Biomedical subjects

C Guidry

Publications and source records attributed to C Guidry.

At least 19 recordsLinked to original sources

PubMed2000

Tractional force generation by porcine Müller cells: paracrine stimulation by retinal pigment epithelium.

PURPOSE: To examine the ability of retinal pigment epithelial (RPE) cells to modulate Müller cell extracellular matrix contraction through secreted promoters. METHODS: Freshly isolated RPE cells were maintained in continuous culture until the morphologic and immunocytochemical changes associated with myofibroblastic dedifferentiation were complete. Secretory products collected from these cells during extended incubations in serum-free medium and at different stages of dedifferentiation were examined for the ability to promote extracellular matrix contraction by Müller cells. The contributions of specific growth factors to RPE-secreted activity were examined with growth factor-neutralizing antibodies. RESULTS: Secretory products from RPE cells throughout dedifferentiation contained biologically active quantities of Müller cell contraction promoters. Secretory activity increased during extended incubation in serum-free medium and during myofibroblastic dedifferentiation. Growth factor-specific neutralizing antibodies enabled the determination that insulin-like growth factor- and platelet-derived growth factor-related proteins were the secreted species to which Müller cells responded. Finally, gene expression of insulin-like growth factor 1 and platelet-derived growth factor A chain by porcine RPE cells was confirmed using reverse transcription-polymerase chain reaction. CONCLUSIONS. RPE cells are a viable source of biologically active quantities of two growth factors that stimulate extracellular matrix contraction by Müller cells. This secretory profile persists for extended periods in an otherwise serum-free environment and is enhanced during myofibroblastic dedifferentiation.

Animals↗

PubMed1998

Antigenic changes of rabbit retinal Müller cells in culture.

PURPOSE: To determine whether dissociated and cultured Müller cells from the avascular rabbit retina undergo the same phenotypic changes as Müller cells that are dissociated and cultured from a vascular retina. METHODS: Müller cells were dissociated from adult rabbit retinas by using an enzymatic digestion-mechanical trituration technique and a cell attachment method that provided Müller cell- enriched cell cultures. Indirect immunofluorescence localization of vimentin, glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), beta-amyloid precursor protein (beta-APP), and (alpha-smooth muscle actin (alpha-SMA) was carried out on Müller cells that were freshly dissociated, on those that had been in culture 2 and 6 days, and on confluent primary cultures and late-passage cultures. The specificity of the antibodies and changes in protein expression were examined by western blot analysis. RESULTS: The expression of vimentin, GFAP, GS, and beta-APP was present 2 days after dissociation and was retained through 6 days in culture, at which time alpha-SMA began to be expressed in a small number of cells. The confluent, primary cultures no longer expressed GS, but vimentin and beta-APP were still expressed, and the expression of alpha-SMA was increased. During the late-passage stage, the morphologic appearance of the Müller cell cultures was large and amorphous, with additional changes in antigenicity. Although there was loss of expression of the intermediate filament proteins GFAP and vimentin, the expression of beta-APP was maintained, whereas alpha-SMA was increased and appeared to be a major cytoskeletal protein. CONCLUSIONS: Dissociated Müller cells that were maintained in culture underwent phenotypic changes that included a large, amorphous appearance; the loss of detectable vimentin, GFAP, and GS expression; the persistent presence of beta-APP; and the de novo appearance of alpha-SMA. The phenotypic and antigenic changes that occurred in cultured Müller cells from an avascular retina were similar but not identical to the changes observed in cultured Müller cells from a vascular retina.

Actins↗

PubMed1997

Quantitative vitreous collection using a modified infusion fluid during vitrectomy.

PURPOSE: To develop a vitreous sampling method that increases yield while reducing risk of harm to the patient. METHODS: Potential negative effects of fluoresceinated BSS infusion solution on biologic activity were assessed by monitoring changes in fibroblast contraction of collagen gels in response to serum or vitreous. Paired vitreous samples were collected from ten patients before and during infusion of fluoresceinated BSS solution. The extent to which the vitreous was diluted was calculated by comparing the levels of fluorescence in the samples with that in fluoresceinated BSS. Protein concentrations and levels of contraction-stimulating activity were measured for each sample. RESULTS: Fluoresceinated BSS did not alter fibroblast morphology, rate, or extent of gel contraction. Differences in corrected protein concentrations and biologic activities of the undiluted and diluted homogenous vitreous samples were not statistically significant. CONCLUSIONS: The use of fluoresceinated BSS infusion yields larger vitreous samples from which the native biochemical characteristics can be determined. Patient safety during collection is enhanced because ocular hypotony and collapse can be avoided.

Body Fluids↗

PubMed1997

Tractional force generation by porcine Müller cells. Development and differential stimulation by growth factors.

PURPOSE: To assess the ability of retinal Müller cells to generate tractional forces during dedifferentiation in culture and to assess their responsiveness to contraction-stimulating growth factors. METHODS: Müller cells were isolated from papain-DNase-digested porcine retina. The identity of the isolated cells was confirmed by immunodetection of carbonic anhydrase II (CA-II), cellular retinaldehyde-binding protein (CRALBP), glial fibrillary acidic protein (GFAP), vimentin, and alpha smooth muscle actin (alpha SMA). Tractional force generation was assessed as a function of Müller cell contraction of collagenous extracellular matrices in vitro. The effects of potential promoters were assessed by addition directly to culture medium. The contributions of specific promoting to the contraction-promoting activity in serum were assessed by adding neutralizing antibodies and measuring loss of stimulatory activity. RESULTS: Freshly isolated Müller cells did not generate substantial matrix contraction. However, this activity increased 150-fold within 12 days in culture and continued to increase during the next 21 days. Development of the capacity for extracellular matrix contraction coincided with the acquisition of immunodetectable alpha SMA and loss of GFAP. Matrix contraction by Müller cells was stimulated in a dose-dependent fashion by human serum, platelet-derived growth factor (PDGF), and insulin-like growth factor-I (IGF-I). Müller cells were not stimulated by transforming growth factor beta 1 (TGF beta 1), transforming growth factor beta 2 (TGF beta 2), or endothelin-1 (E1). Neutralizing antibodies against PDGF and IGF-I reduced the activity in human serum by 37% and 58%, respectively, and 87% when added together. CONCLUSIONS: Porcine Müller cells in culture acquire the ability to contract extracellular matrices and thus generate tractional forces. Acquisition of this activity coincides with alpha SMA expression and loss of GFAP. Further, this activity is dependent on the presence of exogenous promoters, including PDGF or IGF-I.

Animals↗

PubMed1997

Tractional force generation by porcine Müller cells: stimulation by growth factors in human vitreous.

PURPOSE: To examine the levels of Müller cell contraction-stimulating activity in human vitreous, correlate these levels with clinical presentation, and identify, the causative growth factors. METHODS: Human vitreous was collected from patients undergoing pars plana vitrectomy (n = 84). Müller cells were isolated from porcine retina and maintained in tissue culture. Tractional forces generated by cells incubated on three-dimensional collagen gels were measured as changes in gel thickness. Contraction-stimulating activity in vitreous (VA) was calculated from the close-response profiles of gel contraction to vitreous protein. The contributions of individual growth factors to vitreous activity (n = 10) were assessed by inhibition with specific neutralizing antibodies. RESULTS: The mean VA of patients with retinal detachment (3.65) and proliferative vitreoretinopathy stages A, B, and C (2.06) were elevated above that of patients without retinal pathology (vitreous activity = 0.23) or retinal defects alone (0.57). Mean activities in patients with epimacular proliferation (1.22) and vitreous hemorrhage (1.40) were also significantly elevated. The percentage of this activity attributable to insulin-like growth factor 1 (IGF-1) varied from 9.2% to 84.5% with a mean of 61.3%. Similarly, the percent contribution of platelet-derived growth factor (PDGF) ranged from 6.8% to 49.0% with a mean of 26.5%. CONCLUSIONS: The vitreous of patients with retinal detachment, proliferative retinal disease, and vitreous hemorrhage contain varying amounts of growth factors that stimulate tractional force generation by Müller cells. The majority of the activity can be attributed to IGF-1 and a smaller proportion to PDGF.

Animals↗

PubMed1996

[Contraction of extracellular matrix by transdifferentiated retinal pigment epithelial cells, inducers and inhibitors].

Transdifferentiated retinal pigment epithelial cells (RPE) display enhanced contractile potentials and have been implicated in the development of tractional retinal detachment. This study determines the activity of contraction-promoting factors, examines some involved mechanisms and evaluates inhibitors. Using an in vitro contraction assay, we demonstrated that collagen matrix contraction by transdifferentiated RPE cells is effectively stimulated by serum, platelet-derived growth factor and insulin-like growth factor-1. Endothelin-1 and transforming growth factor-beta 1 and -beta 2 have a more discrete or marginal effect. Tractional forces promoted by these peptides are completely protein synthesis dependent. Contraction stimulated by serum is only partly dependent on de novo protein synthesis, suggesting different active factors and/or pathways. Staurosporine, a broad-spectrum kinase inhibitor, effectively inhibited collagen matrix contraction by transdifferentiated RPE cells regardless of the promoter.

Collagen↗

PubMed1996

Isolation and characterization of porcine Müller cells. Myofibroblastic dedifferentiation in culture.

PURPOSE: To characterize phenotypic and antigenic changes in isolated Müller cells during proliferation in extended culture. METHODS: Müller cells were isolated from porcine retina by sequential papain and DNase digestion, trituration, and density gradient centrifugation. The identity of the isolated cells was confirmed by immunodetection of carbonic anhydrase II (CA-II), cellular retinaldehyde-binding protein (CRALBP), glial fibrillary acidic protein (GFAP), vimentin, and delta smooth muscle actin (alpha SMA). Continuously proliferating cells established in culture were examined for changes in the expression of these antigens. RESULTS: Primary cultures of purified Müller cells, incubated under routine culture conditions, were proliferative and lost immunodetectable CRALBP within 2 weeks. The expression of CA-II also diminished with time, but at an apparently lower rate than that of CRALBP. Loss of GFAP expression was even more gradual and was complete by passage 5. De novo expression of alpha SMA was detectable in some cells within 12 days in culture and by all cells by passage 5. During this period, vimentin expression remained qualitatively unchanged. CONCLUSIONS: Isolated porcine Muller cells in culture undergo a phenotypic dedifferentiation to a fibroblast-like cell, which includes loss of detectable CRALBP, CA-II, and GFAP, and they acquire expression of the myoid marker alpha SMA.

Animals↗

PubMed1995

Pathologic human vitreous promotes contraction by fibroblasts. Implications for proliferative vitreoretinopathy.

OBJECTIVE: To establish and quantify the presence of contraction-stimulating activity in pathologic vitreous and correlate this activity with clinical presentation and outcome, especially with proliferative vitreoretinopathy. METHODS: Contraction-stimulating activity of vitreous collected during surgery was quantified with a tissue culture assay using fibroblasts as target cells. The activity of each sample was correlated with patient history, clinical presentation, risk factors, proliferative disease, and postoperative proliferation. RESULTS: Pathologic vitreous contained measurable quantities of contraction-stimulating activity and stimulated contraction in vitro, with elevated activities in samples from patients with proliferative vitreoretinopathy, epimacular proliferation, retinal detachment, retinal defects, pigmented cells in the vitreous, hemorrhage, or uveitis. Patients with postoperative proliferation had significantly elevated mean activities. CONCLUSIONS: Levels of contraction-stimulating activity in pathologic vitreous correlate with some risk factors for the development of proliferative vitreoretinopathy and may ultimately be useful in the assessment of disease severity and the prediction of postoperative proliferation.

Adolescent↗

PubMed1995

Transdifferentiation of retinal pigment epithelial cells from epithelial to mesenchymal phenotype.

PURPOSE: To describe and evaluate retinal pigment epithelial (RPE) cell transdifferentiation in vitro and to determine its importance to the development of proliferative vitreoretinal disorders. METHODS: Porcine RPE cells from single animals were examined at different passages in culture. The authors examined cellular morphology, contraction of a collagenous matrix, and adhesion to fibronectin and type I collagen-coated substrata. These activities were correlated with loss of epithelial characteristics, redistribution of the actin cytoskeleton, and expression of alpha-smooth muscle actin (alpha-SMA), a marker of myoid differentiation. RESULTS: During routine culture on tissue culture plastic, porcine RPE cells lose epithelial characteristics and acquire a mesenchymal cell-like phenotype. The ability of cultured porcine RPE cells to adhere to and exert tractional forces on an extracellular matrix increases with continued passage in vitro and transdifferentiation. This correlates with the loss of the differentiated epithelial morphology, decreased expression of the epithelial marker cytokeratin 18, redistribution of the actin cytoskeleton, and de novo expression of alpha-SMA. CONCLUSION: Results indicate that RPE transdifferentiate in culture and that this transition is accompanied by a shift in biologic activities. Therefore, morphologic and behavioral transdifferentiation of these cells in culture are influencing factors in experimental pathology. The potential relevance of these extensive changes to the biology of proliferative vitreoretinal disorders is discussed.

Actins↗

PubMed1994

Isolation and HPLC of N-epsilon-lithocholyl lysine as its fluorescamine and dimethylaminoazobenzene isothiocyanate derivatives.

N-epsilon-lithocholyl lysine (NELL) is a component of tissue-bound lithocholic acid (TBL). The isolation of NELL from native protein sources was simulated by hydrolysis of lithocholyl-bovine serum albumin (BSA) (synthesized by coupling lithocholyl-N-hydroxysuccinimide to fatty acid-free BSA) by digestion with a mixture of 6N HCl-propionic acid at 70 C for 3 h under partial vacuum. NELL was isolated on a reversed phase Sep-Pak C18 column and converted to either a fluorophor with fluorescamine or to a chromophor with dimethylaminoazobenzene isothiocyanate for subsequent HPLC using appropriate fluorescence or UV/visible absorption detectors. The procedure described here is quantitative, highly sensitive, and not dependent upon the use of Clostridial cholanoylamino acid hydrolase, the activity of which is sometimes blocked by steric hindrance on the substrate. Using this procedure we have demonstrated the presence of TBL in native histones.

Chromatography, High Pressure Liquid↗

PubMed1994

Extracellular matrix contraction by choroidal fibroblasts: inhibition by staurosporine.

PURPOSE: To examine the potential of a broad-spectrum kinase inhibitor as a means of neutralizing the effects of contraction promoters on ocular cells. METHODS: The inhibitory effects of a broad-spectrum kinase inhibitor were examined and characterized using an in vitro assay of extracellular matrix contraction by choroidal fibroblasts. RESULTS: Staurosporine effectively inhibited collagen matrix contraction by choroidal fibroblasts. The inhibitory effects of staurosporine were rapid in onset and reversible upon removal of the inhibitor. Inhibition was observed when fibroblasts were stimulated with serum, transforming growth factor beta 1, transforming growth factor beta 2, platelet-derived growth factor, and endothelin-1. We also observed that platelet-derived growth factor and endothelin-1 stimulated only modest amounts of matrix contraction compared to transforming growth factor beta. CONCLUSIONS: Matrix contraction by cells, as observed in the development of tractional forces, can be modulated by a broad-spectrum kinase inhibitor. The marginal contractile responses of choroidal fibroblasts to platelet-derived growth factor and endothelin-1, both potent promoters of dermal fibroblast contraction, suggest that there are substantive difference in the responses of these two cell types to growth factors.

Alkaloids↗

PubMed1993

Fibroblast contraction of collagen gels requires activation of protein kinase C.

Fibroblasts stimulated to contract collagen gels with serum were completely inhibited by staurosporine, a broad spectrum kinase inhibitor. Further analysis demonstrated that staurosporine is potent (IC50 17 nM), rapid in onset, and completely reversible. Complete inhibition of gel contraction was also observed with calphostin C (IC50 48 nM), an inhibitor specific for protein kinase C (PKC). Similar effects were not observed with KT5926 or KT5720, inhibitors for myosin light chain kinase and cAMP-dependent kinases, respectively. These data suggested that serum-stimulated fibroblast contraction is dependent upon activation of PKC. This was also observed with fibroblast contraction stimulated with endothelin-1, platelet-derived growth factor, and transforming growth factor beta. PKC activated directly with low concentrations of phorbol ester was observed to stimulate fibroblast contraction of collagen gels, in some cases within 30 minutes of exposure.

Alkaloids↗

PubMed1993

Effects of dietary fibre on faecal steroid measurements in baboons (Papio cynocephalus cynocephalus).

A study was conducted in captive baboons to determine (i) the impact of cereal dietary fibre on faecal progestogen excretion, and (ii) whether means of controlling dietary effects could be identified. Blood was collected on 3 days per week and faeces on 5 days per week from four unanesthetized cyclic female baboons, consecutively fed three diets of 5, 10 and 20% fibre for 90 days per diet. A 2 day lag time was detected before progesterone in the blood appeared in the faeces, regardless of diet (mean correlation was 0.62, P = 0.002). Increased dietary fibre had a negative effect on progestogen excretion (P < 0.004). Correspondence between blood and faecal progestogens was consistently greatest and the effect of dietary fibre least when faecal progestogens were expressed g-1 dry faeces. Several means of indexing faecal steroid excretion rates were examined including dehydroepiandrosterone (DHEA) and a number of byproducts of cholesterol metabolism. The cholesterol metabolite, cholestanone, was positively correlated with dietary fibre (r = 0.27; P < 0.04). Multiplying faecal progestogen concentration by the cholestanone g-1 dry faeces concentration increased the correlation between serum and cholestanone-indexed faecal progestogens (r = 0.78, P = 0.0001) compared with nonindexed progestogens (r = 0.71, P = 0.0001). We conclude that expressing faecal progestogens g-1 dry faeces may be sufficient and the most cost-effective method for controlling for most dietary effects when the objective is monitoring longitudinal endocrine status in baboons. However, it may be appropriate to express faecal progestogens by cholestanone concentrations when increased precision is needed to overcome the effects of profound variations in dietary fibre.

Animals↗

PubMed1992

Binding of the proteoglycan decorin to collagen type VI.

We have examined the interactions between the small dermatan sulfate proteoglycan decorin and collagen types I-VI using solid phase binding assays. The results of these studies showed that 125I-decorin bound most efficiently to collagen type VI in a time- and concentration-dependent manner. Furthermore, this interaction was specific and of moderately high affinity (Kd approximately 3 x 10(-7) M). Binding of decorin to collagen type VI appears to involve the decorin core protein rather than the glycosaminoglycan side chains, since the isolated core protein as well as a recombinant fusion protein containing a major segment (65%) of the human decorin core protein inhibited binding of 125I-decorin to collagen type VI. Other related proteoglycans and their respective core proteins also inhibited the binding of 125I-decorin to collagen type VI, whereas unrelated proteins and isolated glycosaminoglycan chains were without effect. In addition to decorin, collagen type II was also shown to bind to immobilized collagen type VI. Both interactions were effectively inhibited by preincubation of the immobilized collagen VI with decorin or collagen type II. These results suggested that the collagen type VI molecule has binding sites for collagen type II and decorin which are located in close proximity on the collagen type VI molecule. Possible functional roles of these interactions are discussed.

Animals↗

PubMed1992

Extracellular matrix contraction by fibroblasts: peptide promoters and second messengers.

Cells contracting connective tissue matrices generate tractional forces in tissues. Studies of fibroblast contraction, using collagen gels in an in vitro model, demonstrate that it involves the actin cytoskeleton, specific extracellular matrix receptors and requires stimulation by exogenous promoters. Fibroblast contraction is stimulated by factors released by platelets and potentially secreted within the contracting tissue. Endothelial cells secrete a potent promoter of fibroblast contraction which has been identified as endothelin 1. The pathway through which fibroblast contraction is stimulated appears to require activation of protein kinase C. Tumor cells can also secrete endothelin. These mechanisms may be relevant to tumor progression.

Animals↗

PubMed1992

Collagen gel contraction by cells associated with proliferative vitreoretinopathy.

The capacities of porcine choroidal fibroblasts, retinal glial cells, and retinal pigment epithelial cells to contract collagen gels in vitro were compared. Experiments with varied cell numbers indicated that glial cells are the most effective, followed by choroidal fibroblasts and retinal pigment epithelial cells. Analysis of the secretory products from cultures of these cell types revealed that retinal pigment epithelial cells synthesize and secrete peptides that promote fibroblast contraction of collagen gels in vitro. The mechanism of action of the retinal pigment epithelial cell-secreted contraction promoter was compared with that found in serum (type A) and secreted by cultured endothelial cells (type B). Like the serum factor, the retinal pigment epithelial cell-secreted factor was not dependent on active protein synthesis by the target cell and must be present continuously to promote contraction.

Animals↗

PubMed1991

Endothelins produced by endothelial cells promote collagen gel contraction by fibroblasts.

Endothelial 1 (E1) is identified as an endothelial cell secreted factor that stimulates collagen gel contraction by fibroblasts. This identification is based on (a) co-localization of stimulatory activity in endothelial cell conditioned media with synthetic E1 in reversed phase analysis; (b) removal of the activity from conditioned media with antiserum directed against E1; and (c) the activity of synthetic E1. Treatment of endothelial cell conditioned media with immobilized anti-E1 antibodies removed 59% of the activity from the pool suggesting that E1 is the major contraction promoter in endothelial cell conditioned medium. The mechanism of action of E1 is shown to be different from serum in that E1-promoted contraction is dependent upon the synthesis of an unknown effector protein. Synthetic E1 is shown to be a potent promoter of gel contraction with half-maximal activity occurring at 32 pM. Two other endothelins, E2 and VIC, are slightly less active than E1. A fourth endothelin species, E3, is substantially less active. A comparison of E1 with other contraction promoting peptides revealed that E1 and platelet-derived growth factor are essentially equal in specific activity, whereas TGF beta is approximately 50-fold more potent.

Cells, Cultured↗

PubMed1990

A second fibronectin-binding region is present in collagen alpha chains.

The interactions of plasma fibronectin with alpha chains or cyanogen bromide fragments of collagen types I and II have been studied using a variety of techniques. Affinity chromatography of cyanogen bromide-cleaved type II collagen on immobilized fibronectin revealed the binding of cyanogen bromide fragment CB12 in addition to the previously characterized CB10. Using fluorescence polarization, we analyzed the interaction between the collagen peptides and fluorescein isothiocyanate-labeled 42-kDa gelatin-binding fragment of fibronectin in solution. Dissociation constants for the binding of CB10 and CB12 to the fibronectin fragment were calculated as 0.38 and 0.94 microM, respectively, indicating a lower affinity for the uncharacterized site. However, as with CB10, CB12 was able to compete effectively with the intact alpha chain for bindinng to fibronectin. Additionally, both CB10 and CB12 absorbed to tissue culture surfaces were each able to support fibronectin-dependent cell adhesion. Finally, the regions of alpha 2(I) homologous to CB12 and CB10 were found to be active in fibronectin binding, demonstrating the presence of two fibronectin-binding regions in this collagen chain.

Amino Acid Sequence↗