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Pedro Gonzalez

Publications and source records attributed to Pedro Gonzalez.

16 recordsLinked to original sources
PubMed2006

Genome-wide expression profile of human trabecular meshwork cultured cells, nonglaucomatous and primary open angle glaucoma tissue.

PURPOSE: To contrast genome-wide gene expression profiles of cultured human trabecular meshwork (HTM) cells to that of control and primary open angle glaucoma (POAG) HTM tissues. METHODS: Cultured HTM cells, HTM tissue dissected from control donors, and HTM tissue from POAG donors receiving medication for glaucoma were fixed in RNA latertrade mark. Total RNA extracted from these samples was linearly amplified with the Ovation Biotin RNA Amplification and Labeling System and individually hybridized to Affymetrix Human Genome U133 Plus 2.0 high density microarrays. Data analysis was performed using GeneSpring Software 7.0. Selected genes showing significant differential expression were validated by quantitative real-time PCR in nonamplified RNA. RESULTS: Cultured HTM cells retained the expression of some genes characteristic of HTM tissue, including chitinase 3-like 1 and matrix Gla protein, but demonstrated downregulation of physiologically important genes such as myocilin. POAG HTM tissue showed relatively small changes compared to that of control donors. These changes included the statistically significant upregulation of several genes associated with inflammation and acute-phase response, including selectin-E (ELAM-I), as well as the downregulation of the antioxidants paraoxonase 3 and ceruloplasmin. CONCLUSIONS: Downregulation in cultured HTM cells of genes potentially relevant for outflow pathway function highlights the importance of developing new conditions for the culture of TM cells capable of preserving the characteristics of TM cells in vivo. Comparative analysis between control and POAG tissues suggests that the upregulation of inflammation-associated genes might be involved in the progression of glaucoma.

Case-Control Studies↗
PubMed2006

An important pool of sucrose linked to starch biosynthesis is taken up by endocytosis in heterotrophic cells.

We have recently shown the occurrence of endocytic sucrose uptake in heterotrophic cells. Whether this mechanism is involved in the sucrose-starch conversion process was investigated by comparing the rates of starch accumulation in sycamore cells cultured in the presence or absence of the endocytic inhibitors wortmannin and 2-(4-morpholynyl-)-8-phenyl-4H-1 benzopyran-4-1 (LY294002). These analyses revealed a two-phase process involving an initial 120 min wortmannin- and LY294002-insensitive starch accumulation period, followed by a prolonged phase that was arrested by the endocytic inhibitors. Both wortmannin and LY294002 led to a strong reduction of the intracellular levels of both sucrose and the starch precursor molecule, ADPglucose. No changes in maximum catalytic activities of enzymes closely linked to starch and sucrose metabolism occurred in cells cultured with endocytic inhibitors. In addition, starch accumulation was unaffected by endocytic inhibitors when cells were cultured with glucose. These results provide a first indication that an important pool of sucrose incorporated into the cell is taken up by endocytosis prior to its subsequent conversion into starch in heterotrophic cells. This conclusion was substantiated further by experiments showing that sucrose-starch conversion was strongly prevented by both wortmannin and LY294002 in both potato tuber discs and developing barley endosperms.

Acer↗
PubMed2005

Induction of IL-6 expression by mechanical stress in the trabecular meshwork.

The trabecular meshwork (TM)/Schlemm's canal (SC) outflow pathway is the tissue responsible for maintaining normal levels of intraocular pressure. In the present study, we investigate the effects of mechanical stress on the expression of IL-6 in the TM meshwork, as well as the effects of this cytokine on outflow pathway function. Application of cyclic mechanical stress to human TM primary cultures resulted in a statistically significant increase in both secretion and transcription of IL-6, compared to nonstressed controls. Addition of TGF-beta1, which has been reported to be upregulated in TM cells under mechanical stress, also induced a significant activation of both the transcription and secretion of IL-6. Moreover, anti-TGF-b1 antibodies partially blocked the stretch-induced IL-6 production. Injection of IL-6 into perfused porcine anterior segments resulted in a 30% increase in outflow facility, as well as increased permeability through SC cell monolayers. These results suggest a role for IL-6 in the homeostatic modulation of aqueous humor outflow resistance.

Animals↗
PubMed2005

Lentiviral mediated gene delivery to the anterior chamber of rodent eyes.

PURPOSE: To study the in vivo efficiency of lentiviral vectors in delivering genes to the trabecular meshwork (TM) of rodent eyes. METHODS: Lentiviral vectors were constructed using the elongation factor 1 alpha (EF-1alpha) promoter driving expression of the green fluorescent protein (GFP) gene. The viral construct was injected intracamerally through the peripheral cornea into the anterior chamber of live rodent eyes. Several variables were evaluated to determine the optimal conditions for TM cell transduction. These parameters included viral concentration, injection volume, needle rotation, and the modulation of anterior chamber current convections. Changes in intraocular pressures (IOPs) were monitored using a Tonopen XL. Signs of inflammation and corneal neovascularization were evaluated by slit lamp observation. Three weeks after injection, the eyes were enucleated and analyzed for GFP expression and distribution. RESULTS: A single intracameral viral dose between 10(7) and 10(8) pfu produced a high and evenly distributed expression of GFP in the TM and corneal endothelial cells. The cornea remained clear and no signs of inflammation were present during the course of the experiment. Moreover, no significant changes in IOPs were observed. CONCLUSIONS: A high transduction efficiency of TM and corneal endothelial cells can be effectively obtained after a single dose of recombinant lentivirus. The EF-1alpha promoter induces high expression of the reporter gene and is a reliable alternative to the CMV promoter when stable, long term expression is desired.

Animals↗
PubMed2005

Extracellular trafficking of myocilin in human trabecular meshwork cells.

Myocilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles.

Adenoviridae↗
PubMed2005

Specific targeting of gene expression to a subset of human trabecular meshwork cells using the chitinase 3-like 1 promoter.

PURPOSE: To compare the gene expression profile of trabecular meshwork (TM) and Schlemm's canal (SC) primary cultures and to identify promoters for targeting gene expression to specific cells in the outflow pathway. METHODS: The differential gene expression profile of four human TM and three SC primary cultures was analyzed by gene microarrays (Affymetrix, Santa Clara, CA) and confirmed by quantitative real-time PCR. Based on the results, a recombinant adenovirus was constructed with the expression of the reporter gene LacZ driven by the 5' promoter region of the chitinase 3-like 1 (Ch3L1) gene (AdCh3L1-LacZ). The expression of the Ch3L1 promoter was analyzed in human TM and SC cells and in human perfused anterior segments infected with AdCh3L1-LacZ. RESULTS: gamma-Sarcoglycan, fibulin-2, and collagen XV were identified as the genes more highly expressed in SC than in TM cells. Ch3L1 showed the highest levels of differential expression in TM versus SC cells. Expression analysis of the Ch3L1 promoter demonstrated specific expression in a subset of the TM cells in cell culture and in perfused anterior segments. CONCLUSIONS: Comparative analysis of gene expression between SC and TM primary cultures identified several genes with promoters potentially capable of targeting gene expression to specific cells within the outflow pathway. Results with the Ch3L1 promoter indicated that two different cell subtypes may be present in the TM. This study provides a new potential tool to investigate the role of these different cell types in both normal and pathophysiological function of the outflow pathway, with implications for possible future glaucoma gene therapy.

Adenoviridae↗
PubMed2005

Epstein-Barr virus in breast carcinoma in Argentina.

CONTEXT: Because the etiology and progression of breast carcinoma remain unclear, novel mechanisms of disease pathogenesis need to be considered. Recent interest has focused on Epstein-Barr virus (EBV), an oncogenic ubiquitous herpesvirus. Investigations of this association could not only broaden understanding of breast cancer etiology but also have implications regarding early detection, treatment, and prevention. OBJECTIVE: To assess EBV presence in breast carcinoma in an Argentine series. DESIGN: Breast biopsy specimens of 69 women with breast carcinoma and fresh tumor tissue of 39 of these women were collected. As controls, 17 biopsy specimens of fibroadenomas, 9 of benign epithelial proliferation, 4 of atypical ductal hyperplasia, and 10 of usual ductal hyperplasia and 8 normal breast tissues from women were studied. The EBV-infected cells were identified by means of immunohistochemical analysis, using a monoclonal antibody against Epstein-Barr virus-encoded nuclear antigen 1 (EBNA-1). Polymerase chain reaction (PCR) was used to amplify EBV DNA, with primers that cover the EBV encoded RNA (EBER) and BamHIW regions. RESULTS: Nuclear expression of EBNA-1 was observed in tumor epithelial cells in 24 (35%) of the 69 cases. We confirmed both positive and negative immunohistochemical results by PCR in those cases where good quality DNA was also available, detecting amplification fragments of 108 base pairs (bp) from the EBER region and 122 bp from the BamHIW region. Neither immunohistochemical analysis nor PCR detected any positive EBV results in the control samples. CONCLUSIONS: Our results demonstrate the presence and expression of EBV restricted to epithelial tumor cells in a subset of breast carcinomas studied. However, no significant association was observed between EBV expression and worse clinical and pathologic patient characteristics.

Aged↗
PubMed2004

Effects of donor age on proteasome activity and senescence in trabecular meshwork cells.

The mechanisms involved in the progressive malfunction of the trabecular meshwork (TM) in glaucoma are not yet understood. To study age-related changes in human TM cells, we isolated primary TM cell cultures from young (ages 9, 14, and 25) and old (ages 66, 70, and 73) donors, and compared levels of oxidized proteins, autofluorescence, proteasome function, and markers for cellular senescence. TM cells from old donors showed a 3-fold increase in oxidized proteins and a 7.5-fold decrease of proteasome activity. Loss of proteasome function was not associated with decreased proteasome content but with partial replacement of the proteolytic subunit PSMB5 with the inducible subunit LMP7. Cells from old donors also demonstrated features characteristic of cellular senescence associated with phosphorylation of p38MAPK but only a modest increase in p53. These data suggest that age-related proteasome inhibition and cellular senescence could contribute to the pathophysiological alterations of the TM in glaucoma.

Adolescent↗
PubMed2004

Expression analysis of the matrix GLA protein and VE-cadherin gene promoters in the outflow pathway.

PURPOSE: To test the ability of promoter fragments from the matrix Gla protein (MGP) and vascular endothelial-cadherin (VE-cad) genes to target gene expression in a specific manner in the cells of the outflow pathway, by using adenoviral-mediated gene transfer in organ culture. METHODS: Perfused anterior segments of human eyes were infected with replication-deficient recombinant adenoviruses expressing the beta-galactosidase reporter gene driven by the cytomegalovirus (CMV; control, n = 6), MGP (n = 6), or VE-cad (n = 12) promoters. Forty-eight hours after infection, the anterior segments were fixed and stained for beta-galactosidase activity. The distribution of beta-galactosidase expression was analyzed in paraffin-embedded sections. RESULTS: The MGP promoter fragment resulted in beta-galactosidase expression by the cells of the conventional outflow pathway and did not show any activity in the corneal endothelium or other cells posterior to the scleral spur. Adenovirus containing the VE-cad promoter fragment showed functionality of the promoter in vascular endothelial cells, but failed to produce any detectable expression in the cells of the outflow pathway. CONCLUSIONS: Directed expression by the MGP gene promoter specifically to the trabecular meshwork (TM) provides a new tool for specific gene transfer to the outflow pathway. Results with the VE-cad promoter fragment indicate possible differences in the regulation of this gene between vascular and Schlemm's canal endothelial cells. Taken together, these data demonstrate the feasibility of targeted gene expression to the outflow pathway cells using tissue specific promoters.

Adenoviruses, Human↗
PubMed2003

Proteasome inhibition by chronic oxidative stress in human trabecular meshwork cells.

The pathophysiologic mechanisms leading to the malfunction of the trabecular meshwork (TM)-Schlemm's canal (SC) outflow pathway in glaucoma are still unclear. We hypothesize that chronic oxidative stress may contribute to the malfunction of the outflow pathway by impairing the intracellular proteasome system of the cells, decreasing the ability of the tissue to modulate outflow resistance. To study the effects of chronic oxidative stress on proteasome function, primary cultures of human TM cells were incubated under 40% oxygen and proteasome activity was analyzed by measuring the accumulation of enhanced green fluorescent protein fused to a PEST motif. Changes in proteasome content, cellular senescence, and cell viability were also monitored. After 10 days of exposure to chronic oxidative stress, TM cells showed a marked decline in proteasome activity that was associated with premature senescence and decreased cell viability. These results suggest that proteasome failure may be involved in glaucoma pathophysiology.

Cell Survival↗
PubMed2003

Malate and malate-channel antibodies inhibit electrogenic and ATP-dependent citrate transport across the tonoplast of citrus juice cells.

Citrus juice cells accumulate high levels of citric acid in their vacuoles when compared to other organic ions including malate. Uptake of citrate into tonoplast vesicles from Citrus juice cells was investigated in the presence of malate, and after incubation with antibodies raised against the vacuolar malate-specific channel of Kalanchoë diagremontiana leaves. Antibodies against the vacuolar malate channel immunoreacted with a protein of similar size in tonoplast extracts from three Citrus varieties differing in citric acid content. Malate channel antibodies inhibited both delta MicroH(+)-dependent and delta MicroH(+)-independent ATP-dependent citrate transport, indicating common domains in both transport systems and to the malate-specific channel of Kalanchoë diagremontiana leaves. Malate strongly inhibited electrogenic citrate transport, whereas ATP-dependent citrate uptake was less affected. Kinetic analysis of citrate transport in the presence of malate confirmed the existence of two citrate transport mechanisms and indicated that both citrate and malate share a common transport channel across the tonoplast of Citrus juice cells.

Adenosine Triphosphate↗
PubMed2003

Evidence for a tonoplast-associated form of sucrose synthase and its potential involvement in sucrose mobilization from the vacuole.

The following work presents new evidence for a tonoplast localization of sucrose synthase and its functional role during the mobilization of vacuolar sucrose. Highly purified tonoplast vesicles were associated with significant SuSy levels as determined by immuno-recognition, enzymatic activity, and by-product measurements. Total tonoplast-bound SuSy was estimated to be approximately 7% of the total tissue activity. SuSy affinity to the tonoplast was confirmed by the lack of SuSy displacement by ionic washes and also by the tonoplast ability to bind to exogenously added SuSy as compared to the cytosolic marker alcohol dehydrogenase. UL-[(14)C]sucrose-loaded vesicles incubated with ATP and UDP produced [(14)C]UDP-Glc as determined by UDP-Glc dehydrogenase and by the ability of the product to bind to DEAE-cellulose and to co-migrate with authentic UDP-Glc on TLC. ATP alone induced sucrose efflux but not the production of [(14)C]ADP-Glc. Kinetic analysis of [(14)C]UDPG formation under conditions of low sucrose availability suggests sucrose channelling between the ATP-dependent sucrose transporter and SuSy, thus corroborating the association of SuSy with the tonoplast and its involvement in sucrose mobilization from the vacuole.

Beta vulgaris↗
PubMedDate not supplied

Cellular senescence in the glaucomatous outflow pathway.

The mechanisms responsible for the progressive malfunction of the trabecular meshwork (TM)-Schlemm's canal (SC) conventional outflow pathway tissue in primary open angle glaucoma (POAG) are still not fully understood. To determine whether POAG is characterized by an accumulation of senescent cells, similar to what has been described in other diseases, we have compared the levels of the senescence marker senescence-associated-beta-galactosidase (SA-beta-gal) in the outflow pathway cells of POAG and age-matched control donors. POAG donors demonstrated a statistically significant fourfold increase in the percentage of SA-beta-gal positive cells. These results suggest a potential role for cellular senescence in the pathophysiology of the outflow pathway.

Aged↗