Expression of matrix metalloproteinase-1 in round ligament and uterosacral ligament tissue from women with pelvic organ prolapse

To evaluate the matrix metalloproteinase-1 (MMP-1) expression in different parts of pelvic connective tissue in postmenopausal women with and without pelvic organ prolapse (POP). Ninety-one samples were obtained from only postmenopausal women (42 with POP and 49 non-POP subjects). All women were evaluated by pelvic organ prolapse quantitation. The POP group had stage 2 or more, and the controls had stage 1 or less. Round ligament (RL) and uterosacral ligament (USL) biopsies were obtained from women with POP and controls. Immunohistochemistry for MMP-1 was performed on formalin-fixed and paraffin-embedded sections. The two groups were matched for age, body mass index, parity and postmenopausal status. MedCalc Statistical Software Programme Version 12.0.5 was used for statistical analysis. Expression of MMP-1 were significantly higher in both RL and USL tissue from postmenopausal women with POP, compared with controls. MMP-1 immunoreactivities were identified in both RL and USL biopsies from all women with and without POP. The expression pattern of MMP-1 were similar in these ligaments and were significantly higher in POP group compared with control subjects. These changes indicate a possible relation between MMP-1 expression of RL and USL in women with and without POP.     

Expression of matrix metalloproteinase-1 in uterosacral ligaments tissue of women with genital prolapse

Collagen metabolism is altered in the pelvic organ tissues of women with genital prolapse. The aim of this study was to compare collagen metabolism by measuring matrix metalloproteinase-1 (MMP-1) expression in uterosacral ligament tissues of postmenopausal women with and without genital prolapse. Uterosacral ligament tissues were obtained at the time of abdominal or vaginal surgery from twenty-four patients with pelvic organ prolapse (POP) and 21 women who underwent gynecologic surgery for benign indications. The tissue samples were analyzed by immunohistochemistry. There were no differences in age, BMI and parity between two groups. The patients with genital prolapse demonstrated significantly higher occurences of MMP-1 expression compared to controls. These findings indicate that increased MMP-1 expression in uterosacral ligaments is associated with genital prolapse. Our data are consistent with the theory that increased collagen breakdown may play an important role in the onset and development of pelvic organ prolapse (POP).     

Protective effects of the nuclear factor kappa B inhibitor pyrrolidine dithiocarbamate in bladder ischemia–reperfusion injury in rats

The aim of the present study was to evaluate the protective effects of the NF-кB inhibition with pyrrolidine-dithiocarbamate (PDTC) in ischemia–reperfusion (I/R) injury in the rat bladder. Twenty-four Sprague-Dawley male rats were divided into three groups. Group I; (n = 8) control, group II; (n = 8) I/R group; group III (n = 8) I/R and PDTC treatment. Superoxide dismutase (SOD), catalase (CAT), and gluatathione-S-transferase (GST) enzymes was studied in bladder tissue. Lipid peroxidation (as TBARS) levels in tissue homogenate were measured with thiobarbituric acid reaction. All the slides were stained with NF-кB, p53 and HSP60 immunohistochemistry for detection genome destruction and tissue stress, respectively. Our results show that the mean TBARS levels were significantly higher in group II (p < 0.05). The TBARS levels were significantly decreased in group III compared with the group II (p < 0.05). CAT, SOD and GST activities were decreased in group II, but these enzymes levels were significantly increased in group III according to the group II (p < 0.05). Under microscopic evaluation NF-кB expression increased significantly in group II compared to the group I (p < 0.05) and then decreased in group III (p < 0.05). HSP60 and p53 expression in group II was increased significantly compared with group I. Under microscopic evaluation we detected that HSP60 and p53 expression was increased significantly in group II compared with group I. In group III PDTC administration was decreased the HSP60 and p53 expression, this difference was statistically significant (p < 0.05). The results of the present study have demonstrated that NF-кB inhibition with PDTC protects and provides beneficial effects on ischemia/reperfusion stress related bladder tissue destruction.     

Indirect co-culture with tenocytes promotes proliferation and mRNA expression of tendon/ligament related genes in rat bone marrow mesenchymal stem cells

Recent evidences have suggested that humoral factors released from the appropriate co-cultured cells influenced the expansion and differentiation of mesenchymal stem cells (MSCs). However, little is known about the proliferation and differentiation of MSCs subjected to co-culture condition with tenocytes. In this study, we aimed to establish a co-culture system of MSCs and tenocytes and investigate the proliferation and tendon/ligament related gene expression of MSCs. MTT assay was used to detect the expansion of MSCs. Semi-quantitative RT-PCR was performed to investigate the expression of proliferation associated c-fos gene and tendon/ligament related genes, including type I collagen (Col I), type III collagen (Col III), tenascin C and scleraxis. Significant increase in MSCs expansion was observed after 3 days of co-culture with tenocytes. The c-fos gene expression was found distinctly higher than for control group on day 4 and day 7 of co-culture. The mRNA expression of four tendon/ligament related genes was significantly up-regulated after 14 days of co-culture with tenocytes. Thus, our research indicates that indirect co-culture with tenocytes promotes the proliferation and mRNA expression of tendon/ligament related genes in MSCs, which suggests a directed differentiation of MSCs into tendon/ligament.     

Changes in the distribution of fibrillar collagens in the collateral and cruciate ligaments of the rabbit knee joint during fetal and postnatal development

Summary The collateral ligaments can be clearly distinguished in the 25-day fetal rabbit knee joint. Types I and V collagens are present in the extracellular matrix between the cells of the lateral and medial collateral ligaments and this distribution persists until the rabbit is skeletally mature. From 8 months onwards type III collagen is also present, particularly around the cells. Type I collagen mRNA is expressed by the cells from the 25-day fetal to 8-month-old adult ligament. The ligament sheath is composed of types III and V collagens. The cruciate ligaments are present between the femur and tibia in the 20-day fetus. The matrix is composed of types I and V collagens from the 25-day fetus until at 12- to 14-weeks postnatal, type III collagen appears in the pericellular regions together with type V. At 8 months and 2 years, the amount of type III collagen has increased. All the cells express the mRNA for type I collagen at 12- to 14-weeks, but only isolated cells express this mRNA at 8 months. Thus, both the collateral and cruciate ligaments undergo changes in their complement of collagens during postnatal development and ageing. The implications of these complex interactions of different types of collagen are discussed in relation to healing and the surgical replacement of torn ligaments by tendons.     

A Quantitative Study of the Relationship between the Distribution of Different Types of Collagen and the Mechanical Behavior of Rabbit Medial Collateral Ligaments

The mechanical properties of ligaments are key contributors to the stability and function of musculoskeletal joints. Ligaments are generally composed of ground substance, collagen (mainly type I and III collagen), and minimal elastin fibers. However, no consensus has been reached about whether the distribution of different types of collagen correlates with the mechanical behaviors of ligaments. The main objective of this study was to determine whether the collagen type distribution is correlated with the mechanical properties of ligaments. Using axial tensile tests and picrosirius red staining-polarization observations, the mechanical behaviors and the ratios of the various types of collagen were investigated for twenty-four rabbit medial collateral ligaments from twenty-four rabbits of different ages, respectively. One-way analysis of variance was used in the comparison of the Young''s modulus in the linear region of the stress-strain curves and the ratios of type I and III collagen for the specimens (the mid-substance specimens of the ligaments) with different ages. A multiple linear regression was performed using the collagen contents (the ratios of type I and III collagen) and the Young''s modulus of the specimens. During the maturation of the ligaments, the type I collagen content increased, and the type III collagen content decreased. A significant and strong correlation () was identified by multiple linear regression between the collagen contents (i.e., the ratios of type I and type III collagen) and the mechanical properties of the specimens. The collagen content of ligaments might provide a new perspective for evaluating the linear modulus of global stress-strain curves for ligaments and open a new door for studying the mechanical behaviors and functions of connective tissues.     

Changes in Muscularis Propria of Anterior Vaginal Wall in Women with Pelvic Organ Prolapse

The objective of this study was to evaluate the morphological and immunohistochemical alterations of tissue removed from the upper third of anterior vaginal wall in a sample group of the female population presenting homogenous risk factors associated with pelvic organ prolapse (POP). The case study consisted of 14 patients with POP and there were 10 patients in the control group. Patient selection was carried on the basis of specific criteria and all of the patients involved in the study presented one or more of the recognized POP risk factors. Samples were taken from POP patients during vaginal plastic surgery following colpohysterectomy, and from control patients during closure of the posterior fornix following hysterectomy. Samples were processed for histological and immunohistochemical analyses for Collagen I and Collagen III, α-Smooth Muscle Actin (α-SMA), Platelet-Derived-Growth-Factor (PDGF), matrix metalloproteinase 3 (MMP3), tissue inhibitors metalloproteinase 1 (TIMP1), Caspase3. Immunofluorescence analyses for Collagen I and III and PDGF were also carried out. In prolapsed specimens our results show a disorganization of smooth muscle cells that appeared to have been displaced by an increased collagen III deposition resulting in rearrangement of the muscularis propria architecture. These findings suggest that the increase in the expression of collagen fibers in muscularis could probably be due to a phenotypic switch resulting in the dedifferentiation of smooth muscle cells into myofibroblasts. These alterations could be responsible for the compromising of the dynamic functionality of the pelvic floor.Key words: Pelvic organ prolapse, immunohistochemistry, smooth muscle cells, collagen, PDGF     

Genetic and environmental influence on lung function impairment in Swedish twins

Background

The purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGF-beta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal tolerance.

Methods

Female New Zealand rabbits (N = 12) were immunized with 1 mg/ml of collagen V in Freund's adjuvant (IM). After 150 days, six immunized animals were tolerated by nasal administration of collagen V (25 μg/day) (IM-TOL) daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for multiple comparison when appropriate and the level of significance was determined to be p < 0.05.

Results

IM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels (0.371 ± 0.118 vs. 0.874 ± 0.282, p < 0.001), bronchioles (0.294 ± 0.139 vs. 0.646 ± 0.172, p < 0.001) and in the septal interstitium (0.027 ± 0.014 vs. 0.067 ± 0.039, p = 0.026). The lung tissue of IM-TOL, when compared to IM, showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 ± 0.07 vs. 1.0 ± 0.528, p = 0.002) and V (1.12 ± 0.42 vs. 4.74 ± 2.25, p = 0.009) collagen, in addition to decreased TGF-beta expression (p < 0.0001).

Conclusions

Collagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment.     

Level of Thyroid-Stimulating Hormone (TSH) in Patients with Acute Schizophrenia,Unipolar Depression or Bipolar Disorder

The aim of this study is to investigate differences in thyroid-stimulating hormone (TSH) level in patients with acute schizophrenia, unipolar depression, bipolar depression and bipolar mania. Serum level of TSH was measured in 1,685 Caucasian patients (1,064 women, 63.1 %; mean age 46.4). Mean serum TSH concentration was: schizophrenia (n = 769) 1.71 μIU/mL, unipolar depression (n = 651) 1.63 μIU/mL, bipolar disorder (n = 264) 1.86 μIU/mL, bipolar depression (n = 203) 2.00 μIU/mL, bipolar mania (n = 61) 1.38 μIU/mL (H = 11.58, p = 0.009). Depending on the normal range used, the overall rate of being above or below the normal range was 7.9–22.3 % for schizophrenia, 13.9–26.0 % for unipolar depression, 10.8–27.6 % for bipolar disorder, 12.2–28.5 % for bipolar depression, and 11.4–24.5 % for bipolar mania. We have also found differences in TSH levels between the age groups (≤20, >20 years and ≤40, >40 years and ≤60 years and >60 years). TSH level was negatively correlated with age (r = ? 0.23, p < 0.001). Weak correlations with age have been found in the schizophrenia (r = ? 0.21, p < 0.001), unipolar depression (r = ? 0.23, p < 0.001), bipolar depression (r = ? 0.25, p = 0.002) and bipolar disorder (r = ? 0.21, p = 0.005) groups. Our results confirm that there may be a higher prevalence of thyroid dysfunctions in patients with mood disorders (both unipolar and bipolar) and that these two diagnostic groups differ in terms of direction and frequency of thyroid dysfunctions.     

Impact of repeated intravenous bone marrow mesenchymal stem cells infusion on myocardial collagen network remodeling in a rat model of doxorubicin-induced dilated cardiomyopathy

Bone marrow mesenchymal stem cells (MSCs) transplantation improved cardiac function and reduced myocardial fibrosis in both ischemic and non-ischemic cardiomyopathies. We evaluated the effects of repeated peripheral vein injection of MSCs on collagen network remodeling and myocardial TGF-β1, AT1, CYP11B2 (aldosterone synthase) gene expressions in a rat model of doxorubicin (DOX)-induced dilated cardiomyopathy (DCM). Thirty-eight out of 53 SD rats survived at 10 weeks post-DOX injection (2.5 mg/kg/week for 6 weeks, i.p.) were divided into DCM blank (without treatment, n = 12), DCM placebo (intravenous tail injection of 0.5 mL serum-free culture medium every other day for ten times, n = 13), and DCM plus MSCs group (intravenous tail injection of 5 × 10⁶ MSCs dissolved in 0.5 mL serum-free culture medium every other day for 10 times, n = 13). Ten untreated rats served as normal controls. At 20 weeks after DOX injection, echocardiography, myocardial collagen content, myocardial expressions of types I and III collagen, TGF-β1, AT1, and CYP11B2 were compared among groups. At 20 weeks post-DOX injection, 8 rats (67 %) survived in DCM blank group, 9 rats (69 %) survived in DCM placebo group while 13 rats (100 %) survived in DCM plus MSCs group. Left ventricular end-diastolic diameter was significantly higher and ejection fraction was significantly lower in DCM blank and DCM placebo groups compared to normal control rats, which were significantly improved in DCM plus MSCs group (all p < 0.05 vs. DCM blank and DCM placebo groups). Moreover, myocardial collagen volume fraction, types I and III collagen, myocardial mRNA expressions of TGF-β1, AT1, CYP11B2, and collagen I/III ratio were all significantly lower in DCM plus MSCs group compared to DCM blank and DCM placebo groups (all p < 0.05). Repeated intravenous MSCs transplantation could improve cardiac function by attenuating myocardial collagen network remodeling possibly through downregulating renin–angiotensin–aldosterone system in DOX-induced DCM rats.     

Comparison of elastic,viscoelastic and failure tensile material properties of knee ligaments and patellar tendon

The knee ligaments and patellar tendon function in concert with each other and other joint tissues, and are adapted to their specific physiological function via geometry and material properties. However, it is not well known how the viscoelastic and quasi-static material properties compare between the ligaments. The purpose of this study was to characterize and compare these material properties between the knee ligaments and patellar tendon.Dumbbell-shaped tensile test samples were cut from bovine knee ligaments (ACL, LCL, MCL, PCL) and patellar tendon (PT) and subjected to tensile testing (n = 10 per ligament type). A sinusoidal loading test was performed at 8% strain with 0.5% strain amplitude using 0.1, 0.5 and 1 Hz frequencies. Subsequently, an ultimate tensile test was performed to investigate the stress-strain characteristics.At 0.1 Hz, the phase difference between stress and strain was higher in LCL compared with ACL, PCL and PT (p < 0.05), and at 0.5 Hz that was higher in LCL compared with all other ligaments and PT (p < 0.05). PT had the longest toe-region strain (p < 0.05 compared with PCL and MCL) and MCL had the highest linear and strain-dependent modulus, and toughness (p < 0.05 compared with ACL, LCL and PT).The results indicate that LCL is more viscous than other ligaments at low-frequency loads. MCL was the stiffest and toughest, and its modulus increased most steeply at the toe-region, possibly implying a greater amount of collagen. This study improves the knowledge about elastic, viscoelastic and failure properties of the knee ligaments and PT.     

The influence of titanium coating of mesh polypropylene endoprostheses on their biocompatibility

A comparative analysis of the proliferative activity of inflammatory infiltrate cells and the distribution of collagen type I and III in granulomas formed in the area of contact of mesh materials made of polypropylene (PP Std Light) and titanium-coated polypropylene (TiMesh) was performed via polarized microscopy and immunohistochemical detection of Ki-67 protein. Mesh materials were implanted in the soft tissues of the lumbar region of rats. The number of proliferative cells around implants made of the standard polypropylene (PP Std Light) 7 days after the operation was detected with the use of antibodies to Ki-67 protein to be less than the number of those around titanium-coated polypropylene (TiMesh) (29.1 ± 5.7 and 33.6 ± 3.1%, respectively, p < 0.001). The similar differences were revealed 1 month after the beginning of the experiment (15.9 ± 4.3 and 26.9 ± 3.6%, respectively, p < 0.001). Various types of collagen fibers in granulomas around the implanted mesh materials were detected in polarized light with the use of sections stained with Sirius Red. The ratios between collagen fibers type I and III in granulomas around PP Std Light and TiMesh materials determined 7 days after the operation were 1.085 ± 0.022 and above 1.107 ± 0.013, respectively (p = 0.017). The ratio increased to 1.174 ± 0.036 and 1.246 ± 0.102, respectively (p = 0.045) 1 month after the operation. The obtained results suggest that the titanium coating of polypropylene stimulates the formation of collagen type I and more mature connective tissue around mesh endoprostheses.     

Iron toxicity mediated by oxidative stress enhances tissue damage in an animal model of diabetes

Although iron is a first-line pro-oxidant that modulates clinical manifestations of various systemic diseases, including diabetes, the individual tissue damage generated by active oxidant insults has not been demonstrated in current animal models of diabetes. We tested the hypothesis that oxidative stress is involved in the severity of the tissues injury when iron supplementation is administered in a model of type 1 diabetes. Streptozotocin (Stz)-induced diabetic and non-diabetic Fischer rats were maintained with or without a treatment consisting of iron dextran ip at 0.1 mL day^?1 doses administered for 4 days at intervals of 5 days. After 3 weeks, an extensive increase (p < 0.001) in the production of reactive oxygen species (ROS) in neutrophils of the diabetic animals on iron overload was observed. Histological analysis revealed that this treatment also resulted in higher (p < 0.05) tissue iron deposits, a higher (p < 0.001) number of inflammatory cells in the pancreas, and apparent cardiac fibrosis, as shown by an increase (p < 0.05) in type III collagen levels, which result in dysfunctional myocardial. Carbonyl protein modification, a marker of oxidative stress, was consistently higher (p < 0.01) in the tissues of the iron-treated rats with diabetes. Moreover, a significant positive correlation was found between ROS production and iron pancreas stores (r = 0.42, p < 0.04), iron heart stores (r = 0.54, p < 0.04), and change of the carbonyl protein content in pancreas (r = 0.49, p < 0.009), and heart (r = 0.48, p < 0.02). A negative correlation was still found between ROS production and total glutathione content in pancreas (r = ?0.50, p < 0.03) and heart (r = ?0.45, p < 0.04). In conclusion, our results suggest that amplified toxicity in pancreatic and cardiac tissues in rats with diabetes on iron overload might be attributed to increased oxidative stress.     

Characterisation of collagen from normal and scoliotic human spinal ligament

Acid0soluble and pepsin-soluble collagens have been isolated from spinal ligaments of normal and scoliotic individuals. Polyacrylamide gel electrophoresis of native and cyanogen bromide-treated collagens, and amino acid analysis, showed that the ligament collagen is almost all of the Type I variety with only trace amounts of Type III present. There was no evidence for abnormal ratios of collagen α-chains, or underhydroxylation of proline and lysine in the scoliotic ligament. These results indicate that collagen biochemistry is normal with respect to type, post-translational modification and cross-linking in spinal ligaments of patients with idiopathic scoliosis. Elastin and proteoglycan were only minor components of the ligaments. The nature of the non-collagenous part of the ligament is unknown, although it contains some proteins with a hydrophobic nature.     

Advanced glycation end products as a source of artifacts in immunoenzymatic methods

The most abundant proteins in the arteries are those of extracellular matrix, ie. collagen and elastin. Due to their long half-lifes these proteins have an increased chance to undergo glycation. The aim of this study was to determine relationship between the content of the main extracellular matrix proteins and the advanced glycation end products (AGEs) in arteries. In this study 103 fragments of aorta were analyzed by ELISA and immunobloting for the content of collagens type I, III and IV and elastin and the content of advanced glycation end-products (AGE). A negative correlation between the content of collagens type III and IV and AGE (r = ?0,258, p = 0,0122, and a weak negative correlation between collagen type III and age of the sample donor (r = 0,218, p = 0,0262) were demonstrated. This result comes as a surprise and it contradicts an intuitive assumption that with more glycation substrate, i.e. matrix proteins, more AGE products are expected. We have concluded that the results of the ELISA tests must have been influenced by the glycation. As a consequence, either modified protein molecules were not being recognized by the antibodies, or the glycation, and formation of crosslinks have blocked access of the antibodies to the antigen. It will conceal the effect of the linear dependence between the result (absorbance/densitometry) from the quantity of protein to which the antibody is directed.     

Identifying metabolite markers for preterm birth in cervicovaginal fluid by magnetic resonance spectroscopy

Introduction

Preterm birth (PTB) may be preceded by changes in the vaginal microflora and metabolite profiles.

Objectives

We sought to characterise the metabolite profile of cervicovaginal fluid (CVF) of pregnant women by 1H NMR spectroscopy, and assess their predictive value for PTB.

Methods

A pair of high-vaginal swabs was obtained from pregnant women with no evidence of clinical infection and grouped as follows: asymptomatic low risk (ALR) women with no previous history of PTB, assessed at 20–22 gestational weeks, g.w., n = 83; asymptomatic high risk (AHR) women with a previous history of PTB, assessed at both 20–22 g.w., n = 71, and 26–28 g.w., n = 58; and women presenting with symptoms of preterm labor (PTL) (SYM), assessed at 24–36 g.w., n = 65. Vaginal secretions were dissolved in phosphate buffered saline and scanned with a 9.4 T NMR spectrometer.

Results

Six metabolites (lactate, alanine, acetate, glutamine/glutamate, succinate and glucose) were analysed. In all study cohorts vaginal pH correlated with lactate integral (r = ?0.62, p < 0.0001). Lactate integrals were higher in the term ALR compared to the AHR (20–22 g.w.) women (p = 0.003). Acetate integrals were higher in the preterm versus term women for the AHR (20–22 g.w.) (p = 0.048) and SYM (p = 0.003) groups; and was predictive of PTB < 37 g.w. (AUC 0.78; 95 % CI 0.61–0.95), and delivery within 2 weeks of the index assessment (AUC 0.84; 95 % CI 0.64–1) in the SYM women, whilst other metabolites were not.

Conclusion

High CVF acetate integral of women with symptoms of PTL appears predictive of preterm delivery, as well as delivery within 2 weeks of presentation.