Biomedical subjects

H Perlman

Publications and source records attributed to H Perlman.

At least 19 recordsLinked to original sources

PubMed2001

Constitutively activated Akt-1 is vital for the survival of human monocyte-differentiated macrophages. Role of Mcl-1, independent of nuclear factor (NF)-kappaB, Bad, or caspase activation.

Recent data from mice deficient for phosphatase and tensin homologue deleted from chromosome 10 or src homology 2 domain-containing 5' inositol phosphatase, phosphatases that negatively regulate the phosphatidylinositol 3-kinase (PI3K) pathway, revealed an increased number of macrophages in these animals, suggesting an essential role for the PI3K pathway for macro-phage survival. Here, we focused on the role of the PI3K-regulated serine/threonine kinase Akt-1 in modulating macrophage survival. Akt-1 was constitutively activated in human macrophages and addition of the PI3K inhibitor, LY294002, suppressed the activation of Akt-1 and induced cell death. Furthermore, suppression of Akt-1 by inhibition of PI3K or a dominant negative (DN) Akt-1 resulted in loss of mitochondrial transmembrane potential, activation of caspases-9 and -3, and DNA fragmentation. The effects of PI3K inhibition were reversed by the ectopic expression of constitutively activated Akt-1 or Bcl-x(L). Inhibition of PI3K/Akt-1 pathway either by LY294002 or DN Akt-1 had no effect on the constitutive or inducible activation of nuclear factor (NF)-kappaB in human macrophages. However, after inhibition of the PI3K/Akt-1 pathway, a marked decrease in the expression of the antiapoptotic molecule Mcl-1, but not other Bcl-2 family members was observed, and Mcl-1 rescued macrophages from LY294002-induced cell death. Further, inhibition of Mcl-1 by antisense oligonucleotides, also resulted in macrophage apoptosis. Thus, our findings demonstrate that the constitutive activation of Akt-1 regulates macrophage survival through Mcl-1, which is independent of caspases, NF-kappaB, or Bad.

Animals↗

PubMed2001

The Fas-FasL death receptor and PI3K pathways independently regulate monocyte homeostasis.

Peripheral blood-derived monocytes spontaneously undergo apoptosis mediated by Fas-Fas ligand (FasL) interactions. Activation of monocytes by LPS or TNF-alpha prevents spontaneous monocyte apoptosis through an unknown mechanism. Here, we demonstrate that LPS and TNF-alpha up-regulate Flip and suppress spontaneous Fas-FasL mediated monocyte apoptosis and caspase 8 and 3 activation. Flip was responsible for this protection, since inhibition of Flip by antisense oligonucleotides in the presence of LPS or TNF-alpha restored monocyte sensitivity to spontaneous apoptosis. We also investigated whether the PI3K pathway contributes to the suppression of spontaneous monocyte apoptosis mediated by LPS and TNF-alpha. Monocytes treated with a reversible PI3K inhibitor (LY294002) displayed enhanced apoptosis, while LPS and TNF-alpha partially protected against apoptosis mediated by LY294002. However, direct suppression of Fas-FasL interactions by addition of neutralizing anti-FasL antibody did not further suppress LY294002-induced apoptosis in the presence of LPS or TNF-alpha. Collectively, these data demonstrate that LPS or TNF-alpha protect monocytes from death receptor-mediated apoptosis through the up-regulation of Flip, but not apoptosis initiated by inhibition of the PI3K pathway.

Animals↗

PubMed2001

Rheumatoid arthritis synovial macrophages express the Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein and are refractory to Fas-mediated apoptosis.

OBJECTIVE: The chronic inflammation and progressive joint destruction observed in rheumatoid arthritis (RA) are mediated in part by macrophages. A paucity of apoptosis has been observed in RA synovial tissues, yet the mechanism remains unknown. The present study sought to characterize the expression of Fas, Fas ligand (FasL), and Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP), and to quantify the apoptosis induced by agonistic anti-Fas antibody, using mononuclear cells (MNC) isolated from the peripheral blood (PB) and synovial fluid (SF) of RA patients. METHODS: The expression of Fas, FasL, and FLIP and apoptosis induced by agonistic anti-Fas antibody in MNC from the PB and SF of RA patients were determined by flow cytometry. Immunohistochemistry employing a monospecific anti-FLIP antibody was performed on RA and osteoarthritis (OA) synovial tissue. RESULTS: CD14-positive monocyte/macrophages from normal and RA PB and from RA SF expressed equivalent levels of Fas and FasL. Furthermore, unlike the CD14-positive PB monocytes, RA SF monocyte/macrophages were resistant to the addition of agonistic anti-Fas antibody. In contrast, both CD14-positive PB and SF monocyte/macrophages were sensitive to apoptosis mediated by a phosphatidylinositol 3-kinase inhibitor. Intracellular staining of the caspase 8 inhibitor, FLIP, in CD14-positive SF monocyte/macrophages revealed a significant up-regulation of FLIP compared with normal and RA PB monocytes. Immunohistochemical analysis of synovial tissue from RA and OA patients revealed increased FLIP expression in the RA synovial lining compared with the OA synovial lining. Furthermore, FLIP expression was observed in the CD68positive population in the RA synovial lining. Forced reduction of FLIP by a chemical inhibitor resulted in RA SF macrophage apoptosis that was enhanced by agonistic anti-Fas antibody, indicating that FLIP is necessary for SF macrophage survival. CONCLUSION: These data suggest that up-regulation of FLIP in RA macrophages may account for their persistence in the disease. Thus, the targeted suppression of FLIP may be a potential therapeutic strategy for the amelioration of RA.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Regulation of IL-6 and IL-8 expression in rheumatoid arthritis synovial fibroblasts: the dominant role for NF-kappa B but not C/EBP beta or c-Jun.

Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) produce IL-6 and IL-8, which contribute to inflammation and joint damage. The promoters of both cytokines possess binding sites for NF-kappaB, C/EBPbeta, and c-Jun, but the contribution of each to the regulation of IL-6 and IL-8 in RA FLS is unknown. We employed adenoviral-mediated gene delivery of a nondegradable IkappaBalpha, or dominant-negative versions of C/EBPbeta or c-Jun, to determine the contribution of each transcription factor to IL-6 and IL-8 expression. Inhibition of NF-kappaB activation significantly reduced the spontaneous and IL-1beta-induced secretion of IL-6 and IL-8 by RA FLS and the IL-1ss-induced production of IL-6 and IL-8 by human dermal fibroblasts. Inhibition of C/EBPbeta modestly reduced constitutive and IL-1beta-induced IL-6 by RA FLS, but not by human dermal fibroblasts, and had no effect on IL-8. Inhibition of c-Jun/AP-1 had no effect on the production of either IL-6 or IL-8. Employing gel shift assays, NF-kappaB, C/EBPbeta, and c-Jun were constitutively activated in RA FLS, but only NF-kappaB and c-Jun activity increased after IL-1beta. The reduction of cytokines by IkappaBalpha was mediated through inhibition of NF-kappaB activation, which resulted in decreased IL-6 and IL-8 mRNA. NF-kappaB was essential for IL-6 expression, because fibroblasts in which both NF-kappaB p50/p65 genes were deleted failed to express IL-6 in response to IL-1. These findings document the importance of NF-kappaB for the regulation of the constitutive and IL-1beta-stimulated expression of IL-6 and IL-8 by RA FLS and support the role of inhibition of NF-kappaB as a therapeutic goal in RA.

Adenoviruses, Human↗

PubMed2000

Bcl-2 expression in synovial fibroblasts is essential for maintaining mitochondrial homeostasis and cell viability.

The regulation of proliferation and cell death is vital for homeostasis, but the mechanism that coordinately balances these events in rheumatoid arthritis (RA) remains largely unknown. In RA, the synovial lining thickens in part through increased proliferation and/or decreased synovial fibroblast cell death. Here we demonstrate that the anti-apoptotic protein, Bcl-2, is highly expressed in RA compared with osteoarthritis synovial tissues, particularly in the CD68-negative, fibroblast-like synoviocyte population. To determine the importance of endogenous Bcl-2, an adenoviral vector expressing a hammerhead ribozyme to Bcl-2 (Ad-Rbz-Bcl-2) mRNA was employed. Ad-Rbz-Bcl-2 infection resulted in reduced Bcl-2 expression and cell viability in synovial fibroblasts isolated from RA and osteoarthritis synovial tissues. In addition, Ad-Rbz-Bcl-2-induced mitochondrial permeability transition, cytochrome c release, activation of caspases 9 and 3, and DNA fragmentation. The general caspase inhibitor zVAD.fmk blocked caspase activation, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation, but not loss of transmembrane potential or viability, indicating that cell death was independent of caspase activation. Ectopically expressed Bcl-xL inhibited Ad-Rbz-Bcl-2-induced mitochondrial permeability transition and apoptosis in Ad-Rbz-Bcl-2-transduced cells. Thus, forced down-regulation of Bcl-2 does not induce a compensatory mechanism to prevent loss of mitochondrial integrity and cell death in human fibroblasts.

Adenoviruses, Human↗

PubMed2000

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

OBJECTIVE: The 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. METHODS: We 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). RESULTS: Forced 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. CONCLUSION: These 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.

Adenoviridae↗

PubMed2000

Macrophages require constitutive NF-kappaB activation to maintain A1 expression and mitochondrial homeostasis.

NF-kappaB is a critical mediator of macrophage inflammatory responses, but its role in regulating macrophage survival has yet to be elucidated. Here, we demonstrate that constitutive NF-kappaB activation is essential for macrophage survival. Blocking the constitutive activation of NF-kappaB with pyrrolidine dithiocarbamate or expression of IkappaBalpha induced apoptosis in macrophagelike RAW 264.7 cells and primary human macrophages. This apoptosis was independent of additional death-inducing stimuli, including Fas ligation. Suppression of NF-kappaB activation induced a time-dependent loss of mitochondrial transmembrane potential (DeltaPsi(m)) and DNA fragmentation. Examination of initiator caspases revealed the cleavage of caspase 9 but not caspase 8 or the effector caspase 3. Addition of a general caspase inhibitor, z-VAD. fmk, or a specific caspase 9 inhibitor reduced DNA fragmentation but had no effect on DeltaPsi(m) collapse, indicating this event was caspase independent. To determine the pathway leading to mitochondrial dysfunction, analysis of Bcl-2 family members established that only A1 mRNA levels were reduced prior to DeltaPsi(m) loss and that ectopic expression of A1 protected against cell death following inactivation of NF-kappaB. These data suggest that inhibition of NF-kappaB in macrophages initiates caspase 3-independent apoptosis through reduced A1 expression and mitochondrial dysfunction. Thus, constitutive NF-kappaB activation preserves macrophage viability by maintaining A1 expression and mitochondrial homeostasis.

Apoptosis↗

PubMed1999

FLICE-inhibitory protein expression during macrophage differentiation confers resistance to fas-mediated apoptosis.

Macrophages differentiated from circulating peripheral blood monocytes are essential for host immune responses and have been implicated in the pathogenesis of rheumatoid arthritis and atherosclerosis. In contrast to monocytes, macrophages are resistant to Fas-induced cell death by an unknown mechanism. FLICE (Fas-associated death domain-like interleukin 1beta-converting enzyme)-inhibitory protein (Flip), a naturally occurring caspase-inhibitory protein that lacks the critical cysteine domain necessary for catalytic activity, is a negative regulator of Fas-induced apoptosis. Here, we show that monocyte differentiation into macrophages was associated with upregulation of Flip and a decrease in Fas-mediated apoptosis. Overexpression of Flip protected monocytes from Fas-mediated apoptosis, whereas acute Flip inhibition in macrophages induced apoptosis. Addition of an antagonistic Fas ligand antibody to Flip antisense-treated macrophages rescued cultures from apoptosis, demonstrating that endogenous Flip blocked Fas-induced cell death. Thus, the expression of Flip in macrophages conferred resistance to Fas-mediated apoptosis, which may contribute to the development of inflammatory disease.

Apoptosis↗

PubMed1999

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.

Adenoviruses, Human↗

PubMed1999

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.

Adenoviridae↗

PubMed1999

C/EBP beta in rheumatoid arthritis: correlation with inflammation, not disease specificity.

Rheumatoid arthritis synovial tissue was examined and compared with osteoarthritis tissue for the presence of the nuclear transcription factor C/EBP beta (NF-IL-6). The region (lining or sublining), cell type, and subcellular distribution (cytoplasmic or nuclear) of the expression of C/EBP beta was characterized. Rheumatoid arthritis synovial fluid and blood and normal peripheral blood were also examined. C/EBP beta was detected in the synovial lining and in sublining cells of synovial tissue from patients with both rheumatoid and osteoarthritis. A significant (P < 0.001 and < 0.05, respectively) increase in the percentage of cells with nuclear staining was seen in the lining layer, compared to cells in the sublining region, in rheumatoid and osteoarthritis. In both diseases a strong correlation (r = 0.79, P < 0.001) was observed between the percentage of cells in the synovial lining that were positive for nuclear C/EBP beta and lining cell depth. Two-color immunohistochemistry demonstrated that both macrophages and fibroblast-like synoviocytes were positive for nuclear C/EBP beta. The presence of C/EBP beta was confirmed by immunohistochemistry and Western blot analysis with isolated synovial fibroblasts. Nuclear C/EBP beta was also detected in rheumatoid synovial fluid monocytes/macrophages, but not in lymphocytes or neutrophils. Western blot analysis confirmed the presence of C/EBP beta in these cells. The intensity of C/EBP beta staining was greater (P < 0.001) in synovial fluid monocytes than in those from normal or rheumatoid peripheral blood. In conclusion, the enhanced nuclear staining for C/EBP beta in the synovial lining, compared to the sublining, suggesting activation in the lining, and the positive correlation of lining layer depth with the percentage of cells in the lining positive for nuclear C/EBP beta, suggest a potential role for C/EBP beta in chronic inflammation. The regulation of the production or activity of C/EBP beta, to inhibit inflammatory mediator expression by synovial macrophages and fibroblasts, offers a novel approach to therapeutic intervention.

Arthritis, Rheumatoid↗

PubMed1999

Regulation of Apoptosis in the Prostate Gland by Androgenic Steroids.

The prostate gland requires androgenic steroids for its appropriate embryological formation and postpubertal growth and, once at adult size, remains dependent on a continuous supply of androgens for its vitality and function. A reduction of the levels of circulating androgens will rapidly induce apoptosis of the cells of the prostate, leading to extensive glandular regression. Studies of rodent models of prostate response to castration have shown that there are some remarkable changes in the gene activity of prostate epithelial cells leading up to apoptosis. There is now evidence for a critical cell signaling pathway, regulated by c-fos expression, necessary for castration-induced apoptosis, as well as evidence that this signaling initiates an abrupt and transient alteration in the synthesis of fas antigen, p53, bax and bcl-2 proteins in the androgen receptor-expressing prostate epithelial cells, the cellular compartment that appears to be the most affected by castration. However, more recent studies suggest that these castration-induced effects on the prostate epithelial cells might be, at least in part, an indirect response to a critical reduction in blood flow to the prostate gland that precedes the onset of epithelial cell apoptosis. The castration effects on blood flow to the prostate gland seem to be related to vascular degeneration associated with apoptosis of a subset of prostate endothelial cells.

Journal Article↗

PubMed1999

An elevated bax/bcl-2 ratio corresponds with the onset of prostate epithelial cell apoptosis.

The prostate gland in adult male rats is highly dependent on androgenic steroids. Castration initiates the regression of this tissue through a process involving the loss of the vast majority of cells by means of apoptosis. We studied this well characterized in vivo model of apoptosis to evaluate how the expression of two particular gene products, bcl-2 and bax, known to be important for the regulation of apoptosis were affected by castration. An RNase protection assay designed to quantify the levels of bax mRNA showed that this transcript was transiently elevated after castration, reaching a peak in expression at 3 days and declining thereafter. In contrast, bcl-2 mRNA expression was continuously elevated over a period of up to 7 days after castration. The distinct changes in the expression of the mRNAs encoding these two genes were confirmed by an in situ hybridization analysis of regressing rat ventral prostate tissues. The elevation in mRNAs were apparently restricted to the secretory epithelial cells of the gland, the cellular compartment of the tissue most affected by castration. Finally, SDS - PAGE/Western blot analysis of bax and bcl-2 protein expression in the regressing rat prostate gland with bax and bcl-2-specific antibodies showed that the changes in the bax and bcl-2 protein levels were similar and consistent to that found for the mRNAs. In summary, the expression of both bax and bcl-2 gene products are uniquely modulated during castration-induced regression of the rat ventral prostate gland. The changes we observed identify a transient but marked increase in the bax/bcl-2 expression ratio of the tissue that peaks on the second and third days after castration, coinciding with the peak periods of prostate cell apoptosis. These data support previous studies done on in vitro systems wherein it was shown that the bax/bcl-2 ratio determines the apoptotic potential of a cell.

Animals↗

PubMed1999

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.

Adenoviridae↗

PubMed1998

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 p21(WAF/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 p21(WAF/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.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

Fas ligand gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated T cell response.

Proliferation of vascular smooth muscle cells (VSMCs) in response to injury plays a key role in the pathogenesis of vascular disorders. Fas ligand (FasL) induces apoptosis in Fas-bearing cells, and its expression on activated T cells contributes to the regulation of the immune response and physiological cell turnover. Here, we show that a replication-defective adenovirus encoding FasL (Ad-FasL) induced apoptosis in Fas-bearing VSMCs. When introduced locally to balloon-injured rat carotid arteries, a well characterized model of a VSMC-derived lesion, Ad-FasL functioned as a potent inhibitor of neointima formation. In rats immunized with an empty adenoviral vector, robust T cell infiltration of the vessel wall was detected after local delivery of a beta-galactosidase-expressing virus (Ad-betagal), whereas T cell infiltrates were not detected after local delivery of Ad-FasL. Prior immunization prevented beta-galactosidase expression from Ad-betagal, whereas the expression of the FasL transgene was unaffected. When Ad-betagal and Ad-FasL were delivered together to preimmunized animals, T cell infiltration was reduced and beta-galactosidase expression was restored. These data demonstrate that Fas ligand gene transfer can effectively inhibit injury-induced vessel lesion formation and can allow adenovirus-harboring cells to evade immune destruction.

Adenoviridae Infections↗

PubMed1997

Adenoviral constructs encoding phosphorylation-competent full-length and truncated forms of the human retinoblastoma protein inhibit myocyte proliferation and neointima formation.

BACKGROUND: The retinoblastoma (Rb) protein is a key cell-cycle regulator that controls entry into the S phase by modulating the activity of the E2F transcription factor. We analyzed the effects of full-length phosphorylation-competent and a mutant truncated form of human Rb for their effects on vascular smooth muscle cell (VSMC) proliferation and neointima formation. METHODS AND RESULTS: A number of mutant forms, both phosphorylation competent and incompetent, of human Rb protein were evaluated for their ability to inhibit E2F activity. The results of these assays indicated that a phosphorylation competent, amino-terminal-truncated Rb protein (Rb56) was a particularly potent inhibitor of E2F-mediated transcription relative to the full-length Rb construct (Rb110). Adenoviral constructs containing Rb56 or Rb110 expressed their respective Rb forms in VSMCs, as determined by Western immunoblot analysis, and were similar in their abilities to arrest the cell cycle, as determined by assays of 3H-thymidine incorporation and by flow cytometric analyses. When examined for their effect on neointima formation after balloon injury of the rat carotid artery, both full-length and truncated forms of Rb inhibited formation of this VSMC-derived lesion. CONCLUSIONS: These analyses revealed that the maintenance of high levels of phosphorylation-competent human Rb, either full-length or truncated forms, in VSMCs is an effective method of modulating the extent of intimal hyperplasia that occurs after balloon-induced vascular injury.

Adenoviridae↗