muscle actin in the anterior vaginal wall (×100). Anterior vaginal wall biopsy from a patient with anterior vaginal wall prolapse was immunostained with antibody to actin

Fig. 4 Immunohistochemical analysis of caldesmon in the anterior vaginal wall (×100). Anterior vaginal wall biopsy from a patient with anterior vaginal wall prolapse was immunostained with antibody to caldesmon

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from women with prolapse was significantly decreased compared to women without prolapse [mean ± SD (0.22± 0.08 vs 0.33±0.15, P=0.04)]. In addition, the fractional area of nonvascular caldesmon staining in the vagina of women with anterior vaginal wall prolapse was significantly decreased compared to women without prolapse [mean ± SD (0.09±0.04 vs 0.16±0.09, P=0.03), Figs. 3 and 4].

Discussion Although the exact mechanism of POP is not completely understood, structural defects in the endopelvic fascia and muscles supporting the pelvic viscera are thought to play a major role in its development. The morphologic changes taking place in women with POP are well described [5–9]. The fractional area of smooth muscle cells has been shown to be significantly lower in patients with prolapse, independent of the stage of prolapse or the age of women [5–7]. Besides alterations in structural components, there are also changes in functional properties, facilitated by the smooth muscle regulatory proteins, like caldesmon [5]. The thin filament associate protein, caldesmon, inhibits contraction and actin-activated ATPase activity [12]. In vitro studies have suggested two possible mechanisms by which the inhibitory actions of caldesmon can be reversed: by the binding of calcium-calmodulin and phosphorylation [17]. Previous experiments with bladder hypertrophy have shown that phosphorylation, and therefore, a greater expression of caldesmon, results in inhibition of the actinmyosin interaction and inhibition of contraction [18]. In another study, Mannikarottu et al. demonstrated that bladder hypertrophy secondary to diabetes results in a fourfold increase in caldesmon, further elucidating the role of increased actin binding proteins in hypertrophy [19]. We have compared 22 women undergoing surgery for either POP repair or benign gynecological condition. It was found that anterior vaginal wall prolapse was associated with a significant decrease in the fraction of smooth muscle area. Additionally, we have also demonstrated a marked decrease in h-caldesmon in the smooth muscle of the anterior vaginal wall in POP patients. These findings suggest a decrease in the inhibitory effects of caldesmon as a possible adaptation the vagina undergoes to compensate for the decrease in smooth muscle. Our finding of a decrease in the fractional smooth muscle area in the muscularis layer is consistent with previous studies [6, 7]. Additionally, we have noted that not only is the total smooth muscle cell cross-sectional area decreased, but there are also irregular smooth muscle bundles seen under microscopy. Our study has demonstrated a significant decrease in h-caldesmon expression in women with POP. This data

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conflicts with a previous study by Boreham et al. demonstrating an increase in h-caldesmon in POP patients [5]. The different findings between the two studies may in part be explained by the difference in the collection techniques of the vaginal wall samples. Differences in collection techniques may result in variation of the fraction of smooth muscle in the vaginal muscularis. All of our specimens were collected at the time of hysterectomy from the anterior mid portion of the vagina in both groups of patients. Boreham et al., however, collected their specimens from women with prolapse at the time of vaginal or abdominal anterior repair or colpocleisis [5]. In addition, Boreham et al. normalized the tissue content of each protein to the smooth muscle content of the vagina and we have not, due to the fact that our aim was to determine the total caldesmon content rather than the relative expression. Furthermore, changes in caldesmon mRNA levels mirrored its protein expression. We theorize that the decrease in the inhibitory action of h-caldesmon on the myosin ATPase activity is a compensatory mechanism to attempt to increase contractile force during atrophy and apoptosis. Further studies investigating contractility and other regulatory proteins should provide valuable information concerning the atrophy well described in POP patients. Our study groups were similar with respect to menopausal status, hormone therapy use, and age. Limitations to this study include the relatively small sample size and the inability to distinguish whether the prolapse is a cause or effect of smooth muscle changes. More patients will need to be enrolled to further clarify the relationship between tissue remodeling during atrophy and the role of actin binding regulatory proteins, such as h-caldesmon on the vagina.

Conflicts of interest None.

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