Antisense Oligonucleotides to the P65 Subunit of NF-κb Inhibit Human Vascular Smooth Muscle Cell Adherence and Proliferation and Prevent Neointima Formation in Rat Carotid Arteries

doi:10.1006/bbrc.1995.2204

Biochemical and Biophysical Research Communications

Volume 213, Issue 3, 24 August 1995, Pages 827-836

https://doi.org/10.1006/bbrc.1995.2204 Get rights and content

Abstract

Neointima formation associated with vascular restenosis is a complex local inflammatory process actively involving the major cellular component of the atherosclerotic lesion, the vascular smooth muscle cell. NF-kB is a pleotrophic transactivator of a diverse group of genes whose activation has been strongly associated with the cellular response to inflammation. We treated human vascular smooth muscle cells (VSMC) with phosphorothio antisense oligonucleotides to the p65 subunit of NF-kB and report that addition of p65 antisense oligonucleotides (1-20 μM), but not sense or p50, inhibit human VSMC adherence and proliferation in a concentration-dependent manner. Additionally, administration of p65 antisense significantly inhibited neointima formation in balloon angioplasty treated rat carotid arteries, indicating that the p65 subunit of NF-kB transactivates genes whose expression is important in VSMC pathobiology. These results suggest that abrogation of p65 reduces neointima formation by inhibition of smooth muscle cell proliferation and adherence.

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Cited by (119)

Vascular smooth muscle cell proliferation as a therapeutic target. Part 1: molecular targets and pathways
2018, Biotechnology Advances
Citation Excerpt :
The NF-κB inhibitors salicylate and acetylsalicylic acid (aspirin), in concentrations between 1 and 5 mM, effectively attenuated VSMC proliferation and DNA synthesis without inducing cellular toxicity or apoptosis (Marra and Liao, 2001). The use of antisense oligonucleotides to the p65 subunit of NF-κB reduced cell proliferation of cultured VSMCs as well as intimal thickening of injured arteries in rats (Autieri et al., 1995). Microinjection of the inhibitor IκBα blocked NF-κB activation and attenuated VSMC proliferation (Bellas et al., 1995).
Cardiovascular diseases are a major cause of human death worldwide. Excessive proliferation of vascular smooth muscle cells contributes to the etiology of such diseases, including atherosclerosis, restenosis, and pulmonary hypertension. The control of vascular cell proliferation is complex and encompasses interactions of many regulatory molecules and signaling pathways. Herein, we recapitulated the importance of signaling cascades relevant for the regulation of vascular cell proliferation. Detailed understanding of the mechanism underlying this process is essential for the identification of new lead compounds (e.g., natural products) for vascular therapies.
Perivascular delivery of resolvin D1 inhibits neointimal hyperplasia in a rat model of arterial injury
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In this study, we demonstrate that RvD1 has direct effects on RASMCs, consistent with those of a candidate antirestenosis agent. Inhibition of p65 nuclear translocation is of particular importance in that it is implicated not only in inflammatory signaling but also in vascular smooth muscle cell proliferation, migration, and oxidative signaling pathways.10,32-34 Importantly, no cytotoxicity was observed across a wide range of RvD1 concentrations (0.01-100 nM) in vitro.
Lipid mediators derived from omega-3 polyunsaturated fatty acids such as resolvin D1 (RvD1) accelerate the resolution of inflammation and have potential as vascular therapeutics. The objective of this study was to evaluate local perivascular delivery of RvD1 as a means to attenuate neointimal hyperplasia in a rat model of arterial injury.
Smooth muscle cells were harvested from rat aortas to study the effects of RvD1 on rat arterial vascular smooth muscle cell responses in vitro, with focus on inflammation, proliferation, migration, cytoskeletal changes, and cytotoxicity. The safety and efficacy of perivascular delivery of RvD1 through thin biodegradable three-layered poly(lactic-co-glycolic acid) wraps or 25% Pluronic F127 gels were studied in a rat model of carotid angioplasty. A total of 200 ng of RvD1 was loaded into each construct for perivascular delivery after injury. Morphometric and histologic analyses were performed 3 and 14 days after injury.
RvD1 attenuated rat arterial vascular smooth muscle cell inflammatory pathways, proliferation, migration, and mitogen-induced cytoskeletal changes in vitro, without evidence of cytotoxicity. RvD1-loaded wraps reduced neointimal formation after carotid angioplasty by 59% vs no-wrap controls (P = .001) and by 45% vs vehicle-wrap controls (P = .002). RvD1-loaded Pluronic gels similarly reduced neointimal formation by 49% vs no-gel controls (P = .02) and by 52% vs vehicle-gel controls (P = .02). No group was associated with infection, thrombosis, or negative vessel remodeling. Wraps were found to be easier to apply than gel constructs. Ki67 proliferation index was significantly lower in RvD1-loaded wrap-treated arteries compared with both no-wrap and vehicle-wrap controls at both 3 and 14 days after injury (65% vs no-wrap group and 70% vs vehicle-wrap group at day 3, 49% vs both control groups at day 14; P < .05). Similarly, oxidative stress (30% and 29%; P < .05) and nuclear factor κB activation (42% and 45%; P < .05) were significantly lower in the RvD1-loaded wrap group compared with both no-wrap and vehicle-wrap controls at 3 days after injury.
Local perivascular delivery of RvD1 attenuates formation of neointimal hyperplasia without associated toxicity in a rat model of carotid angioplasty. This effect is likely due to attenuation of inflammatory pathways as well as decreased arterial smooth muscle cell proliferation and migration.
Urinary trypsin inhibitor reduced inflammatory response after stent injury in minipig
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Citation Excerpt :
NF-κB could mediate the expression of many inflammatory cytokines [10], which played an important role in smooth muscle cell proliferation and differentiation. Antisense oligonucleotide to the P65 subunit of NF-κB was found to inhibit human VSMC adherence and proliferation and to prevent neointima formation in rat carotid arteries [4]. MCP-1 and IL-6 were the inflammatory factors regulated by the NF-κB signaling pathway.
This study investigated whether urinary trypsin inhibitor (UTI) inhibits neointimal formation by reducing inflammatory response after stent injury.
Twenty minipigs having undergone oversized bare material stent implantation in the left anterior descending artery were randomly subdivided into two groups: a UTI group (n = 10) and a control group (n = 10). Two systemic markers of inflammation (serum macrophage chemoattractant protein-1 and interleukin-6 levels measured by ELISA) were increased after stent implantation, and two days after stem implantation, their levels were positively correlated with the maximal percentage of area stenosis on day 28 (r² = 0.889 and 0.743, respectively). This effect was abolished by UTI administration. Twenty-eight days after implantation, morphometric analysis of the stented arteries revealed significantly reduced luminal stenosis (38 ± 6% vs. 64 ± 12%, P < 0.05), a neointimal area (3.22 ± 0.57 mm² vs. 5.21 ± 1.04 mm², P < 0.05), neointimal thickness (0.31 ± 0.13 mm vs. 0.46 ± 0.16 mm, P < 0.05), and an inflammatory score of 1.02 ± 0.05 vs. 1.30 ± 0.08 in UTI-treated animals as compared with controls. Twenty-eight days after stenting, arterial nuclear factor-κB expression was 36.93 ± 7.16% in all of the cells in controls and 23.32 ± 4.54% in UTI-treated minipigs.
UTI could reduce neointimal formation after stenting by inhibiting the local and the systemic inflammatory response. Percutaneous coronary intervention could benefit from precocious anti-inflammatory treatment.
Divergent roles of NF-κB and Egr-1 in flow-dependent restenosis after angioplasty and stenting
2011, Atherosclerosis
Restenosis after both angioplasty and stenting is flow dependent. The effects of flow are preventable with the antioxidant pyrrolidine dithiocarbamate (PDTC) after angioplasty but not after stenting. We examined to what extent these observations could be explained by the effect of PDTC on NF-κB and Egr-1, two transcription factors which are both flow- and redox-sensitive.
In a flow-modified rabbit carotid model of angioplasty and stenting, we assessed the effects of altered flow, injury and PDTC on expression of Egr-1 and nuclear binding activity of NF-κB. We also examined the effects of local delivery of decoy oligodeoxynucleotides (ODN) specific for NF-κB and Egr-1 on morphology at 28 days in normal and low flow.
The activity of both transcription factors was enhanced by injury (stent > balloon alone) and was further augmented by low flow. PDTC markedly attenuated the activity of NF-κB but not Egr-1. Specific decoy ODN for Egr-1 attenuated intima formation in both stented and balloon injured vessels in both normal and low flow but had no effect on remodelling. In contrast while NF-κB decoy ODN caused a modest but significant reduction in intima formation, there was a striking effect on remodelling in low flow vessels only.
We conclude that Egr-1 plays a pivotal role in intima formation under all flow conditions and that NF-κB plays a key role in flow-sensitive remodelling after angioplasty and that NF-κB inhibition likely accounts for a significant part of the morphological effects of PDTC after vessel injury.
Human proinsulin C-peptide reduces high glucose-induced proliferation and NF-κB activation in vascular smooth muscle cells
2008, Atherosclerosis
Citation Excerpt :
The activated p65 subunit has been found in macrophages, endothelial cells, and VSMCs within human atherosclerotic lesions [9]. Furthermore, administration of anti-sense oligonucleotides to the p65 subunit of NF-κB blocked human VSMC proliferation [10]. Other studies mostly point to a role of NF-κB in regulating apoptosis of VSMCs and fine-tuning of the inflammatory response present in the injured vessel wall [11].
Excessive proliferation of vascular smooth muscle cells (VSMCs) is one of the primary lesions in atherosclerosis development during diabetes. High glucose triggers VSMC proliferation and initiates activation of the transcription factor nuclear factor (NF)-κB. Recently, clinical studies have demonstrated that replacement therapy with C-peptide, a cleavage product of insulin, to type 1 diabetic (T1D) patients is beneficial on a variety of diabetes-associated vascular complications. However, the mechanisms underlying the beneficial activity of C-peptide on the vasculature in conditions of hyperglycemia are largely unknown. The effects of C-peptide on the proliferation of human umbilical artery smooth muscle cell (UASMC) and aortic smooth muscle cell (AoSMC) lines cultured under high glucose for 48 h were tested. To gain insights on potential intracellular signaling pathways affected by C-peptide, we analyzed NF-κB activation in VSMCs since this pathway represents a key mechanism for the accelerated vascular disease observed in diabetes. High glucose conditions (25 mmol/L) stimulated NF-κB-dependent VSMC proliferation since the addition of two NF-κB-specific inhibitors, BAY11-7082 and PDTC, prevented proliferation. C-peptide at the physiological concentrations of 0.5 and 1 nmol/L decreased high glucose-induced proliferation of VSMCs that was accompanied by decreased phosphorylation of IκB and reduced NF-κB nuclear translocation. These results suggest that in conditions of hyperglycemia C-peptide reduces proliferation of VSMCs and NF-κB nuclear translocation. In patients with T1D, physiological C-peptide levels may exert beneficial effects on the vasculature that, under high glucose conditions, is subject to progressive dysfunction.
Differential regulation of Mn-superoxide dismutase in neurons and astroglia by HIV-1 gp120: Implications for HIV-associated dementia
2007, Free Radical Biology and Medicine
HIV-associated dementia, like several other neurodegenerative diseases, is characterized by selective degeneration of neurons amidst survival of glial cells like astroglia. The molecular basis of such selective susceptibility within the same milieu remains largely unknown. Neurons are rarely infected by the virus. However, they are vulnerable to viral products, like HIV-1 coat protein gp120. Interestingly, gp120 induced oxidative stress in neurons, but not in astroglia. This led us to postulate that astroglia were armed with a more efficient antioxidant system than neurons. Here, we report that the constitutive level of MnSOD (SOD2), the major cellular antioxidant enzyme, is significantly higher in astroglia than in neurons. Furthermore, gp120 treatment enhanced MnSOD levels in astroglia but decreased the same in neurons. This increase in astroglial MnSOD was dependent on NF-κB, the crucial transcription factor required for sod2 gene transcription. Blocking NF-κB with p65-antisense, p65-si-RNA, or a specific inhibitor, NBD peptide, led to reduced MnSOD levels and enhanced vulnerability of astroglia to gp120. Additionally, neurons were found to have a lower constitutive level of NF-κB p65 than astrocytes. Overexpression of p65 increased the level of MnSOD in neurons. This, in turn, elicited greater neuronal resistance to gp120. Taken together, our study suggests that astroglia manifest a higher threshold for gp120-induced lethality than neurons due to greater MnSOD availability, which is demonstrated due to greater level of NF-κB p65.

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Regular ArticleAntisense Oligonucleotides to the P65 Subunit of NF-κb Inhibit Human Vascular Smooth Muscle Cell Adherence and Proliferation and Prevent Neointima Formation in Rat Carotid Arteries

Abstract

Regular Article
Antisense Oligonucleotides to the P65 Subunit of NF-κb Inhibit Human Vascular Smooth Muscle Cell Adherence and Proliferation and Prevent Neointima Formation in Rat Carotid Arteries