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Biomedical subjects

I M Herman

Publications and source records attributed to I M Herman.

At least 19 recordsLinked to original sources
PubMed1999

Inhibition of calpain blocks platelet secretion, aggregation, and spreading.

Previous studies have indicated that the Ca(2+)-dependent protease, calpain, is activated in platelets within 30-60 s of thrombin stimulation, but specific roles of calpain in platelets remain to be identified. To directly test the functions of calpain during platelet activation, a novel strategy was developed for introducing calpain's specific biological inhibitor, calpastatin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrating signal sequence from Kaposi's fibroblast growth factor connected to a calpain-inhibiting consensus sequence derived from calpastatin. Calpastat specifically inhibits thrombin peptide (SFLLR)-induced alpha-granule secretion (IC(50) = 20 microM) during the first 30 s of activation, thrombin-induced platelet aggregation (IC(50) = 50 microM), and platelet spreading on glass surfaces (IC(50) = 34 microM). Calpastat-Ala, a mutant peptide in which alanine is substituted at conserved calpastatin residues, lacks calpain inhibitory activity and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secretion, aggregation and spreading, but require 3-10-fold higher concentrations than calpastat for biological activity. Together, these findings demonstrate that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation following thrombin receptor stimulation than had been previously detected.

Amino Acid Sequence↗
PubMed1998

Calpain regulates actin remodeling during cell spreading.

Previous studies suggest that the Ca2+-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3- derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading.

3T3 Cells↗
PubMed1998

Sorting of beta-actin mRNA and protein to neurites and growth cones in culture.

The transport of mRNAs into developing dendrites and axons may be a basic mechanism to localize cytoskeletal proteins to growth cones and influence microfilament organization. Using isoform-specific antibodies and probes for in situ hybridization, we observed distinct localization patterns for beta- and gamma-actin within cultured cerebrocortical neurons. beta-Actin protein was highly enriched within growth cones and filopodia, in contrast to gamma-actin protein, which was distributed uniformly throughout the cell. beta-Actin protein also was shown to be peripherally localized after transfection of beta-actin cDNA bearing an epitope tag. beta-Actin mRNAs were localized more frequently to neuronal processes and growth cones, unlike gamma-actin mRNAs, which were restricted to the cell body. The rapid localization of beta-actin mRNA, but not gamma-actin mRNA, into processes and growth cones could be induced by dibutyryl cAMP treatment. Using high-resolution in situ hybridization and image-processing methods, we showed that the distribution of beta-actin mRNA within growth cones was statistically nonrandom and demonstrated an association with microtubules. beta-Actin mRNAs were detected within minor neurites, axonal processes, and growth cones in the form of spatially distinct granules that colocalized with translational components. Ultrastructural analysis revealed polyribosomes within growth cones that colocalized with cytoskeletal filaments. The transport of beta-actin mRNA into developing neurites may be a sequence-specific mechanism to synthesize cytoskeletal proteins directly within processes and growth cones and would provide an additional means to deliver cytoskeletal proteins over long distances.

Actins↗
PubMed1998

beta-Actin is confined to structures having high capacity of remodelling in developing and adult rat cerebellum.

Neurons undergo complex morphological changes during differentiation and in cases of plasticity. A major determinant of cell morphology is the actin cytoskeleton, which in neurons is comprised of two actin isoforms, non-muscle gamma- and beta-actin. To better understand their respective roles during differentiation and plasticity, their cellular and subcellular localization was examined in developing and adult cerebellar cortex. It was observed that gamma-actin is expressed at a constant level throughout development, while the level of beta-actin expression rapidly decreases with age. At the light microscopic level, gamma-actin staining is ubiquitous and the only developmental change observed is a relative reduction of its concentration in cell bodies and white matter. In contrast, beta-actin staining almost completely disappears from the cytoplasm of cell bodies, primary dendrites and axons. In young cerebellar cultures, gamma-actin is found in the cell body, neurites and growth cones, while beta-actin is mainly found in growth cones, as previously reported in other primary neuronal culture systems [Kaech et al. (1997), J. Neuroscience, 17, 9565-9572; Bassell et al., (1998), J. Neuroscience, 18, 251-265]. Electron microscopy of post-embedding immunogold-labelled tissue confirms the widespread distribution of gamma-actin, and also reveals an increased concentration of gamma-actin in dendritic spines in the adult. During development, beta-actin accumulation is observed in actively growing structures, e.g., growth cones, filopodia, cell bodies and axonal tracts. In the adult cerebellar cortex, beta-actin is preferentially found in dendritic spines, structures which are known to retain their capacity for morphological modifications in the adult brain. This differential subcellular localization and developmental regulation of the two actin isoforms point to their different roles in neurons.

Actins↗
PubMed1998

The mechanics of vascular cell motility.

Alterations in vascular cell shape and motility occur during developmental processes and in response to injury. Similarly, during tumor vascularization and atherogenesis, endothelial and smooth muscle cells undergo motile and proliferative responses to extracellular cues. Recent inroads into our understanding of signal transduction have identified several candidate pathways by which the extracellular matrix- and growth factor-mediated stimulation of vascular cell motility may be mediated. The multiple and divergent extracellular stimuli that stimulate vascular motile responses may converge on the cytoskeleton via a family of ras-related GTPases. Biochemical analyses as well as examination of cytoskeletal dynamics in vivo indicate that actin polymerization at the forward aspects of spreading cytoplasm is capable of driving forward protrusion formation in the absence of a conventional actin motor. Actin polymerization at the plasma membrane of leading lamellae may be mediated both by de novo nucleation of actin filaments and the generation of free filament ends by uncapping the barbed ends of existing actin filaments. This review summarizes the most recent findings in extracellular-cytoskeletal-signal transduction, therein, providing a framework to explain the remarkable remodeling seen in the vasculature during developmental and disease-related processes.

Actin Cytoskeleton↗
PubMed1997

Measurement of glucose consumption by hybridoma cells growing in hollow fiber cartridge bioreactors: use of blood glucose self-monitoring devices.

Two different types of blood glucose self-monitoring device, designed for use by diabetics (Accu-Chek Advantage* and Accu-Chek III*), were evaluated for the purpose of monitoring glucose concentration in tissue culture media. The Accu-Chek Advantage* meter was found to systematically overestimate the glucose concentration in a variety of commonly used tissue culture media by 50-90%, in comparison with their formulated glucose concentration. The Accu-Chek III* meter reliably estimated glucose concentrations from 100 to 300 mg/dl and overestimated glucose concentrations above 300 mg/dl. The systematic overestimation of glucose concentration in tissue culture fluids by the Accu-Chek Advantage* meter was further investigated. A standard curve was constructed and the meter reading was found to be linearly related to the actual glucose concentration and the best linear fit was given by the formula y = -43.504 + 1.9246x, where y is the meter reading and x is the actual glucose concentration. Rearranging the equation to make x the subject gave the following algorithm x = (y + 43.504) divided by 1.9246 which could be used to correct the 'raw' meter reading. The mean corrected glucose concentrations deviated from the formulated glucose concentration by less than 3.5% in five media tested, indicating that this meter is more than adequate for monitoring glucose consumption by cells growing in hollow fiber cartridge bioreactors, when used in conjunction with this correction factor.

Animals↗
PubMed1996

Phalloidin binding and rheological differences among actin isoforms.

Actin is a highly conserved protein in eukaryotes, yet different isoforms of this protein can be found within the same cell. To begin to explore whether isoactin sequence diversity leads to functional differences in actin filaments, we have examined the phalloidin binding kinetics and the bulk rheologic properties of purified actin isoforms from a variety of eukaryotic sources. We observe differences in the phalloidin association kinetics between muscle alpha- and cytoplasmic actins. Phalloidin dissociates from all mammalian actin isoforms tested at the same slow rate, while dissociation from yeast actin is 1 order of magnitude more rapid. The actin isoforms form viscoelastic gels to varying degrees with skeletal muscle alpha-actin gels being the most elastic, smooth muscle alpha- and gamma-actins being less elastic, and beta-actin not forming elastic structures under our experimental conditions. The sequence variation among isoforms is discussed in light of these biophysical and biochemical differences.

Actins↗
PubMed1996

Beta cap73: a novel beta actin-specific binding protein.

Whereas actin-binding proteins (ABPs) regulate network formation during the cell cycle, it is not known whether ABPs also function to sequester or target isoactins to specific subcellular compartments. Recently, we have shown that ezrin indirectly associates with beta, but not alpha actin filaments in a calcium- and cytochalasin-sensitive manner [Shuster and Herman, 1995: J. Cell Biol. 128:837-848]. To identify the beta actin-specific binding protein that fosters ezrin-beta actin interactions, we developed an isoactin affinity fractionation and F-isoactin overlay/Western blotting technique. Results reveal that a 73 kd polypeptide that co-precipitates with ezrin and beta actin [Shuster and Herman, 1995: J. Cell Biol. 128:837-848] can also binds directly to filaments of beta, but not alpha actin by isoactin overlay. In an effort to establish whether p73 plays a role in regulating beta actin dynamics in cells, we produced monoclonal antibodies by immunizing BALB/c mice with p73-containing lamellar lysates or high salt elutions from beta actin affinity columns. Two monoclonal antibodies were cloned that react with p73 present in fractions released from beta actin Sepharose-4B or purified to homogeneity by DEAE chromatography. Anti-p73 Western blots reveal that there is a 16-fold difference in p73 binding to beta actin vs. alpha actin affinity columns when experiments are performed in physiological salts. To characterize p73-beta actin binding in vitro and establish whether p73 binds along the lengths or at the barbed end of the beta actin filament, we asked whether cytochalasin D (CD) could displace p73 pre-bound to beta actin-Sepharose 4B. Anti-p73 Western blotting reveals that nanomolar concentrations of CD are capable of selectively eluting p73 and ezrin from beta actin Sepharose 4B, indicating that p73 binds beta actin via the barbed end. Simultaneous double antibody localization studies using anti-beta actin IgG and anti-p73 IgM reveal that p73 and beta actin are co-localized in the forward aspects of motile cytoplasmic domains, in close proximity to the plasma membrane. Because of its isoform-specific interactions with the barbed end of beta actin filaments, we have named this molecule beta cap73. These results indicate that isoform-specific actin-binding proteins can be identified from cortical cytoplasm, and suggest that beta cap73 may not only act to spatially regulate the intracellular distribution of isoactins, but may also facilitate forward protrusion formation through the regulated release of free filament ends during cell motility.

Actins↗
PubMed1995

Indirect association of ezrin with F-actin: isoform specificity and calcium sensitivity.

Whereas it has been demonstrated that muscle and nonmuscle isoactins are segregated into distinct cytoplasmic domains, the mechanism regulating subcellular sorting is unknown (Herman, 1993a). To reveal whether isoform-specific actin-binding proteins function to coordinate these events, cell extracts derived from motile (Em) versus stationary (Es) cytoplasm were selectively and sequentially fractionated over filamentous isoactin affinity columns prior to elution with a KCl step gradient. A polypeptide of interest, which binds specifically to beta-actin filament columns, but not to muscle actin columns has been conclusively identified as the ERM family member, ezrin. We studied ezrin-beta interactions in vitro by passing extracts (Em) over isoactin affinity matrices in the presence of Ca(2+)-containing versus Ca(2+)-free buffers, with or without cytochalasin D. Ezrin binds and can be released from beta-actin Sepharose-4B in the presence of Mg2+/EGTA and 100 mM NaCl (at 4 degrees C and room temperature), but not when affinity fractionation of Em is carried out in the presence of 0.2 mM CaCl2 or 2 microM cytochalasin D. N-acetyl-(leucyl)2-norleucinal and E64, two specific inhibitors of the calcium-activated protease, calpain I, protect ezrin binding to beta actin in the presence of calcium. Moreover, biochemical analysis of endothelial lysates reveals that a calpain I cleavage product of ezrin emerges when cell locomotion is stimulated in response to monolayer injury. Immunofluorescence analysis of leading lamellae reveals that anti-ezrin and anti-beta-actin IgGs can be simultaneously co-localized, extending the results of isoactin affinity fractionation of Em-derived extracts and suggesting that ezrin and beta-actin interact in vivo. To test the hypothesis that ezrin binds directly to beta-actin, we performed three sets of studies under a wide range of physiological conditions (pH 7.0-8.5) using purified pericyte ezrin and either alpha- or beta-actin. These included co-sedimentation, isoactin affinity fractionation, and co-immunoprecipitation. Results of these experiments reveal that purified ezrin does not directly bind to beta-actin filaments, either in solution or while isoactins are covalently cross-linked to Sepharose-4B. This is in contrast to our finding that ezrin and beta-actin could be co-immunoprecipitated or co-sedimented from Em-derived cell lysates. To explore whether calcium transients occur in cellular domains enriched in ezrin and beta-actin, we mapped cellular free calcium in endothelial monolayers crawling in response to injury.(ABSTRACT TRUNCATED AT 400 WORDS)

Actin Cytoskeleton↗
PubMed1994

Optic nerve injury alters basic fibroblast growth factor localization in the retina and optic tract.

Basic fibroblast growth factor (bFGF) is thought to be a trophic factor for several classes of neurons. Its distribution changes in response to cortical neural injury. We have determined the effect of injury to the optic nerve on localization of bFGF in the rodent retina and visual pathways. Our observations were confirmed by using different antisera and monoclonal antibodies. While photoreceptors normally contain virtually no bFGF, crushing the optic nerve causes a striking increase, over a period of several weeks, in the amount of bFGF in retinal photoreceptors. Since photoreceptors do not synapse directly upon the injured ganglion cells, intermediary cells must participate in the cascade of events that results in the elevated bFGF. In light of the observation that exogenous bFGF protects photoreceptors from photodamage (Faktorovich et al., 1992), this increase in bFGF in photoreceptors may explain, in part, why crushing the optic nerve protects photorecptors against photodamage (Bush and Williams, 1991). Whereas bFGF is constitutively found in glia in the optic nerve, little bFGF is found in glia in the optic tract. However, damage to the optic nerve increases bFGF in astrocytes in the optic tract. This change occurs within days, suggesting that a relatively direct signal may intervene between the injured axon and the adjacent glial cells. Thus, despite the fact that the optic nerve and optic tract are contiguous structures through which axons of retinal ganglion cells project, the glial elements in these structures express distinct properties, because of differences in either glial subclasses or microenvironment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Pericyte growth and contractile phenotype: modulation by endothelial-synthesized matrix and comparison with aortic smooth muscle.

We compared the effects of endothelial-synthesized matrix and purified matrix molecules on pericyte (PC) and aortic smooth muscle cell (SMC) growth, heparin sensitivity, and contractile phenotype in vitro. When PC are plated on endothelial-synthesized (EC) matrix, cell number is, on average, 3.1-fold higher than identical populations grown on plastic. Under the same conditions, SMC proliferation is stimulated 1.6-fold. Purified matrix molecules, such as collagen type IV (COLL) or fibronectin (FN), both major components of the EC matrix, stimulate PC/SMC growth 1.2-1.7-fold. Heparin (100 micrograms/ml), which inhibits the growth of early passage SMC by 60%, inhibits PC growth approximately 50%, when cells were plated on plastic. However, PC plated on EC matrix in the presence of heparin (100 micrograms/ml) grow as well as parallel cultures grown on plastic (in the absence of heparin). Concomitant with matrix-stimulated proliferation, we observed a marked reduction in PC containing alpha vascular smooth muscle actin (alpha VSMA), as seen by immunofluorescence using affinity-purified antibodies (173/615 positive pericytes on DOC matrix (28%) vs. 221/285 (77%) positive on glass). SMC respond similarly. Whereas alpha VSMA protein is markedly altered when PC and SMC are cultured on EC matrix, similar reductions in mRNA are not observed. However, Northern blotting does reveal that PC contain 17-30 times the steady-state levels of alpha VSMA mRNA compared to SMC. When SMC and PC cultures on plastic are treated with heparin, the steady-state levels of vascular smooth muscle actin mRNA increase 5 and 1.5 fold, respectively. Similarly, heparin treatment of PC grown on plastic induces a 1.8 fold increase in nonmuscle actin mRNA. These heparin-induced alterations in isoactin mRNA levels are not seen when PC are cultured on EC matrix. We also observed reductions in alpha VSMA and beta actin mRNA levels when PC are plated on FN, where they maintain a ratio of 13:1 (alpha:beta). Similar ratios are found in SMC present in rat and bovine aortae in vivo. These steady-state isoactin mRNA ratios are slightly different from those seen in cultured PC (8-10:1; alpha:beta). These results suggest that selective synthesis and remodelling of the endothelial basal lamina may signal alterations in pericyte growth and contractile phenotype during normal vascular morphogenesis, angiogenesis, or during the microvascular remodelling that accompanies hypertensive onset.

Actins↗
PubMed1993

Actin isoforms.

The actin supergene family encodes a number of structurally related, but perhaps functionally distinct, protein isoforms that regulate contractile potential in muscle tissues and help to control the shape as well as the motility of non-muscle cells. In spite of the documented conservation amongst isoactin genes and their encoded proteins, recent results of biochemical, antibody localization, molecular mutagenesis and isoactin gene replacement studies lend credence to the notion that functional differences amongst muscle and non-muscle actin isoforms exist. Furthermore, the discovery of a new class of actin isoforms, the actin-related proteins, reveals that the actin gene and protein isoform family is more complex than was previously believed.

Actins↗
PubMed1993

Molecular mechanisms regulating the vascular endothelial cell motile response to injury.

Vascular endothelial cell (EC) wound healing was characterized on an EC-synthesized extracellular matrix (ECM) previously treated with enzymes and antibodies specific for ECM components. Using a computer-assisted video-microscope recording system capable of automatic EC recognition, we learned whether components of the EC-synthesized matrix influenced post-injury migration and wound healing in vitro. Localization of actin and its encoded mRNA using isoform-specific antibodies and labeled cDNA probes allowed for a direct correlation of living-cell behavior with cytoskeletal form and distribution. Results of these studies indicate that the computer-assisted EC tracking system allows for an automatic and reproducible analysis of EC behavior following injury in vitro. EC migrate fastest immediately following injury and then achieve a new, slower migration rate that is maintained until EC from one edge of 200- to 300-microns-wide wound zone contact EC from the other wound face. Treatment of EC-synthesized matrices with antibodies against fibronectin and laminin has no effect on EC migration following injury (-0.25 microns/min) or on cytoskeletal array. Similarly, digestion of these matrices with heparinase and hyaluronidase has no effect on wound healing rates. Slowly spreading EC cytoplasm, which borders the intact and antibody-treated EC matrices, is rich in actin but lacks myosin II. Two different preparations of collagenase (bacterial and mammalian) each potentiate EC wound healing in vitro. Bacterial collagenase treatment of the EC-synthesized matrices potentiates EC migration fivefold (1 micron/min) while treatment of EC-matrices with mammalian cell collagenase stimulates EC migration following injury some twofold (0.4 micron/min) over control values. Whereas EC on control matrices migrate in unison as a tissue-like sheet, EC on the collagenase-treated EC matrices migrate as individuals. Concomitant with the increased rates of migration following injury on the collagenase-treated EC-matrices is a two- to fourfold increase in the steady-state levels of beta-actin mRNA. This increase in actin mRNA abundance is observable by its preferential localization (seen by in situ hybridization) in the lamellae bordering the wound edge in association with beta-actin, which is exclusively localized there. Because beta-actin and its encoded mRNA are positioned together in association with the plasma membrane in regions of moving cytoplasm, it seems likely that beta-actin filament assembly is required for motility following endothelial injury.

Actins↗
PubMed1993

Bone morphogenetic protein expression in human atherosclerotic lesions.

Artery wall calcification associated with atherosclerosis frequently contains fully formed bone tissue including marrow. The cellular origin is not known. In this study, bone morphogenetic protein-2a, a potent factor for osteoblastic differentiation, was found to be expressed in calcified human atherosclerotic plaque. In addition, cells cultured from the aortic wall formed calcified nodules similar to those found in bone cell cultures and expressed bone morphogenetic protein-2a with prolonged culture. The predominant cells in these nodules had immunocytochemical features characteristic of microvascular pericytes that are capable of osteoblastic differentiation. Pericyte-like cells were also found by immunohistochemistry in the intima of bovine and human aorta. These findings suggest that arterial calcification is a regulated process similar to bone formation, possibly mediated by pericyte-like cells.

Animals↗
PubMed1992

Preparation of fluorescent basic fibroblast growth factor: localization in living retinal microvascular endothelial cells.

A biologically active fluorescent derivative of recombinant human basic fibroblast growth factor (bFGF) was prepared by immobilization on heparin-Sepharose 4B (HS) and derivatization with the fluorophore, Texas Red (TR). TR-bFGF was separated from free dye and carrier protein by elution from HS using 1.5 M NaCl. TR-bFGF contained an average of two dye molecules bound per bFGF, retained its mitogenic activity and was visible using a fluorescence microscope equipped with silicon intensified target camera (SIT). TR-bFGF stimulated the growth of bovine aortic endothelial cells (BAEC), microvessel endothelial cells (MVEC) and BHK-21 cells grown in culture. BAEC, MVEC and BHK-21 cells treated with 20 ng ml-1 (1 nM) TR-bFGF for 72 hr were stimulated over serum controls by 87, 26 and 6%, respectively. TR-bFGF stimulated EC growth was inhibited in a dose-dependent fashion when cells were coincubated with microM chloroquine. When EC were treated with TR-bFGF at 4 degrees C and then monitored at 37 degrees C, bright, focal, cytoplasmic spots were observed, which accumulated as punctate, perinuclear fluorescence. EC internalization of TR-bFGF was inhibited 80% by the addition of 100-fold molar excess unlabeled bFGF or by maintaining cultures at 4 degrees C. TR-bFGF colocalized with an EC lysosomal marker, but TR-bFGF was not detected in the nucleus. Results of these localization studies suggest that TR-bFGF stimulates EC proliferation without entering the nucleus.

Cell Division↗
PubMed1992

Radial keratotomy. II. Role of the myofibroblast in corneal wound contraction.

The cellular mechanism of corneal wound contraction after radial keratotomy (RK) was studied in a feline eye model. A total of 10 cat eyes were evaluated at various times from 0-30 days after surgery. Changes in the distribution of intracellular filamentous actin, nonmuscle myosin, alpha-actinin, surface membrane alpha 5 beta 1 integrin, and extracellular fibronectin were studied using immunofluorescence and laser confocal and electron microscopy. From day 3-7, staining for fibronectin increased along the wound margin. By day 7, keratocytes adjacent to the wound margin showed increased f-actin staining with intense staining for fibronectin compared with normal keratocytes. Myosin and alpha 5 beta 1 integrin expression was very weak at this time; alpha-actinin was not found. By day 14, fibroblasts within the wound formed f-actin microfilament bundles (stress fibers) which colocalized with fibronectin. Wound-healing fibroblasts also stained positively for alpha 5 beta 1 integrin, myosin, and alpha-actinin (the latter two were colocalized). The presence of myosin and alpha-actinin in the wound fibroblasts and the re-organization of f-actin into stress fibers by day 14 correlated with the development of wound contraction. A comparison of the cellular distribution of actin, myosin, and alpha-actinin with alpha 5 beta 1 integrin 14 days after injury suggested that integrin was localized along stress fiber bundles during wound contraction. The data from this study suggest that modulation of wound gape during healing of RK wounds may involve transformation of the corneal keratocyte to a myofibroblast-like cell and the subsequent formation of intracellular stress fibers composed of f-actin, nonmuscle myosin, and alpha-actinin. Based on the colocalization of fibronectin filaments and f-actin filaments and the unique distribution of alpha 5 beta 1 integrin, these findings support the hypothesis that the tension within the wound is generated by the formation of intracellular stress fibers and the interactions between stress fibers and the extracellular matrix, mediated by specific membrane receptor molecules.

Animals↗
PubMed1992

Density-dependent accumulation of basic fibroblast growth factor in the subendothelial matrix.

Recent evidence indicates that basic fibroblast growth factor (bFGF), which lacks a conventional signal recognition sequence, is a component of the subendothelial matrix. However, the molecular mechanisms regulating its cellular release and subsequent matrix deposition remain equivocal. To examine the cellular and subcellular mechanisms regulating bFGF release and subendothelial sequestration, we generated polyclonal antibodies against a chemically cross-linked bFGF. We then used anti-bFGF IgG in conjunction with 3T3 cell [3H]thymidine incorporation assays, enzyme immunoassays and immunofluorescence to learn whether bFGF accumulation in the subendothelial matrix is dependent upon endothelial cell (EC)-cell contact, which coincides with growth arrest. In contrast to subconfluent cultures, which lacked any detectable extracellular matrix bFGF localization, bovine aortic and microvascular EC plated at confluent densities displayed a punctate extracellular staining pattern that was abolished when EC were pretreated with 10 micrograms/ml cycloheximide. Additionally, when EC were treated with either 1 mM beta-D xyloside, an inhibitor of proteoglycan assembly, or 100 micrograms/ml heparin, there was a 40% reduction in matrix-associated bFGF (quantified by image analysis of antibody stained cultures). 3T3 [3H]thymidine incorporation assays indicated that the beta-D xyloside-induced reduction of matrix-associated bFGF coincided with a significant increase in bFGF activity in the conditioned media. Neither sparsely-plated nor confluent EC cultures possessed specific bFGF localization of the nuclear compartment when cells were fixed using cold methanol; however, when EC were fixed in formaldehyde and lysed in isotonic buffers containing 0.1% Triton X-100 or absolute acetone, there was a marked decrease in anti-bFGF staining of the postconfluent extracellular matrix and a concomitant increase in nuclear fluorescence. Because bFGF-stimulated vascular cell growth has been implicated in controlling neointimal cell proliferation, we screened normal and atherosclerotic coronary blood vessels for bFGF, but we were unable to detect it either in lesioned or normal intima. In contrast, significant bFGF levels were observed in association with the EC and mesangial cells of the renal corpuscle, where heparan sulfate accumulates within the glomerular basement membrane. Our in vitro results suggest that bFGF accumulates within the proteoglycan-containing subendothelial matrix concomitant with the formation of cell-cell contacts. In situ, the composition of the microvascular matrix and the cellular phenotype may facilitate the selective accumulation of bFGF that we observed. This, in turn, may influence vascular morphogenesis and remodeling during angiogenesis.

3T3 Cells↗