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Biomedical subjects

D L Epstein

Publications and source records attributed to D L Epstein.

At least 19 recordsLinked to original sources
PubMed2001

Modulation of aqueous humor outflow facility by the Rho kinase-specific inhibitor Y-27632.

PURPOSE: The goal of this study was to investigate the role of Rho kinase in the modulation of aqueous humor outflow facility. Rho kinase, a critical downstream effector of Rho GTPase is recognized to control the formation of actin stress fibers, focal adhesions, and cellular contraction. METHODS: Expression of Rho GTPase, Rho kinase, and other downstream targets of Rho GTPase were determined in human trabecular meshwork (HTM) and Schlemm's canal (SC) primary cell cultures by Western blot analysis. The Rho kinase-specific inhibitor (Y-27632)-induced changes in actin stress fibers, focal adhesions, and protein phosphotyrosine status were evaluated by staining with rhodamine-phalloidin, anti-paxillin, and anti-phosphotyrosine antibodies, respectively. Myosin light-chain phosphorylation was determined by Western blot analysis. Y-27632-induced changes in SC cell monolayer permeability were quantitated using a colorimetric assay to evaluate horseradish peroxidase diffusion through SC cell monolayers grown in transwell chambers. Aqueous humor outflow facility was measured using enucleated porcine eyes and a constant-pressure perfusion system. RESULTS: Treatment of HTM and SC cells with Y-27632 (10 microM) led to significant but reversible changes in cell shape and decreases in actin stress fibers, focal adhesions, and protein phosphotyrosine staining. SC cell monolayer permeability increased (by 80%) in response to Y-27632 (10 microM) treatment, whereas myosin light-chain phosphorylation was decreased in both HTM and SC cells. Aqueous humor outflow facility increased (40%-80%) in enucleated porcine eyes perfused with Y-27632 (10-100 microM), and this effect was associated with widening of the extracellular spaces, particularly the optically empty area of the juxtacanalicular tissue (JCT). The integrity of inner wall of aqueous plexi, however, was observed to be intact. CONCLUSIONS: Based on the Rho kinase inhibitor-induced changes in myosin light-chain phosphorylation and actomyosin organization, it is reasonable to conclude that cellular relaxation and loss of cell-substratum adhesions in HTM and SC cells could result in either increased paracellular fluid flow across Schlemm's canal or altered flow pathway through the JCT, thereby lowering resistance to outflow. This study also suggests Rho kinase as a potential therapeutic target for the development of drugs to modulate intraocular pressure in glaucoma patients.

Actins↗
PubMed2001

Expression of aquaporin-1 in human trabecular meshwork cells: role in resting cell volume.

PURPOSE: Drainage of aqueous humor from the human eye appears dependent on intracellular volume of trabecular meshwork (TM) cells, the predominant cell type of the human outflow pathway. Thus, the modulation of water and solute flux across the plasma membrane of TM cells is predicted to be an important factor in regulating outflow facility. Aquaporin (AQP)-1 is a hexahelical integral membrane protein that functions as a regulated channel for water and cations in fluid-secreting and -absorbing tissues. AQP1 is present in many tissues of the human eye, including the TM; however, its role in outflow facility is unknown. The purpose of the present study was twofold: to evaluate the prospect of manipulating AQP1 protein levels in TM cells using sense and antisense mRNA and to investigate the functional role of AQP1 in TM cells. METHODS: An adenovirus (AV) expression system was used to alter AQP1 protein levels. AQP1 protein expression was monitored using immunoblot analysis, and resting cell volume was measured by forward light scatter, electronic cell sizing, and [(14)C]-sucrose/urea equilibration. Permeability of TM monolayers to [(14)C]-sucrose was also assessed as an indirect evaluation of cell volume. RESULTS: AV-mediated gene transfer of AQP1 cDNA to TM cells resulted in a titer-dependent increase in recombinant AQP1, whereas transfer of antisense cDNA decreased native AQP1 protein by 71.7% +/- 5.5% (P < 0.01) after 5 days. A novel finding of this study is that mean resting volumes of AQP1(s) AV-infected TM cells in suspension were 8.7% +/- 3.0% greater (P < 0.05) than control cells. Conversely, AQP1 antisense (as) AV-infected cells had resting volumes 7.8% +/- 2.9% less than control cells (P < 0.05). Similar effects of AQP1 expression on resting cell volume were observed in TM monolayers. Consistent with this finding, paracellular permeability of AQP1(s) AV-infected TM monolayers to [(14)C]-sucrose decreased by 8.0% +/- 1.4% (P < 0.001). CONCLUSIONS: In addition to influencing the osmotic permeability of TM plasma membranes, the level of AQP1 protein expression influences resting intracellular volume and thus paracellular permeability of TM cell monolayers in vitro. These data suggest that AQP1 expression may affect outflow facility in vivo.

Adenoviruses, Human↗
PubMed2000

Antithrombin III, a serpin family protease inhibitor, is a major heparin binding protein in porcine aqueous humor.

Our hypothesis is that the proteins in aqueous humor may be involved in the regulation of outflow facility through the trabecular meshwork and uveoscleral meshwork. In this study, we analyzed the profile of heparin-binding proteins present in porcine aqueous humor to identify and characterize secretory proteins with a binding affinity for heparin. A single step involving heparin-sepharose affinity chromatography of porcine aqueous humor yielded a approximately 60 kDa protein as the major heparin-binding species. This protein was specifically eluted from the column by heparin. The N-terminal sequence and immunological cross reactivity of this protein confirmed its identity as antithrombin III. Aqueous humor from different species, as well as cells from human trabecular meshwork, Schlemm's canal, and lens epithelium, contained detectable amounts of antithrombin III. Based on its known anticoagulative function in endothelial cells and effects on the production of prostacyclin, it is reasonable to speculate that antithrombin III present in aqueous humor might influence the physiology of the trabecular and uveoscleral meshwork and thereby regulate intraocular pressure.

Amino Acid Sequence↗
PubMed2000

Genes upregulated in the human trabecular meshwork in response to elevated intraocular pressure.

PURPOSE: To identify genes upregulated in perfused, intact human trabecular meshwork (TM) in response to elevated intraocular pressure (IOP). METHOD: Two pairs of anterior segments of normal human eyes from postmortem donors were placed in culture and perfused 24 hours at constant flow (3 microl/min). After reaching baseline, the flow of one eye from each pair was raised to obtain an incremental pressure (deltaP) of 50 mm Hg for 6 hours. The anterior segments were then quickly frozen in liquid nitrogen, and their TMs were dissected for RNA extraction. SMART cDNA libraries were generated from control and high-pressure human TM RNAs and hybridized to sets of identical high-density cDNA gene arrays. These arrays contained 18,376 human expressed sequence tags (ESTs), corresponding to both characterized and unknown genes. Differentially expressed genes were identified by different-intensity hybridization signals and confirmed by semi-quantitative polymerase chain reaction. RESULTS: Eleven genes were found to be consistently upregulated in the human TM by elevated IOP: interleukin-6, preprotachykinin-1, secretogranin-II, cathepsin-L, stromelysin-1, thymosin-beta4, alpha-tubulin, alphaB-crystallin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein and Cu/Zn superoxide dismutase. The products of these genes are involved in vascular permeability, secretion, extracellular matrix remodeling, cytoskeleton reorganization, and reactive oxygen species scavenging. CONCLUSIONS: Elevated IOP induced specific upregulation of 11 physiologically relevant genes. On the basis of their known activities, the products of each of these genes might predict homeostatic mechanisms similar to those involved in the regulation of blood vessel permeability. We hypothesize that similar mechanisms might be involved in regulating flow through Schlemm's Canal endothelium.

Aged↗
PubMed2000

Isolation of primary open-angle glaucomatous trabecular meshwork cells from whole eye tissue.

PURPOSE: Isolation and culture of human trabecular meshwork (TM) cells from primary open-angle glaucomatous (POAG) tissue has proven difficult. The objective of this study was to directly compare the utility of two different isolation methods to obtain viable human TM cells from POAG whole eye tissue. METHODS: Using a blunt dissection technique, human TM tissue was obtained from four pairs of donor eyes (67, 77, 81 and 82 years) with a documented history of POAG. TM tissue from one eye was explanted into tissue culture. TM from the contralateral eye was digested with a collagenase mixture and seeded onto culture plates. RESULTS: Primary cell isolates were obtained from all donors with both techniques. However, only cells obtained using the digestion method (3 of 4 TMs) could be passaged for expansion and freeze-downs (3 x 107 second passage cells/donor). None of the cells obtained from explanted TMs could be passaged. Cells from successful isolations were of uniform size, possessed typical TM morphology and had doubling times < 48 hours. CONCLUSION: These results demonstrate a clear advantage to digesting the extracellular matrix of glaucomatous TM tissue to obtain sufficient numbers of healthy cells for use in experiments. In contrast to cells obtained from explants, cells liberated from POAG TM tissue by digestion appear indistinguishable morphologically and behaviorally from "normal" TM cells.

Aged↗
PubMed2000

Characterization of gene expression in human trabecular meshwork using single-pass sequencing of 1060 clones.

PURPOSE: To study the gene expression profile of the human trabecular meshwork (HTM). METHODS: A polymerase chain reaction (PCR)-amplified cDNA library was constructed using RNA from the TM of a 67-year-old normal, perfused human eye. A total of 1060 clones were randomly selected for sequencing of one end. These sequences were searched against nonredundant GenBank and dbEST databases for similarity comparison by using a FASTA file and the BLASTcl3 program. Relative expression patterns of those clones that matched other expressed sequence tags (ESTs) were determined using the National Center for Biotechnology Information (NCBI) Unique Human Gene Sequence Collection (UniGene) database. RESULTS: Of the 1060 clones analyzed, 519 (48.9%) had sequences identical with known genes, 125 (11.8%) matched ESTs, and 189 (17.8%) did not match any database sequences. Of the remaining clones, 31 (3%) corresponded to mitochondrial transcripts and 196 (18.5%) to repetitive and noninformative sequences. It is notable that some of the genes highly represented in this library are not ubiquitously expressed in other tissues, which suggests a potentially important role in the HTM. As evidence for the presence of true novel genes in the library, one of the clones was fully sequenced. This clone comprised a complete open reading frame of 966 nucleotides, and its deduced amino acid sequence corresponded to a protein 33% similar to the MAS-related G-protein-coupled receptor. CONCLUSIONS: The identification of the more highly expressed genes in HTM and the discovery of novel genes expressed in this tissue provides basic information for further research on the physiology of the TM and for the identification of glaucoma candidate genes.

Adult↗
PubMed1999

A new eyedrop dispensing bottle.

PURPOSE: To report a new eyedrop dispensing container that allows administration of eyedrops while the head of the patient is in the usual upright position. METHODS: An eyedrop dispensing container with a dip tube extending to the bottom, an automatic venting mechanism, and an angulated dispensing tube was developed. RESULTS: The problem of unwanted efflux of fluid was solved by incorporating the automatic venting mechanism. The final design includes an adjustable, angulated dispensing tube and "single structure" construction incorporating the cap. A removable cheek rest is also provided. The design allows eyedrops to be instilled while the patient's head is in the upright position and even when spectacles are being worn. A prototype 15-ml bottle delivered an eyedrop volume of 19.7+/-1.2 microl (mean +/- SD) (n = 75). CONCLUSION: This new eyedrop bottle should allow precise self-administration of eyedrops by a patient in front of a mirror, without a need to change head position.

Drug Delivery Systems↗
PubMed1999

Adenoviral reporter gene transfer to the human trabecular meshwork does not alter aqueous humor outflow. Relevance for potential gene therapy of glaucoma.

Obstruction of the aqueous humor outflow from the anterior chamber of the eye leads to an elevation of intraocular pressure in glaucoma, the second major cause of blindness worldwide. Our goal is to be able to modulate aqueous humor outflow resistance by gene transfer to the cells of the trabecular meshwork (TM). We have previously shown that adenoviral vectors are able to transfer a reporter gene to the TM of postmortem human donors. However, assessing gene therapy for glaucoma requires models that can monitor changes in aqueous humor outflow facility (C = flow/pressure). In this study we used four replication-deficient adenoviruses in two such perfusion models. In the first model, whole porcine eyes were infected, perfused at constant pressure and flow changes recorded for 5 h. In the second one, anterior segments from human eyes were infected, perfused at constant flow and pressure changes recorded for 3 days. A single dose of 10(8) adenovirus plaque forming units (pfu) causes a reduction in C while single doses of 10(7), 10(6) and 10(5) p.f.u. do not affect outflow facility and retain positive gene transfer. These findings indicate that adenovirus, at effective doses, could become useful vectors for gene therapy of glaucoma.

Adenoviridae↗
PubMed1999

Glucocorticoids regulate transendothelial fluid flow resistance and formation of intercellular junctions.

The regulation of transendothelial fluid flow by glucocorticoids was studied in vitro with use of human endothelial cells cultured from Schlemm's canal (SCE) and the trabecular meshwork (TM) in conjunction with computer-linked flowmeters. After 2-7 wk of 500 nM dexamethasone (Dex) treatment, the following physiological, morphometric, and biochemical alterations were observed: a 3- to 5-fold increase in fluid flow resistance, a 2-fold increase in the representation of tight junctions, a 10- to 30-fold reduction in the mean area occupied by interendothelial "gaps" or preferential flow channels, and a 3- to 5-fold increase in the expression of the junction-associated protein ZO-1. The more resistive SCE cells expressed two isoforms of ZO-1; TM cells expressed only one. To investigate the role of ZO-1 in the aforementioned Dex effects, its expression was inhibited using antisense phosphorothioate oligonucleotides, and the response was compared with that observed with the use of sense and nonsense phosphorothioate oligonucleotides. Inhibition of ZO-1 expression abolished the Dex-induced increase in resistance and the accompanying alterations in cell junctions and gaps. These results support the hypothesis that intercellular junctions are necessary for the development and maintenance of transendothelial flow resistance in cultured SCE and TM cells and are likely involved in the mechanism of increased resistance associated with glucocorticoid exposure.

Body Fluids↗
PubMed1999

Identification and isolation of differentially expressed genes from very small tissue samples.

Identification of differentially expressed genes from tissue samples weighing only a few milligrams has remained a major challenge. Here, we describe a novel and simple strategy that uses standard molecular biology equipment and commercially available kits. The approach combines isolation of total RNA by silica-gel binding, reverse transcription using anchored modified, 5' end enhancers oligonucleotides, exponential amplification of the single-stranded cDNA and hybridization to high-density cDNA filter arrays. The method was tested by comparing genes expressed on freshly isolated human trabecular meshwork tissue with those expressed in corresponding primary cells at third passage. Validation was achieved by using two biological properties: (i) hybridization, to identify the differentially expressed genes, and (ii) PCR amplification, to confirm their distinct expression. The strategy presented allows the identification of differentially expressed genes and/or uncharacterized expressed sequence tags (ESTs) in very small tissue samples, including those from clinical specimens.

Biotechnology↗
PubMed1999

Hydraulic pressure stimulates adenosine 3',5'-cyclic monophosphate accumulation in endothelial cells from Schlemm's canal.

PURPOSE: Fluid flow across various endothelia results in a variety of intracellular and extracellular adaptations. In the living eye, aqueous humor flows across the surface of endothelial cells on trabecular meshwork (TM) beams and in the juxtacanalicular tissue and through or between a continuous monolayer of endothelial cells that line Schlemm's canal (SC). This study was undertaken to test the hypothesis that fluid flow induces biochemical changes in the endothelial cells of the outflow pathway that may modify outflow resistance. METHODS: Trabecular meshwork and SC cells isolated from the outflow pathway of human cadaveric eyes were seeded onto porous filters, placed in Ussing-type chambers, and subjected to fluid flow driven by a pressure head of 15 mm Hg on their apical surface. Cell lysates were prepared and analyzed for adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Barrier function of cell monolayers was examined using transendothelial electrical resistance measurements. RESULTS: Three different SC cell strains in 14 independent experiments responded with at least a threefold increase in cAMP that was both time and pressure dependent. Conversely, flow-treated TM cells failed to respond in six independent experiments in which five different TM cell strains were used. Electrical resistance across cell monolayers positively correlated with cAMP accumulation and was calcium sensitive. CONCLUSIONS: cAMP signaling is affected by pressure differentials across SC cell monolayers and provides evidence for the participation of SC cells in the regulation of aqueous outflow.

Anterior Eye Segment↗
PubMed1999

Modulation of myocilin/TIGR expression in human trabecular meshwork.

PURPOSE: To study factors that modulate myocilin/trabecular meshwork inducible glucocorticoid response protein (TIGR) mRNA expression in human trabecular meshwork (TM). METHODS: mRNA from fresh TM of four human donors, from perfused anterior segment organ cultured TM of three donors, and from four primary TM cell lines of different donors was isolated. The full length cDNA of myocilin/TIGR was cloned from TM mRNA using a polymerase chain reaction approach and used as probe for northern blot analysis hybridization. Trabecular meshwork cell cultures were treated with transforming growth factor (TGF)-beta1 (1 ng/ml), dexamethasone (10(-7) M), and mechanical stretch (10%). RESULTS: mRNA for myocilin/TIGR could be readily detected by northern blot analysis hybridization in 2 to 3 microg of total RNA from all fresh and all organ-cultured TM samples. In contrast, no mRNA for myocilin/TIGR could be detected in 20 microg of total RNA isolated from three different primary TM cell lines. Only one TM cell line had a baseline expression of myocilin/TIGR, which was 35- to 55-fold lower than that of fresh or organ-cultured TM samples. Treatment of TM cell cultures with dexamethasone for 1 day markedly increased expression of myocilin/TIGR mRNA, an effect that was even more pronounced after 3 days of treatment. Treatment with TGF-beta1 for 24 hours had no effect; however, after 3 and 12 days of treatment a 3.8- and 4-fold increase in myocilin/TIGR mRNA expression was observed. Expression of myocilin/TIGR mRNA was also increased after 10% mechanical stretch; however, in contrast to the effects of TGF-beta-1, this effect was observed much earlier (8-24 hours) after treatment. CONCLUSIONS: Dynamic mechanical stimuli maintain myocilin/TIGR expression in TM in situ and lack of these stimuli in monolayer cell cultures might be involved in downregulation of myocilin/TIGR expression.

Adult↗
PubMed1999

Acto-myosin drug effects and aqueous outflow function.

PURPOSE: Previous studies have identified the cytoskeletal proteins actin and tubulin as potential cellular targets in the trabecular meshwork for novel glaucoma therapy. The authors and others have hypothesized that acto-myosin interactions may be important for outflow function. The current study was conducted to evaluate 2,3-butanedione 2-monoxime (BDM), a compound that interferes with acto-myosin function through the myosin adenosine triphosphatase (ATPase) reaction; 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), a proposed myosin light-chain kinase inhibitor; and the direct actin disrupter, latrunculin B, in an outflow pathway cell culture and perfused excised eye model system. METHODS: Freshly enucleated porcine eyes were perfused using the constant-pressure method at 15 mm Hg and 25 degrees C. Human trabecular meshwork (HTM) cells and Schlemm's canal (SC) cells were grown in culture, treated with BDM, H-7, and latrunculin B, and then fixed, stained for beta-tubulin and filamentous actin, and observed by epifluorescence. RESULTS: Twenty millimolar BDM, 100 microM H-7, and 1 microM latrunculin B increased outflow facility 36%, 63%, and 72%, respectively, compared to sham-treated controls, 13%, 15%, and 4% (n=7, 8, and 8; P=0.01, 0.0001, and 0.0002), respectively. In cultured HTM and SC cells, 100 microM H-7 caused a rapid loss of filamentous actin staining but did not produce a change in cell shape or cell- cell attachment. In contrast, 20 mM BDM induced a loss of cell- cell attachment and a change in cell shape that was associated with a 50% to 60% loss of filamentous actin staining, often in a distinct stick-and-ball pattern. Latrunculin B caused a severe loss of actin staining and cell shape changes. No drug altered beta-tubulin staining. CONCLUSIONS: Interference with myosin function can cause a secondary loss of actin organizational structure. Our study indicates that myosin, perhaps through its various phosphorylation reactions, may have a potential regulatory role in outflow function.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1998

Tyrphostins disrupt stress fibers and cellular attachments in endothelial monolayers.

Endothelial permeability, which plays a critical role in many physiologic and pathologic processes, depends on the integrity of intercellular and cell-substrate attachments and the actin cytoskeleton. The proteins located at the cytoplasmic face of adherens and focal contact junctions are rich in sites of tyrosine phosphorylation. To better understand the role of tyrosine phosphorylation in regulating endothelial cell shape, actin stress fibers, and cell junctions, we treated confluent calf pulmonary artery endothelial cells with 14 different tyrphostins, a class of specific tyrosine kinase inhibitors. Using immunofluorescence microscopy to assess cell shape, phosphotyrosine levels, actin stress fibers, and focal contact and junctional proteins, we found that the effects of the tyrphostins could be grouped into three categories. Four tyrphostins had no discernible effect on stress fibers or cell attachments. Seven tyrphostins produced cell retraction with concomitant disruption of both stress fibers and cell-substrate attachments. One member of this group, tyrphostin 25, showed greater specificity for cell-cell junctions than the others, causing cell separation without significantly affecting actin stress fibers or focal contacts. The third group of tyrphostins had the opposite effect, completely disrupting stress fibers and focal contacts without causing cell separation. The ability of specific tyrphostins to disrupt cell-cell or cell-substrate attachments and/or actin stress fibers implies that a certain steady-state level of tyrosine phosphorylation is necessary to maintain these structures and that there may be independent tyrosine kinase signaling pathways controlling them. Comparison of the phosphotyrosinated proteins affected by each group of tyrphostins should provide a useful new approach toward understanding the regulation of endothelial cell-cell and cell-substrate junctions.

Actins↗
PubMed1998

Microtubule disruption leads to cellular contraction in human trabecular meshwork cells.

PURPOSE: To determine whether microtubule- and actin-altering drugs, which have been shown to increase aqueous humor outflow, cause cellular contraction in human trabecular meshwork (HTM) cells. METHODS: HTM cells were plated in culture dishes containing a polymerized deformable silicone substrate. After 48 hours, the dishes were placed on an inverted microscope and treated with ethacrynic acid, colchicine, vinblastine, cytochalasin B, or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and then recorded on videotape for 15 minutes. An increase in silicone substrate wrinkle size and/or number indicated a contraction. Sham controls were used. RESULTS: Cellular contraction was observed with ethacrynic acid, colchicine, and vinblastine in the 10(-5) to 10(-4) M dosage range. Pretreatment with H-7 blocked these effects. Cytochalasin B did not produce cellular contraction. CONCLUSIONS: Microtubule disruption causes cellular contraction in HTM cells, and this effect depends on an intact actin cytoskeleton network. Contraction of trabecular meshwork cells in response to various stimuli is an attractive hypothesis for possible homeostatic mechanisms in the outflow pathway, and this may serve as a focus for novel glaucoma drug development.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1998

Mechanical stretch alters the actin cytoskeletal network and signal transduction in human trabecular meshwork cells.

PURPOSE: Human trabecular meshwork (HTM) cells were mechanically stretched in vitro as a potential model for the distension of this tissue that can occur in vivo in response to increased pressure gradients. Cell morphology and certain components of the signal transduction pathways, including the mitogen-activated protein kinase (MAPK) and c-Jun N-terminal protein kinase (JNK) pathways, were evaluated for stretch-induced alterations. METHODS: Primary HTM cells grown in tissue culture were subjected to a mechanical stretch lasting from 10 seconds to 4 days. The actin cytoskeletal network was visualized by phalloidin staining. Proteins phosphorylated on their tyrosine residues were isolated using an immunoaffinity system and were analyzed by gel electrophoresis and immunostaining. Mitogen-activated protein kinase activity was evaluated using an in-gel assay system, and the mRNA levels of c-fos and c-jun were determined by quantitation of competitive reverse transcription-polymerase chain reaction. In addition, the amount of c-Fos protein was estimated by chemiluminescent immunoblot analysis. RESULTS: On stretching, the HTM cells elongated but regained their normal morphologic characteristics within 24 hours. Unstretched HTM cells displayed a diffuse F-actin microfilament network, whereas stretched cells exhibited complex geodesic patterns. Ten seconds after stretching began, the level of tyrosine phosphorylation on the six major phosphoproteins significantly decreased between 80% and 100%, whereas the level of paxillin tyrosine phosphorylation significantly increased 39%. Stretching caused MAPK activity and the amount of mRNA and protein of the immediate-early gene c-fos to decrease more than 60% within 2 minutes, but within 15 to 30 minutes they increased above or equivalent to normal levels. The level of c-jun mRNA was unchanged by stretching. CONCLUSIONS: In response to a mechanical stretch, major cytoskeletal alterations occur in HTM cells, which involve changes in the levels of tyrosine phosphorylation. Mechanotransduction (signal transduction by mechanical stimulation) through the MAPK signaling pathway was significantly depressed immediately after stretching; however, the JNK pathway appeared to be unaffected. The data suggest that HTM cells adapt to mechanical stress by altering the cytoskeletal network and signaling cascades.

Actin Cytoskeleton↗