Mechanobiology of the endothelium in vascular health and disease: in vitro shear stress models.

In recent years, there has been growing evidence that vascular pathologies arise in sites experiencing an altered haemodynamic environment. Fluid shear stress (FSS) is an important contributor to vascular homeostasis and regulates endothelial cell (EC) gene expression, morphology, and behaviour through specialised mechanosensitive signalling pathways. The presence of an altered FSS profile is a pathological characteristic of many vascular diseases, with the most established example being the preferential localisation of atherosclerotic plaque development. However, the precise haemodynamic contributions to other vascular pathologies including coronary artery vein graft failure remains poorly defined. To evaluate potential novel therapeutics for the treatment of vascular diseases via targeting EC behaviour, it is important to undertake in vitro experiments using appropriate culture conditions, particularly FSS. There are a wide range of in vitro models used to study the effect of FSS on the cultured endothelium, each with the ability to generate FSS flow profiles through which the investigator can control haemodynamic parameters including flow magnitude and directionality. An important consideration for selection of an appropriate model of FSS exposure is the FSS profile that the model can generate, in comparison to the physiological and pathophysiological haemodynamic environment of the vessel of interest. A resource bringing together the haemodynamic environment characteristic of atherosclerosis pathology and the flow profiles generated by in vitro methods of applying FSS would be beneficial to researchers when selecting the appropriate model for their research. Consequently, here we summarise the widely used methods of exposing cultured endothelium to FSS, the flow profile they generate and their advantages and limitations in investigating the pathological contribution of altered FSS to vascular disease and evaluating novel therapeutic targets for the treatment and prevention of vascular disease.

A novel small diameter nanotextile arterial graft is associated with surgical feasibility and safety and increased transmural endothelial ingrowth in pig.

Globally, millions of patients are affected by myocardial infarction or lower limb gangrene/amputation due to atherosclerosis. Available surgical treatment based on vein and synthetic grafts provides sub-optimal benefits. We engineered a highly flexible and mechanically robust nanotextile-based vascular graft (NanoGraft) by interweaving nanofibrous threads of poly-L-lactic acid to address the unmet need. The NanoGrafts were rendered impervious with selective fibrin deposition in the micropores by pre-clotting. The pre-clotted NanoGrafts (4 mm diameter) and ePTFE were implanted in a porcine carotid artery replacement model. The fibrin-laden porous milieu facilitated rapid endothelization by the transmural angiogenesis in the NanoGraft. In-vivo patency of NanoGrafts was 100% at 2- and 4-weeks, with no changes over time in lumen size, flow velocities, and minimal foreign-body inflammatory reaction. However, the patency of ePTFE at 2-week was 66% and showed marked infiltration, neointimal thickening, and poor host tissue integration. The study demonstrates the in-vivo feasibility and safety of a thin-layered vascular prosthesis, viz., NanoGraft, and its potential superiority over the commercial ePTFE.

Suppression of neointima formation by targeting ß-catenin/TCF pathway.

Coronary artery disease is treated by vein grafting and stent implantation. Late vein graft failure and restenosis of stented arteries reduce the success rates of these approaches and are caused by neointima formation. We have previously shown that Wnt proteins are up-regulated during intimal thickening, and have speculated that these lead to activation of downstream genes with ß-catenin/T-cell factor (TCF)-responsive promoters. In the present study, we aimed to provide evidence that ß-catenin/TCF signalling promotes neointima formation and assess whether targeting this pathway has potential for reducing neointima formation. We utilized a gene therapy approach selectively targeting cells in which the ß-catenin/TCF pathway is activated by using a recombinant adenovirus Ad-TOPTK, which carries a herpes simplex virus thymidine kinase (HSV-TK) gene under the control of a ß-catenin/TCF-response promoter. Cells with activated ß-catenin will therefore be selectively killed. Ad-TOPTK and ganciclovir (GCV) treatment significantly suppressed the growth of the neointima in a murine model of left carotid artery ligation. In summary, we demonstrated that Wnt/ß-catenin/TCF signalling promotes neointima formation, by showing that the selective death of cells with activated ß-catenin suppressed neointima formation. This highlights the therapeutic potential for reducing late vein graft failure and in-stent restenosis by targeting ß-catenin/TCF signalling.

The Effect of Ageing on Vascular Smooth Muscle Cell Behaviour--A Mini-Review.

Ageing is a prominent risk factor for atherosclerosis and cardiovascular disease. Vascular smooth muscle cells (VSMCs) are an integral part of atherosclerotic plaque formation, progression and subsequent rupture. Emerging evidence suggests that VSMC behaviour is modified by age, which in turn may affect disease outcome in the elderly. In this review, we discuss the effect of age on VSMC behaviour, proliferation, migration, apoptosis, inflammation, extracellular matrix synthesis and calcification. In addition, we discuss the multiple signalling factors underlying these behavioural changes including angiotensin-II, matrix metalloproteinases, monocyte chemotactic protein-1, and transforming growth factor-ß1. Understanding the molecular processes underpinning altered VSMC behaviour with age, may lead to the identification of novel therapeutic targets for suppressing atherosclerosis in the elderly population.

Wnt5a-induced Wnt1-inducible secreted protein-1 suppresses vascular smooth muscle cell apoptosis induced by oxidative stress.

OBJECTIVE: Apoptosis of vascular smooth muscle cells (VSMCs) contributes to thinning and rupture of the atherosclerotic plaque fibrous cap and is thereby associated with myocardial infarction. Wnt protein activation of ß-catenin regulates numerous genes that are associated with cell survival. We therefore investigated Wnt/ß-catenin survival signaling in VSMCs and assessed the presence of this pathway in human atherosclerotic plaques at various stages of the disease process. APPROACH AND RESULTS: Wnt5a induced ß-catenin/T-cell factor signaling and retarded oxidative stress (H2O2)-induced apoptosis in mouse aortic VSMCs. Quantification of mRNA levels revealed a >4-fold (P<0.05; n=9) increase in the expression of the Wnt/ß-catenin responsive gene, Wnt1-inducible secreted protein-1 (WISP-1), which was dependent on cAMP response element-binding protein and sustained in the presence of H2O2. Exogenous WISP-1 significantly reduced H2O2-induced apoptosis by 43% (P<0.05; n=3) and was shown using silencing small interfering RNA, to be important for Wnt5a-dependent survival responses to H2O2 (P<0.05; n=3). WISP-1 protein levels were significantly lower (≈50%) in unstable atherosclerosis compared with stable plaques (n=11 and n=14). CONCLUSIONS: These results indicate for the first time that Wnt5a induces ß-catenin survival signaling in VSMCs via WISP-1. The deficiency of the novel survival factor, WISP-1 in intimal VSMCs of unstable coronary plaques, suggests that there is altered Wnt/ß-catenin/ T-cell factor signaling with progressive atherosclerosis, and restoration of WISP-1 protein might be an effective stabilization factor for vulnerable atherosclerotic plaques.

Wnt signalling in smooth muscle cells and its role in cardiovascular disorders.

Vascular smooth muscle cells (SMCs) are the major cell type within blood vessels. SMCs exhibit low rates of proliferation, migration, and apoptosis in normal blood vessels. However, increased SMC proliferation, migration, and apoptosis rates radically alter the composition and structure of the blood vessel wall and contribute to cardiovascular diseases, such as atherosclerosis, and restenosis that occur after coronary artery vein grafting and stent implantation. Consequently, therapies that modulate SMC proliferation, migration, and apoptosis may be useful for treating cardiovascular diseases. The family of Wnt proteins, which were first identified in the wingless drosophila, has a well-established role in embryogenesis and development. It is now emerging that Wnt proteins also regulate SMC proliferation, migration, and survival. In this review article, we discuss recently emerging research that has revealed that Wnt proteins are important regulators of SMC behaviour via activation of ß-catenin-dependent and ß-catenin-independent Wnt signalling pathways.

Inhibition of N-cadherin retards smooth muscle cell migration and intimal thickening via induction of apoptosis.

OBJECTIVES: Inhibition of vascular smooth muscle cell (VSMC) migration is a potential strategy for reducing intimal thickening during in-stent restenosis and vein graft failure. In this study, we examined the effect of disrupting the function of the VSMC adhesion molecule, N-cadherin, using antagonists, neutralizing antibodies, and a dominant negative, on VSMC migration and intimal thickening. Migration was assessed by the scratch-wound assay of human saphenous vein VSMCs and in a human saphenous vein ex vivo organ culture model of intimal thickening. RESULTS: Inhibition of cadherin function using a pan-cadherin antagonist, significantly reduced migration by 53%±8% compared with the control peptide (n=3; P<.05). Furthermore, inhibition of N-cadherin function with an N-cadherin antagonist, neutralizing antibodies, and adenoviral expression of dominant negative N-cadherin (RAd dn-N-cadherin), significantly reduced migration by 31%±2%, 23%±1% and 32%±7% compared with controls, respectively (n=3; P<.05). Inhibition of cadherin function significantly increased apoptosis by between 1.5- and 3.3-fold at the wound edge. In an ex vivo model of intimal thickening, inhibition of N-cadherin function by infection of human saphenous vein segments with RAd dn-N-cadherin significantly reduced VSMC migration by 55% and increased VSMC apoptosis by 2.7-fold. As a result, intimal thickening was significantly suppressed by 54%±14%. Importantly, there was no detrimental effect of dn-N-cadherin on endothelial coverage; in fact, it was significantly increased, as was survival of cultured human saphenous vein endothelial cells. CONCLUSIONS: Under the condition of this study, cell-cell adhesion mediated by N-cadherin regulates VSMC migration via modulation of viability. Interestingly, inhibition of N-cadherin function significantly retards intimal thickening via inhibition of VSMC migration and promotion of endothelial cell survival. We suggest that disruption of N-cadherin-mediated cell-cell contacts is a potential strategy for reducing VSMC migration and intimal thickening.

Regulation of cell-matrix contacts and beta-catenin signaling in VSMC by integrin-linked kinase: implications for intimal thickening.

Vascular smooth muscle cell (VSMC) proliferation and migration is responsible for intimal thickening that occurs in restenosis and atherosclerosis. Integrin-linked kinase (ILK) is a serine/threonine protein kinase implicated in signaling pathways involved in cell proliferation and migration. We studied the involvement of ILK in intimal thickening. ILK expression was significantly increased in two models of intimal thickening: balloon-injured rat carotid arteries and human saphenous vein organ cultures. Over-expression of a dominant negative ILK (DN-ILK) significantly reduced intimal thickening by approximately 50% in human saphenous vein organ cultures, demonstrating an important role in intimal thickening. ILK protein and activity was reduced on laminin and up-regulated on fibronectin, indicating ILK protein expression is modulated by extracellular matrix composition. Inhibition of ILK by siRNA knockdown and DN-ILK significantly decreased VSMC proliferation and migration while wild type ILK significantly increased proliferation and migration on laminin, confirming an essential role of ILK in both processes. Localization of paxillin and vinculin and protein levels of FAK and phospho-FAK indicated that inhibition of ILK reduced focal adhesion formation. Additionally, inhibition of ILK significantly attenuated the presence of the cell-cell complex proteins N-cadherin and beta-catenin, and beta-catenin signaling. We therefore suggest ILK modulates VSMC proliferation and migration at least in part by acting as a molecular scaffold in focal adhesions as well as modulating the stability of cell-cell contact proteins and beta-catenin signaling. In summary, ILK plays an important role in intimal thickening by modulating VSMC proliferation and migration via regulation of cell-matrix and cell-cell contacts and beta-catenin signaling.

Differential alterations in the expression and activity of matrix metalloproteinases 2 and 9 after transient cerebral ischemia in mice.

Abnormal expression and activity of matrix metalloproteinases (MMPs) may contribute to the pathophysiology of cerebral disease such as ischemic injury. In this study, we compared the cellular localization, expression, and activity of MMP-2 and -9 in relation to the evolution of neuronal damage 24 and 72 h after transient global ischemia. In response to ischemia, there was a generalized increase in cellular MMP-2 immunoreactivity at 24-h reperfusion (in neurons, glia and vessels) whereas at 72-h reperfusion the increase in MMP-2 was predominantly in glia. These glial alterations contributed to a significant increase in pro MMP-2 levels in ischemic regions (P < 0.01) as measured by zymography. In contrast, MMP-9 was predominantly upregulated in neurons and this was significantly different to shams at 24- and 72-h reperfusion after ischemia (P < 0.05). Notably, a dramatic increase in proteolytic activity in neurons was observed 24 h after ischemia and this response was absent at 72 h post-ischemia. The present data are supportive of a role for MMPs in contributing to neuronal injury after ischemia.

MMPs, cadherins, and cell proliferation.

Proliferation of vascular smooth muscle cells plays a key role in the development of pathologic processes characterized by intimal thickening. Cadherins are adhesion receptors involved in homophilic cell-cell interactions, but they can also act as signaling molecules that affect cell behavior, including proliferation, migration. and survival. The matrix-degrading metalloproteinases (MMPs) are a family of proteolytic enzymes that modulate tissue remodeling, proliferation, and migration by degradation of extracellular matrix proteins as well as non-matrix proteins, including cadherins. This review discusses the key links between cadherins and MMPs in cell proliferation. A greater understanding of these processes may aid with the design of new clinical therapeutic approaches for atherosclerosis and restenosis.