Osteopontin Activation and Microcalcification in Venous Grafts Can Be Modulated by Dexamethasone.

BACKGROUND: Osteopontin has been implicated in vascular calcification formation and vein graft intimal hyperplasia, and its expression can be triggered by pro-inflammatory activation of cells. The role of osteopontin and the temporal formation of microcalcification in vein grafts is poorly understood with a lack of understanding of the interaction between haemodynamic changes and the activation of osteopontin. METHODS: We used a porcine model of vein interposition grafts, and human long saphenous veins exposed to ex vivo perfusion, to study the activation of osteopontin using polymerase chain reaction, immunostaining, and 18F-sodium fluoride autoradiography. RESULTS: The porcine model showed that osteopontin is active in grafts within 1 week following surgery and demonstrated the presence of microcalcification. A brief pretreatment of long saphenous veins with dexamethasone can suppress osteopontin activation. Prolonged culture of veins after exposure to acute arterial haemodynamics resulted in the formation of microcalcification but this was suppressed by pretreatment with dexamethasone. 18F-sodium fluoride uptake was significantly increased as early as 1 week in both models, and the pretreatment of long saphenous veins with dexamethasone was able to abolish its uptake. CONCLUSIONS: Osteopontin is activated in vein grafts and is associated with microcalcification formation. A brief pretreatment of veins ex vivo with dexamethasone can suppress its activation and associated microcalcification.

Inhibition of Intimal Thickening By PRH (Proline-Rich Homeodomain) in Mice.

BACKGROUND: Late vein graft failure is caused by intimal thickening resulting from endothelial cell (EC) damage and inflammation which promotes vascular smooth muscle cell (VSMC) dedifferentiation, migration, and proliferation. Nonphosphorylatable PRH (proline-rich homeodomain) S163C:S177C offers enhanced stability and sustained antimitotic effect. Therefore, we investigated whether adenovirus-delivered PRH S163C:S177C protein attenuates intimal thickening via VSMC phenotype modification without detrimental effects on ECs. METHODS: PRH S163C:S177C was expressed in vitro (human saphenous vein-VSMCs and human saphenous vein-ECs) and in vivo (ligated mouse carotid arteries) by adenoviruses. Proliferation, migration, and apoptosis were quantified and phenotype was assessed using Western blotting for contractile filament proteins and collagen gel contraction. EC inflammation was quantified using VCAM (vascular cell adhesion protein)-1, ICAM (intercellular adhesion molecule)-1, interleukin-6, and monocyte chemotactic factor-1 measurement and monocyte adhesion. Next Generation Sequencing was utilized to identify novel downstream mediators of PRH action and these and intimal thickening were investigated in vivo. RESULTS: PRH S163C:S177C inhibited proliferation, migration, and apoptosis and promoted contractile phenotype (enhanced contractile filament proteins and collagen gel contraction) compared with virus control in human saphenous vein-VSMCs. PRH S163C:S177C expression in human saphenous vein-ECs significantly reduced apoptosis, without affecting cell proliferation and migration, while reducing TNF (tumor necrosis factor)-α-induced VCAM-1 and ICAM-1 and monocyte adhesion and suppressing interleukin-6 and monocyte chemotactic factor-1 protein levels. PRH S163C:S177C expression in ligated murine carotid arteries significantly impaired carotid artery ligation-induced neointimal proliferation and thickening without reducing endothelial coverage. Next Generation Sequencing revealed STAT-1 (signal transducer and activator of transcription 1) and HDAC-9 (histone deacetylase 9) as mediators of PRH action and was supported by in vitro and in vivo analyses. CONCLUSIONS: We observed PRH S163C:S177C attenuated VSMC proliferation, and migration and enhanced VSMC differentiation at least in part via STAT-1 and HDAC-9 signaling while promoting endothelial repair and anti-inflammatory properties. These findings highlight the potential for PRH S163C:S177C to preserve endothelial function whilst suppressing intimal thickening, and reducing late vein graft failure.

Monitoring Cellular Proliferation, Migration, and Apoptosis Associated with Atherosclerosis Plaques In Vitro.

Bromodeoxyuridine/5-bromo-2'-deoxyuridine (BrdU) is a nucleoside analog of thymidine and its incorporation into DNA during replication within S-phase of the cell cycle is used to quantify cell proliferation. Quantification of incorporated BrdU is considered the most direct measure of cell proliferation, and here we describe BrdU incorporation into cultured vascular smooth muscle cells (VSMCs) and endothelial cells in vitro. Incorporation of fluorescent-labeled ethynyldeoxyuridine/5-ethynyl-2'-deoxyuridine (EdU) is a novel alternative to BrdU assays and presents significant advantages. This method of detection of EdU based on a simple "click" chemical reaction, which covalently bonds EdU to a fluorescent dye is also outlined in this chapter with a protocol for quantitative analysis of EdU incorporation using a Fiji-based macro. We also describe how proliferation can be assessed by quantification of classical proliferative markers such as phopsho-Ser807/811 retinoblastoma (Rb), proliferating cell nuclear antigen (PCNA) and cyclin D1 by Western blotting. As these markers are involved in different aspects of the cell cycle regulation, examining their expression levels can not only reveal the relative population of proliferating cells but can also improve our understanding of the mechanism of action of a given treatment or intervention. The scratch wound assay is a simple and cost-effective technique to quantify cell migration. A protocol which involves creating a wound in a cell cultured monolayer and measuring the distance migrated by the cells after a predefined time period is also described. Gap creation can also be achieved via physical cell exclusion where cells are seeded in distinct reservoirs of a cell culture insert which reveal a gap upon removal. Cell migration may then be quantified by monitoring the rate of gap closure. The presence of cleaved caspase-3 is a marker of programmed cell death (apoptosis). To detect cleaved caspase-3 in vitro, immunocytochemistry and fluorescence can be performed as outlined in this chapter.

Aneurysm severity is suppressed by deletion of CCN4.

Patients with abdominal aortic aneurysms are frequently treated with high-risk surgery. A pharmaceutical treatment to reverse aneurysm progression could prevent the need for surgery and save both lives and healthcare resources. Since CCN4 regulates cell migration, proliferation and apoptosis, processes involved in aneurysm progression, it is a potential regulator of aneurysm progression. We investigated the role of CCN4 in a mouse aneurysm model, using apolipoprotein-E knockout (ApoE-/-) mice fed high fat diet and infused with Angiotensin II (AngII). Blood pressure was similarly elevated in CCN4-/-ApoE-/- mice and CCN4+/+ApoE-/- mice (controls) in response to AngII infusion. Deletion of CCN4 significantly reduced the number of ruptured aortae, both thoracic and abdominal aortic area, and aneurysm grade score, compared to controls. Additionally, the frequency of vessel wall remodelling and the number of elastic lamina breaks was significantly suppressed in CCN4-/-ApoE-/- mice compared to controls. Immunohistochemistry revealed a significantly lower proportion of macrophages, while the proportion of smooth muscle cells was not affected by the deletion of CCN4. There was also a reduction in both proliferation and apoptosis in CCN4-/-ApoE-/- mice compared to controls. In vitro studies showed that CCN4 significantly increased monocyte adhesion beyond that seen with TNFα and stimulated macrophage migration by more than threefold. In summary, absence of CCN4 reduced aneurysm severity and improved aortic integrity, which may be the result of reduced macrophage infiltration and cell apoptosis. Inhibition of CCN4 could offer a potential therapeutic approach for the treatment of aneurysms.

Role of smooth muscle cells in coronary artery bypass grafting failure.

Atherosclerosis is the underlying pathology of many cardiovascular diseases. The formation and rupture of atherosclerotic plaques in the coronary arteries results in angina and myocardial infarction. Venous coronary artery bypass grafts are designed to reduce the consequences of atherosclerosis in the coronary arteries by diverting blood flow around the atherosclerotic plaques. However, vein grafts suffer a high failure rate due to intimal thickening that occurs as a result of vascular cell injury and activation and can act as 'a soil' for subsequent atherosclerotic plaque formation. A clinically-proven method for the reduction of vein graft intimal thickening and subsequent major adverse clinical events is currently not available. Consequently, a greater understanding of the underlying mechanisms of intimal thickening may be beneficial for the design of future therapies for vein graft failure. Vein grafting induces inflammation and endothelial cell damage and dysfunction, that promotes vascular smooth muscle cell (VSMC) migration, and proliferation. Injury to the wall of the vein as a result of grafting leads to the production of chemoattractants, remodelling of the extracellular matrix and cell-cell contacts; which all contribute to the induction of VSMC migration and proliferation. This review focuses on the role of altered behaviour of VSMCs in the vein graft and some of the factors which critically lead to intimal thickening that pre-disposes the vein graft to further atherosclerosis and re-occurrence of symptoms in the patient.

Targeting Wnt/ß-Catenin Activated Cells with Dominant-Negative N-cadherin to Reduce Neointima Formation.

Approximately 50% of coronary artery bypass grafts using the autologous saphenous vein fail within 10 years due to intimal thickening. This study examined whether a gene therapy approach that selectively kills Wnt/ß-catenin/T cell factor (TCF) activated vascular smooth muscle cells (VSMCs) using dominant-negative N-cadherin (dn-N-cadherin) reduced intimal thickening. Cultured human VSMCs infected with an adenovirus (Ad) encoding dn-N-cadherin via the TCF promoter (Ad-TOP-dn-N-cadherin) specifically expressed dn-N-cadherin in response to activation of the Wnt/ß-catenin/TCF pathway. Infection with Ad-TOP-dn-N-cadherin significantly increased VSMC apoptosis (3 ± 0.2% versus 9 ± 0.7%; p < 0.05, n = 6) and significantly inhibited VSMC migration by 83 ± 15% (p < 0.05, n = 6), but did not affect VSMC proliferation (p > 0.05, n = 5). In an ex vivo human saphenous vein organ culture model, luminal delivery of Ad-TOP-dn-N-cadherin significantly increased VSMC apoptosis after 7 days of culture (4 ± 1.4% versus 9 ± 1.6%; p < 0.01, n = 6) and suppressed intimal thickening by 75 ± 7% (p < 0.05, n = 5), without a detrimental effect on endothelial cell coverage. In vivo, Ad-TOP-dn-N-cadherin significantly reduced intimal thickening at day 21 (n = 10) in comparison to the Ad-ß-galactosidase (Ad-ß-gal) control virus (n = 12, p < 0.05) in the mouse carotid artery ligation model. In summary, we have developed a novel approach to selectively reduce intimal thickening, which may be beneficial in reducing late vein graft failure.

Misregulation of the proline rich homeodomain (PRH/HHEX) protein in cancer cells and its consequences for tumour growth and invasion.

The proline rich homeodomain protein (PRH), also known as haematopoietically expressed homeobox (HHEX), is an essential transcription factor in embryonic development and in the adult. The PRH protein forms oligomeric complexes that bind to tandemly repeated PRH recognition sequences within or at a distance from PRH-target genes and recruit a variety of PRH-interacting proteins. PRH can also bind to other transcription factors and co-regulate specific target genes either directly through DNA binding, or indirectly through effects on the activity of its partner proteins. In addition, like some other homeodomain proteins, PRH can regulate the translation of specific mRNAs. Altered PRH expression and altered PRH intracellular localisation, are associated with breast cancer, liver cancer and thyroid cancer and some subtypes of leukaemia. This is consistent with the involvement of multiple PRH-interacting proteins, including the oncoprotein c-Myc, translation initiation factor 4E (eIF4E), and the promyelocytic leukaemia protein (PML), in the control of cell proliferation and cell survival. Similarly, multiple PRH target genes, including the genes encoding vascular endothelial growth factor (VEGF), VEGF receptors, Endoglin, and Goosecoid, are known to be important in the control of cell proliferation and cell survival and/or the regulation of cell migration and invasion. In this review, we summarise the evidence that implicates PRH in tumourigenesis and we review the data that suggests PRH levels could be useful in cancer prognosis and in the choice of treatment options.