Peter I. Bonta

Anti–MCP-1 Gene Therapy Inhibits Vascular Smooth Muscle Cells Proliferation and Attenuates Vein Graft Thickening Both In Vitro and In Vivo

Objective—Because late vein graft failure is caused by intimal hyperplasia (IH) and accelerated atherosclerosis, and these processes are thought to be inflammation driven, influx of monocytes is one of the first phenomena seen in IH, we would like to provide direct evidence for a role of the MCP-1 pathway in the development of vein graft disease. Methods and Results—MCP-1 expression is demonstrated in various stages of vein graft disease in a murine model in which venous interpositions are placed in the carotid arteries of hypercholesterolemic ApoE3Leiden mice and in cultured human saphenous vein (HSV) segments in which IH occurs. The functional involvement of MCP-1 in vein graft remodeling is demonstrated by blocking the MCP-1 receptor CCR-2 using 7ND-MCP-1. 7ND-MCP1 gene transfer resulted in 51% reduction in IH in the mouse model, when compared with controls. In HSV cultures neointima formation was inhibited by 53%. In addition, we demonstrate a direct inhibitory effect of 7ND-MCP-1 on the proliferation of smooth muscle cell (SMC) in HSV cultures and in SMC cell cultures. Conclusion—These data, for the first time, prove that MCP-1 has a pivotal role in vein graft thickening due to intimal hyperplasia and accelerated atherosclerosis.

Activation of Nuclear Receptor Nur77 by 6-Mercaptopurine Protects Against Neointima Formation

Background— Restenosis is a common complication after percutaneous coronary interventions and is characterized by excessive proliferation of vascular smooth muscle cells (SMCs). We have shown that the nuclear receptor Nur77 protects against SMC-rich lesion formation, and it has been demonstrated that 6-mercaptopurine (6-MP) enhances Nur77 activity. We hypothesized that 6-MP inhibits neointima formation through activation of Nur77. Methods and Results— It is demonstrated that 6-MP increases Nur77 activity in cultured SMCs, which results in reduced [3H]thymidine incorporation, whereas Nur77 small interfering RNA knockdown partially restores DNA synthesis. Furthermore, we studied the effect of 6-MP in a murine model of cuff-induced neointima formation. Nur77 mRNA is upregulated in cuffed arteries, with optimal expression after 6 hours and elevated expression up to 7 days after vascular injury. Local perivascular delivery of 6-MP with a drug-eluting cuff significantly inhibits neointima formation in wild-type mice. Locally applied 6-MP does not affect inflammatory responses or apoptosis but inhibits expression of proliferating cell nuclear antigen and enhances protein levels of the cell-cycle inhibitor p27Kip1 in the vessel wall. An even stronger inhibition of neointima formation in response to local 6-MP delivery was observed in transgenic mice that overexpressed Nur77. In contrast, 6-MP does not alter lesion formation in transgenic mice that overexpress a dominant-negative variant of Nur77 in arterial SMCs, which provides evidence for the involvement of Nur77-like factors. Conclusions— Enhancement of the activity of Nur77 by 6-MP protects against excessive SMC proliferation and SMC-rich neointima formation. We propose that activation of the nuclear receptor Nur77 is a rational approach to treating in-stent restenosis.

NR4A nuclear orphan receptors: protective in vascular disease?

Purpose of review The nuclear orphan receptors Nur77 (NR4A1), Nurr1 (NR4A2) and NOR-1 (NR4A3) are known to be involved in T-cell apoptosis, brain development, and the hypothalamic–pituitary–adrenal axis. Here, we review our current understanding of the NR4A nuclear receptors in processes that are relevant to vascular disease. Recent findings NR4A nuclear receptors have recently been described to play a role in metabolism by regulating gluconeogenesis, lipolysis, energy expenditure, and adipogenesis. The function of NR4A nuclear receptors has also extensively been investigated in cells crucial in vascular lesion formation, such as macrophages, endothelial cells and smooth muscle cells. Summary The involvement of NR4A nuclear receptors in both metabolism and in processes in the vessel wall supports a substantial role for NR4A nuclear receptors in the development of vascular disease.

Blood flow-dependent arterial remodelling is facilitated by inflammation but directed by vascular tone

AIMS Altered blood flow affects vascular tone, attracts inflammatory cells, and leads to microvascular remodelling. We tested the hypothesis that inflammation facilitates the remodelling response, but that vascular tone determines its direction (inward or outward). METHODS AND RESULTS Mouse mesenteric resistance arteries were ligated to create either increased blood flow or low blood flow in vivo. In vivo microscopy was used to determine changes in vascular tone. Structural remodelling was studied after 2 days, with or without macrophage depletion. In order to characterize the inflammatory response, immunostaining, confocal microscopy, and real-time PCR were used. To address the role of vascular tone in remodelling, arteries were treated with the vasodilator amlodipine during organ culture. Vessels exposed to high blood flow dilated, whereas low flow induced constriction. After 1 day, inflammatory markers showed a complex but remarkably similar increase in expression during high flow and low flow. Both high-flow and low-flow vessels showed an increase in the number of adventitial macrophages. Depletion of macrophages eliminated flow-induced remodelling. Manipulation of vascular tone reversed inward remodelling in response to low blood flow. CONCLUSION Altered blood flow triggers an inflammatory response that facilitates remodelling. Vascular tone is a crucial determinant of the direction of the remodelling response.

Endothelial CD81 is a marker of early human atherosclerotic plaques and facilitates monocyte adhesion.

AIMS In a recent report, we established at the genome-wide level those genes that are specifically upregulated in the endothelium of atherosclerotic plaques in human arteries. As the transcriptome data revealed that mRNA for the tetraspanin family member CD81 is significantly and specifically upregulated in the endothelium overlying early atheroma, we set out to validate these results on the protein level, and investigate the functional consequences of CD81 upregulation. METHODS AND RESULTS Immunohistochemical analysis in an independent set of donor arteries verified in the endothelium of early human atherosclerotic lesions the enhanced expression of CD81, which appears oxidative stress-dependent. Using lentiviral overexpression and silencing in human umbilical endothelial cells, we established in an in vitro flow adhesion assay that elevated endothelial CD81 is associated with increased monocyte adhesion to non-activated CD81-transduced endothelial cells, approaching the levels normally only attained after tumour necrosis factor alpha stimulation. The CD81 effect was dependent on both intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as it was abolished in the presence of a mixture of anti-ICAM-1 and anti-VCAM-1 antibodies. Flow cytometry revealed that increased CD81 levels did not increase total ICAM-1 and VCAM-1 surface expression. Instead, it concentrated the available adhesion molecules into membrane clusters, as indicated by confocal and electron microscopy. CD81 also colocalized with ICAM-1 and VCAM-1 in the adhesion rings around bound monocytes. CONCLUSION Endothelial CD81 upregulated in early human atheroma has the potential to play a crucial role in the initial stages of atherosclerotic plaque formation by increasing monocyte adhesion prior to the full-blown inflammatory response.

Nuclear receptor Nurr1 is expressed in and is associated with human restenosis and inhibits vascular lesion formation in mice involving inhibition of smooth muscle cell proliferation and inflammation.

Background— Restenosis is the major drawback of percutaneous coronary interventions involving excessive activation and proliferation of vascular smooth muscle cells (SMCs). The nuclear receptor Nurr1 is an early response gene known mainly for its critical role in the development of dopamine neurons. In the present study, we investigated Nurr1 in human and experimental vascular restenosis. Methods and Results— In a prospective cohort of 601 patients undergoing percutaneous coronary intervention, including stent placement, we found a strong association between Nurr1 haplotypes and in-stent restenosis risk. Furthermore, Nurr1 is specifically expressed in human in-stent restenosis and induced in cultured human SMCs in response to serum or tumor necrosis factor-&agr;. Lentivirus-mediated gain- and loss-of-function experiments in SMCs demonstrated that overexpression of Nurr1 inhibited proliferation, consistent with increased expression of the key cell-cycle inhibitor p27Kip1, whereas Nurr1 silencing enhanced SMC growth. The tumor necrosis factor-&agr;–induced proinflammatory response of SMCs is inhibited by Nurr1, as reflected by reduced interleukin-1&bgr;, tumor necrosis factor-&agr;, and monocyte chemoattractant protein-1 expression. Consistent with our in vitro data, endogenous Nurr1 reduced wire injury–induced proliferation and vascular lesion formation in carotid arteries of ApoE−/− mice. Conclusion— Nurr1 haplotypes are associated with human restenosis risk, and Nurr1 is expressed in human in-stent restenosis. In SMCs, Nurr1 inhibits proliferation and inflammatory responses, which explains the inhibition of SMC-rich lesion formation in mice. The recently identified small-molecule drugs that enhance the activity of Nurr1 reveal this nuclear receptor as an attractive novel target for (local) intervention in restenosis.

Plasminogen activator inhibitor-1 regulates neutrophil influx during acute pyelonephritis

Acute pyelonephritis, frequently caused by Escherichia coli, is a substantial health problem. Plasminogen activator inhibitor type-1 (PAI-1) not only inhibits plasminogen activation but is also involved in cell migration. To determine if it has a role in host defense, we induced pyelonephritis in PAI-1 gene knockout and wild-type mice by intravesical inoculation with uropathogenic E. coli 1677. Bacterial growth was determined on blood agar plates in portions of the kidneys homogenized in sterile saline. Kidney levels of PAI-1 were increased in infected compared to control mice, suggesting a physiological role for PAI-1 during pyelonephritis. The knockout mice had significantly more bacterial outgrowth in kidney homogenates compared to the wild-type mice. Strikingly, higher colony-forming units were accompanied by increased levels of the cytokines TNF-alpha, IL-1beta, and IL-6 in the kidneys of knockout mice, but levels of the chemokines KC and MIP-2 were not different. Remarkably, plasma levels of KC were higher, but renal neutrophil influx was significantly lower, in the knockout than in the wild-type mice. Our study shows that PAI-1 is critically involved in host defense against E. coli-induced acute pyelonephritis, in part, by modulating neutrophil influx.

p27kip1–838C>A Single Nucleotide Polymorphism Is Associated With Restenosis Risk After Coronary Stenting and Modulates p27kip1 Promoter Activity

Background— The cyclin-dependent kinase inhibitor p27kip1 is a key regulator of smooth muscle cell and leukocyte proliferation in vascular disease, including in-stent restenosis. We therefore hypothesized that common genetic variations or single nucleotide polymorphisms in p27kip1 may serve as a useful tool in risk stratification for in-stent restenosis. Methods and Results— Three single nucleotide polymorphisms concerning the p27kip1 gene (−838C>A, rs36228499; −79C>T, rs34330; +326G>T, rs2066827) were determined in a cohort of 715 patients undergoing coronary angioplasty and stent placement. We discovered that the p27kip1-838C>A single nucleotide polymorphism is associated with clinical in-stent restenosis; the −838AA genotype decreases the risk of target vessel revascularization (hazard ratio, 0.28; 95% confidence interval, 0.10 to 0.77). This finding was replicated in another cohort study of 2309 patients (hazard ratio, 0.61; 95% confidence interval, 0.40 to 0.93). No association was detected between this end point and the p27kip1-79C>T and +326G>T single nucleotide polymorphisms. We subsequently studied the functional importance of the −838C>A single nucleotide polymorphism and detected a 20-fold increased basal p27kip1 transcriptional activity of the −838A allele containing promoter. Conclusions— Patients with the p27kip1-838AA genotype have a decreased risk of in-stent restenosis corresponding with enhanced promoter activity of the −838A allele of this cell-cycle inhibitor, which may explain decreased smooth muscle cell proliferation.

6-Mercaptopurine Inhibits Atherosclerosis in Apolipoprotein E*3-Leiden Transgenic Mice Through Atheroprotective Actions on Monocytes and Macrophages

Objective—6-Mercaptopurine (6-MP), the active metabolite of the immunosuppressive prodrug azathioprine, is commonly used in autoimmune diseases and transplant recipients, who are at high risk for cardiovascular disease. Here, we aimed to gain knowledge on the action of 6-MP in atherosclerosis, with a focus on monocytes and macrophages. Methods and Results—We demonstrate that 6-MP induces apoptosis of THP-1 monocytes, involving decreased expression of the intrinsic antiapoptotic factors B-cell CLL/Lymphoma-2 (Bcl-2) and Bcl2-like 1 (Bcl-xL). In addition, we show that 6-MP decreases expression of the monocyte adhesion molecules platelet endothelial adhesion molecule-1 (PECAM-1) and very late antigen-4 (VLA-4) and inhibits monocyte adhesion. Screening of a panel of cytokines relevant to atherosclerosis revealed that 6-MP robustly inhibits monocyte chemoattractant chemokine-1 (MCP-1) expression in macrophages stimulated with lipopolysaccharide (LPS). Finally, local delivery of 6-MP to the vessel wall, using a drug-eluting cuff, attenuates atherosclerosis in hypercholesterolemic apolipoprotein E*3-Leiden transgenic mice (P<0.05). In line with our in vitro data, this inhibition of atherosclerosis by 6-MP was accompanied with decreased lesion monocyte chemoattractant chemokine-1 levels, enhanced vascular apoptosis, and reduced macrophage content. Conclusion—We report novel, previously unrecognized atheroprotective actions of 6-MP in cultured monocytes/macrophages and in a mouse model of atherosclerosis, providing further insight into the effect of the immunosuppressive drug azathioprine in atherosclerosis.

Transfusion of 35-Day Stored RBCs in the Presence of Endotoxemia Does Not Result in Lung Injury in Humans.

Objective:Transfusion-related acute lung injury is the leading cause of transfusion-related mortality. Preclinical studies have shown that aged RBCs can induce transfusion-related acute lung injury in the presence of a “first hit” (e.g., sepsis). Clinical studies, however, show conflicting results on this matter. We tested whether maximally stored RBCs are able to induce lung injury in the presence of a “first hit” in humans (Dutch Trial Register: NTR4455). Design:Open-label, randomized controlled trial. Patients:Healthy male volunteers. Interventions:Eighteen healthy male volunteers donated one unit of autologous RBCs 2 or 35 days before the experiment. The experiment was started by infusion of 2 ng/kg lipopolysaccharide (“first hit”). After 2 hours, volunteers received normal saline (n = 6), 2-day stored transfusion (n = 6), or 35-day stored transfusion (n = 6) (“second hit”). Blood was sampled hourly. Six hours after transfusion, the diffusion capacity of the lungs for carbon monoxide was tested and volunteers underwent spirometry, chest x-ray study, and a bronchoalveolar lavage. Measurements and Main Results:All volunteers fulfilled sepsis criteria after lipopolysaccharide injection. The stored blood transfusion did not result in significant changes in either hemodynamic or respiratory variables compared with the control groups. Furthermore, chest x-rays, lung function, and PaO2/FIO2 ratios did not differ between groups. Transfusion of stored autologous RBCs did not result in an increased level of protein in the lungs or neutrophil influx. Conclusions:Transfusion of 35-day stored autologous RBCs in the presence of endotoxemia does not result in lung injury in humans.