Harris Perlman

Cell cycle exit upon myogenic differentiation

Myogenic precursor cells withdraw irreversibly from the cell cycle as they differentiate into mature myotubes. Cell cycle exit occurs early during the differentiation program and is required for normal expression of the contractile phenotype. Differentiated myocytes also display a decreased propensity to undergo apoptotic cell death. The upregulation of the cyclin-dependent kinase inhibitor p21 and the dephosphorylation of retinoblastoma protein (pRb) appear to be critical regulatory events for the establishment of both the postmitotic and apoptosis-resistant states. The coordinated regulation of cell proliferation and death provides the developing embryo with a mechanism for controlling muscle mass and thereby the size of individual motor units.

Evidence for the Rapid Onset of Apoptosis in Medial Smooth Muscle Cells After Balloon Injury

BACKGROUNDnVascular myocyte apoptotic cell death has been reported in human atherectomy and endarterectomy specimens and for neointimal smooth muscle cells (SMCs) in balloon-injured rat carotid arteries between 7 and 30 days after injury. However, the immediate effect of balloon injury on medial SMC viability has not been examined.nnnMETHODS AND RESULTSnRat carotid arteries were harvested at the time of balloon injury (T = 0) and at 0.5, 1, 2, and 4 hours after injury. Uninjured vessels or vessels harvested at the time of injury (T = 0) did not display evidence of apoptosis. However, as early as 30 minutes after injury, 70% of medial SMCs appeared apoptotic by TdT-mediated dUTP nick end labeling (TUNEL) analysis and by the appearance of condensed chromatin. High frequencies of TUNEL-positive cells were also observed at 1 and 2 hours after injury but not at 4 hours. Transmission electron microscopy revealed many cells with morphological characteristics of apoptosis in the injured sections. A marked decrease in bcl-X expression was detected in the most luminal layers of the media. To corroborate these findings in a second animal model, rabbit external iliac arteries were analyzed after balloon angioplasty. Apoptotic cell death was evident in rabbit arteries at 30 minutes and at 4 hours after injury.nnnCONCLUSIONSnAs early as 30 minutes after balloon injury, myocytes appear to undergo apoptotic cell death at a high frequency as shown by TUNEL staining, chromatin condensation, and the appearance of morphological features in electron micrographs. The induction of apoptosis coincides with a marked downregulation of bcl-X expression.

GATA-6 induces p21(Cip1) expression and G1 cell cycle arrest.

GATA transcription factors represent a family of highly conserved zinc finger proteins with tissue-specific expression patterns. Previous studies have shown that GATA-6 is expressed in vascular smooth muscle cells (VSMCs) and rapidly down-regulated when VSMCs are induced to proliferate. Here we investigated whether the GATA-6 transcription factor can modulate cellular proliferation. Transient transfection with a GATA-6 expression vector inhibited S-phase entry in VSMCs and in mouse embryonic fibroblasts (MEFs) lacking both p53 alleles. The GATA-6-induced growth arrest correlated with a marked increase in the expression of the general cyclin-dependent kinase (Cdk) inhibitor p21. In contrast to p53-deficient MEFs and VSMCs, MEFs null for both p21 alleles were refractory to the GATA-6-induced growth inhibition. These data demonstrate that elevated GATA-6 expression can promote the quiescent phenotype in VSMCs.

GATA-6 Induces p21Cip1 Expression and G1Cell Cycle Arrest

GATA transcription factors represent a family of highly conserved zinc finger proteins with tissue-specific expression patterns. Previous studies have shown that GATA-6 is expressed in vascular smooth muscle cells (VSMCs) and rapidly down-regulated when VSMCs are induced to proliferate. Here we investigated whether the GATA-6 transcription factor can modulate cellular proliferation. Transient transfection with a GATA-6 expression vector inhibited S-phase entry in VSMCs and in mouse embryonic fibroblasts (MEFs) lacking both p53 alleles. The GATA-6-induced growth arrest correlated with a marked increase in the expression of the general cyclin-dependent kinase (Cdk) inhibitor p21. In contrast to p53-deficient MEFs and VSMCs, MEFs null for both p21 alleles were refractory to the GATA-6-induced growth inhibition. These data demonstrate that elevated GATA-6 expression can promote the quiescent phenotype in VSMCs.

A recombinant defective adenoviral agent expressing anti-bcl-2 ribozyme promotes apoptosis of bcl-2-expressing human prostate cancer cells.

Over‐expression of bcl‐2, a potent anti‐apoptosis protein, is likely to be one of the genetic mechanisms through which human prostate cancer cells develop resistance to hormonal and other forms of therapy. To develop a therapeutic agent for hormone‐resistant prostate cancer based on suppression of bcl‐2 expression, we had previously designed and synthesized a dual‐hammerhead ribozyme capable of recognizing and specifically cleaving human bcl‐2 mRNA in vitro as well as in vivo. To increase the efficiency by which the anti‐bcl‐2 ribozyme can be delivered to target cells, we have created a recombinant replication‐deficient (defective) adenoviral agent capable of expressing the anti‐bcl‐2 ribozyme upon infection. This viral agent effectively reduces intracellular levels of bcl‐2 mRNA and protein in cultured LNCaP prostate cancer cells following standard infection procedures. Likewise, the defective adenovirus‐anti‐bcl‐2 ribozyme induces extensive apoptosis in several androgen‐sensitive (LNCaP) and androgen‐insensitive (LNCaP/bcl‐2 and PC‐3) human prostate cancer cell lines that express differing amounts of bcl‐2 protein. One androgen‐insensitive prostate cancer cell line, DU‐145, lacking in bcl‐2 expression, was found to be completely refractory to the effects of the virus ribozyme, supporting the concept that the cytotoxic effects of the ribozyme are based solely on its effects on bcl‐2 expression. Our results support further development of this adenovirus/anti‐bcl‐2 ribozyme for potential gene therapeutic purposes in certain forms of hormone‐resistant prostate cancer where over‐expression of bcl‐2 proto‐oncogene is indicated. Int. J. Cancer 82:846–852, 1999.

Bax‐mediated cell death by the Gax homeoprotein requires mitogen activation but is independent of cell cycle activity

Tissues with the highest rates of proliferation typically exhibit the highest frequencies of apoptosis, but the mechanisms that coordinate these processes are largely unknown. The homeodomain protein Gax is down‐regulated when quiescent cells are stimulated to proliferate, and constitutive Gax expression inhibits cell proliferation in a p21WAF/CIP‐dependent manner. To understand how mitogen‐induced proliferation influences the apoptotic process, we investigated the effects of deregulated Gax expression on cell viability. Forced Gax expression induced apoptosis in mitogen‐activated cultures, but quiescent cultures were resistant to cell death. Though mitogen activation was required for apoptosis, neither the cdk inhibitor p21WAF/CIP nor the tumor suppressor p53 was required for Gax‐induced cell death. Arrest in G1 or S phases of the cell cycle with chemical inhibitors also did not affect apoptosis, further suggesting that Gax‐mediated cell death is independent of cell cycle activity. Forced Gax expression led to Bcl‐2 down‐regulation and Bax up‐regulation in mitogen‐activated, but not quiescent cultures. Mouse embryonic fibroblasts homozygous null for the Bax gene were refractive to Gax‐induced apoptosis, demonstrating the functional significance of this regulation. These data suggest that the homeostatic balance between cell growth and death can be controlled by mitogen‐dependent pathways that circumvent the cell cycle to alter Bcl‐2 family protein expression.

Adenovirus-encoded hammerhead ribozyme to Bcl-2 inhibits neointimal hyperplasia and induces vascular smooth muscle cell apoptosis

OBJECTIVEnThe balance between apoptosis and cell proliferation is vital for cellular homeostasis, yet little is known about the mechanisms that coordinate these two cell fates, particularly in the vessel wall. It is well established that the members of Bcl-2-gene family are regulators of apoptosis, but their role in cellular proliferation is less clear.nnnMETHODSnWe analyzed the effects of disrupting Bcl-2 expression in vascular smooth muscle cells (VSMCs) by adenoviral-mediated delivery of a hammerhead ribozyme against bcl-2 mRNA (Ad-Rbz-Bcl-2).nnnRESULTSnForced ablation of Bcl-2 in balloon-injured rat carotid arteries reduced cell number and inhibited neointimal hyperplasia. In vitro, VSMCs transduced with the Ad-Rbz-Bcl-2 underwent apoptosis as indicated by a reduction in cell number and DNA fragmentation. Ad-Rbz-Bcl-2-transduced cells also exhibited aberrations in both G1- and S-phases of the cell cycle. However, forced perturbations in cell cycle activity by serum-stimulation or treatment with chemical inhibitors did not affect Ad-Rbz-Bcl-2-induced cell death, indicating that these cell cycle changes are not essential for apoptosis.nnnCONCLUSIONnThese data show that physiological levels of Bcl-2 are essential for VSMC viability and that ablation of Bcl-2 alters cell cycle activity through the execution of the apoptotic process.

Adenovirus-mediated delivery of the Gax transcription factor to rat carotid arteries inhibits smooth muscle proliferation and induces apoptosis

Adenovirus-mediated gene delivery in animal models of vascular injury has provided insights into the mechanisms underlying vessel wall pathologies. We have previously demonstrated that overexpression of the Gax transcription factor inhibits neointimal formation in rat and rabbit models of arterial injury. Here, we evaluate potential mechanisms for the reduction in stenotic lesion size due to Gax overexpression. At 3, 7 and 14 days after injury the Ad-Gax-infected arteries displayed a marked decrease in medial vascular smooth muscle cell number (3 days, 54% reduction P < 0.01; 7 days, 41% reduction p < 0.003; 14 days, 49% reduction p < 0.02). at 3 days after injury, pcna expression was attenuated in the ad-gax-treated vessels compared with control vessels (65% reduction p < 0.02), indicating a reduction in cellular proliferation. at 7 days and 14 days after injury ad-gax-infected arteries exhibited elevated number of tunel-positive medial vsmcs compared with control-treated arteries (7 days, 9.2-fold increase p < 0.03; 14 days, 17.2-fold increase p < 0.03), indicating an induction of apoptotic cell death. these data suggest that deregulated gax expression induces first cell cycle arrest and then apoptosis in the vascular smooth muscle cells that contribute to the neointimal layer. therefore, the efficacy of this therapeutic strategy appears to result from the ability of the gax transcriptional regulator to modulate multiple cellular responses.

Early cell loss after angioplasty results in a disproportionate decrease in percutaneous gene transfer to the vessel wall.

Acute cell loss has been documented following angioplasty of normal rat and rabbit arteries. Here we analyzed the effects of balloon injury intensity on early cellular loss in single- and double-injury models and how it influences the efficiency of percutaneous gene delivery to the vessel wall. Rabbits underwent bilateral iliac angioplasties (n = 52) with 2.5-mm (balloon-to-artery [B/A] ratio, 1.08 to 1.13) and 3.0-mm (B/A ratio, 1.29 to 1.34) balloons. In the single-injury model, the 3.0-mm balloon induced a 61% reduction in medial cellularity at 3 days postinjury (p < 0.001) while the 2.5-mm balloon did not produce significant cell loss. In the double-injury model, the effects were more pronounced, with 35% (p < 0.01) and 91% (p < 0.001) reductions in medial cellularity at 3 days with the 2.5- and 3.0-mm balloons, respectively, but neointimal cellularity was decreased only with the 3.0-mm balloon (37% reduction, p = 0.025). Adenovirus-mediated beta-galactosidase gene delivery with a channel balloon (n = 24) revealed that larger balloon-to-artery ratios decreased both absolute levels and relative frequencies of transgene expression in the vessel wall. In the single-injury model, gene transfer efficiency was 4.2+/-1.1 and 1.3+/-0.25% (p < 0.05) for the small and large balloons, respectively. In the double-injury model, gene transfer efficiency was 6.6+/-1.6 and 2.3+/-0.8% (p < 0.05) in the neointima and 4.1+/-1.2 and 2.6+/-1.2% (p = NS) in the media for the small and large balloon, respectively. We conclude that early cell loss is dependent on the intensity of the injury in both single- and double-injury models of balloon angioplasty, with greater frequencies of cell loss occurring in the media than in the neointima. In both models, larger balloon-to-artery ratios result in disproportionate reductions in percutaneous adenovirus-mediated gene delivery.

Regulation of Vascular Smooth Muscle Differentiation and Cell Cycle

Publisher Summary Mature vascular smooth muscle cells (VSMCs) differ from cardiac and skeletal myocytes in that they are not terminally differentiated and they retain their ability to reenter the cell cycle in response to appropriate environmental stimuli. Expression of Cell cycle regulatory proteins in the VSMCs of the vessel wall is temporally and spatially regulated during injury induced lesion formation. Analyses of the kinetics and activity of cell cycle regulatory factors have revealed a marked induction of the cdk2 cyclin-dependent kinase and its cyclin E regulatory subunit within the eight hours of injury. The importance of cell cycle regulatory molecules during intimal hyperplasia is underscored by the observation that injury-induced lesion formation is inhibited by molecules that can interfere with cell-cycle progression. Recently, the expression of the cdk inhibitors p21 and p27 in VSMCs has been shown to be regulated by the composition of the extracellular matrix. Thus, extracellular matrix may play a key role in maintaining homeostasis of the vasculature, whereby integrins provide a sensing mechanism utilized by VSMCs to monitor tissue integrity within the artery. Endothelial release of nitric oxide (NO) is believed to function as a physiological regulator of VSMC proliferation.