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Biomedical subjects

F Grinnell

Publications and source records attributed to F Grinnell.

At least 19 recordsLinked to original sources
PubMed1999

Release of mechanical tension triggers apoptosis of human fibroblasts in a model of regressing granulation tissue.

In an in vitro model of granulation tissue, early passage human diploid fibroblasts under mechanical tension showed little or no apoptosis. Release of mechanical tension triggered an apoptotic response that occurred within 3-6 h and reached a plateau by 24 h. The percentage of apoptotic cells (approximately 15%) remained constant up to 7 days, and after 3 days, total cell number declined. Identification of mechanical unloading as a stimulus for apoptosis, without application of pharmacologic or genetic intervention, is a novel observation that permits us to model similar events that occur during wound healing. Studies on the mechanism regulating apoptosis under these conditions established that the apoptotic response does not require differentiation of cells into myofibroblasts but is governed by a combination of mechanical tension and growth factors in the collagen matrix.

Actins↗
PubMed1999

Differences in the regulation of fibroblast contraction of floating versus stressed collagen matrices.

To learn more about the regulation of contraction of collagen matrices by fibroblasts, we compared the ability of lysophosphatidic acid (LPA) and platelet-derived growth factor (PDGF) to stimulate contraction of floating and stressed collagen matrices. In floating collagen matrices, PDGF and LPA stimulated contraction with similar kinetics, but appeared to utilize complementary signaling pathways since contraction obtained by the combination of growth factors exceeded that observed with saturating concentrations of either alone. The PDGF-simulated pathway was selectively inhibited by the protein kinase inhibitor KT5926. In stressed collagen matrices, PDGF and LPA stimulated contraction with different kinetics, with LPA acting rapidly and PDGF acting only after an approximately 1-h lag period. Pertussis toxin, known to block signaling through the Gi class of heterotrimeric G-proteins, inhibited LPA-stimulated contraction of floating but not stressed matrices, suggesting that LPA-stimulated contraction depends on receptors coupled to different G-proteins in floating and stressed matrices. On the other hand, the Rho inhibitor C3 exotransferase blocked contraction of both floating and stressed collagen matrices. These results suggest the possibility that distinct signaling mechanisms regulate contraction of floating and stressed collagen matrices.

ADP Ribose Transferases↗
PubMed1998

Decreased PDGF receptor kinase activity in fibroblasts contracting stressed collagen matrices.

Fibroblasts cultured in mechanically stressed collagen matrices proliferate, whereas cells in floating collagen matrices become quiescent. Previous research indicated that one factor contributing to cell quiescence in floating matrices was reduced receptor autophosphorylation in response to PDGF stimulation (i.e., PDGF receptor desensitization). To learn more about the mechanism of PDGF receptor desensitization, we analyzed changes in PDGF receptor autophosphorylation and receptor kinase activity after stressed collagen matrices were switched to floating conditions, which results in rapid cell contraction and dissipation of mechanical stress. PDGF receptor desensitization occurred during contraction stimulated by serum but not in the absence of serum, and desensitization was prevented by inhibitors of contraction but not by inhibitors of the contraction-activated cyclic AMP signaling pathway. Receptor desensitization resulted from decreased receptor kinase activity rather than from elevated protein tyrosine phosphatase activity, and only receptors unoccupied at the time of contraction were affected. After contraction, radiolabeled PDGF binding to the cells was decreased, which suggested that receptor desensitization resulted from a contraction-dependent change in receptor availability or affinity.

Cells, Cultured↗
PubMed1998

Identification of proteinase 3 as the major caseinolytic activity in acute human wound fluid.

Wound fluid contains several proteinases that are important in the repair process. In this study, we analyzed caseinolytic activity in wound fluid obtained from acute (burn) wounds. Caseinolytic activity in wound fluid increased markedly 2 d after injury and appeared on casein zymographs as a series of bands or a smear ranging from 30 to 100 kDa. Most of the enzyme activity was inhibited by the synthetic human neutrophil elastase inhibitor MDL 27,367 but not by the naturally occurring inhibitor of elastase, human secretory leukoproteinase inhibitor. Fractionation of wound fluid indicated that a single enzyme accounted for approximately 80% of the caseinolytic activity. This enzyme degraded the elastase substrate methoxysuccinyl-ala-ala-pro-val-p-nitroanilide at a slow rate. The above findings suggested that the enzyme responsible for caseinolytic activity might be proteinase 3, an elastase-related enzyme whose physiologic functions are poorly understood. Consistent with the above possibility, we found that monoclonal antibodies against proteinase 3 removed caseinolytic activity from wound fluid, and that purified proteinase 3 had a similar caseinolytic profile and inhibitor sensitivity to burn fluid.

Antibodies, Monoclonal↗
PubMed1998

Collagenase-1 complexes with alpha2-macroglobulin in the acute and chronic wound environments.

The purpose of this study was to examine the appearance and activation of collagenase-1 (MMP-1) in the wound environment. We found that MMP-1 accumulates in the fluid phase of the burn wound environment within 2 d of injury and reaches maximal levels by day 4. Two forms of the enzyme were evident; one that corresponded to proMMP-1 and another that corresponded to a group of high molecular mass (approximately 200 kDa and >200 kDa doublet) MMP-1 containing complexes. ProMMP-1 and MMP-1 containing complexes also occurred in wound fluid from venous stasis ulcers, but neither was detected in mastectomy fluid or in plasma. Levels of the proteinase inhibitor alpha2-macroglobulin in burn fluid and chronic ulcer wound fluid were almost as high as in plasma, and the high molecular mass MMP-1 containing complexes in burn fluid appeared to result from binding between alpha2-macroglobulin and activated MMP-1. These observations provide direct evidence that active MMP-1 in the fluid phase of the wound environment becomes complexed to alpha2-macroglobulin.

Acute Disease↗
PubMed1998

Increased c-fos mRNA expression by human fibroblasts contracting stressed collagen matrices.

We studied early changes in gene expression during fibroblast contraction of stressed collagen matrices. The level of c-fos mRNA increased dramatically and peaked 50 to 60 min after matrix contraction was initiated. This response did not require serum and could not be accounted for simply by disruption of the actin cytoskeleton. Increased c-fos mRNA levels required Ca2+ influx but not the cyclic AMP or extracellular signal-regulated kinase (ERK 1/2) signaling pathways, both of which are activated when fibroblasts contract stressed collagen matrices. The levels of two other immediate-early genes, fosb and c-jun, also increased transiently after fibroblast contraction, whereas the levels of fra-1, fra-2, c-myc, and the transcription factor NF-kappaB remained the same, indicating that fibroblast contraction caused changes in a selective group of genes. The increase in c-fos mRNA during contraction of stressed collagen matrices may reflect a unique role for c-fos in mechanoregulated events at the end of wound repair.

Biomechanical Phenomena↗
PubMed1998

Disinfection of latex gloves with ethyl alcohol.

The ability to disinfect latex gloves successfully between procedures would save time and be cost-effective. An in vitro study examined the efficacy of using an ethyl alcohol/bactericide compound to disinfect latex gloves contaminated with five common bacteria.

Anti-Bacterial Agents↗
PubMed1997

Fibroblasts contracting collagen matrices form transient plasma membrane passages through which the cells take up fluorescein isothiocyanate-dextran and Ca2+.

When fibroblasts contract collagen matrices, the cells activate a Ca(2+)-dependent cyclic AMP signaling pathway. We have found that contraction also stimulates uptake of fluorescein isothiocyanate-dextran molecules from the medium. Our results indicate that fluorescein isothiocyanate-dextran enters directly into the cell cytoplasm through 3- to 5-nm plasma membrane passages. These passages, which reseal in less than 5 s in the presence of divalent cations, also are likely sites of Ca2+ uptake during contraction and the first step in contraction-activated cyclic AMP signaling. The formation of plasma membrane passages during fibroblast contraction may reflect a general cellular response to rapid mechanical changes.

Binding Sites↗
PubMed1996

Studies on the biocompatibility of materials: fibroblast reorganization of substratum-bound fibronectin on surfaces varying in wettability.

The ability of human fibroblasts to remove and reorganize fibronectin (FN) bound on material surfaces was studied as a novel feature of material surface biocompatibility. Other traditional parameters of biocompatibility analyzed included cell spreading, clustering of fibronectin receptors into focal adhesions, development of stress fibers, and cell growth. Five different materials with surface wettability ranging from hydrophilic (underwater contact angle 25 degrees) to hydrophobic (underwater contact angle 111 degrees) were used, i.e., clean glass (GLASS), aminopropylsilane (APS), octadecylsilane (ODS), polylactate (PL), and silicone (SI). When cells were cultured on these materials in serum-containing medium, formation of FN receptor-rich focal adhesions and actin stress fibers were more evident on the hydrophilic surfaces (GLASS and APS) compared to the hydrophobic ones (PL, ODS, and SI). Cell growth showed a similar pattern, that is, increased cell proliferation with increasing material surface wettability. Preadsorption of FN on the material surfaces increased subsequent cell spreading and cytoskeletal reorganization on hydrophobic surfaces except SI. Removal and reorganization of FN from the material surfaces into extracellular matrixlike structures occurred on GLASS but not on less wettable surfaces, suggesting that this removal/reorganization process may be more sensitive to changes in surface wettability than other parameters of biocompatibility.

Actins↗
PubMed1996

Fibronectin degradation in chronic wounds depends on the relative levels of elastase, alpha1-proteinase inhibitor, and alpha2-macroglobulin.

The goal of our studies was to learn about the mechanism of fibronectin degradation in chronic ulcers. We found that the appearance of fibronectin fragments in chronic ulcer wound fluid correlated with elevated levels of elastase and cleavage of the proteinase inhibitors alpha2-macroglobulin (alpha2-M) and alpha 1-proteinase inhibitor (alpha1-P1). Some wound fluid samples retained the capacity to degrade fibronectin in vitro. Degradation of fibronectin by these samples was blocked by specific inhibitors of neutrophil elastase but not by inhibitors of metalloproteinases. Addition of human neutrophil elastase to mastectomy fluid, an acute wound fluid, resulted in formation of alpha1-PI and alpha2-M complexes and cleavage products resembling those observed in chronic wound fluid. Moreover, degradation of fibronectin and processing of matrix metalloproteinase MMP-9 occurred under these conditions. Taken together, our findings suggest that elevated levels of neutrophil elastase are responsible for fibronectin degradation in the chronic wound environment.

Adult↗
PubMed1995

Non-muscle myosin II heavy chain has a cryptic cell-adhesion domain.

We have discovered a cryptic cell-adhesion domain in non-muscle myosin II heavy chain. A 205 kDa cell-adhesion-promoting polypeptide (p205) was extracted from BHK cells by Nonidet P-40 or Dounce homogenization. Adhesion to p205 was specifically inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro, indicating a role for the Arg-Gly-Asp cell-adhesion motif. Purified p205 was identified as non-muscle myosin II heavy chain, based on sequence analysis and on the cross-reactivity of p205 with anti-(bovine trachea myosin) antibodies. Further experiments showed that the heavy chain of purified myosin II has cell-adhesion-promoting activity in a cell-blotting assay, and cross-reacted with anti-p205 antibodies. Finally, the adhesion domain was located in the tail portion of myosin II heavy chain, where an Arg-Gly-Asp-containing sequence can be found.

Amino Acid Sequence↗
PubMed1995

Fibronectin receptor internalization and AP-2 complex reorganization in potassium-depleted fibroblasts.

Potassium-depleted fibroblasts are unable to develop polarized morphology and lack coated pits. Experiments were carried out to measure internalization of fibronectin receptors (FNR) in potassium-depleted cells and possible association of FNR with AP-2 complexes after adding potassium back to the cells, which restores cell polarization. AP-2 complexes are the cell surface component of coated pits that contain both clathrin and membrane receptor binding domains. Potassium-depleted fibroblasts endocytosed antibody-tagged FNR and also internalized fluorescent fibronectin that previously had been adsorbed to the substratum. During cell polarization, antibody-tagged FNR reorganized into fibrillar structures along stress fibers beginning from nucleation sites at cell margins. Plasma membrane AP-2 complexes, which were undetectable in potassium-depleted cells, reappeared at the cell surface above the nucleus and then spread toward the cell margins. The results show that endocytosis of FNR can occur at least partially by a coated pit-independent mechanism.

Actins↗
PubMed1995

Treatment of human fibroblasts with vanadate and platelet-derived growth factor in the presence of serum inhibits collagen matrix contraction.

Human fibroblasts cultured in an anchored collagen matrix reorganize the matrix and develop stress. Upon experimental release of the matrix, cells contract the matrix, a phenomenon that has been studied as an in vitro model of wound contraction. We have found that treatment of fibroblasts in stressed collagen matrices with vanadate and platelet-derived growth factor (PDGF) in serum-containing medium inhibits the ability of cells to contract the matrix. Vanadate/PDGF/serum stimulation did not block contraction immediately, but rather resulted in generation of an inhibitory signal that developed over a period of 80 min. The signal was highly specific since other factors such as lysophosphatidic acid and epidermal growth factor were unable to replace PDGF or serum. The presence of vanadate also blocked dephosphorylation of p-Tyr-PDGF receptors after PDGF/serum stimulation and caused accumulation of tyrosine-phosphorylated proteins in the cells. In parallel experiments, fibroblasts in monolayer culture were found to undergo reorganization of their actin cytoskeleton when treated with vanadate in the presence of PDGF and serum-containing medium. Our results suggest that a p-Tyr signaling pathway is important in the regulation of wound contraction.

Actins↗
PubMed1995

Role of phospholipase D in the cAMP signal transduction pathway activated during fibroblast contraction of collagen matrices.

Fibroblast contraction of stressed collagen matrices results in activation of a cAMP signal transduction pathway. This pathway involves influx of extracellular Ca2+ ions and increased production of arachidonic acid. We report that within 5 min after initiating contraction, a burst of phosphatidic acid release was detected. Phospholipase D was implicated in production of phosphatidic acid based on observation of a transphosphatidylation reaction in the presence of ethanol that resulted in formation of phosphatidylethanol at the expense of phosphatidic acid. Activation of phospholipase D required extracellular Ca2+ ions and was regulated by protein kinase C. Ethanol treatment of cells also inhibited by 60-70% contraction-dependent release of arachidonic acid and cAMP but had no effect on increased cAMP synthesis after addition of exogenous arachidonic acid or on phospholipase A2 activity measured in cell extracts. Moreover, other treatments that inhibited the burst of phosphatidic acid release after contraction--chelating extracellular Ca2+ or down-regulating protein kinase C--also blocked contraction activated cyclic AMP signaling. These results were consistent with the idea that phosphatidic acid production occurred upstream of arachidonic acid in the contraction-activated cAMP signaling pathway.

Arachidonic Acid↗