Gadiparthi N. Rao

Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression.

Vascular smooth muscle cells (VSMCs) proliferate in response to arterial injury. Recent findings suggest that, in addition to platelet-derived growth factors, growth factors from inflammatory cells and endothelial cells at the site of injury may contribute to VSMC proliferation. We hypothesized that a common mechanism by which endothelial cells and inflammatory cells stimulate VSMC growth could be the active oxygen species (i.e., O2-, H2O2, and .OH) generated during arterial injury. Using xanthine/xanthine oxidase to generate active oxygen species, we studied the effects of these agents on VSMC growth. Xanthine/xanthine oxidase (100 microM xanthine and 5 microunits/ml xanthine oxidase) stimulated DNA synthesis in growth-arrested VSMCs by 180% over untreated cells. Administration of the scavenging enzymes superoxide dismutase and catalase demonstrated that H2O2 was primarily responsible for xanthine/xanthine oxidase-induced VSMC DNA synthesis. H2O2 directly increased VSMC DNA synthesis and cell number (maximal at 200 microM) but decreased DNA synthesis of endothelial cells and fibroblasts. This effect was protein kinase C independent: sphingosine, a potent protein kinase C inhibitor, failed to block H2O2-induced VSMC DNA synthesis. H2O2 (200 microM) stimulated c-myc and c-fos mRNA levels by fourfold and 20-fold, respectively, as compared with quiescent levels. In contrast to DNA synthesis, H2O2 induction of c-myc and c-fos mRNA was primarily protein kinase C dependent. These findings show that H2O2 specifically increases VSMC DNA synthesis and suggest a role for this oxidant in intimal proliferation, especially after arterial injury.

Induction of Vascular Endothelial Growth Factor in Balloon-Injured Baboon Arteries: A Novel Role for Reactive Oxygen Species in Atherosclerosis

Neovascularization is a hallmark of neointimal formation in atherosclerotic plaques and restenotic lesions. Vascular endothelial growth factor (VEGF) promotes neovascular growth, whereas oxidative stress is a potent factor in vascular cell proliferation. To investigate the mechanisms of neovascular formation, we treated human and rat vascular smooth muscle cells (VSMCs) with H2O2. Northern blot analysis demonstrated a dose- and time-dependent increase in VEGF mRNA, with a maximum of 4-fold at 3 hours (200 mumol/L). As determined by immunoblotting and enzyme-linked immunosorbent assay, VEGF protein expression and secretion were similarly increased. Human umbilical vein endothelial cells were treated with conditioned medium from VSMCs incubated with 200 mumol/L H2O2. DNA synthesis, measured by thymidine incorporation, was increased 4-fold compared with control, an effect that was blocked by a neutralizing anti-VEGF antibody. The lipid peroxidation product 4-hydroxynonenal (1 mumol/L), an endogenous reactive oxygen species present in human atherosclerotic lesions, also increased VEGF secretion in VSMCs in a similar time-dependent fashion. Immunohistochemical staining and in situ hybridization of aortic sections from balloon-injured baboons demonstrated increased VEGF expression in discrete areas of the neointima and media compared with control sections, and expression correlated with the generation of 4-hydroxynonenal. Regulators of VEGF expression, such as reactive oxygen species, may enhance neovascularization of atherosclerotic and restenotic arteries.

Hydrogen peroxide activation of cytosolic phospholipase A2 in vascular smooth muscle cells.

We have reported previously that hydrogen peroxide induces arachidonic acid release from prelabeled vascular smooth muscle cells. Here, we studied the effect of hydrogen peroxide on the phosphorylation of cytosolic phospholipase A2 in these cells. Hydrogen peroxide induced a rapid, time-dependent increase in the phosphorylation of cytosolic phospholipase A2. Hydrogen peroxide also increased arachidonic acid release from prelabeled cells in a time-dependent manner similar to that of phosphorylation of cytosolic phospholipase A2. Protein kinase C depletion significantly inhibited the hydrogen peroxide-stimulated cytosolic phospholipase A2 phosphorylation and arachidonic acid release. Hydrogen peroxide caused a time-dependent increase in mitogen activated protein kinase activity. Taken together, these findings suggest that cytosolic phospholipase A2 may, at least in part, contribute to arachidonic acid release induced by hydrogen peroxide and this effect appears to be mediated by protein kinase C and mitogen activated protein kinase.

Molecular cloning and complete nucleotide sequence of the cDNA encoding a bovine lens intrinsic membrane protein (MP19)

Recently, we reported the partial characterization of bovine lens intrinsic membrane proteins having apparent SDS-PAGE derived molecular mass of 19, 21, and 23 kDa, and determined that they contained identical NH2- terminal amino acid sequences for the first 20 amino acids. From this amino acid sequence information, a mixed synthetic oligonucleotide was constructed and used to screen a calf lens lambda gt11 cDNA library in order to isolate and characterize the cDNA coding for this membrane polypeptide(s). Two separate cDNA clones were isolated and sequenced, and were found to have an identical sequence of 883 bases with an open reading frame coding for a polypeptide of 173 amino acids, having a molecular mass of 19,683 Daltons. The first 20 amino acids of the translated sequence were identical to that determined by our laboratory previously, and the last seven amino acids were identical to that recently determined by another laboratory from analysis of the extracted polypeptides, indicating that this cDNA is the authentic molecule coding for MP19.

Regulation of CAD gene expression in mouse fibroblasts during the transition from the resting to the growing state

We have analyzed the steady-state levels of CAD mRNA and ATCase activity in BALB/c 3T3 mouse fibroblasts at quiescence and at various time points following the initiation of serum stimulation. Steady-state levels of CAD mRNA in 3T3 cells following 12 h of serum stimulation increased 10-fold over levels measured at quiescence. In contrast to the observed increase in steady-state levels of CAD mRNA, its rate of transcription increased only 3-fold, suggesting that the expression of CAD gene in these cells is regulated at both the transcriptional and post-transcriptional levels, to a major extent by the latter. These increases in CAD mRNA in serum-stimulated cells were followed by parallel increases in ATCase activity as well. When comparing DNA synthesis [( 3H]thymidine uptake) to the accumulation of CAD mRNA and ATCase activity, it was observed that this accumulation occurred during the mid- to late-G1 phase of the cell cycle. These results suggest that the expression of CAD gene is cell growth dependent and may be a prerequisite to DNA synthesis.

Bovine Lens Membrane Proteins: MP70, MP64, and MP38 are Products of the Same Gene

We have carried out limited microsequence analysis of bovine lens intrinsic membrane proteins having molecular weights of 70, 64, and 38 kD. These three polypeptides all have an identical amino acid terminal sequence, at least for the first 17 amino acid residues, indicating a common origin. When calf lens RNA was hybridized with a labeled antisense oligonucleotide common to the amino acid sequence of these three polypeptides, a single message with an apparent molecular size of 2.6 kb was detected. Together, these results indicate that bovine lens MP70, MP64, and MP38 are products of the same gene and that the lower molecular weight polypeptides are the result of degradation (processing) of lens MP70 at its COOH-terminal end.

Bovine lens 23, 21 and 19 kDa intrinsic membrane proteins have an identical amino‐terminal amino acid sequence

We have isolated bovine lens intrinsic membrane proteins (MP) having molecular masses of 19, 21 and 23 kDa. Limited amino acid sequence analysis of the amino‐terminal portion of each of these polypeptides revealed a 100% homology in sequence for the number of residues determined (20 amino acids). Northern blot analysis of bovine lens mRNA using a labeled antisense oligonucleotide probe common to the amino acid sequence of these three peptides revealed a single band having an apparent molecular size of 0.8 kb. Taken together, these findings suggest a genetic commonality between these polypeptides.

Regulation of expression of c-myc protooncogene in a clonal line of mouse lens epithelial cells by serum growth factors

Steady-state levels of c-myc mRNA were determined in a clonal line of mouse lens epithelial cells in quiescent and growth-stimulated states. Steady-state mRNA levels for c-myc increased rapidly from an undetectable amount in quiescent cells to the maximum level (8-fold) in growth-stimulated cells. In contrast to its steady-state mRNA levels, its rate of transcription increased by only 3.4-fold in serum-stimulated cells versus quiescent cells, indicating that the abundance of c-myc transcripts in lens epithelial cells during the serum-induced transition from quiescence to proliferation is regulated by both transcriptional and post-transcriptional mechanisms. Serum stimulation in combination with cycloheximide caused superinduction in the steady-state levels of c-myc mRNA in lens epithelial cells. These additive increases in c-myc mRNA levels in the presence of cycloheximide could be due to a decrease in the apparent turnover rate of c-myc mRNA, which, in fact, was observed in actively growing cells. DNA synthesis, as revealed by [3H]thymidine uptake, began 18 h after the addition of serum to quiescent cells and peaked at 24 h. From these results it is concluded that the expression of c-myc gene in mouse lens epithelial cells in response to serum induction is growth dependent.

Posttranscriptional regulation of the expression of CAD gene during differentiation of F9 teratocarcinoma cells by induction with retinoic acid and dibutyryl cyclic AMP

We have studied the regulation of expression of the carbamoyl‐phosphate synthetase II‐aspartate transcarbamylase‐dihydroorotase gene in F9 teratocarcinoma cells during their differentiation into parietal endoderm cells by induction with a combination of retinoic acid and dibutyryl cyclic AMP. Steady‐state levels of CAD mRNA decreased by 7‐fold in F9 cells following 120 h of retinoic acid and dibutyryl cyclic AMP induction as compared to levels in uninduced cells. Conversely, no apparent changes were found in the steady‐state levels of β‐actin mRNA between induced and uninduced cells. Despite a 7‐fold decrease in the steady‐state levels of CAD mRNA, its rate of transcription remained the same between induced and uninduced cells, indicating a role for posttranscriptional mechanisms for its down regulation during retinoic acid‐ and dibutyryl cyclic AMP‐induced differentiation of F9 cells. The cellular growth rate of F9 cells as determined by [3H]thymidine uptake and parallel cell counting decreased markedly during their induction with retinoic acid and dibutyryl cyclic AMP. Taken together, it is apparent that the expression of the CAD gene is cell‐growth‐dependent and its regulation in this system is at the posttranscriptional level.

Expression of c-myc protooncogene in primary cultures of human corneal stromal cells

We have determined steady-state levels of c-myc mRNA in quiescent and serum-stimulated human corneal stromal cells. Steady-state levels of c-myc mRNA increased 6-fold following 2 hours of serum stimulation over levels observed at quiescence. A parallel increase in the rate of c-myc gene transcription was observed in serum-stimulated cells as compared to quiescent cells, indicating that the abundance of c-myc transcripts in corneal stromal cells during the transition from quiescence to proliferation is regulated mainly at the transcriptional level. These findings indicated that the expression of c-myc gene in human corneal stromal cells is regulated in a cell growth dependent manner in response to serum induction.