Rouel S. Roque

Retina-derived microglial cells induce photoreceptor cell death in vitro

In animals with retinal degeneration, the presence of activated microglial cells in the outer retina during the early stages of injury suggests that they may be involved in the ensuing photoreceptor cell death. In the following study, we investigated the effects of rat retina-derived microglial cells on a photoreceptor cell line (661w) using cell culture techniques. The difficulty of obtaining pure populations of photoreceptor cells necessitated our use of the 661w photoreceptor cells generated from retinas of transgenic mice. 661w Cells were incubated for 24-48 h in basal medium or basal medium conditioned by activated microglial cells (MGCM) or Müller cells (MCCM), and tested for cell death using lactate dehydrogenase (LDH) assay. The induction of apoptosis in the 661w cells by MGCM was investigated using Terminal deoxynucleotidyl Transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and DNA laddering. Treatment of 661w cells with MGCM for 48 h resulted in approximately 73% of cells dead as compared with 19-20% of cells grown in either basal medium or MCCM. Serum supplementation or pretreatment with heat did not abolish the cytotoxicity of MGCM. More TUNEL-positive cells were observed in MGCM-treated cultures as compared with those in basal medium. Bands in multiples of approximately 180 bp formed DNA ladders in MGCM-treated but not in basal medium-treated samples. Our study shows that microglial cells release soluble product(s) that induce degeneration of cultured photoreceptor cells. Moreover, the mechanism of microglia-induced photoreceptor cell death may involve apoptosis similar to that observed in animals with retinal degeneration.

Tissue Transglutaminase Expression and Activity in Normal and Glaucomatous Human Trabecular Meshwork Cells and Tissues

PURPOSE Glaucoma is a leading cause of irreversible visual impairment and blindness in the world. A major risk factor for glaucoma is elevated intraocular pressure due to increased resistance of aqueous humor outflow through the trabecular meshwork (TM). In the glaucomatous TM, there is increased accumulation of extracellular matrix (ECM) material due to a disruption of the normal balance between ECM deposition and degradation. Tissue transglutaminase (TGM2) belongs to a family of calcium-dependent enzymes that catalyze the posttranslational modification of the ECM by cross-linking proteins, thus making these proteins resistant to enzymatic and physical degradation. It is possible that the increase in ECM proteins in the glaucomatous TM is due to increased cross-linking activity of TGM2. The purpose of this study was to determine whether there are differences in expression and activity of TGM2 between normal and glaucoma TM cells and tissues. METHODS Normal (n = 3 NTM) and glaucomatous (n = 3 GTM) human TM cell lines were grown until confluent. Western immunoblot analysis of cell lysates was used to compare TGM2 protein levels in NTM and GTM cells. TGM2 enzyme activity between NTM and GTM cells was studied by using a biotin cadaverine assay. In addition, immunohistochemistry of three normal and three glaucomatous TM tissues was used to evaluate the in vivo expression of TGM2, fibronectin (FN) and epsilon-(gamma-glutamyl) lysine (GGEL) proteins. RESULTS Western blot analysis and immunohistochemistry demonstrated the presence of TGM2 protein in both NTM and GTM cells. There was an increase in TGM2 protein in GTM cells compared with NTM cells, and GTM cells also had increased in TGM2 enzyme activity compared with NTM cells. Immunohistochemical results demonstrated increased expression of TGM2 and FN in GTM tissues. FN and GGEL proteins were colocalized in GTM tissues, indicating significant cross-linking of FN by TGM2. CONCLUSIONS This study demonstrated that both NTM and GTM cells express TGM2. In addition, TGM2 protein levels and enzyme activities were elevated in GTM cells. There was also an increase in colocalization of FN and GGEL protein in GTM tissues. These results indicate that TGM2 may play an important role in the pathogenesis of glaucoma by cross-linking ECM proteins such as FN and thus making the ECM more resistant to degradation.

3D‐model of adult cardiac stem cells promotes cardiac differentiation and resistance to oxidative stress

The regenerative inadequacy of the injured myocardium leads to adverse remodeling, cardiac dysfunction, and heart disease. Stem cell‐replacement of damaged myocardium faces major challenges such as inappropriate differentiation, cellular uncoupling, scar formation, and accelerated apoptosis of transplanted cells. These challenges can be met by engineering an in vitro system for delivering stem cells capable of cardiac differentiation, tissue integration, and resistance to oxidative stress. In this study, we describe the formation of three‐dimensional (3D) cell aggregates (“cardiospheres”) by putative stem cells isolated from adult dog myocardium using poly‐L‐ornithine. De novo formation of cardiospheres in growth factor‐containing medium occurred over a period of 2–3 weeks, but accelerated to 2–3 days when seeded on poly‐L‐ornithine. Older cardiospheres developed foci of “beating” cells upon co‐culture with rat neonatal cardiomyocytes. Cardiospheres contained cells that exhibited characteristics of undifferentiated cells; differentiating cardiomyocytes with organized contractile machinery; and vascular cells capable of forming “vessel‐like” networks. They exhibited strong resistance to elevated concentrations of hydrogen peroxide in culture and survived subcutaneous injections without undergoing neoplastic transformation up to 3 weeks post‐transplantation. These findings suggest that cardiospheres are potentially useful for delivering functional stem cells to the damaged heart. J. Cell. Biochem. 105: 612–623, 2008.

dystroglycan isoforms are differentially distributed in adult rat retina

α‐Dystroglycan (α ‐DG) is a laminin/agrin receptor expressed in skeletal muscle as well as in nervous system and other tissues. Glycosylation of the core protein of α‐DG is extensive, variable from tissue to tissue, and functionally relevant. To address differential glycosylation of α‐DG in the retina, we have investigated the distribution of this protein using two different antibodies: 1B7 directed against the core protein of α‐dystroglycan, and IIH6 directed against a carbohydrate moiety (Ervasti and Campbell [1993] J Cell Biol 122:809–823). Monoclonal antibody 1B7 recognizes a broader band than IIH6, which seems to recognize only a subset of α‐DG forms in retina. These data reflect the existence of differentially glycosylated isoforms of α‐DG. Monoclonal antibody 1B7 shows an extensive staining for α‐DG in the inner limiting membrane as well as in the ganglion cell and inner plexiform layers labeling Müller cell processes, whereas monoclonal antibody IIH6 staining is restricted to the inner limiting membrane and blood vessels. Our data indicate that there are distinct isoforms of α‐DG that are localized in apposition to basal lamina in the inner limiting membrane and blood vessels or within the parenchyma of the retina along Müller glia. Both isoforms are expressed in a Müller cell line in culture and coimmunoprecipitate with β‐dystroglycan. These data suggest that DGs may participate in organizing synapses and basement membrane assembly in the retina. J. Comp. Neurol. 420:182–194, 2000.

RPE secreted proteins and antibody influence photoreceptor cell survival and maturation.

Proteins in media conditioned by retinal pigment epithelial cells (RPE-CM) and an antibody against these proteins (RPE-SP) were tested for their respective effects on rat retinal development in vitro and in vivo. Proteins of RPE-CM were separated in denaturing gels and evaluated by Western blot analysis. Retinal explants from postnatal day 2 (P2) rats were cultured in RPE-CM only or CM diluted with the RPE-SP antibody and, after 7 days, the explants were dissociated into single cells that were immunostained for opsin. RPE-CM or antibody was also injected into the vitreous of postnatal day 7 (P7) Long-Evans rats and analyzed 7 and 21 days later. Electrophoretic analysis of RPE-CM predominantly showed 60-70 kDa proteins; when these proteins were probed with RPE-SP antibody by Western blot, immunoreactive proteins were restricted to this narrow molecular weight range. In P2 retinal explant cultures supplemented with RPE-CM, long ganglion cell-like neurites were detected in 3 days. This activity was nullified in explant cultures grown in RPE-CM titrated with antibody, and these explants appeared to degenerate within 5 days. Over 80% of dissociated retinal cells from explants 7 days after treatment with RPE-CM expressed opsin, compared to only 20% of cells from explants grown in defined medium or serum. Retinas of P14 rats injected intravitreally with RPE-CM at P7 had increased numbers of ectopic photoreceptor cells within the inner nuclear layer when compared to retinas of sham-injected eyes. In contrast, retinas of eyes injected intravitreally with RPE-SP antibody exhibited shorter outer (OS) and inner (IS) segments and thinner outer nuclear (ONL) and outer plexiform (OPL) layers than retinas of sham-injected eyes. In conclusion, proteins in RPE-CM appeared to accelerate and maximize the development of rat photoreceptor cells in vitro, while intravitreal injections of its antibody caused an apparent retardation of outer segment maturation. These results suggest that a protein(s) secreted by RPE plays a key role in normal retinal development, particularly in photoreceptor cell survival and outer segment maturation.

Vascular endothelial growth factor is increased following coronary artery occlusion in the dog heart

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible factor expressed into several molecular forms in mammalian tissues of which VEGF120, VEGF164, and VEGF188 are the major isoforms. While VEGF164 is the predominant isoform in most tissues, VEGF188 has been reported to be dominant in cardiac tissues such as that in guinea pig, rat, and mouse. In the dog heart, an important model for studies of myocardial ischemia and angiogenesis, the expression of VEGF remains to be established. We investigated the expression of the various isoforms of VEGF in normal and ischemic dog heart tissues using Reverse transcription-Polymerase chain reaction, Ribonuclease Protection Assay and Western blotting. The nucleotide sequences of the major isoforms of VEGF were also determined using homology cloning techniques. Our study showed that the nucleotide sequences of dog VEGF were highly homologous to human VEGF especially in the c-terminal region spanning exons 5-8. A single amino acid-deletion (Glu5 in human VEGF), similar to that reported in other animal species, was observed in the major isoforms resulting in monomers of 120, 164, and 188 amino acids. A novel splice site similar to that in human VEGF183 was also identified in the dog heart, resulting in the 182 amino acid-containing isoform (VEGF182). Moreover, VEGF164 was expressed at a higher level as compared with VEGF182 or VEGF188 in both normal and ischemic tissues. The identification of the nucleotide sequences of VEGF isoforms in the dog heart should prove useful in investigating the molecular expression of VEGF in canine tissues.

Photic injury promotes cleavage of p75NTR by TACE and nuclear trafficking of the p75 intracellular domain

The p75 neurotrophin receptor (p75NTR) is a member of the tumor necrosis factor receptor superfamily that paradoxically mediates neuronal survival and differentiation or apoptotic cell death. Cleavage of p75NTR by a constitutively active metalloprotease could result in shedding of its extracellular domain (p75ECD) and generation of a pro-apoptotic intracellular domain (p75ICD). In this study, we established that exposure of a transgenic mouse photoreceptor cell line to intense light upregulated the expression of p75NTR and of the disintegrin metalloprotease tumor necrosis factor-converting enzyme (TACE) and resulted in apoptotic cell death. Light damage promoted TACE cleavage of p75NTR resulting in shedding of the soluble p75ECD and nuclear translocation of the p75ICD. Overexpression of TACE and p75NTR-induced p75NTR cleavage and secretion of p75ECD, but not nuclear transport of p75ICD. Light-induced cleavage of p75NTR, nuclear localization of p75ICD, and apoptosis were inhibited by IC-3, a metalloprotease inhibitor. Increased levels of p75NTR and TACE were observed in photoreceptor cells of animals with photic injury. Our findings support a role for TACE in the proteolytic cleavage of p75NTR and light-induced apoptosis.

Expression of p75NTR in photoreceptor cells of dystrophic rat retinas

Although a gene mutation in the Royal College of Surgeons (RCS) dystrophic rat results in defective phagocytosis and in accumulation of debris in the subretinal space, the molecular mechanisms leading to photoreceptor cell death remain unclear. In this study, the expression of p75(NTR), the low-affinity neurotrophin receptor incriminated in the apoptosis of developing neurons, was investigated at various stages of retinal degeneration in dystrophic rats using immunohistochemistry, in situ reverse transcription polymerase chain reaction (RT-PCR), Western blot, and relative RT-PCR. In normal adult retinas, p75(NTR) immunolabeling was observed mainly in the outer limiting membrane, with punctate labeling in the inner nuclear and ganglion cell layers. In 18- to 30-day-old dystrophic retinas, the immunostaining appeared to increase especially in the photoreceptor outer and inner segments. Dense staining was also observed in the retinal pigment epithelium (RPE) and choroid. In 60-day-old dystrophic rat retinas, the density of immunolabeling for p75(NTR) increased dramatically in the remaining inner retina, especially in the inner nuclear, inner plexiform, and ganglion cell layers. Post-embedding immunogold labeling of normal retinas verified the distribution of p75(NTR) in photoreceptor cells within the inner segments, cell bodies, and outer segments. The apparent increased intensity in p75(NTR) immunostaining in dystrophic retinas was verified by Western blots and densitometric analyses. In situ RT-PCR and relative RT-PCR further established increased synthesis of p75(NTR) in dystrophic retinas. The increased levels of p75(NTR) in the RPE and photoreceptor cells, the initial sites of injury, during retinal degeneration in dystrophic rats strongly suggest that altered expression of p75(NTR) may be directly involved in photoreceptor death.

Enhancement of cisplatin sensitivity by NSC109268 in budding yeast and human cancer cells is associated with inhibition of S-phase progression

PurposeNSC109268 has been described previously as inhibitor of proteasomal degradation and of phosphatase 2Cα. In a yeast screen, we isolated NSC109268 as an agent altering sensitivity to DNA-damaging agents. We found that NSC109268 and the related compound NSC109272 enhance cellular sensitivity to cis- and transplatin but reduce sensitivity to nitrogen mustard. We explored if similar effects could be found in human cancer cells and if cell cycle analysis could hint at the underlying molecular mechanism.MethodsHaploid yeast cells were treated in suspension with platinum agents and nitrogen mustard alone or in combination with NSC compounds, and survival was measured by colony-formation assays. Sensitivity of ovarian and prostate cancer cells toward these treatments was evaluated using the MTS assay. Cell cycle progression was determined by flow cytometry.ResultsThe enhancement of cisplatin sensitivity by NSC109268 found in yeast was confirmed in cisplatin-sensitive and cisplatin-resistant human ovarian cancer lines and in prostate cancer cells. In yeast and in human carcinoma cells, a correlation of enhanced sensitivity with delaying S-phase progression was revealed.ConclusionThe known activities of NSC109268 as proteasome or phosphatase inhibitor could explain the phenotype of S-phase delay by assuming a higher initial DNA damage load, inhibition of DNA translesion synthesis or extended checkpoint arrest.

RPE-derived factors modulate photoreceptor differentiation: A possible role in the retinal stem cell niche

A photoreceptor cell line, designated 661W, was tested for its response to growth factors secreted by retinal pigment epithelial cells including basic fibroblast growth factor, epidermal growth factor, and nerve growth factor. Early passaged 661W cells expressed high levels of retinal progenitor markers such as nestin and Pax6, but not opsin or glial fibrillary acidic protein. 661W cells grown in FGF-2 or EGF exhibited a multiple-process morphology with small phase-bright nuclei similar to neurons, whereas cells cultured in nerve growth factor (NGF) or retinal pigment epithelium (RPE)-conditioned medium (RPE-CM) displayed rounded profiles lacking processes. 661W cells grown in FGF-2 were slightly elevated, but not significantly above, control cultures; but cells treated with RPE-CM or NGF were fewer, ∼63% and 49% of control, respectively. NGF immunodepletion of RPE-CM strongly suppressed the inhibitory activity of RPE-CM on cell proliferation. Cells treated with FGF-2, but not NGF, upregulated their expression of opsin. All treatment conditions resulted in almost 100% viability based on calcium AM staining. Cells grown on extracellular matrix proteins laminin, fibronectin, and/or collagen resembled those grown on untreated dishes. This study showed that early passaged 661W cells displayed characteristics of retinal progenitor cells. The 661W cells proliferated and appeared to mature morphologically expressing rod photoreceptor phenotype in response to FGF-2. In contrast, NGF and RPE-CM inhibited proliferation and morphological differentiation of 661W cells, possibly inducing cell cycle arrest. These findings are consistent with reports that the RPE modulates photoreceptor differentiation and retinal progenitor cells via secreted factors and may play a role in the regulation of the retinal stem cell niche.