WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts' Activation and Collagen Processing.

Hypertension induces cardiac fibrotic remodelling characterised by the phenotypic switching of cardiac fibroblasts (CFs) and collagen deposition. We tested the hypothesis that Wnt1-inducible signalling pathway protein-1 (WISP-1) promotes CFs' phenotypic switch, type I collagen synthesis, and in vivo fibrotic remodelling. The treatment of human CFs (HCFs, n = 16) with WISP-1 (500 ng/mL) induced a phenotypic switch (α-smooth muscle actin-positive) and type I procollagen cleavage to an intermediate form of collagen (pC-collagen) in conditioned media after 24h, facilitating collagen maturation. WISP-1-induced collagen processing was mediated by Akt phosphorylation via integrin ß1, and disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS-2). WISP-1 wild-type (WISP-1+/+) mice and WISP-1 knockout (WISP-1-/-) mice (n = 5-7) were subcutaneously infused with angiotensin II (AngII, 1000 ng/kg/min) for 28 days. Immunohistochemistry revealed the deletion of WISP-1 attenuated type I collagen deposition in the coronary artery perivascular area compared to WISP-1+/+ mice after a 28-day AngII infusion, and therefore, the deletion of WISP-1 attenuated AngII-induced cardiac fibrosis in vivo. Collectively, our findings demonstrated WISP-1 is a critical mediator in cardiac fibrotic remodelling, by promoting CFs' activation via the integrin ß1-Akt signalling pathway, and induced collagen processing and maturation via ADAMTS-2. Thereby, the modulation of WISP-1 levels could provide potential therapeutic targets in clinical treatment.

Pharmacological Inhibition of MMP-12 Exerts Protective Effects on Angiotensin II-Induced Abdominal Aortic Aneurysms in Apolipoprotein E-Deficient Mice.

Human abdominal aortic aneurysms (AAAs) are characterized by increased activity of matrix metalloproteinases (MMP), including MMP-12, alongside macrophage accumulation and elastin degradation, in conjunction with superimposed atherosclerosis. Previous genetic ablation studies have proposed contradictory roles for MMP-12 in AAA development. In this study, we aimed to elucidate if pharmacological inhibition of MMP-12 activity with a phosphinic peptide inhibitor protects from AAA formation and progression in angiotensin (Ang) II-infused Apoe-/- mice. Complimentary studies were conducted in a human ex vivo model of early aneurysm development. Administration of an MMP-12 inhibitor (RXP470.1) protected hypercholesterolemia Apoe-/- mice from Ang II-induced AAA formation and rupture-related death, associated with diminished medial thinning and elastin fragmentation alongside increased collagen deposition. Proteomic analyses confirmed a beneficial effect of MMP-12 inhibition on extracellular matrix remodeling proteins combined with inflammatory pathways. Furthermore, RXP470.1 treatment of mice with pre-existing AAAs exerted beneficial effects as observed through suppressed aortic dilation and rupture, medial thinning, and elastin destruction. Our findings indicate that pharmacological inhibition of MMP-12 activity retards AAA progression and improves survival in mice providing proof-of-concept evidence to motivate translational work for MMP-12 inhibitor therapy in humans.

The contribution of matrix metalloproteinases and their inhibitors to the development, progression, and rupture of abdominal aortic aneurysms.

Abdominal aortic aneurysms (AAAs) account for up to 8% of deaths in men aged 65 years and over and 2.2% of women. Patients with AAAs often have atherosclerosis, and intimal atherosclerosis is generally present in AAAs. Accordingly, AAAs are considered a form of atherosclerosis and are frequently referred to as atherosclerotic aneurysms. Pathological observations advocate inflammatory cell infiltration alongside adverse extracellular matrix degradation as key contributing factors to the formation of human atherosclerotic AAAs. Therefore, macrophage production of proteolytic enzymes is deemed responsible for the damaging loss of ECM proteins, especially elastin and fibrillar collagens, which characterise AAA progression and rupture. Matrix metalloproteinases (MMPs) and their regulation by tissue inhibitors metalloproteinases (TIMPs) can orchestrate not only ECM remodelling, but also moderate the proliferation, migration, and apoptosis of resident aortic cells, alongside the recruitment and subsequent behaviour of inflammatory cells. Accordingly, MMPs are thought to play a central regulatory role in the development, progression, and eventual rupture of abdominal aortic aneurysms (AAAs). Together, clinical and animal studies have shed light on the complex and often diverse effects MMPs and TIMPs impart during the development of AAAs. This dichotomy is underlined from evidence utilising broad-spectrum MMP inhibition in animal models and clinical trials which have failed to provide consistent protection from AAA progression, although more encouraging results have been observed through deployment of selective inhibitors. This review provides a summary of the supporting evidence connecting the contribution of individual MMPs to AAA development, progression, and eventual rupture. Topics discussed include structural, functional, and cell-specific diversity of MMP members; evidence from animal models of AAA and comparisons with findings in humans; the dual role of MMPs and the requirement to selectively target individual MMPs; and the advances in identifying aberrant MMP activity. As evidenced, our developing understanding of the multifaceted roles individual MMPs perform during the progression and rupture of AAAs, should motivate clinical trials assessing the therapeutic potential of selective MMP inhibitors, which could restrict AAA-related morbidity and mortality worldwide.

A Nrf2-OSGIN1&2-HSP70 axis mediates cigarette smoke-induced endothelial detachment: implications for plaque erosion.

AIMS: Endothelial erosion of plaques is responsible for ∼30% of acute coronary syndromes (ACS). Smoking is a risk factor for plaque erosion, which most frequently occurs on the upstream surface of plaques where the endothelium experiences elevated shear stress. We sought to recreate these conditions in vitro to identify potential pathological mechanisms that might be of relevance to plaque erosion. METHODS AND RESULTS: Culturing human coronary artery endothelial cells (HCAECs) under elevated flow (shear stress of 7.5 Pa) and chronically exposing them to cigarette smoke extract (CSE) and tumour necrosis factor-alpha (TNFα) recapitulated a defect in HCAEC adhesion, which corresponded with augmented Nrf2-regulated gene expression. Pharmacological activation or adenoviral overexpression of Nrf2 triggered endothelial detachment, identifying Nrf2 as a mediator of endothelial detachment. Growth/Differentiation Factor-15 (GDF15) expression was elevated in this model, with protein expression elevated in the plasma of patients experiencing plaque erosion compared with plaque rupture. The expression of two Nrf2-regulated genes, OSGIN1 and OSGIN2, was increased by CSE and TNFα under elevated flow and was also elevated in the aortas of mice exposed to cigarette smoke in vivo. Knockdown of OSGIN1&2 inhibited Nrf2-induced cell detachment. Overexpression of OSGIN1&2 induced endothelial detachment and resulted in cell cycle arrest, induction of senescence, loss of focal adhesions and actin stress fibres, and disturbed proteostasis mediated in part by HSP70, restoration of which reduced HCAEC detachment. In ACS patients who smoked, blood concentrations of HSP70 were elevated in plaque erosion compared with plaque rupture. CONCLUSION: We identified a novel Nrf2-OSGIN1&2-HSP70 axis that regulates endothelial adhesion, elevated GDF15 and HSP70 as biomarkers for plaque erosion in patients who smoke, and two therapeutic targets that offer the potential for reducing the risk of plaque erosion.

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.

Monoclonal Autoantibody Against a Cryptic Epitope on Tissue-Adherent Low-Density Lipoprotein for Molecular Imaging in Atherosclerosis.

BACKGROUND: Antibody-based constructs for molecular imaging and therapeutic delivery provide promising opportunities for the diagnosis and treatment of atherosclerosis. OBJECTIVES: The authors aimed to generate and characterize immunoglobulin (Ig)G monoclonal autoantibodies in atherosclerosis for targeting of novel molecular determinants. METHODS: The authors created hybridomas from an unimmunized low-density lipoprotein (LDL) receptor-deficient (Ldlr-/-) mouse and selected an IgG2b isotype autoantibody, LO9, for further characterization. RESULTS: LO9 reacted well with native LDL bound to immobilized matrix components and less well to oxidized LDL. LO9 binding to immobilized native LDL was not neutralized by fluid-phase native LDL, indicating an adhesion-dependent epitope. The authors localized the epitope to a 20 amino-acid peptide sequence (P5) in the globular amino-terminus of apolipoprotein B. LO9 reacted with antigen in mouse atherosclerosis and in both human stable and ruptured coronary atherosclerosis. Furthermore, in vivo near-infrared fluorescence molecular tomographic imaging, and ex vivo confocal microscopy showed that intravenously injected LO9 localized beneath endothelium of the aortic arch in Ldlr-/- mice, in the vicinity of macrophages. CONCLUSIONS: The authors believe LO9 is the first example of an IgG autoantibody that reacts with a native LDL epitope revealed by adherence to tissue matrix. Antibodies against adherent native LDL have potential as molecular targeting agents for imaging of and therapeutic delivery to atherosclerosis.

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.

Monitoring Cellular Proliferation and Apoptosis in Atherosclerosis Plaques and Intimal Thickenings.

Immunohistochemistry for specific proteins characteristic of proliferative or apoptotic cells allows for monitoring of these cell behaviors in biological tissues samples, including atherosclerotic plaques and intimal thickenings. Proliferating cell nuclear antigen (PCNA) and Ki-67 are widely used markers of cell proliferation and cleaved caspase-3 is a well-established marker of apoptosis that can be detected in tissue samples using immunohistochemistry. This technique enables quantification of the abundance of these proteins and provides information on the distribution of these biomarkers in tissues. By combining with immunohistochemistry for specific cell type markers, it is also possible to determine which cell types are proliferating or undergoing apoptosis. Here, we detail protocols for immunohistochemistry of PCNA, Ki-67, and cleaved caspase-3 for evaluation of cellular proliferation and apoptosis in atherosclerotic plaques in vivo. In addition, we outline methods for the quantification and localization of cell proliferation using bromodeoxyuridine/5-bromo-2'-deoxyuridine (BrdU) and ethynyldeoxyuridine/5-ethynyl-2 ́-deoxyuridine(EdU) labeled tissue samples collected from animals exposed to BrdU or EdU.

Investigation of Atherosclerotic Plaque Vulnerability.

Histochemical and immunohistochemical approaches permit the detection and evaluation of proteins and cell types within murine brachiocephalic artery atherosclerotic plaques, that can be subsequently analyzed to provide inferences on atherosclerotic plaque vulnerability. Here we describe the specific histochemical techniques deployed to examine the expression of elastin, fibrillar collagens, and neutral lipids, alongside immunohistochemistry protocols for the identification of macrophages (CD68) and vascular smooth muscle cells (α-smooth muscle actin). We will also describe how analyses derived from these methods can be combined to determine evidence of previous plaque rupture and susceptibility to rupture.

Use of Mouse Carotid Artery Ligation Model of Intimal Thickening to Probe Vascular Smooth Muscle Cell Remodeling and Function in Atherosclerosis.

The thickening of the intima is a critical underlying component of atherosclerosis. Consequently, robust and reproducible animal models of intimal thickening are essential for a greater understanding of the mechanisms underlying the process of intimal thickening and to evaluate new approaches for the reduction of intimal thickening and thereby atherosclerosis. The ligation of the carotid artery in the mouse causes the thickening of the intimal layer of the artery. This model is relatively simple and is reproducible and therefore is a preferred and well-established model of intimal thickening. Here, we detail a protocol for carotid artery ligation in the mouse and methods for histological examination and quantification of intimal thickening.

Pro-inflammatory role of Wnt/ß-catenin signaling in endothelial dysfunction.

Background: Endothelial dysfunction is a critical component of both atherosclerotic plaque formation and saphenous vein graft failure. Crosstalk between the pro-inflammatory TNF-α-NFκB signaling axis and the canonical Wnt/ß-catenin signaling pathway potentially plays an important role in regulating endothelial dysfunction, though the exact nature of this is not defined. Results: In this study, cultured endothelial cells were challenged with TNF-α and the potential of a Wnt/ß-catenin signaling inhibitor, iCRT-14, in reversing the adverse effects of TNF-α on endothelial physiology was evaluated. Treatment with iCRT-14 lowered nuclear and total NFκB protein levels, as well as expression of NFκB target genes, IL-8 and MCP-1. Inhibition of ß-catenin activity with iCRT-14 suppressed TNF-α-induced monocyte adhesion and decreased VCAM-1 protein levels. Treatment with iCRT-14 also restored endothelial barrier function and increased levels of ZO-1 and focal adhesion-associated phospho-paxillin (Tyr118). Interestingly, inhibition of ß-catenin with iCRT-14 enhanced platelet adhesion in cultured TNF-α-stimulated endothelial cells and in an ex vivo human saphenous vein model, most likely via elevating levels of membrane-tethered vWF. Wound healing was moderately retarded by iCRT-14; hence, inhibition of Wnt/ß-catenin signaling may interfere with re-endothelialisation in grafted saphenous vein conduits. Conclusion: Inhibition of the Wnt/ß-catenin signaling pathway with iCRT-14 significantly recovered normal endothelial function by decreasing inflammatory cytokine production, monocyte adhesion and endothelial permeability. However, treatment of cultured endothelial cells with iCRT-14 also exerted a pro-coagulatory and moderate anti-wound healing effect: these factors may affect the suitability of Wnt/ß-catenin inhibition as a therapy for atherosclerosis and vein graft failure.

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.

Effective decellularisation of human saphenous veins for biocompatible arterial tissue engineering applications: Bench optimisation and feasibility in vivo testing.

Human saphenous vein (hSV) and synthetic grafts are commonly used conduits in vascular grafting, despite high failure rates. Decellularising hSVs (D-hSVs) to produce vascular scaffolds might be an effective alternative. We assessed the effectiveness of a detergent-based method using 0% to 1% sodium dodecyl sulphate (SDS) to decellularise hSV. Decellularisation effectiveness was measured in vitro by nuclear counting, DNA content, residual cell viability, extracellular matrix integrity and mechanical strength. Cytotoxicity was assessed on human and porcine cells. The most effective SDS concentration was used to prepare D-hSV grafts that underwent preliminary in vivo testing using a porcine carotid artery replacement model. Effective decellularisation was achieved with 0.01% SDS, and D-hSVs were biocompatible after seeding. In vivo xeno-transplantation confirmed excellent mechanical strength and biocompatibility with recruitment of host cells without mechanical failure, and a 50% patency rate at 4-weeks. We have developed a simple biocompatible methodology to effectively decellularise hSVs. This could enhance vascular tissue engineering toward future clinical applications.

A Protocol for a Novel Human Ex Vivo Model of Aneurysm.

Aortic aneurysm rupture is a significant cause of premature mortality worldwide. Although animal models exist, some frequently experience aortic rupture and sudden death. An alternative approach is therefore required that would use human material to aid translation. Accordingly, we present an optimized and validated protocol to isolate human umbilical cord arteries and their subsequent deployment within a bioreactor. Consequently, this reproducible ex vivo human model of aneurysm can be used for pathogenesis studies and accompanying assessment of potential novel therapeutics.

NF-κB inhibition prevents acute shear stress-induced inflammation in the saphenous vein graft endothelium.

The long saphenous vein (LSV) is commonly used as a conduit in coronary artery bypass grafting. However, long term patency remains limited by the development of vascular inflammation, intimal hyperplasia and accelerated atherosclerosis. The impact of acute exposure of venous endothelial cells (ECs) to acute arterial wall shear stress (WSS) in the arterial circulation, and the subsequent activation of inflammatory pathways, remain poorly defined. Here, we tested the hypothesis that acute exposure of venous ECs to high shear stress is associated with inflammatory responses that are regulated by NF-κB both in-vitro and ex-vivo. Analysis of the LSV endothelium revealed that activation of NF-κB occurred within 30 min after exposure to arterial rates of shear stress. Activation of NF-κB was associated with increased levels of CCL2 production and enhanced binding of monocytes in LSVECs exposed to 6 h acute arterial WSS. Consistent with this, ex vivo exposure of LSVs to acute arterial WSS promoted monocyte interactions with the vessel lumen. Inhibition of the NF-κB pathway prevented acute arterial WSS-induced CCL2 production and reduced monocyte adhesion, both in vitro and in human LSV ex vivo, demonstrating that this pathway is necessary for the induction of the acute arterial WSS-induced pro-inflammatory response. We have identified NF-κB as a critical regulator of acute endothelial inflammation in saphenous vein in response to acute arterial WSS. Localised endothelial-specific inhibition of the NF-κB pathway may be beneficial to prevent vein graft inflammation and consequent failure.

Galectin-3 Identifies a Subset of Macrophages With a Potential Beneficial Role in Atherosclerosis.

OBJECTIVE: Galectin-3 (formerly known as Mac-2), encoded by the LGALS3 gene, is proposed to regulate macrophage adhesion, chemotaxis, and apoptosis. We investigated the role of galectin-3 in determining the inflammatory profile of macrophages and composition of atherosclerotic plaques. Approach and Results: We observed increased accumulation of galectin-3-negative macrophages within advanced human, rabbit, and mouse plaques compared with early lesions. Interestingly, statin treatment reduced galectin-3-negative macrophage accrual in advanced plaques within hypercholesterolemic (apolipoprotein E deficient) Apoe-/- mice. Accordingly, compared with Lgals3+/+:Apoe-/- mice, Lgals3-/-:Apoe-/- mice displayed altered plaque composition through increased macrophage:smooth muscle cell ratio, reduced collagen content, and increased necrotic core area, characteristics of advanced plaques in humans. Additionally, macrophages from Lgals3-/- mice exhibited increased invasive capacity in vitro and in vivo. Furthermore, loss of galectin-3 in vitro and in vivo was associated with increased expression of proinflammatory genes including MMP (matrix metalloproteinase)-12, CCL2 (chemokine [C-C motif] ligand 2), PTGS2 (prostaglandin-endoperoxide synthase 2), and IL (interleukin)-6, alongside reduced TGF (transforming growth factor)-ß1 expression and consequent SMAD signaling. Moreover, we found that MMP12 cleaves macrophage cell-surface galectin-3 resulting in the appearance of a 22-kDa fragment, whereas plasma levels of galectin-3 were reduced in Mmp12-/-:Apoe-/- mice, highlighting a novel mechanism where MMP12-dependent cleavage of galectin-3 promotes proinflammatory macrophage polarization. Moreover, galectin-3-positive macrophages were more abundant within plaques of Mmp12-/-:Apoe-/- mice compared with Mmp12+/+:Apoe-/- animals. CONCLUSIONS: This study reveals a prominent protective role for galectin-3 in regulating macrophage polarization and invasive capacity and, therefore, delaying plaque progression.

Non-coding RNAs in cardiovascular cell biology and atherosclerosis.

Atherosclerosis underlies the predominant number of cardiovascular diseases and remains a leading cause of morbidity and mortality worldwide. The development, progression and formation of clinically relevant atherosclerotic plaques involves the interaction of distinct and over-lapping mechanisms which dictate the roles and actions of multiple resident and recruited cell types including endothelial cells, vascular smooth muscle cells, and monocyte/macrophages. The discovery of non-coding RNAs (ncRNAs) including microRNAs, long non-coding RNAs, and circular RNAs, and their identification as key mechanistic regulators of mRNA and protein expression has piqued interest in their potential contribution to atherosclerosis. Accruing evidence has revealed ncRNAs regulate pivotal cellular and molecular processes during all stages of atherosclerosis including cell invasion, growth, and survival; cellular uptake and efflux of lipids, expression and release of pro- and anti-inflammatory intermediaries, and proteolytic balance. The expression profile of ncRNAs within atherosclerotic lesions and the circulation have been determined with the aim of identifying individual or clusters of ncRNAs which may be viable therapeutic targets alongside deployment as biomarkers of atherosclerotic plaque progression. Consequently, numerous in vivo studies have been convened to determine the effects of moderating the function or expression of select ncRNAs in well-characterized animal models of atherosclerosis. Together, clinicopathological findings and studies in animal models have elucidated the multifaceted and frequently divergent effects ncRNAs impose both directly and indirectly on the formation and progression of atherosclerosis. From these findings' potential novel therapeutic targets and strategies have been discovered which may pave the way for further translational studies and possibly taken forward for clinical application.

Elucidating the contributory role of microRNA to cardiovascular diseases (a review).

Cardiovascular diseases encompassing atherosclerosis, aortic aneurysms, restenosis, and pulmonary arterial hypertension, remain the leading cause of morbidity and mortality worldwide. In response to a range of stimuli, the dynamic interplay between biochemical and biomechanical mechanisms affect the behaviour and function of multiple cell types, driving the development and progression of cardiovascular diseases. Accumulating evidence has highlighted microRNAs (miRs) as significant regulators and micro-managers of key cellular and molecular pathophysiological processes involved in predominant cardiovascular diseases, including cell mitosis, motility and viability, lipid metabolism, generation of inflammatory mediators, and dysregulated proteolysis. Human pathological and clinical studies have aimed to identify select microRNA which may serve as biomarkers of disease and their progression, which are discussed within this review. In addition, I provide comprehensive coverage of in vivo investigations elucidating the modulation of distinct microRNA on the pathophysiology of atherosclerosis, abdominal aortic aneurysms, restenosis, and pulmonary arterial hypertension. Collectively, clinical and animal studies have begun to unravel the complex and often diverse effects microRNAs and their targets impart during the development of cardiovascular diseases and revealed promising therapeutic strategies through which modulation of microRNA function may be applied clinically.

Aging differentially modulates the Wnt pro-survival signalling pathways in vascular smooth muscle cells.

We previously reported pro-survival effects of Wnt3a and Wnt5a proteins in vascular smooth muscle cells (VSMCs). Wnt5a achieved this through induction of Wnt1-inducible signalling pathway protein-1 (WISP-1) consequent to ß-catenin/CREB-dependent, TCF-independent, signalling. However, we found that as atherosclerosis advances, although Wnt5a protein was increased, WISP-1 was reduced. We hypothesized this disconnect could be due to aging. In this study, we elucidate the mechanism underlying Wnt3a pro-survival signalling and demonstrate the differential effect of age on Wnt3a- and Wnt5a-mediated survival. We show Wnt3a protein was expressed in human atherosclerotic coronary arteries and co-located with macrophages and VSMCs. Meanwhile, Wnt3a stimulation of primary mouse VSMCs increased ß-catenin nuclear translocation and TCF, but not CREB, activation. Wnt3a increased mRNA expression of the pro-survival factor WISP-2 in a TCF-dependent manner. Functionally, ß-catenin/TCF inhibition or WISP-2 neutralization significantly impaired Wnt3a-mediated VSMC survival. WISP-2 was upregulated in human atherosclerosis and partly co-localized with Wnt3a. The pro-survival action of Wnt3a was effective in VSMCs from young (2 month) and old (18-20 month) mice, whereas Wnt5a-mediated rescue was impaired with age. Further investigation revealed that although Wnt5a induced ß-catenin nuclear translocation in VSMCs from both ages, CREB phosphorylation and WISP-1 upregulation did not occur in old VSMCs. Unlike Wnt5a, pro-survival Wnt3a signalling involves ß-catenin/TCF and WISP-2. While Wnt3a-mediated survival was unchanged with age, Wnt5a-mediated survival was lost due to impaired CREB activation and WISP-1 regulation. Greater understanding of the effect of age on Wnt signalling may identify targets to promote VSMC survival in elderly patients with atherosclerosis.