Daniel Sedding

Vascular proliferation and atherosclerosis: New perspectives and therapeutic strategies

In atherosclerosis, the vascular smooth muscle cell (VSMC) contributes to vessel wall inflammation and lipoprotein retention, as well as to the formation of the fibrous cap that provides stability to the plaque. The VSMC can undergo a proliferative response that underlies the development of in-stent restenosis, bypass graft occlusion and transplant vasculopathy. Although the benefit/risk of therapeutic inhibition of VSMC proliferation in atherosclerosis is unclear, experimental and human evidence strongly suggests the therapeutic potential of antiproliferative therapy for in-stent restenosis, bypass graft failure and other vascular proliferative disorders.

Extracellular RNA constitutes a natural procoagulant cofactor in blood coagulation

Upon vascular injury, locally controlled haemostasis prevents life-threatening blood loss and ensures wound healing. Intracellular material derived from damaged cells at these sites will become exposed to blood components and could contribute to blood coagulation and pathological thrombus formation. So far, the functional and mechanistic consequences of this concept are not understood. Here, we present in vivo and in vitro evidence that different forms of eukaryotic and prokaryotic RNA serve as promoters of blood coagulation. Extracellular RNA was found to augment (auto-)activation of proteases of the contact phase pathway of blood coagulation such as factors XII and XI, both exhibiting strong RNA binding. Moreover, administration of exogenous RNA provoked a significant procoagulant response in rabbits. In mice that underwent an arterial thrombosis model, extracellular RNA was found associated with fibrin-rich thrombi, and pretreatment with RNase (but not DNase) significantly delayed occlusive thrombus formation. Thus, extracellular RNA derived from damaged or necrotic cells particularly under pathological conditions or severe tissue damage represents the long sought natural “foreign surface” and provides a procoagulant cofactor template for the factors XII/XI-induced contact activation/amplification of blood coagulation. Extracellular RNA thereby reveals a yet unrecognized target for antithrombotic intervention, using RNase or related therapeutic strategies.

Caveolin-1 Facilitates Mechanosensitive Protein Kinase B (Akt) Signaling In Vitro and In Vivo

Mechanotransduction represents an integral part of vascular homeostasis and contributes to vascular lesion formation. Previously, we demonstrated a mechanosensitive activation of phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt) resulting in p27Kip1 transcriptional downregulation and cell cycle entry of vascular smooth muscle cells (VSMC). In this study, we further elucidated the signaling from outside-in toward PI3-K/Akt in vitro and in an in vivo model of elevated tensile force. When VSMC were subjected to cyclic stretch (0.5 Hz at 125% resting length), PI3-K, Akt, and Src kinases were found activated. Disrupting caveolar structures with &bgr;-cyclodextrin or transfection of VSMC with caveolin-1 antisense oligonucleotides (ODN) prevented PI3-K and Akt activation and cell cycle entry. Furthermore, PI3-K and Akt were resistant to activation when Src kinases were inhibited pharmacologically or by overexpression of a kinase-dead c-Src mutant. &agr;V&bgr;3 integrins were identified to colocalize with PI3-K/caveolin-1 complexes, and blockade of &agr;V&bgr;3 integrins prevented Akt activation. The central role of caveolin-1 in mechanotransduction was further examined in an in vivo model of elevated tensile force. Interposition of wild-type (WT) jugular veins into WT carotid arteries resulted in a rapid Akt activation within the veins that was almost abolished when veins of caveolin-1 knockout (KO) mice were used. Furthermore, late neointima formation within the KO veins was significantly reduced. Our study provides evidence that PI3-K/Akt is critically involved in mechanotransduction of VSMC in vitro and within the vasculature in vivo. Furthermore, caveolin-1 is essential for the integrin-mediated activation of PI3-K/Akt.

Correlation of Vasa Vasorum Neovascularization and Plaque Progression in Aortas of Apolipoprotein E−/−/Low-Density Lipoprotein−/− Double Knockout Mice

Objective—We hypothesized that apolipoprotein E (apoE)−/−/low-density lipoprotein (LDL)−/− double knockout mice might develop vasa vasorum (VV) in association with advanced lesion formation. Methods and Results—Aortas from apoE−/−/LDL−/− mice aged 16, 18, 20, or 80 weeks were infused in situ with Microfil, harvested, and scanned with micro-computed tomography (CT). We characterized plaque volume and CT “density” as well as VV luminal volume along the aorta using Analyze 6.0 software. Results were complemented by a detailed histological plaque classification according to American Heart Association guidelines. From 16 to 80 weeks, plaque volume and VV opacified lumen volume increased with age (P<0.001). The 3-dimensional micro-CT images of arterial and venous VV trees allowed perfusion territories to be delineated. The spatial location and magnitude of VV density and adventitial inflammation were strongly correlated in advanced atherosclerotic lesions (r=0.91) and identified as an independent correlate to advanced lesions. At age 80 weeks, VV luminal volume was increased 20-fold compared with animals at age 16 weeks (P<0.001). Micro-CT showed that adventitial VV communicate with intraplaque microvessels. Conclusion—Our results show that apoE−/−/LDL−/− double knockout mice develop VV and advanced atheromas along the aorta. Lesion volume was closely associated with amount of neovascularization in advanced atheromas.

Pro-proliferative and inflammatory signaling converge on FoxO1 transcription factor in pulmonary hypertension.

Pulmonary hypertension (PH) is characterized by increased proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs). Forkhead box O (FoxO) transcription factors are key regulators of cellular proliferation. Here we show that in pulmonary vessels and PASMCs of human and experimental PH lungs, FoxO1 expression is downregulated and FoxO1 is inactivated via phosphorylation and nuclear exclusion. These findings could be reproduced using ex vivo exposure of PASMCs to growth factors and inflammatory cytokines. Pharmacological inhibition and genetic ablation of FoxO1 in smooth muscle cells reproduced PH features in vitro and in vivo. Either pharmacological reconstitution of FoxO1 activity using intravenous or inhaled paclitaxel, or reconstitution of the transcriptional activity of FoxO1 by gene therapy, restored the physiologically quiescent PASMC phenotype in vitro, linked to changes in cell cycle control and bone morphogenic protein receptor type 2 (BMPR2) signaling, and reversed vascular remodeling and right-heart hypertrophy in vivo. Thus, PASMC FoxO1 is a critical integrator of multiple signaling pathways driving PH, and reconstitution of FoxO1 activity offers a potential therapeutic option for PH.

Time-Course Analysis on the Differentiation of Bone Marrow-Derived Progenitor Cells Into Smooth Muscle Cells During Neointima Formation

Objective—Bone marrow-derived progenitor cells have been implicated to contribute to neointima formation, but the time course and extent of their accumulation and differentiation into vascular cells and, most importantly, the long-term contribution of bone marrow-derived progenitor cells to the vascular lesion remain undefined. Methods and Results—Wire-induced injury of the femoral artery was performed on chimeric C57BL/6 mice transplanted with bone marrow from transgenic mice expressing enhanced green fluorescence protein, and vessels were harvested at 3 days, 1, 2, 3, 4, 6, and 16 weeks after dilatation (n=8 animals per time point). Using high-resolution microscopy, we unexpectedly found that the expression of smooth muscle cell or endothelial cell markers in enhanced green fluorescence protein positive cells was a very rare event. Indeed, most of the enhanced green fluorescence protein positive cells that accumulated during the acute inflammatory response were identified as monocytes/macrophages, and their number declined at later time points. In contrast, a substantial fraction of highly proliferative stem cell antigen-1 and CD34+ but enhanced green fluorescence protein negative and thus locally derived cells were detected in the adventitia. Conclusion—These data provide evidence that the differentiation of bone marrow-derived progenitor cells into smooth muscle cell or endothelial cell lineages seems to be an exceedingly rare event. Moreover, the contribution of bone marrow-derived cells to the cellular compartment of the neointima is limited to a transient period of the inflammatory response.

The G534E polymorphism of the gene encoding the factor VII–activating protease is associated with cardiovascular risk due to increased neointima formation

The G534E polymorphism (Marburg I [MI]) of factor VII–activating protease (FSAP) is associated with carotid stenosis and cardiovascular disease. We have previously demonstrated that FSAP is present in atherosclerotic plaques and it is a potent inhibitor of vascular smooth muscle proliferation and migration in vitro. The effect of wild-type (WT)- and MI-FSAP on neointima formation in the mouse femoral artery after wire-induced injury was investigated. Local application of WT-FSAP led to a 70% reduction in the neointima formation, and this effect was dependent on the protease activity of FSAP. MI-FSAP did not inhibit neointima formation in vivo. This is due to a reduced proteolytic activity of MI-FSAP, compared to WT-FSAP, toward platelet-derived growth factor BB, a key mediator of neointima development. The inability of MI-FSAP to inhibit vascular smooth muscle accumulation explains the observed linkage between the MI-polymorphism and increased cardiovascular risk. Hence, FSAP has a protective function in the vasculature, and analysis of MI polymorphism is likely to be clinically relevant in restenosis.

Mechanosensitive p27Kip1 Regulation and Cell Cycle Entry in Vascular Smooth Muscle Cells

Background—Cyclic stretch plays an important role in the homeostasis of vessel structure. Increased forces might, however, contribute to remodeling processes, resulting in vascular proliferative diseases. The initial molecular events necessary for mechanosensitive cell cycle entry of quiescent smooth muscle cells are poorly understood. Methods and Results—In this study, we demonstrate that mechanical strain resulted in a rapid, integrin-dependent but mitogen-independent activation of phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt) in quiescent vascular smooth muscle cells. Subsequently, downstream ALL 1 fused gene from chromosome X (AFX)-like forkhead transcription factors were inactivated, leading to transcriptional downregulation of p27Kip1. This contrasted with the posttranscriptional protein reduction of p27Kip1 in cells stimulated with serum mitogens. Stretch-mediated p27Kip1 downregulation was accompanied by activation of cyclin-dependent kinase 2, hyperphosphorylation of retinoblastoma protein, and proliferation. Forkhead transcription factor inactivation and p27Kip1 downregulation were prevented by the PI3-K inhibitors wortmannin and LY294002. Pharmacological blockade of other kinases, such as p42/44, p38, and protein kinase A or C, did not influence the mechanosensitive gene regulation. p27Kip1 downregulation and cell cycle entry were, however, prevented by overexpression of a constitutively inactive form of Akt or constitutively active forms of forkhead transcription factors. Conclusions—Our data demonstrate that the earliest cell cycle events can occur in a solely mechanosensitive fashion. Vascular smooth muscle cells are, furthermore, able to use transcriptional or posttranscriptional mechanisms to regulate p27Kip1, depending on the stimulus to which they are exposed. This observation has novel implications for understanding of vascular proliferative diseases.

Cell Cycle–Dependent Regulation of Smooth Muscle Cell Activation

Objective—Although numerous diseases involving cellular proliferation are also associated with phenotypic changes, there has been little direct evidence that cell phenotype and the cells response to external stimuli are modified during passage through different phases of the cell cycle. In this study, we demonstrate that an association exists between cell cycle progression and the expression of genes involved in cellular activation. Methods and Results—Early cell cycle arrest of aortic smooth muscle cells was found to inhibit the tumor necrosis factor α (TNFα)-induced upregulation of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, important markers of vascular cell activation in diseases such as atherosclerosis. A combination of immunocytochemistry and flow cytometry were used to document that TNFα-induced adhesion molecule upregulation was inhibited during G1-phase and S-phase, but not in G0-phase or G2/M-phase cells. The inhibition of adhesion molecule expression occurred at the level of transcription, as demonstrated by changes in the patterns of mRNA and protein accumulation in cycling and arrested cells. Conclusions—Early cell cycle phases may represent states in which the responses to a variety of stimuli that influence cell fate can be modulated, and these observations may have novel implications for the prevention and/or therapy of vascular proliferative, neoplastic, and inflammatory diseases.

Factor VII-activating protease (FSAP): Vascular functions and role in atherosclerosis

FSAP is a plasma serine protease for which a potential role in the regulation of coagulation and fibrinolysis is postulated, based on its property to activate factor VII (FVII) as well as pro-urokinase (uPA). In clinical studies, the G534E single nucleotide polymorphism (Marburg I) of FSAP has been linked to late complications of atherothrombosis and is associated with a low proteolytic activity, particularly, towards pro-uPA. This has stimulated much interest in a search for additional functions of FSAP in the cardiovascular system. FSAP is a potent inhibitor of vascular smooth muscle cell proliferation and migration in vitro and local application of FSAP (but not Marburg I variant) in animal models reduces neointima formation. This is due to a reduced proteolytic activity of the variant isoform towards platelet derived growth factor-BB, a key mediator of neointima development. Moreover, appreciable quantities of FSAP are localized to unstable atherosclerotic plaques and may contribute to plaque instability. These data indicate that the cellular regulatory effects of FSAP may be more important than its influence on haemostasis. In this review the contribution of FSAP to vascular fibroproliferative inflammatory diseases in the context of pericellular proteolysis of the extracellular matrix, growth factor activity and haemostasis will be highlighted.