Immunochemical and urodynamic outcomes after polypropylene mesh explant from the pelvic wall of rats.

INTRODUCTION AND HYPOTHESIS: To analyze the immunochemical and urodynamic outcomes after partial versus complete excision of transvaginal polypropylene mesh (PPM) from pelvic walls of rats. METHODS: Forty-eight female Sprague-Dawley (SD) rats were randomly distributed into seven groups: control, mesh total removal 60 days (M-T 60D), mesh total removal 180 days (M-T 180D), mesh partial removal 60 days (M-H 60D), mesh partial removal 180 days (M-H 180D), sham 60 days (Sham 60D), and sham 180 days (Sham 180D). In the mesh groups, PPM was inserted and partially (0.3 × 0.3 cm) or completely removed 30 days later. In the Sham group, the space between the vagina and bladder was dissected without placing or removing the synthetic mesh at day 1 and day 30 later. Urodynamic studies, immunochemical analysis, and Western blot were done at days 60 and 180. RESULTS: The M-T 60D voiding pressure was significantly decreased compared to the Sham 60D and M-H 60D. The voiding interval of M-T 60D was significantly shorter than that of M-H 60D. In the M-T 60D and M-T 180D groups, the leak point pressure was significantly less than in their corresponding sham groups. IL-1 and TNF-α were significantly more intense in M-T 60D compared to M-H 60D and Sham 60D. NGF was significantly greater in M-T 60D compared to Sham 60D. There were no significant differences in MMP-2 and CD-31s throughout the group. CONCLUSION: Total mesh excision incites a host inflammatory response and transitory lower urinary tract dysfunction. Despite the good outcomes after total excision, the invasiveness and surgical risk associated with repeated procedures should not be underestimateded.

Immunochemical analysis on polypropylene mesh: does mesh size make a difference?

INTRODUCTION AND HYPOTHESIS: The aim of the study is to demonstrate the impact of the size of implanted mesh in relation to its immunohistochemical reaction implanted into animal models. METHODS: An experimental study utilizing 54 female Sprague Dawley (SD) rats was divided into five groups: control, sham, and study groups (mesh-small [M-S], mesh-medium [M-M], mesh-large [M-L]). The M-S group used a mesh size of 0.2 × 0.2 cm, the M-M group a mesh size of 0.5 × 0.5 cm, and the M-L a mesh size of 0.7 × 1.0 cm. The sham group underwent vaginal dissection with no mesh implantation. The rats were sacrificed using isoflurane overdose on days 7 and 30. The mesh with the surrounding vaginal and bladder wall tissues were removed and processed for histochemical and western blot analysis. RESULTS: There is a significant increase in IL-1 and TNF-α immunoreactivity in the M-M and M-L groups on day 7 when compared with the sham group with p values of 0.001 and < 0.001 respectively. M-L showed significantly higher immunoreactivity to TNF-α persisting until day 30. All study groups presented a significantly higher immunoreactivity to MMP-2 and NGF on day 7. However, reactivity to NGF does not persist to day 30 in all groups. Immunoreactivity to CD 31 on days 7 and 30 appears significantly greater in the M-M and M-L groups, with the reaction in the M-L group continuing until day 30. CONCLUSION: Mesh size is directly proportional to the inflammatory reaction in the host tissue. The prolonged inflammatory process leads to delayed tissue remodeling and angiogenesis, which could delay mesh-tissue integration.

Dissecting of the paravesical space associated with lower urinary tract dysfunction - a rat model.

To determine the association of opening the paravesical space in relation to its occurrence of de novo SUI in an animal model. Thirty five female Sprague Dawley rats were divided into 5 groups of 7 rats each: Control group, Sham groups(F, H), and Study groups(MF, MH). Groups labeled with "F" had the paravesical space opened, "H" had tissue dissection with no opening of the space, and "M" had mesh implanted inside the vaginal wall. Urodynamic studies, immunohistochemical analysis, and western blot were done at day 40. The mean weight and age of 35 rats were 302.1 ± 25.1 grams and 12.8 ± 1.2 weeks old. No significant differences were noted among the control, Sham F, Sham H, Study MF, and Study MH on the voiding pressure and voided volume. The Sham F and Study MF (opened paravesical space) groups had significantly lower values on leak point pressures (LPP) (p = 0.026; p < 0.001) and shorter voiding intervals (p = 0.032; p = 0.005) when compared to other groups. Immunohistochemical analysis showed IL-1 and TNF-α to be intensely increased for the Study MF group (p = 0.003; p = <0.001). MMP-2 and CD 31 markers were also significantly higher in the Study MH and MF group. NGF expression was significantly increased in the Study MF and Sham F groups. Thus, opening of the paravesical space causes an increased inflammatory reaction, which leads to tissue destruction and lower urinary tract dysfunction, exemplified in the study with low leak point pressure and shortened voiding intervals.

Association of urodynamics and lower urogenital tract nerve growth factor after synthetic vaginal mesh implantation on a rat model.

AIM: By investigating the association of urodynamics and urogenital nerve growth factor (NGF) levels in vaginal mesh surgery, we may be able to associate the likelihood of postoperative lower urinary tract symptoms developing as a result of synthetic mesh implanted for pelvic floor reconstructive surgery. METHODS: Thirty-eight female Sprague-Dawley rats were divided into three groups: mesh, sham (no mesh), and control. Urodynamic study and NGF analysis of the urogenital tissues were done and results were compared among all groups. The urodynamic studies of the mesh and sham groups were further divided into the 4th and 10th days. A P-value < 0.05 was considered statistically significant. RESULTS: All rats survived and no complications were observed during the post-implantation period. Histological evaluation showed intense acute inflammatory reaction on days 4 and 7 in the mesh and sham groups when compared to the control. The mesh group showed a larger area of inflammation as compared to the sham. The NGF levels increased significantly in the mesh and sham groups on the 4th and 10th days when compared to the control (P < 0.001, P < 0.001, respectively). Both the mesh and sham groups had shorter voiding interval and lower voiding volume on days 4 and 10 when compared to the control group (P < 0.001, P < 0.001, respectively). The magnitude on increasing NGF level and decreasing voiding interval and voiding volume was significantly more on the mesh group than the sham group. CONCLUSION: A higher level of NGF in the early days post-transvaginal mesh implantation is associated with a shorter voiding interval and a smaller bladder capacity, which represents abnormal lower urinary tract symptoms following transvaginal mesh implantation.

The immunohistochemical and urodynamic evaluation towards the collagen-coated and non-coated polypropylene meshes implanted in the pelvic wall of the rats.

Our aim is to study the inflammatory response towards the collagen-coated and non-coated polypropylene meshes in rats and the urodynamic investigation post-operatively. Forty-two female Sprague Dawley were divided into 7 groups of 6 rats; Control, Day 7 and 30 for Sham, Avaulta Plus (MPC), Perigee (MP). UDS were taken at days 7 and 30. Mesh with the vagina and bladder wall was removed and sent for immunohistochemical examination. Results showed intense inflammatory reaction on day 7 in the study groups which decreased on day 30. IL-1, TNF-α, MMP-2 and CD31 were observed to decrease from day 7 to day 30. NGF was almost normal on day 30 in all groups. UDS showed no difference in voiding pressure. Both Study and Sham groups had shorter voiding interval (VI) on day 7 but significantly lower in MPC. VI had significantly increased on day 30 in all groups. Voided volume was significantly lower in the mesh groups even when an increase was seen on day 30. In conclusion, the higher levels of IL-1, TNF-α and MMP-2 in collagen-coated polypropylene mesh imply greater inflammation than the non-coated polypropylene mesh. Mesh implantation can lead to shorter voiding interval and smaller bladder capacity.

Correction: Krüppel-like Factor 4, a Tumor Suppressor in Hepatocellular Carcinoma Cells Reverts Epithelial Mesenchymal Transition by Suppressing Slug Expression.

[This corrects the article DOI: 10.1371/journal.pone.0043593.].

Effect of suburethral prolene mesh for suburethral function and histology in a stress urinary incontinence mouse model.

OBJECTIVES: To create a mouse model pertaining to mesh-elicited suburethral functional and histological changes after vaginal distention, and to examine the possible mechanism behind these complications. METHODS: We divided 48 virgin female C57BL/6 mice into four groups: vaginal distention alone, vaginal distention followed by prolene mesh implantation, vaginal distention followed by sham mesh implantation and untreated control. Each group was divided into two subgroups for conscious cystometrogram, leak-point pressure testing and harvesting of suburethral tissue 4 or 10 days after vaginal distention. The suburethral tissues underwent immunohistochemistry and western blot analysis of nerve growth factor, matrix metalloproteinase 2 and tissue inhibitor of metalloproteinase 2. Urodynamic results were compared among groups using one way ANOVA, with Tukey's multiple comparisons post-test for pair wise comparisons. RESULTS: Leak-point pressure in the vaginal distention and vaginal distention + sham mesh groups were significantly lower than in the control and vaginal distention + mesh groups at day 4. Leak-point pressure in the vaginal distention + mesh group were significantly higher than in the other three groups at both day 4 and 10. Immunohistochemistry and western blotting showed increased matrix metalloproteinase 2 and tissue inhibitor of metalloproteinase 2 levels in the vaginal distention + mesh group at day 4 and 10. Furthermore, nerve growth factor expression was increased in the same area and same group at 10 days. CONCLUSIONS: Increased suburethral tissue matrix metalloproteinase and nerve growth factor expression might be related to tissue remodeling after prolene mesh implantation for stress urinary incontinence.

Reciprocal regulation of microRNA-99a and insulin-like growth factor I receptor signaling in oral squamous cell carcinoma cells.

BACKGROUND: MicroRNAs (miRNAs), small noncoding RNA molecules can function as oncogenes or tumor suppressors in tumorigenesis. Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers worldwide with a 5-year survival rate of approximately 50%. METHODS: The expression of microRNA-99a (miR-99a) in OSCC tissues and cell lines was investigated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The functions of miR-99a in migration/invasion and lung colonization were determined by transwell and tail vein injection assays, respectively. Specific targets of miR-99a were determined by software prediction, correlation with target protein expression, and luciferase reporter assay. The signaling pathways involved in regulation of miR-99a were investigated using the kinase inhibitors. RESULTS: We observed reduced levels of miR-99a, identified as one of the most downregulated miRNA in OSCC and all tested OSCC cell lines compared to normal oral keratinocytes. Ectopic miR-99a expression in OSCC cells markedly reduced migration and invasion in vitro as well as lung colonization in vivo. When evaluating the specific targets of miR-99a, we found that ectopic miR-99a expression downregulates insulin-like growth factor 1 receptor (IGF1R) protein and that the expression of miR-99a correlates negatively with IGF1R protein in OSCC cells. Insertion of the 3'UTR of IGF1R mRNA into the 3'UTR of a reporter gene markedly reduced luciferase activity in OSCC cells expressing miR-99a, suggesting that miR-99a reduces luciferase activity by targeting the 3'UTR of IGF1R mRNA. When evaluating the mechanisms of miR-99a downregulation, we observed the upregulation of miR-99a expression in serum-starved conditions and its suppression in response to insulin-like growth factor (IGF1) stimulation. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) kinase inhibited IGF1-induced suppression of miR-99a, suggesting the negative regulation of miR-99a expression by IGF1R signaling. CONCLUSION: Overall, results indicate that miR-99a functions as a tumor metastasis suppressor in OSCC cells and mutually regulates IGF1R expression in a reciprocal regulation.

Differentiated epithelial- and mesenchymal-like phenotypes in subcutaneous mouse xenografts using diffusion weighted-magnetic resonance imaging.

Epithelial-mesenchymal transition (EMT) is important for tumor metastasis. Detection of EMT protein expression and observation of morphological changes are commonly used to identify EMT. Diffusion-weighted magnetic resonance imaging (DW-MRI) and measuring apparent diffusion coefficient (ADC) values are noninvasive techniques for characterizing tumor microenvironments. We investigated the difference in ADC values between epithelial- and mesenchymal-like subcutaneous mouse xenografted tumors using DW-MRI. Epithelial-like MM189 PB-Klf4 and BL322 PB-Klf4 cells were generated from tumor suppressive Kruppel-like factor 4 (Klf4)-expressing mesenchymal-like MM189 and BL322 cells. The ADC values of xenografted tumors from epithelial-like MM189 PB-Klf4 and BL322 PB-Klf4 were significantly lower than those from their mesenchymal-like counterparts (p < 0.05 and p < 0.01, respectively). Our results suggested that DW-MRI is a potential tool for observing mesenchymal- or epithelial-like characteristics of subcutaneous xenografted tumors.

p16 Stimulates CDC42-dependent migration of hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. Tumor dissemination to the extra-hepatic region of the portal vein, lymph nodes, lungs or bones contributes to the high mortality seen in HCC; yet, the molecular mechanisms responsible for HCC metastasis remain unclear. Prior studies have suggested a potential link between accumulated cytoplasm-localized p16 and tumor progression. Here we report that p16 enhances metastasis-associated phenotypes in HCC cells - ectopic p16 expression increased cell migration in vitro, and lung colonization after intravenous injection, whereas knockdown of endogenous p16 reduced cell migration. Interestingly, analysis of p16 mutants indicated that the Cdk4 interaction domain is required for stimulation of HCC cell migration; however, knockdown of Cdk4 and Cdk6 showed that these proteins are dispensable for this phenomenon. Intriguingly, we found that in p16-positive HCC samples, p16 protein is predominantly localized in the cytoplasm. In addition, we identified a potential role for nuclear-cytoplasmic shuttling in p16-stimulated migration, consistent with the predominantly cytoplasmic localization of p16 in IHC-positive HCC samples. Finally, we determined that p16-stimulated cell migration requires the Cdc42 GTPase. Our results demonstrate for the first time a pro-migratory role for p16, and suggest a potential mechanism for the observed association between cytoplasmic p16 and tumor progression in diverse tumor types.

Krüppel-like factor 4, a tumor suppressor in hepatocellular carcinoma cells reverts epithelial mesenchymal transition by suppressing slug expression.

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor that plays an important role in differentiation and pathogenesis. KLF4 has been suggested to act as an oncogene or tumor suppressor in different tumor types. However, the role of KLF4 in hepatocellular carcinoma (HCC) remains unclear. Here, we demonstrate that forced expression of Klf4 in murine HCC cell lines reduced anchorage-independent growth in soft agar as well as cell migration and invasion activities in vitro. Ectopic Klf4 expression impaired subcutaneous tumor growth and lung colonization in vivo. By contrast, Klf4 knockdown enhanced HCC cell migration. Interestingly, ectopic expression of Klf4 changed the morphology of murine HCC cells to a more epithelial phenotype. Associated with this, we found that expression of Slug, a critical epithelial mesenchymal transition (EMT)-related transcription factor, was significantly down-regulated in Klf4-expressing cells. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays showed that Klf4 is able to bind and repress the activity of the Slug promoter. Furthermore, ectopic Slug expression partially reverts the Klf4-mediated phenotypes. Consistent with a role as a tumor suppressor in HCC, analysis of the public microarray databases from Oncomine revealed reduced KLF4 expression in human HCC tissues in comparison with normal liver tissues in 3 out of 4 data sets. By quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we found reduced KLF4 mRNA in 50% of HCC tissues. Importantly, an inverse correlation between the expression of KLF4 and SLUG was found in HCC tissues. Our data suggest that KLF4 acts as a tumor suppressor in HCC cells, in part by suppressing SLUG transcription.