Mast cell enzymes alter the connective tissue phenotype of bladder wall fibroblasts and smooth muscle cells.
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Chymases↗
Biomedical subjects
P S Howard
Publications and source records attributed to P S Howard.
Maintenance of oxidative phosphorylation protects cells from Actinobacillus actinomycetemcomitans leukotoxin-induced apoptosis.
Subnanomolar concentrations (3 x 10(-10) M) of Actinobacillus actinomycetemcomitans leukotoxin (Ltx) trigger apoptosis of JY cells, as shown by a decrease in mitochondrial transmembrane potential (DeltaPsim), hyperproduction of reactive oxygen species (ROS) and release of cytochrome c from the intermembrane space. When compared with heat-inactivated leukotoxin (DeltaI Ltx) controls, ATP levels in Ltx-treated JY cells continued to decrease during a 24 h experiment while cytoplasmic ADP concentrations were increasing. These results suggest that a blockage occurred in ATP/ADP exchange. To maintain ATP/ADP exchange, JY cells were transfected with bcl-2 and bcl-xL and incubated with Ltx. ATP levels of the transfected cells decreased to 67% (JY/bcl-2) and 73% (JY/bcl-xL) after the experiment. Furthermore, cytochrome c remained localized to the mitochondrial fraction of Ltx-treated JY/bcl-2 and JY/bcl-xL cells, whereas its presence in the cytoplasmic fraction of JY/gen cells suggests an uncoupling of electron transport. Expression of bcl-2 and bcl-xL in cells inhibited downstream apoptotic events such as cleavage of poly(ADP-ribose) polymerase, DNA fragmentation and activation of a family of caspases. The results indicate that Ltx induces apoptosis through a mitochondrial pathway that involves decreased levels of the ADP in the mitochondrial matrix, a lack of substrate for ATP synthetase and arrest of oxidative phosphorylation.
Adenosine Triphosphate↗
Extracellular matrix and nuclear localization of beta ig-h3 in human bladder smooth muscle and fibroblast cells.
The extracellular matrix (ECM) plays an essential role in bladder structure and function. In this study, expression of beta ig-h3, a recently identified extracellular matrix protein, was investigated in human bladder tissue, and human bladder smooth-muscle (SMC) and fibroblast cells in vitro. SMCs secreted greater than three times the level of this protein compared with fibroblasts. The relative levels of beta ig-h3 mRNA in the two cell types reflected the protein expression. Immunohistochemical analysis demonstrated protein deposition in the ECM as well as cytoplasmic localization and, unexpectedly, nuclei. Anti-beta ig-h3 antibodies also stained the matrix surrounding the detrusor SMCs and nuclei of bladder fibroblasts, SMCs, and urothelium in intact bladder tissue. Western blot analyses of medium and matrix fractions obtained from cells in vitro revealed protein of approximately 70-74 kDa, whereas nuclear extracts contained a 65-kDa reactive protein band. We propose that although this protein is a structural component of bladder ECM, its nuclear localization suggests that it has other regulatory and/or structural functions.
Amino Acid Sequence↗
Expression of betaig-h3 by human bronchial smooth muscle cells: localization To the extracellular matrix and nucleus.
Bronchial smooth muscle cells play a central role in normal lung physiology by controlling airway tone. In addition, airway smooth muscle hyperplasia and hypertrophy are important factors in the pathophysiology of asthma. In this study, expression of betaig-h3, a recently identified component of the extracellular matrix (ECM), was investigated in primary human bronchial smooth muscle (HBSM) cells. Northern blot analysis demonstrated that treatment of cultured HBSM cells with transforming growth factor-beta1 resulted in a 4- to 5-fold increase in the steady-state level of betaig-h3 messenger RNA. Western blot analysis of secreted proteins using monospecific antibodies generated against peptide sequences found in the N- and C-terminal regions of the protein identified several isoforms having apparent mass of 70-74 kD. Immunohistochemical analysis of human lung localized betaig-h3 to the vascular and airway ECM, and particularly to the septal tips of alveolar ducts and alveoli, suggesting that it may have a morphogenetic role. Analysis of cultured HBSM cells identified betaig-h3 in both the ECM as well as the cytoplasm, and surprisingly also in the nucleus. These results demonstrate that betaig-h3 is produced by resident lung cells, is a component of lung ECM, and may play an important role in lung structure and function. The presence of this protein in nuclei suggests that it may have regulatory functions in addition to its role as a structural component of lung ECM.
Amino Acid Sequence↗
Mechanical stretch induces platelet-activating factor receptor gene expression through the NF-kappaB transcription factor.
In this study, we used the platelet-activating factor (PAF) receptor gene as a model of a mechano-sensitive gene to investigate how mechanical stimuli regulate gene expression and cell function. We utilized a culture system of pulmonary artery smooth muscle cells and a well-defined in vitro mechanical device that imparts an equibiaxial strain repeatedly to cells attached to an elastomeric membrane. Northern blot and immunohistochemical analyses revealed increased PAF receptor expression at both the mRNA and protein levels after 1 h exposure of the cells to a 5% strain at a frequency of 1 Hz. To investigate the mechanism of activation of this gene by stretch, we performed transfection experiments with a luciferase reporter gene linked to segments of the 5' flanking region of the receptor gene promoter. Expression of the transfected reporter gene bearing a 1.1-kb fragment of the promoter was enhanced in mechanically stretched cells indicating a direct effect on transcriptional activity. When truncated to leave the nucleotides between -610 to +27, the promoter-reporter construct lost stretch inducibility suggesting that the region between -1099 and -610 was required for stretch responsiveness. This region contains four copies of NF-kappaB binding sites. These elements are in close proximity to one another and can form a complex with nuclear proteins derived from stretched cells as demonstrated by gel mobility shift assay. Moreover, in experiments using cycloheximide, we found that de novo protein synthesis was not necessary for the induction of the PAF receptor gene expression by mechanical stretch. Conversely, preincubation of the cells with protein kinase C inhibitors suppressed mechanical stretch-induced PAF receptor gene expression at the mRNA levels and abrogated upstream events of NF-kappaB activation in the cytoplasm. These data strongly suggest that stretch-induced PAF receptor gene expression is mediated by NF-kappaB binding to the PAF receptor gene promoter and that protein kinase C activation is among the molecular features of NF-kappaB activation and translocation into the nucleus in mechanically stretched cells.
Animals↗
Biochemical and physiological characterization of the urinary bladder in Ehlers-Danlos syndrome.
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Child↗
The role of collagen in bladder filling.
While this model is speculative, it is attractive in that it can account for the physiologic properties of the bladder. It also implies that connections must exist between the tension generating elements, i.e., the smooth muscle cells, and the other components of the bladder. In bladders that become noncompliant, it is likely that there is some interference with the ability of the collagen fibers to elastically and reversibly alter their tortuosity. This, predictably, would reduce total bladder capacity. Further studies will be required to establish the relationship between compliance changes and the passive mechanical elements of the bladder wall that comprise its structural protein matrix.
Animals↗
Identification of stretch-responsive genes in pulmonary artery smooth muscle cells by a two arbitrary primer-based mRNA differential display approach.
Physical forces induce profound changes in cell phenotype, shape and behavior. These changes can occur in vascular structures as a result of pressure overload and their effects can be seen in atherosclerotic vessels in which smooth muscle cells have undergone hyperplastic and hypertrophic changes. At the molecular level, mechanical stimuli are converted into chemical ones and lead to modulation of gene expression and/or the activation of a new repertoire of genes whose encoded proteins help the cells to adapt to their microenvironment. In this study, we have used a two primer-based mRNA differential display technique to identify candidate mechano-responsive genes in pulmonary artery smooth muscle cells. As compared to the original method described by Liang and Pardee, this technique uses two arbitrary primers instead of an anchored oligo(dt) plus an arbitrary primer in the polymerase chain reaction. The chief advantages of these modifications are an increase in the efficiency of the amplification and in the identification of differentially expressed clones. Using this approach, we compared the pattern of expressed genes in cells cultured under static conditions with those in cells that were mechanically stretched (1 Hz) for 24 h in a well-defined in vitro mechanical system. Three candidate genes that showed reproducible differences were chosen for further characterization and cloning. One clone was under expressed in stretched cells and had a DNA sequence with 90% homology to the human fibronectin gene. Two other clones were highly expressed in stretched cells and had a 92% and a 83% sequence homology with human platelet-activating factor (PAF) receptor and rat insulin-like growth factor-I (IGF-I) genes respectively. Northern blot analysis confirmed low levels of fibronectin mRNA transcripts in stretched cells. In contrast, accumulation of PAF receptor mRNA occurred 30 min after mechanical stretch was initiated whereas IGF-I mRNA levels peaked at 8 h. Both mRNA levels were sustained for up to 24 h of mechanical stretching. These results demonstrate the usefulness of the two primer-based mRNA differential display that enabled us to identify and characterize alterations at the level of gene expression among matrix proteins, G-protein coupled receptors and growth factors, each of whose response to mechanical strain is different. A more complete understanding of these responses will provide further insight into the pathologic processes associated with hypertension and atherosclerosis.
Animals↗
Roles of the lamina propria and the detrusor in tension transfer during bladder filling.
In this study, structural changes within the lamina propria and detrusor layers were analysed during development as a function of bladder filling. Second-, third- and full-term foetal bovine bladders were filled to 0%, 25%, 50%, 75% and 100% of their total capacity and snap frozen. The bladders were analysed histochemically and the relative thicknesses of the lamina propria and detrusor were measured. In all gestational stages examined, the total thickness of the bladder wall decreased during bladder filling. The lamina propria of the full-term bladder thinned at a consistently faster rate than did the detrusor. The lamina propria of second and third trimester bladders followed the same thinning pattern, except when the bladders were filled from 25% to 50% of their capacities. At these gestational stages, the detrusor thinned at a faster rate than the lamina propria. Our results demonstrate that the detrusor layer carries tension only during a specific portion of the filling cycle and only during the second and third trimesters. We conclude that the lamina propria acts as the capacitance layer, while the detrusor functions as the "limiting" or "girding" layer to prevent over-distension of the bladder wall.
Animals↗
The role of endoscopy and implant texture in transaxillary submuscular breast augmentation.
In 1996 the authors reported their experience with 92 consecutive patients undergoing traditional (nonendoscopic) transaxillary submuscular breast augmentation. They reported a capsular contraction rate of 1.1% using textured saline implants, as well as an implant malposition rate of 8.6%. At that time they hypothesized that "the endoscope will lessen the rate of implant malposition." To clarify the role of implant texture and to justify the use of endoscopic techniques in transaxillary submuscular breast augmentation, the authors have studied an additional 58 consecutive patients with two significant changes. First, the endoscope was used to dissect the implant pocket in each patient. Second, smooth-wall implants were substituted for the previously used textured implants. Their rate of implant malposition dropped significantly, from 8.6% to 2.0% (p = 0.10), and their capsular contraction rate remained low, at 2.0% (p = 0.63). They conclude that direct endoscopic control improves implant positioning in the transaxillary submuscular operation, and implant texture does not improve capsular contraction rates when the prosthesis is placed in the submuscular position.
Breast Implants↗
Role of type III collagen in bladder filling.
The function of the urinary bladder is to store urine at low pressure and expel it periodically. To accomplish this, it must have the appropriate structural properties to accommodate slow but continuous volume changes. While much is presently known about the functional measurements of compliance, relatively little is known about the structural basis of compliance. In the present study, immunohistochemistry has been used to localize type III collagen fibers in the bladder wall at different intravesical volumes. To improve the resolution of these fibers, confocal microscopy was utilized to determine the changes in type III collagen fiber orientation and correlate them with the degree of mechanical distension of the bladder wall at partial and full capacity. We demonstrate that there were significant changes in both the orientation and conformation of type III collagen fibers during bladder filling. These observations support the view that volume accommodation in the bladder is achieved by changes in the arrangement of type III collagen. These data suggest that abnormal deposition or arrangement of type III collagen fibers can have an impact on normal bladder function.
Animals↗
Mechanical forces alter extracellular matrix synthesis by human periodontal ligament fibroblasts.
Periodontal ligament fibroblasts (PDLFs) are a heterogeneous population of cells that are involved in the normal maintenance, repair and regeneration of both the ligament and adjacent hard tissues. Additionally, the ability of these cells to respond to mechanical stimulation suggests that they have a central role in mediating the osseous remodeling that underlies physiological and orthodontic tooth movement. To characterize their role further in this process, the current study evaluated the effect of tensional stress on the biosynthesis of extracellular matrix (ECM) proteins by human PDLFs. Cell strains were established from extracted human premolars and third molars. Cells exposed to 5% biaxial deformation (strain) at a frequency of 30 times/min for 24 hr exhibited statistically significant increases in type I collagen and fibronectin synthesis, and a statistically significant decrease in tropoelastin production relative to unstretched controls. Cells exposed to 10% strain exhibited similar responses for fibronectin and tropoelastin while the amount of type I collagen synthesized by stretched cells did not differ from control levels. These results indicate that mechanical stimulation of PDLFs alters type I collagen, tropoelastin and fibronectin production and that these cells are differentially responsive to varying levels of mechanical stress. The ability of these cells to alter ECM protein synthesis in response to specific magnitudes of tensional stress may in part explain how PDLFs regulate ligament and hard tissue remodeling.
Cells, Cultured↗
Temporal expression of elastic fiber components in bladder development.
Fetal and postnatal bovine bladders were examined for expression of elastic fiber components by immunohistochemistry as well as by measurement of steady state mRNA levels. Expression of fibrillin-1, microfibril-associated glycoprotein (MAGP) and elastin during the fetal period were compared with that of postnatal two year old animals (heifers) and adults. Each bladder was separated into two distinct tissue samples: 1) the outer smooth muscle layer (detrusor) and 2) the inner epithelium (urothelium) lined lamina propria (urotherial-lamina propria). Each of these samples was analyzed separately. Distribution of the elastic fiber components, determined by immunohistochemistry with matrix-specific antibodies, was different depending upon the region of the bladder wall examined and its developmental stage. In particular, MAGP and fibrillin-1 were conspicuously present in the urothelium during the later fetal stages. RNA products of elastic fiber genes were detectable both in the detrusor smooth muscle and urothelial-lamina propria fractions. The highest level of expression occurred in the urothelial-lamina propria fraction during the late second-early third trimester. Elastin expression was different from that of MAGP and fibrillin-1. The highest levels of steady-state elastin mRNA occurred at the earliest developmental stages examined and then progressively decreased through term. A high level of elastin expression occurred within the inner or lamina propria layer of the bladder. Since this layer is the functional capacitance layer within the bladder, its flexibility is likely related to the structural integration of elastin and associated microfibrillar components.
Animals↗
Molecular analysis of collagens in bladder fibrosis.
PURPOSE: Fibrosis of bladder tissue is characterized by an abnormal deposition of connective tissue within different layers of the bladder wall, resulting in a low volume, high pressure vesical which may ultimately contribute to renal scarring and failure. These bladders are functionally referred to as "non-compliant" and may result from different etiologies: neurogenic, which encompasses myelodysplasia and spinal cord injury, or non-neurogenic, owing to obstruction or radiation therapy. To examine the molecular mechanisms responsible for this fibrosis, we have analyzed a well-characterized pediatric patient population for alteration(s) in collagen types I and III regulation at the protein and nucleic acid levels. MATERIALS AND METHODS: Immunohistochemical localization of collagen subtypes (I, III, and IV) was carried out using type specific monoclonal antibodies. Total collagen was determined by hydroxyproline analysis, and subtype specific expression of collagenous proteins, following cyanogen bromide extraction procedures, was quantified by competitive ELISA. Total RNA was extracted by guanidinium/phenol/chloroform, and slot blot hybridization analyses with radiolabeled human cDNA probes were quantified by densitometry of resulting autoradiograms. RESULTS: Connective tissue infiltration of detrusor smooth muscle bundles was specific for type III collagen. Protein analyses demonstrated: 1) an increase in total collagen, 2) a statistically significant increase in the type III: type I collagen ratio, and 3), an absolute increase in type III collagen protein in non-compliant bladder tissue. At the mRNA level, there was a coordinate increase in both collagen I and III steady-state mRNAs in non-neurogenic bladder tissue, whereas neurogenic bladder tissue was characterized by an increase in the type III: type I mRNA transcript ratio. CONCLUSIONS: These data suggest that regulation of collagen synthesis in bladder fibrosis is complex and is characterized by both transcriptional and post-transcriptional mechanisms, depending upon the etiology of the fibrosis.
Adolescent↗
A modification of the primary nasal correction in the rotation-advancement unilateral cleft lip repair.
Primary correction of the unilateral cleft lip nasal deformity remains a challenge to the cleft surgeon. We wish to present a method of primary nasal correction utilizing presurgical orthopedics with the Latham dentomaxillary advancement appliance, concomitant rotation-advancement cleft lip repair, gingivoperiosteoplasty, and immediate correction of the nasal cartilage distortion with an "open technique" utilizing the rotation-advancement incisions. Direct elevation and suture fixation of the cleft side alar cartilage is performed as well as recreation of the normal upper lateral/lower lateral cartilage relationship, repair of nasal web, and release of the vestibular lining utilizing an L-flap pedicled to the alar base. Details of the operative technique, observations of unilateral cleft nasal anatomy as visualized with the "open technique," and preliminary results are illustrated in patients followed as long as 5 years.
Cartilage↗
Developmental expression of interstitial collagen genes in fetal bladders.
Bovine bladders at 3 stages during fetal development were examined for expression of collagens by immunohistochemistry as well as by measurement of steady state mRNA levels. Expression of type I and type III collagens during the fetal period was compared with that of adult cows as well as a young animal (heifer). Each bladder was separated into a detrusor and a urothelial-lamina propria sample which were then analyzed separately. Distribution and fiber arrangement of types I and III collagens were different depending upon the region of the bladder wall examined. Type III collagen, in particular, has a "coiled" appearance which is especially prominent within the lamina propria. Collagen gene expression showed a distinctive pattern which was different for both type I and type III collagen. While type I collagen gene expression peaked during the late second to early third trimester, type III collagen expression progressively decreased throughout the fetal period. In addition, expression of both collagens was greater in the urothelial-lamina propria fractions. These data demonstrate that the pattern of collagen gene expression in the developing bladder is developmentally regulated and is unique to each of the two major structural layers.
Animals↗
Type III collagen messenger RNA is modulated in non-compliant human bladder tissue.
PURPOSE: Previous studies have demonstrated that the non-compliant bladder is characterized histologically by an increased deposition of extracellular matrix protein, especially type III collagen, in the muscle wall. We sought to determine if an increased tissue level of type III collagen messenger RNA (mRNA) parallels the observed increase in protein expression. MATERIALS AND METHODS: Using a reverse transcription-polymerase chain reaction (RT-PCR) quantitative technique we measured and compared the bladder tissue concentration of type III collagen mRNA between an experimental group of patients (n = 7) with urodynamically proven non-compliant bladders (< 12 cc/cm. H2O) and a control group (n = 8) with normal bladder compliance (> 20 cc/cm. H2O). RESULTS: The mean (+/-S.E.M.) of type III collagen mRNA level in the non-compliant group was 11.72 +/- 2.56 attomole/milligram (amol/mg.) which was almost threefold higher than the level 4.26 +/- 0.74 amol/mg measured in the control group. This difference was statistically significant (p = 0.027). CONCLUSIONS: Type III collagen mRNA levels are increased in the human non-compliant bladder. Therefore the accumulation of type III collagen protein is, in part, transcriptionally regulated.
Adolescent↗
The collagens and their urologic significance.
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Animals↗