Objective
To compare smooth muscle regulatory protein expression in the uterosacral ligament (USL) of women with and without uterine prolapse.
Study Design
USLs ligament were sampled in women with (n = 9) or without (n = 9) uterine prolapse. Caldesmon, smooth muscle actin (SMA), myosin heavy chain, and zinc finger protein messenger RNA expression was assessed by quantitative real-time polymerase chain reaction. Immunohistochemistry and digital image analysis were used to determine protein expression.
Results
Caldesmon messenger RNA expression and the ratio of caldesmon-SMA messenger RNA expression was significantly increased in the USL from women with uterine prolapse compared with women without prolapse (caldesmon mean ± standard deviation messenger RNA, 0.81 ± 0.46 vs 0.39 ± 0.16; P = .01 and caldesmon-SMA messenger RNA ratio, mean ± standard deviation, 0.11 ± 0.04 vs 0.07 ± 0.02; P = .01). In addition, the ratio of caldesmon-SMA staining was significantly increased in women with uterine prolapse compared with women without prolapse (mean ± standard deviation, 0.44 ± 0.28 vs 0.28 ± 0.16; P = .03).
Conclusion
Uterine prolapse is associated with an increased ratio of caldesmon-SMA actin expression.
Pelvic organ prolapse (POP) is a prevalent disorder affecting up to 2.9% of nonpregnant women in the United States. The lifetime risk for requiring surgical intervention for prolapse or incontinence is 11%. The cause of POP is not clearly understood; however, several factors thought to play a role in the development of POP, including abnormal degradation/synthesis of collagen in the connective tissue, site-specific fascial defects, and denervation of the pelvic floor. Recently, Takacs et al and others have proposed that alterations in the smooth muscle content and function may contribute to development of POP.
The uterosacral ligaments (USL) are an important part of the pelvic support system and establish the level 1 support to the cervix and the upper vagina. In vitro studies have shown that the cervical portions of the USL support >17 kg of weight before failure. The USL contains a considerable amount of smooth muscle that is partially responsible for its strength. Almost one-third of the entire USL is comprised of smooth muscle that is surrounded by moderately dense collagenous connective tissue with numerous blood vessels. In POP, the smooth muscle content of the USLs is decreased and the rate of smooth muscle apoptosis is increased. This suggests a possible role of smooth muscle in the pathogenesis of POP. Additional support of the pelvic organs is attributed to the paravaginal attachments and the anterior vaginal wall. In POP, the rate of smooth cell apoptosis is increased, although the caldesmon expression and the smooth content is decreased.
Myosin phosphorylation and calcium-calmodulin binding are the 2 key regulatory factors in smooth muscle cell contraction. Appropriate myosin phosphorylation requires a fine tuning mechanism via thin filament regulation and a major part of this regulatory mechanism is the actin-binding protein, caldesmon heavy isoform (h-caldesmon). This isoform is expressed entirely in smooth muscle cells and inhibits actomyosin crossbridge cycling by inhibiting actin-activated myosin ATPase activity, essentially serving as an “on/off” switch for the thin filaments. 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. There is substantial in vitro and in vivo evidence that caldesmon-based regulation is involved in modifying smooth muscle calcium sensitivity and relaxation. Interestingly, morphologic changes that occur in the smooth muscle of the bladder in diabetic rabbits demonstrate an increase in h-caldesmon, although showing a decrease in force production and an increase in dedifferentiation with dysfunction. Recently, zinc finger proteins (1 of the most common DNA-binding proteins) were reported as important regulators of smooth muscle differentiation and function.
Based on these previous reports, we have hypothesized that USL caldesmon expression is decreased and other smooth muscle regulatory proteins are differentially expressed in POP. Diminished uterosacral ligament h-caldesmon in POP may increase the contractility of the USLs, compensating for the loss of smooth muscle cells commonly found in POP.
To test our hypothesis, we have studied complete cross-sections of USLs from women with or without uterine prolapse to determine the caldesmon, myosin heavy chain, and zinc finger protein expression.
Materials and Methods
Tissue samples of the USLs were obtained from women undergoing abdominal or vaginal hysterectomy for benign reasons at the University of Miami, Miller School of Medicine, Jackson Memorial Hospital, between Dec. 1, 2005, and Oct. 31, 2006. Women with endometriosis, immunologic and connective tissue diseases, recent use of vaginal hormones, and women with prior pessary use were excluded. Institutional review board approval was obtained before the start of the study and every patient signed an informed consent form before surgery, allowing the excision of tissue samples and their use for research purposes. For the morphometric, histologic measurements, 9 patients with and 9 patients without POP were enrolled. Age and parity matching was performed within the specimen collection timeframe. The site of tissue collection was standardized because the fraction of smooth muscle in the USL may vary throughout its length. Approximately 10-mm thick slices were obtained intraoperatively from the cervical portion of the USL between 2 surgical clamps using a scalpel, as previously described by Gabriel et al and Bai et al. 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 Pelvic Organ Prolapse Quantification system. Demographic and pertinent clinical information was recorded prospectively and stored in a dedicated database.
Tissue preparation
USL samples were fixed in Tissue-Tek Xpress Molecular Fixative, (Sakura Finetek Torrance, CA) and then processed by a recently described automated microwave-based rapid tissue-processing instrument (Tissue-Tek Xpress; Sakura Finetek).
Immunohistochemistry
Paraffin sections (4 μM) 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 antimouse immunoglobulin, and streptavidin-biotin-peroxidase complex (LSAB+/HRP kit; Dako, Carpinteria, CA). Diaminobenzidine was used as chromogen in the presence of hydrogen peroxide. Slides were then counterstained with hematoxylin and eosin. All reactions were carried out at room temperature (22°C). To identify the smooth muscle cells antismooth muscle actin antibody was used (monoclonal mouse, 1:250, 30 minutes incubation, clone 1A4, catalog 0851 Dako). Caldesmon expression was studied using a monoclonal mouse antibody, 1:100, 30 minutes incubation, (clone h-CD catalog M3557 Dako). An antigen retrieval step was used for caldesmon using a citrate buffer and a steamer for 30 minutes. 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; National Institutes of Health, Bethesda, MD). The selection technique of similar features on digitized immunohistochemical images has been described previously. The USL (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 h-caldesmon 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 relative to the total area of nonvascular muscularis. 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) and subsequent homogenization of 50-μm thick sections of paraffin blocks with a tissue tearor (Biospec Product Inc, Bartlesville, OK). From the homogenized tissue, RNA was extracted with chloroform, followed by isopropyl precipitation on ice. The RNA pellets were resuspended in 300 μL diethylpyrocarbonate (DEPC)-treated water. RNA concentrations were measured on ND-1000 Spectrophotometer V.3.2.1. (NanoDrop Technologies, Wilmington, DE).
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), zinc finger proteins (ZNF322, ZNF417, ZNF493, ZNF606, ZNF644), and control genes were obtained from Applied Biosystems (TaqMan Gene Expression Assay, Foster City, CA). The expression levels of the 2 endogenous control genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and peptidylprolyl isomerase A (PPIA, or cyclophilin A), were measured by real-time quantitative PCR. All probes were labeled with FAM dye and MGB. First-strand complementary DNA (cDNA) was synthesized using a High-Capacity cDNA Archive Kit according to the manufacturer’s instructions (Applied Biosystems) from 500 ng of total RNA. PCR was performed using 1 μL cDNA template in a 20 μL reaction volume, with TaqMan Universal PCR Master Mix, on the iCycler Thermal Cycler (Bio-Rad, Hercules, CA). PCR conditions had an initial AmpliTaq Gold DNA Polymerase activation at 95°C for 10 minutes, 40 cycles of denaturation at 95°C for 15 seconds, and annealing and extension at 60°C for 1 minute. 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 t test, if the distribution of samples was normal, or the Mann-Whitney U test, if the sample distribution was asymmetric. Differences were considered significant when P value was less than .05. All statistical calculations were performed using the SigmaStat software (SPSS Inc, Chicago, IL).
Results
Demographic characteristics of the women with and without uterine prolapse are described in Table 1 . There were no significant differences in age, parity, menopausal status, or hormone replacement therapy between the 2 groups ( Table 1 ). Caldesmon messenger RNA (mRNA) expression was significantly increased in the USL from women with uterine prolapse compared with women without prolapse (caldesmon mean ± standard deviation [SD] mRNA expression in relative units, 0.81 ± 0.46 vs 0.39 ± 0.16; P = .01; Table 2 ). In addition, the ratio of caldesmon-smooth muscle actin (SMA) mRNA expression was significantly increased in uterine prolapse compared with women without prolapse (caldesmon-SMA mRNA ratio, mean ± SD, 0.11 ± 0.04 vs 0.07 ± 0.02; P = .01; Table 2 ). The fractional area of nonvascular caldesmon staining in the USL of women with uterine prolapse was not significantly different compared with women without prolapse (mean ± SD, 0.12 ± 0.09 vs 0.08 ± 0.05; P = NS). However, the ratio of caldesmon-SMA staining was significantly increased in women with uterine prolapse compared with women without prolapse (mean ± SD, 0.44 ± 0.28 vs 0.28 ± 0.16; P = .03). There were no differences between the mRNA expressions of SMA, myosin heavy chain and zinc finger proteins ( Table 2 ).
