Changes of proapoptotic and antiapoptotic genes affect sensitivity to apoptotic stimuli in impaired contractility due to long term bladder outlet obstruction.

INTRODUCTION: The normal biological process that necessitates cell removal greatly depends on apoptosis. Long term bladder outlet obstruction (BOO) causes damaged smooth muscle cells to undergo apoptosis. However, smooth muscle cell apoptosis that BOO causes is not well known in impaired bladder contractility. Therefore, we designed this study to investigate whether long-term BOO could induce apoptosis activities and to obtain an expression profile of apoptosis related genes. MATERIALS AND METHODS: We used 10 Sprague-Dawley six-week-old female rats. We separated them equally into two groups: a sham intervention group (group 1) and an eight-week BOO group (group 2). We conducted cystometric evaluation eight weeks following BOO onset, with processing of bladder tissue for PCR array. Every array comprised 84 genes, which were established to contribute to an apoptosis response, cell differentiation and metabolism, and 12 sequences were established for the regulation of loading and the quality of cDNA. We performed real-time PCR. Changes in gene expression presented as a fold increase/decrease. Alterations of more than two-fold constituted the cut-off determining expression. RESULTS: Group 2 had a greater bladder weight and Impaired bladder contractility. Immunofluorescent staining with CAS3, TUNEL showed increased in the BOO group. In comparison to group 1, group 2 exhibited an at least two-fold upregulation in five genes, the Bcl-2 (15.1), Birc5 (5.8), Cd40lg (7.5), Il10 (16.2), and Naip2 (13.2). They also demonstrated at least a two-fold downregulation in the PRLR (-18.1) gene. Genes Bcl2ald, Circ5, Cd40lg, Il10, Naip2, and PRLR were among the genes with activity against apoptosis. TNF, STAT3 and TP53 mediated the effect that genes had on one another. CONCLUSION: This study demonstrated that the relative ratios of pro- and antiapoptotic genes determine bladder cell sensitivity cells to apoptotic stimuli in impaired contractility caused by long term BOO. Although we cannot confirm whether this finding is the result of the decompensated phase of the bladder or the process, the gene expression profiles could explain molecular mechanisms of apoptosis in impaired bladder contractility caused by long-term BOO with further studies.

Improved bladder contractility after transplantation of human mesenchymal stem cells overexpressing hepatocyte growth factor into underactive bladder from bladder outlet obstruction models of rats.

INTRODUCTION: An underactive bladder can lead to difficulty in voiding that causes incomplete emptying of the bladder, suggesting the need for a new strategy to increase bladder contractility in such patients. This study was performed to investigate whether human mesenchymal stem cells (hMSCs) were capable of restoring bladder contractility in rats with underactive bladder due to bladder outlet obstruction (BOO) and enhancing their effects by overexpressing hepatocyte growth factor (HGF) in hMSCs. MATERIALS AND METHODS: The hMSCs were transplanted into the bladder wall of rats. Fifty female Sprague-Dawley rats at six weeks of age were divided into five groups: group 1: control; group 2: sham intervention; group 3: eight-week BOO; group 4: BOO rats transplanted with hMSCs; and group 5: BOO rats transplanted with hMSCs overexpressing HGF. Two weeks after the onset of BOO in groups 4 and 5, hMSCs were injected into the bladder wall. Cystometry evaluation was followed by Masson's trichrome staining of bladder tissues. Realtime PCR and immunohistochemical staining were performed to determine for hypoxia, apoptosis, and angiogenesis. RESULTS: Collagen deposition of bladder increased in BOO but decreased after transplantation of hMSCs. The increased inter-contraction interval and residual urine volume after BOO was reversed after hMSCs transplantation. The decreased maximal voiding pressure after BOO was restored by hMSCs treatment. The mRNA expression of bladder collagen1 and TGF-ß1 increased in BOO but decreased after hMSCs transplantation. The decrease in vWF-positive cells in the bladder following BOO was increased after hMSCs transplantation. Caspase 3 and TUNEL-positive apoptosis of bladder cells increased in BOO but decreased after transplantation of hMSCs. These effects were enhanced by overexpressing HGF in hMSCs. CONCLUSION: Transplantation of hMSCs into bladder wall increased the number of micro-vessels, decreased collagen deposition and apoptosis of detrusor muscle, and improved bladder underactivity. The effects were enhanced by overexpressing HGF in hMSCs. Our findings suggest that the restoration of underactive bladder using hMSCs may be used to rectify micturition disorders in patients following resolution of BOO. Further studies are needed before hMSCs can be used in clinical applications.

Superoxide Dismutase 3 Inhibits LL-37/KLK-5-Mediated Skin Inflammation through Modulation of EGFR and Associated Inflammatory Cascades.

The expressions of LL-37 and KLK-5 were found to be altered in various dermatoses, including atopic dermatitis, psoriasis, and rosacea. However, the downstream inflammatory effect of LL-37 and KLK-5 is not as well studied. In addition, there is little high-quality evidence for the treatment of LL-37- and KLK-5-mediated inflammation. In this study, we investigated the effect of superoxide dismutase 3 (SOD3) on LL-37- or KLK-5-induced skin inflammation in vitro and in vivo and its underlying anti-inflammatory mechanisms. Our data showed that SOD3 significantly reduced both LL-37- and KLK-5-induced expression of pro-inflammatory mediators and suppressed the activation of EGFR, protease-activated receptor 2, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3, and p38/extracellular signal-regulated kinase signaling pathways in human keratinocytes. Moreover, SOD3 suppressed LL-37-induced expression of inflammatory mediators, reactive oxygen species production, and p38/extracellular signal-regulated kinase activation in mast cells. In addition, subcutaneous injection of KLK-5 in SOD3 knockout mice exhibited erythema with increased epidermal thickness, mast cell and neutrophil infiltration, expression of inflammatory mediators, and activation of EGFR, protease-activated receptor 2, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3, and downstream mitogen-activated protein kinase pathways. However, treatment with SOD3 in SOD3 knockout mice rescued KLK-5-induced inflammatory cascades. Similarly, KLK-5-induced inflammation in wild-type mice was also ameliorated when treated with SOD3. Taken together, our data suggest that SOD3 is a potentially effective therapy for both LL-37-and KLK-5-induced skin inflammation.

A novel voxel-wise lesion segmentation technique on 3.0-T diffusion MRI of hyperacute focal cerebral ischemia at 1 h after permanent MCAO in rats.

To assess hyperacute focal cerebral ischemia in rats on 3.0-Tesla diffusion-weighted imaging (DWI), we developed a novel voxel-wise lesion segmentation technique that overcomes intra- and inter-subject variation in apparent diffusion coefficient (ADC) distribution. Our novel technique involves the following: (1) intensity normalization including determination of the optimal type of region of interest (ROI) and its intra- and inter-subject validation, (2) verification of focal cerebral ischemic lesions at 1 h with gross and high-magnification light microscopy of hematoxylin-eosin (H&E) pathology, (3) voxel-wise segmentation on ADC with various thresholds, and (4) calculation of dice indices (DIs) to compare focal cerebral ischemic lesions at 1 h defined by ADC and matching H&E pathology. The best coefficient of variation was the mode of the left hemisphere after normalization using whole left hemispheric ROI, which showed lower intra- (2.54 ± 0.72%) and inter-subject (2.67 ± 0.70%) values than the original. Focal ischemic lesion at 1 h after middle cerebral artery occlusion (MCAO) was confirmed on both gross and microscopic H&E pathology. The 83 relative threshold of normalized ADC showed the highest mean DI (DI = 0.820 ± 0.075). We could evaluate hyperacute ischemic lesions at 1 h more reliably on 3-Tesla DWI in rat brains.

Effectiveness of Rehabilitative Balloon Swallowing Treatment on Upper Esophageal Sphincter Relaxation and Pharyngeal Motility for Neurogenic Dysphagia.

OBJECTIVE: To investigate the relationship between dysphagia severity and opening of the upper esophageal sphincter (UES), and to assess the effect of balloon size on functional improvement after rehabilitative balloon swallowing treatment in patients with severe dysphagia with cricopharyngeus muscle dysfunction (CPD). METHODS: We reviewed videofluoroscopic swallowing studies (VFSS) conducted in the Department of Physical Medicine and Rehabilitation, Myongji Hospital from January through December in 2012. All subjects diagnosed with CPD by VFSS further swallowed a 16-Fr Foley catheter filled with barium sulfate suspension for three to five minutes. We measured the maximum diameter of the balloon that a patient could swallow into the esophagus and subsequently conducted a second VFSS. Then, we applied a statistical technique to correlate the balloon diameter with functional improvement after the balloon treatment. RESULTS: Among 283 inpatients who received VFSS, 21 subjects were diagnosed with CPD. It was observed that the degree of UES opening evaluated by swallowing a catheter balloon had inverse linear correlations with pharyngeal transit time and post-swallow pharyngeal remnant. Videofluoroscopy guided iterative balloon swallowing treatment for three to five minutes, significantly improved the swallowing ability in terms of pharyngeal transit time and pharyngeal remnant (p<0.005 and p<0.001, respectively). Correlation was seen between balloon size and reduction in pharyngeal remnants after balloon treatment (Pearson correlation coefficient R=-0.729, p<0.001), whereas there was no definite relationship between balloon size and improvement in pharyngeal transit time (R=-0.078, p=0.738). CONCLUSION: The maximum size of the balloon that a patient with CPD can swallow possibly indicates the maximum UES opening. The iterative balloon swallowing treatment is safe without the risk of aspiration, and it can be an effective technique to improve both pharyngeal motility and UES relaxation.

Improvement in Spinal Cord Injury-Induced Bladder Fibrosis Using Mesenchymal Stem Cell Transplantation Into the Bladder Wall.

Experiments on spinal cord injury (SCI) have largely focused on the transplantation of stem cells into injured spinal cords for motor recovery while neglecting to investigate bladder dysfunction. The present study was performed to investigate the effect of B10 human mesenchymal stem cells (hMSCs) directly transplanted into the bladder wall of SCI rats and to determine whether they are capable of inhibiting collagen deposition and improving cystometric parameters in SCI rats. Forty 6-week-old female Sprague-Dawley rats were divided into four groups (group 1: control, group 2: sham operated, group 3: SCI, group 4: SCI rats that received B10 cells). B10 cells were labeled with fluorescent magnetic nanoparticles (MNPs). Four weeks after the onset of SCI, MNP-labeled B10 cells were injected to the bladder wall. Serial magnetic resonance (MR) images were taken immediately after MNP-B10 injection and at 4 weeks posttransplantation. Voiding function was assessed at 4 weeks posttransplantation, and the bladder was harvested. Improvements in bladder fibrosis and bladder function were monitored by molecular MR imaging. Transplantation of B10 cells into the SCI rats markedly reduced their weights and collagen deposition. MR images showed a clear hypointense signal induced by the MNP-labeled B10 cells at 4 weeks posttransplantation. Transplanted B10 cells were found to differentiate into smooth muscle cells. The intercontraction interval decreased, and the maximal voiding pressure increased after SCI but recovered after B10 cell transplantation. Survival of B10 cells was found at 4 weeks posttransplantation using anti-human mitochondria antibody staining and MR imaging. The transplanted B10 cells inhibited bladder fibrosis and ameliorated bladder dysfunction in the rat SCI model. MSC-based cell transplantation may be a novel therapeutic strategy for bladder dysfunction in patients with SCI.

Expression and regulation of SNAP-25 and synaptotagmin VII in developing mouse ovarian follicles via the FSH receptor.

Soluble-NSF attachment protein receptor (SNARE) proteins play a role in vesicle fusion, exocytosis, and intracellular trafficking in neuronal cells as well as in fertilization and embryogenesis. We investigated the expression patterns of two SNARE proteins, SNAP-25 and synaptotagmin VII (SytVII), and their regulation by pregnant mare serum gonadotropin (PMSG) during mouse ovarian follicular development. Ovaries were obtained at 0, 12, 24, 36, and 48 h post-PMSG injection of immature mice. SNAP-25 and SytVII mRNA expression levels increased gradually in a time-dependant manner. However, protein levels revealed different patterns of expression, suggesting different translational regulation following PMSG stimulation. SNAP-25 and SytVII expression was closely associated with thickening of the granulosa cell (GC) layer and follicle morphological changes from a flattened to a cuboidal shape. To explore follicle stimulating hormone receptor (FSHR)-mediated regulation of their expression, GCs from preantral follicles were cultured to examine the effects of FSHR siRNA knockdown. FSHR siRNA abolished upregulation of the SNAREs in both PMSG and FSH-stimulated GCs. This abolished gene expression was rescued by adding dibutyryl cyclic AMP to the cultures. These results suggest that SNAP-25 and SytVII expression is regulated via the FSHR-cAMP pathway during follicular development.

Association of matrix metalloproteinase-3 with cardiogenic activity during Noggin-induced differentiation of mouse embryonic stem cells.

BACKGROUND: Despite the pluripotency of embryonic stem (ES) cells, their clinical applications have been hindered due to the lack of reliable differentiation methods. Recently, it was shown that Noggin could effectively induce cardiomyocyte differentiation by transient treatment of ES cells. METHODS: To determine how Noggin may induce cardiac differentiation, we compared differentially expressed genes during Noggin-induced differentiation of ES cells using microarray analysis. We found Matrix metalloproteinase-3 (Mmp-3) expression was highly up-regulated by Noggin treatment. To understand the role of Mmp-3 in the cardiac differentiation of ES cells, we inhibited Mmp-3 activity by treating with a specific Mmp-3 inhibitor during Noggin-induced cardiac differentiation of ES cells. We also analyzed the expression levels of cardiac markers and the ratio of spontaneously beating embryoid bodies (EBs) in the presence of the Mmp-3 inhibitor. RESULTS: We analyzed EB samples from zero, two, and four days with or without Noggin treatment, and found that the expression levels of 2 (0 day), 56 (2 days), and 805 (4 days) genes were altered with Noggin treatment. Up-regulation of Mmp-3 was closely associated with relative increases of cardiogenic, vasculogenic, and hematopoietic genes in EB treated with Noggin. By inhibiting Mmp-3 activity, we verified that Mmp-3 activation is partly responsible for both the expression of cardiac markers and the elevated ratio of spontaneously beating to non-beating EBs. CONCLUSIONS: The concurrent expression of Mmp-3 with many cardiogenic genes and the specific inhibition of Mmp-3 revealed a critical role for Mmp-3 in Noggin-induced cardiac differentiation of ES cells.

Effects of conjugated linoleic acid supplementation on fatty acid composition in the plasma, liver, and epididymal fat pads of male-Sprague Dawley rats.

The purpose of this study was to investigate the effects of conjugated linoleic acid (CLA) on the fatty acid composition of plasma, liver, and epididymal fat pads in rats. Seventy-two male Sprague-Dawley rats were divided into four groups and received beef tallow (BT), fish oil (FO), beef tallow with CLA (BTC), and fish oil with CLA (FOC). All the rats were fed an experimental diet containing 12% (wt/wt) total fat, including CLA at 1%, for 30 weeks. The fatty acid analyses of the plasma, liver, and epididymal fat pads were performed by the one-step methylation method, followed by gas chromatography. Monounsaturated fatty acid (MUFA) levels in the plasma were significantly reduced in the BTC group as compared to the BT group. The levels of C18:1 and C20:4 in the liver were significantly lower in the BTC group than in the BT group (P < .05). CLA was detected in the epididymal fat pads of the rats that did not receive supplemental CLA (the BT and FO groups), indicating de novo synthesis. CLA caused significant decreases in C18:1 fatty acid levels in both the BTC and FOC groups in epididymal fat pads. C22:6 was significantly higher in the liver microsomes of the BTC group, as compared to the BT group. MUFA was stored at higher levels in the epididymal fat pads and was significantly lower in level in the CLA-supplemented (BTC and FOC) groups as compared to the BT and FO groups (P < .05). In conclusion, dietary CLA supplementation in rats altered fatty acid composition in a tissue-specific manner. CLA was stored mainly in the epididymal fat pad. The results also suggested that CLA may inhibit the conversions of C18:0 to C18:1 and C18:2 to C20:4 fatty acid, resulting in significantly lower levels of C18:1 and C20:4 in the livers of the BTC group as compared to the BT group.

Label-free, microfluidic separation and enrichment of human breast cancer cells by adhesion difference.

A label-free microfluidic method for separation and enrichment of human breast cancer cells is presented using cell adhesion as a physical marker. To maximize the adhesion difference between normal epithelial and cancer cells, flat or nanostructured polymer surfaces (400 nm pillars, 400 nm perpendicular, or 400 nm parallel lines) were constructed on the bottom of polydimethylsiloxane (PDMS) microfluidic channels in a parallel fashion using a UV-assisted capillary moulding technique. The adhesion of human breast epithelial cells (MCF10A) and cancer cells (MCF7) on each channel was independently measured based on detachment assays where the adherent cells were counted with increasing flow rate after a pre-culture for a period of time (e.g., one, two, and four hours). It was found that MCF10A cells showed higher adhesion than MCF7 cells regardless of culture time and surface nanotopography at all flow rates, resulting in label-free separation and enrichment of cancer cells. For the cell types used in our study, an optimum separation was found for 2 hours pre-culture on the 400 nm perpendicular line pattern followed by flow-induced detachment at a flow rate of 200 microl min(-1). The fraction of MCF7 cells was increased from 0.36 +/- 0.04 to 0.83 +/- 0.04 under these optimized conditions.