Biomedical subjects

Leon Kohen

Publications and source records attributed to Leon Kohen.

16 recordsLinked to original sources

PubMed2006

Signaling pathways involved in PDGF-evoked cellular responses in human RPE cells.

We examined whether PDGF may directly stimulate the expression of VEGF by retinal pigment epithelial (RPE) cells in vitro, and the involvement of three signal transduction pathways in the regulation of PDGF-evoked cell proliferation, migration, and production of VEGF-A was investigated. PDGF stimulated the gene and protein expression of VEGF-A by RPE cells, and increased cell proliferation and chemotaxis. PDGF activated all signaling pathways investigated, as determined by increased phosphorylation levels of ERK1/2, p38, and Akt proteins. The three signaling pathways were involved in the mediation of PDGF-evoked cell proliferation, while p38 and PI3K mediated cell migration, and PI3K mediated secretion of VEGF-A. In addition to VEGF-A, the cells expressed mRNAs for various members of the VEGF family and for their receptors, including VEGF-B, -C, -D, flt-1, and KDR. The data indicate that PDGF selectively stimulates the expression of VEGF-A in RPE cells. PDGF evokes at least three signal transduction pathways which are differentially involved in various cellular responses.

Cell Movement↗

PubMed2006

Expression of HB-EGF by retinal pigment epithelial cells in vitreoretinal proliferative disease.

The heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been implicated in wound-healing processes of various tissues. However, it is not known whether HB-EGF may represent a factor implicated in overstimulated wound-healing processes of the retina during proliferative retinopathies. Therefore, we investigated whether human retinal pigment epithelial (RPE) cells, which are crucially involved in proliferative retinopathies, express and respond to HB-EGF. RPE cells express mRNAs for various members of the EGF-related growth factor family, among them for HB-EGF, as well as for the EGF receptors ErbB1, -2, -3, and -4. The gene expression of HB-EGF is stimulated in the presence of transforming and basic fibroblast growth factors and by oxidative stress and is suppressed during chemical hypoxia. Exogenous HB-EGF stimulates proliferation and migration of RPE cells and the gene and protein expression of the vascular endothelial growth factor (VEGF). HB-EGF activates at least three signal transduction pathways in RPE cells including the extracellular signal-regulated kinases (involved in the proliferation-stimulating action of HB-EGF), p38 (mediates the effects on chemotaxis and secretion of VEGF), and the phosphatidylinositol-3 kinase (necessary for the stimulation of chemotaxis). In epiretinal membranes of patients with proliferative retinopathies, HB-EGF immunoreactivity was partially colocalized with the RPE cell marker, cytokeratins; this observation suggests that RPE cell-derived HB-EGF may represent one factor that drives the uncontrolled wound-healing process of the retina. The stimulating effect on the secretion of VEGF may suggest that HB-EGF is also implicated in the pathological angiogenesis of the retina.

Aged↗

PubMed2005

Human retinal epithelium produces and responds to placenta growth factor.

BACKGROUND: Placenta growth factor (PlGF) is an important co-factor in retinal neovascularization. To examine whether retinal pigment epithelial (RPE) cells may represent a source for PlGF during retinopathy, we determined whether human RPE cells in vitro produce and respond to PlGF. In addition, we determined whether the cells express receptors for PlGF, i.e. flt-1 and neuropilins. METHODS: Cultured human RPE cells of passages 3-5 were used. The regulation of the PlGF gene and protein expression by growth factors and cytokines was evaluated by quantitative PCR and ELISA. Proliferation rates and chemotaxis were determined by a bromodeoxyuridine and a Boyden chamber assay. RESULTS: Human RPE cells express mRNAs for various members of the vascular endothelial growth factor family and for their receptors, including mRNAs for PlGF, flt-1, KDR, and neuropilins-1 and -2. The expression levels of the mRNAs for neuropilins-1 and -2 were significantly higher than those for flt-1 and KDR. Members of the transforming growth factor (TGF)-beta superfamily of growth factors (BMP-4, TGF-beta1, and beta2) were strong inducers of PlGF gene expression, and evoked secretion of PlGF-2 protein by RPE cells. Exogenous PlGF-2 induced chemotaxis in RPE cells and reduced slightly the cell proliferation at high concentrations. CONCLUSION: The findings that RPE cells produce and respond to PlGF indicate that the factor exerts an autocrine/paracrine action on these cells. It is suggested that increased expression of TGF-beta-related growth factors during diabetic retinopathy may cause PlGF secretion by RPE cells contributing to the stimulation of cell migration as a critical component of the progression of fibrovascular membranes.

Blotting, Western↗

PubMed2005

Changes in retinal gene expression in proliferative vitreoretinopathy: glial cell expression of HB-EGF.

PURPOSE: To compare the gene expression pattern of control postmortem retinas with retinas from patients with proliferative vitreoretinopathy (PVR), to determine the expression of the heparin binding epidermal growth factor-like growth factor (HB-EGF) by glial cells in fibroproliferative membranes, and to examine whether cells of the human Müller cell line, MIO-M1, respond to HB-EGF with proliferation, migration, and secretion of the vascular endothelial growth factor (VEGF). METHODS: To identify genes that were differently expressed in PVR and control retinas, the RNA from the neural retinas of seven postmortem donors and of two patients with PVR were analyzed for differential gene expression, by hybridization of labeled cRNA probes to an Affymetrix human genome microarray set. The results were validated by real time PCR experiments investigating RNA from 6 postmortem retinas and 4 PVR retinas. Epiretinal PVR membranes were immunohistochemically stained for colocalization of HB-EGF and the glial cell marker, glial fibrillary acidic protein (GFAP). The HB-EGF evoked proliferation of cultured Müller cells was investigated by a bromodeoxyuridine immunoassay, chemotaxis was assessed with a migration assay, and the release of VEGF was evaluated by ELISA. RESULTS: Out of the 12,600 genes and expressed sequence tags investigated, the levels of 80 showed an increased expression, and 21 were expressed at decreased levels, in the retinas of PVR patients compared to the control retinas. The upregulated signals include genes for nuclear and cell cycle related proteins, extracellular secretory proteins, cytosolic signaling proteins, and proteins of the membrane and the extracellular matrix. The genes of the hepatocyte growth factor and of HB-EGF were found to be expressed in PVR retinas but not in control retinas. In epiretinal membranes of patients with PVR, HB-EGF immunoreactivity partially colocalized with GFAP. In cultured Müller cells, HB-EGF stimulated both proliferation and chemotaxis, and the secretion of VEGF, via activation of the extracellular signal regulated kinases 1 and 2 and of the phosphatidylinositol-3 kinase. CONCLUSIONS: The development of PVR is accompanied by complex changes of the gene expression in the neural retina, with an upregulation of genes that support cell proliferation, cell signaling, cell motility, and extracellular matrix remodeling. HB-EGF is one of the factors that are significantly upregulated in PVR retinas. HB-EGF expression in fibroproliferative tissue and its stimulatory effect on glial cell proliferation, chemotaxis, and VEGF secretion suggest that HB-EGF may be a factor mediating glial cell responses during PVR.

Adolescent↗

PubMed2005

Diversity of aquaporin mRNA expressed by rat and human retinas.

Muller glial cells of the sensory retina mediate K+ and water fluxes that are facilitated by aquaporin-4 (AQP4) water channels and by Kir4.1-K+ channels. However, it is not known which subtypes of aquaporins are expressed in the mammalian retina. Using RT-PCR, we found that both human and rat retinas express mRNA for a diversity of water channel proteins. The human retina expresses mRNAs for AQP0 to AQP12 proteins. Using real-time PCR, we found that the mRNAs for AQP4 and Kir4.1 are downregulated in retinas that were obtained from patients with proliferative retinopathy compared with post-mortem controls. The data suggest that the development of proliferative gliosis is accompanied by disturbed transglial water and ion movements.

Animals↗

PubMed2004

Characterization of the basic fibroblast growth factor-evoked proliferation of the human Müller cell line, MIO-M1.

BACKGROUND: Basic fibroblast growth factor (bFGF) has been suggested to mediate activation of Müller glial cells in the ischemic-hypoxic retina. However, the intracellular pathways activated by bFGF in human Müller cells have been little explored. We characterized the signaling transduction pathways which are involved in the control and growth factor-evoked proliferation of a recently described human Müller cell line, MIO-M1. In addition, we investigated whether bFGF evoked the release of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) from the cells. METHODS: The growth factor-evoked proliferation of cultured MIO-M1 cells was estimated by means of a bromodeoxyuridine immunoassay, in the absence and presence of blockers of mitogen-activated protein kinases (MAPKs) and of the phosphatidylinositol-3 kinase (PI3K). The activation state of the p44/p42 MAPK was determined by Western blotting, and the bFGF-evoked release of VEGF and HGF was evaluated by ELISA. RESULTS: bFGF evoked a concentration-dependent increase of the cell proliferation, with an EC50 of approximately 1 ng/ml, via activation of both the p44/p42 MAPK and the p38 MAPK. In contrast, the mitogenic effects of the platelet-derived and the heparin-binding epidermal growth factors were dependent on p44/p42 MAPK activation and independent of activation of p38 MAPK. The transforming growth factors beta1 and beta2 also evoked cell proliferation which was independent of activation of the MAPKs investigated. bFGF evoked a release of VEGF and of HGF by the cells; these effects were independent of MAPK activation and were possibly mediated by activation of the PI3K signaling pathway. CONCLUSION: bFGF evokes multiple intracellular signaling pathways in human Müller cells which underlie the gliotic cell responses upon ischemic-hypoxic insults in the retina. Beside the stimulation of cell proliferation, which is dependent on activation of p44/p42 and p38 MAPKs, bFGF induces the secretion of VEGF and HGF by Müller cells.

Blotting, Western↗

PubMed2004

Selective staining by vital dyes of Müller glial cells in retinal wholemounts.

Müller glial cells within the retina may respond to different signaling molecules with an elevation of their intracellular free calcium. To prove the localization of the recorded calcium responses in Müller cells within acutely isolated retinal wholemounts, retinal pieces from adult animals and humans were exposed to different vital dyes just after the calcium imaging records were finished. The dyes, Mitotracker Orange, Mitotracker Green, Celltracker Orange, Celltracker Green, and monochlorobimane, are all selectively taken up by Müller glial cells, while neuronal cells remain largely devoid of the dyes. By using this method, it can be demonstrated that the free calcium alterations within the wholemounts indeed occur within Müller cells. Moreover, the cross-sectional areas of (dye-filled) Müller glial cell bodies, as well as of (dye-free) neuronal cell bodies, can be measured in retinal wholemounts, and the spatial densities of both types of cells can be determined. The vital dye loading of Müller cells may facilitate investigations of stimulus-induced alterations of retinal glial cell physiology and morphology.

Animals↗

PubMed2004

Glial cell expression of hepatocyte growth factor in vitreoretinal proliferative disease.

The hepatocyte growth factor (HGF) has been crucially implicated in the development of proliferative retinal diseases; however, it is unclear whether retinal glial cells express or respond to HGF. Therefore, we examined the expression of HGF and of the receptor for HGF, c-Met, by immunohistochemical costaining with glial fibrillary acidic protein (GFAP) in epiretinal membranes of patients with proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), respectively. Furthermore, it was determined whether cells of the human retinal glial cell line, MIO-M1, secrete HGF protein, and whether HGF stimulates proliferation and chemotaxis, and secretion of the vascular endothelial growth factor (VEGF). Neuroretinas of patients with PVR express elevated mRNA level for HGF in comparison to control retinas. In epiretinal membranes of patients with PVR or PDR, immunoreactivity for HGF and for c-Met, respectively, partially colocalized with immunoreactivity for GFAP. Fetal bovine serum and basic fibroblast growth factor, but not heparin-binding epidermal or platelet-derived growth factors, evoked HGF secretion by cultured retinal glial cells. HGF displayed only a marginal effect on cell proliferation while it stimulated chemotaxis. HGF promoted the secretion of VEGF, via activation of the phosphatidylinositol-3 kinase. It is concluded that glial cells in epiretinal membranes express both HGF protein and c-Met receptors. The results suggest an autocrine/paracrine role of HGF in glial cell responses during proliferative vitreoretinal disorders as well as in retinal neovascularization, by stimulating of VEGF release.

Adolescent↗

PubMed2004

Contrary effects of cytokines on mRNAs of cell cycle- and ECM-related proteins in hRPE cells in vitro.

PURPOSE: Retinal pigment epithelial (RPE) cells play a pivotal role in the pathogenesis of proliferative vitreoretinopathy (PVR). As a result of a breakdown of the blood-retina barrier, growth factors obtain access to the subretinal space and stimulate several retinal cell types. The aim of the present study was to analyze the effect of several growth factors on the proliferation of human (h)RPE cells, and on the mRNA expression of transcription factors, cell cycle proteins, and extracellular matrix (ECM) proteins. METHODS: hRPE cells were incubated in the presence of TGF-beta1, TGF-beta2, PDGF, VEGF, or bFGF for 24-72 h. Cell proliferation was assessed by determinating BrdU incorporation. Changes in mRNA expression of c-fos, c-myc, PCNA, FEN1, Ki67, collagen III, and collagen IV were investigated by ribonuclease protection assay (RPA). RESULTS: RPE cell proliferation was significantly increased by exposure to PDGF and bFGF for 48 h, and was decreased by application of TGF-beta1 and TGF-beta2 for 48 and 72 h. All the tested growth factors significantly elevated the amounts of c-fos mRNA (after 1 h) and of c-myc mRNA (after 24 h). PDGF and bFGF up-regulated the expression of Ki67 mRNA, and down-regulated that of collagen III and collagen IV mRNA after 24 h. TGF-beta1 and TGF-beta2 decreased the expression of Ki67 mRNA, and increased that of collagen III and collagen IV mRNA. CONCLUSION: Our results show that distinct cytokines may induce contrary effects with respect to proliferation of, and ECM formation by, hRPE cells in vitro. This knowledge may be useful for the development of improved therapeutic approaches.

Cell Cycle Proteins↗

PubMed2004

Expression of LRP1 in retinal pigment epithelial cells and its regulation by growth factors.

PURPOSE: The retinal pigment epithelial (RPE) cells are mitotically inactive under normal conditions, but play a pivotal role in the pathogenesis of proliferative vitreoretinopathy (PVR). Triggered by changes in the concentrations of growth factors, RPE cells reenter the cell cycle, proliferate, and migrate onto the retinal surface, into the subretinal space, and into the vitreous. The receptor for alpha(2)-macroglobulin (low-density lipoprotein receptor-related protein [LRP1], or CD91) is known to be involved in the processes of cell migration and invasion, as well as in the regulation of growth factor homeostasis. The purpose of this study was to investigate the expression of this receptor and its regulation, at the protein and mRNA levels, in human (h)RPE cells. METHODS: The cell surface expression of the receptor was studied by immunocytochemistry and flow cytometry. The endocytosis-related activity of LRP1 in hRPE cells was examined by assessing the uptake of FITC-labeled, methylamine (MA)-treated alpha(2)-M (alpha(2)-M-MA). LRP1 mRNA expression was analyzed by means of the RNase protection assay (RPA) after the hRPE cells were stimulated with the growth factors TGF-beta1, TGF-beta2, PDGF, VEGF (each 10 ng/mL), or bFGF (5 ng/mL). RESULTS: hRPE cells expressed LRP1 on their cell surface. The receptor mediated rapid binding and endocytosis of FITC-labeled alpha(2)-M-MA. The expression of LRP1 mRNA strongly increased on stimulation of the cells with TGF-beta1, TGF-beta2, or VEGF, whereas PDGF or bFGF elicited only minor effects. CONCLUSIONS: The expression of functionally active LRP1 in hRPE cells suggests that the receptor may be involved in cell migration and invasion, as reported for other LRP1-expressing cells. Thus, certain growth factors may control RPE cell migration and invasion in vivo through a regulation of LRP1 expression. As LRP1 mediates the clearance of alpha(2)-M, known to regulate the homeostasis of many cytokines and growth factors, this receptor may be a promising target for therapeutic intervention in PVR.

Aged↗

PubMed2003

Elevated proto-oncogene and collagen mRNA expression in PVR retinas.

PURPOSE: Retinal detachment is often accompanied by proliferation and migration of retinal cells and by increased synthesis of structural proteins, known as proliferative vitreoretinopathy (PVR). Herein we investigate the messenger RNA (mRNA) expression of proto-oncogenes responsible for cell proliferation and of structural proteins that have a role in membrane formation. METHODS: Retinal samples were obtained from patients undergoing vitreoretinal surgery for the treatment of retinal detachment complicated by PVR. Normal human control retinas were obtained from cornea donors. The mRNA expression of the proto-oncogenes c- myc, c- fos and the proliferation marker Ki67, as well as of collagen type III and type IV, were investigated using the ribonuclease protection assay. RESULTS: Ki67 mRNA expression was not detectable in either sample type, but c- fos and c- myc mRNA expression was found in normal and PVR retinas. Whereas the expression of c- myc showed a marginal increase, the up-regulation in c- fos expression was strongly significant (5.07-fold). The mRNA of collagen type III was detectable at widely varying levels in all the PVR retinas but was found in only 2 of the 16 analysed normal samples. Collagen type IV mRNA was expressed in both PVR and control samples but was higher (2.21-fold) in the PVR retinas. CONCLUSIONS: These results indicate that an up-regulation of the proto-oncogene c- fos occurs in human PVR retinas. An increase in mRNA expression of collagen types III and IV takes place simultaneously. These changes in mRNA expression appear to be mainly connected to the initiation of cell proliferation, dedifferentiation and formation of tractional membranes.

Adolescent↗

PubMed2003

Age-related decrease of potassium currents in glial (Müller) cells of the human retina.

BACKGROUND: Age-dependent alterations have been investigated far less in retinal glial cells than in retinal neurons. We investigated age-dependent alterations of inwardly rectifying potassium (Kir) currents in Müller glial cells of the human retina. METHODS: Müller cells were isolated immediately post mortem from donors without a reported history of eye disease, and the amplitudes of Kir currents and of currents through high-voltage-activated (HVA) calcium channels were measured by whole-cell patch clamping. RESULTS: The amplitude of the Kir currents was lower in the cells from donors older than 50 years than in the cells of younger donors; the decrease was strongly correlated with the donor's age (p < 0.001). The current amplitude in the cells from donors older than 60 years was about 40% lower than the amplitude in the cells from donors younger than 50 years. The amplitude of the HVA currents was greater in the cells from donors older than 55 years than in the cells from younger donors; the increase, up to about 500%, was strongly age-dependent (p < 0.001). INTERPRETATION: The age-related decrease in Kir-current amplitude in Müller cells may reflect the neuron loss in the aged retina. Our findings also indicate that retinal glial cells have enhanced cytoplasmic calcium signals in the course of aging.

Adult↗

PubMed2002

Patch-clamp recording of Müller glial cells after cryopreservation.

Human and other primate retinal Müller cells display dominating K(+) currents as well as other membrane conductances that may change in cases of retinal pathology. Because the use of human and primate tissue is limited by reasons of availability, a method for long-term storage of these cells is desirable. We describe a cryopreservation method in which isolated Müller cells are stored in liquid nitrogen. After thawing, the cells can be used for patch-clamp experiments immediately, without culturing. We show that the main electrophysiological properties are not altered by this method and that voltage- and ligand-gated currents can be recorded from cryopreserved cells even after 2-years storage.

Adenosine Triphosphate↗

PubMed2002

Activation of P2Y receptors stimulates potassium and cation currents in acutely isolated human Müller (glial) cells.

The ability of various neurotransmitters/neuroactive substances to induce fast, transient rises of Ca(2+)-activated K(+) currents (I(BK)) caused by release of Ca(2+) from intracellular stores was investigated in Müller glial cells of the human retina. Müller cells were enzymatically isolated from retinas of healthy donors or of patients with proliferative vitreoretinopathy, and the transmembrane ionic currents were recorded using the whole-cell and the cell-attached patch-clamp techniques. The results of the screening experiments indicate that human Müller cells express, in addition to GABA(A) and perhaps glutamatergic and cholinergic receptors, predominantly P2 receptors. ATP and other nucleotides exerted two effects on membrane currents: repetitive transient increases of the I(BK) amplitude and, in a subpopulation of cells investigated, the appearance of a transient cation conductance at negative potentials. ATP and UTP increased dose-dependently the I(BK) amplitude with half-maximal effects at 0.33 and 0.50 microM, respectively. Since several different P2 receptor agonists increased the I(BK), it is assumed that human Müller cells express a mixture of different types of P2Y receptors. In cell-attached patches, extracellular application of ATP or UTP transiently increased the open probability of single putative BK channels. The increase of I(BK) and the appearance of the cation conductance in whole-cell records were abolished when intracellular Ca(2+) was buffered by a high-EGTA pipette solution or when IP(3) was included in the pipette solution. The expression of agonist-evoked transient cation currents was found to be stronger in cells from patients as compared to cells from healthy donors. It is concluded that human Müller glial cells express P2Y receptors that, via IP(3) formation, cause intracellular Ca(2+) release. The increased intracellular Ca(2+) concentration stimulates the activity of BK channels and may induce opening of cation channels. Both the ATP-induced activity of BK channels and the increased expression of Ca(2+)-gated cation channels may be important in respect to proliferative Müller cell gliosis.

Adenosine Triphosphate↗

PubMed2002

Membrane conductance of Müller glial cells in proliferative diabetic retinopathy.

BACKGROUND: It is not known whether the membrane features of human Müller cells are altered in proliferative diabetic retinopathy (PDR). We performed a study to investigate the expression of several distinct forms of membrane conductance in Müller glial cells from a patient with PDR compared to cells from healthy donors (control cells). METHODS: Müller cells were isolated 2 hours after vitreoretinal surgery in the case of the patient and within 24 hours in the case of the autopsy eyes. Whole-cell voltage-clamp recordings were made. The results for the two groups were compared with the Mann-Whitney U test. RESULTS: As assayed by the whole-cell membrane capacitance, the cells from the patient with PDR showed hypertrophy in comparison to the control cells (mean 85.1 pF [standard deviation (SD) 19.7 pF] vs. 54.3 pF [SD 13.8 pF]). The cells from the patient displayed strong downregulation of inwardly rectifying potassium ion (Kir) currents (mean 0.41 [SD 0.24] pA/pF, compared to 3.43 [SD 1.86] pA/pF for the control cells). The Kir current downregulation was accompanied by a less negative membrane potential (-57.3 mV [SD 16.9 mV], compared with -82.3 mV [SD 5.3 mV] for the control cells). Both the number and the amplitude of voltage-gated sodium ion currents were enhanced in cells from the patient. When P2X7 receptors were activated by 2'-/3'-O-(4-benzoylbenzoyl)-adenosine triphosphate, cells in both groups displayed opening of a cation conductance and, simultaneously, an increase in currents through calcium ion-activated potassium ion channels. INTERPRETATION: Changes in Müller cell membrane conductance in PDR are similar to those described in proliferative vitreoretinopathy. The down-regulation of active Kir channels and the membrane depolarization likely disturb voltage-dependent Müller cell functions, such as regulation of local ion concentrations and uptake of neurotransmitters. The enhanced entry of calcium ions from the extracellular space and the subsequent stimulation of calcium-activated potassium channels support Müller cell proliferation in PDR.

Adenosine Triphosphate↗

PubMedDate not supplied

Influence of interleukin 10 and transforming growth factor-beta on T cell stimulation through allogeneic retinal pigment epithelium cells in vitro.

The effect of interleukin 10 (IL-10) and transforming growth factor-beta (TGF-beta) on the immunological capability of human retinal pigment epithelium (RPE) cells was tested by measuring the proliferation of allogeneic T cells after stimulation through the RPE cells in vitro. For this, human RPE cells were activated with different concentrations of interferon-gamma (0, 50, 100 and 250 U/ml) used as stimulator cells. The following T cell proliferation was measured with a tetrazolium-based colorimetric assay over a period of up to 4 days. Our results show that RPE cells are able to stimulate allogeneic T cells in the presence or absence of professional antigen-presenting cells (macrophages). Additionally, the proliferation could be significantly decreased by IL-10 or TGF-beta. The maximum of suppression, especially by TGF-beta, was found after 48 and 72 h of incubation. This shows that the antigen-presenting properties of human RPE cells can be altered in culture.

Antigen-Presenting Cells↗