Bhooma Srinivasan

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.

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.

Ectodomain shedding of VEGF183, a novel isoform of vascular endothelial growth factor, promotes its mitogenic activity in vitro

We have previously reported the discovery of VEGF183, a novel isoform of vascular endothelial growth factor (VEGF), whose nucleotide sequence revealed an 18-bp deletion in the exon 6A-encoded region of VEGF. The following study was done to characterize VEGF183 and to determine its biological activity in vitro and in vivo. Recombinant human VEGF183 was expressed in Escherichia coli (rhVEGF183) or in human 293 embryonic kidney cells (293-VEGF183) and tested for stimulation of permeability of dermal vessels in normal rats as well as for mitogenic activity and phosphorylation of mitogen-activated protein kinases (MAPK) in cultured human umbilical vein endothelial cells (HUVECs). While small amounts of VEGF183 were secreted into the conditioned media (CM) of 293 cells expressing VEGF183 (293-VEGF183 cells), most of the VEGF183 remained cell surface-bound and could be released into the CM following treatment with plasmin or heparin. CM from 293-VEGF183 cells treated with heparin or plasmin induced about a twofold increase in cell numbers and stimulated MAPK phosphorylation in HUVECs as compared with CM from untreated 293-VEGF183 cells or from heparin- or plasmin-treated 293 cells containing the vector alone. Intradermal injections of rhVEGF183 promoted increased permeability of dermal vessels to Evans blue dye. Our study shows that VEGF183 is predominantly a cell-anchored protein that promotes increased vascular permeability in vivo but requires extracellular cleavage or release by heparin or plasmin to promote its mitogenic activity in vitro.