Junxi Wu

miRNA-21 is dysregulated in response to vein grafting in multiple models and genetic ablation in mice attenuates neointima formation

Aims The long-term failure of autologous saphenous vein bypass grafts due to neointimal thickening is a major clinical burden. Identifying novel strategies to prevent neointimal thickening is important. Thus, this study aimed to identify microRNAs (miRNAs) that are dysregulated during neointimal formation and determine their pathophysiological relevance following miRNA manipulation. Methods and results We undertook a microarray approach to identify dysregulated miRNAs following engraftment in an interpositional porcine graft model. These profiling experiments identified a number of miRNAs which were dysregulated following engraftment. miR-21 levels were substantially elevated following engraftment and these results were confirmed by quantitative real-time PCR in mouse, pig, and human models of vein graft neointimal formation. Genetic ablation of miR-21 in mice or grafted veins dramatically reduced neointimal formation in a mouse model of vein grafting. Furthermore, pharmacological knockdown of miR-21 in human veins resulted in target gene de-repression and a significant reduction in neointimal formation. Conclusion This is the first report demonstrating that miR-21 plays a pathological role in vein graft failure. Furthermore, we also provided evidence that knockdown of miR-21 has therapeutic potential for the prevention of pathological vein graft remodelling.

Mast cells and vascular diseases

Mast cells are increasingly being recognized as effector cells in many cardiovascular conditions. Many mast-cell-derived products such as tryptase and chymase can, through their enzymic action, have detrimental effects on blood vessel structure while mast cell-derived mediators such as cytokines and chemokines can perpetuate vascular inflammation. Mice lacking mast cells have been developed and these are providing an insight into how mast cells are involved in cardiovascular diseases and, as knowledge increase, mast cells may become a viable therapeutic target to slow progression of cardiovascular disease.

Ablation of the androgen receptor from vascular smooth muscle cells demonstrates a role for testosterone in vascular calcification.

Vascular calcification powerfully predicts mortality and morbidity from cardiovascular disease. Men have a greater risk of cardiovascular disease, compared to women of a similar age. These gender disparities suggest an influence of sex hormones. Testosterone is the primary and most well-recognised androgen in men. Therefore, we addressed the hypothesis that exogenous androgen treatment induces vascular calcification. Immunohistochemical analysis revealed expression of androgen receptor (AR) in the calcified media of human femoral artery tissue and calcified human valves. Furthermore, in vitro studies revealed increased phosphate (Pi)-induced mouse vascular smooth muscle cell (VSMC) calcification following either testosterone or dihydrotestosterone (DHT) treatment for 9 days. Testosterone and DHT treatment increased tissue non-specific alkaline phosphatase (Alpl) mRNA expression. Testosterone-induced calcification was blunted in VSMC-specific AR-ablated (SM-ARKO) VSMCs compared to WT. Consistent with these data, SM-ARKO VSMCs showed a reduction in Osterix mRNA expression. However, intriguingly, a counter-intuitive increase in Alpl was observed. These novel data demonstrate that androgens play a role in inducing vascular calcification through the AR. Androgen signalling may represent a novel potential therapeutic target for clinical intervention.

Sertoli Cells Modulate Testicular Vascular Network Development, Structure, and Function to Influence Circulating Testosterone Concentrations in Adult Male Mice

The testicular vasculature forms a complex network, providing oxygenation, micronutrients, and waste clearance from the testis. The vasculature is also instrumental to testis function because it is both the route by which gonadotropins are delivered to the testis and by which T is transported away to target organs. Whether Sertoli cells play a role in regulating the testicular vasculature in postnatal life has never been unequivocally demonstrated. In this study we used models of acute Sertoli cell ablation and acute germ cell ablation to address whether Sertoli cells actively influence vascular structure and function in the adult testis. Our findings suggest that Sertoli cells play a key role in supporting the structure of the testicular vasculature. Ablating Sertoli cells (and germ cells) or germ cells alone results in a similar reduction in testis size, yet only the specific loss of Sertoli cells leads to a reduction in total intratesticular vascular volume, the number of vascular branches, and the numbers of small microvessels; loss of germ cells alone has no effect on the testicular vasculature. These perturbations to the testicular vasculature leads to a reduction in fluid exchange between the vasculature and testicular interstitium, which reduces gonadotropin-stimulated circulating T concentrations, indicative of reduced Leydig cell stimulation and/or reduced secretion of T into the vasculature. These findings describe a new paradigm by which the transport of hormones and other factors into and out of the testis may be influenced by Sertoli cells and highlights these cells as potential targets for enhancing this endocrine relationship.

Modulation of neointimal lesion formation by endogenous androgens is independent of vascular androgen receptor

Aims Low androgen levels have been linked with an increased risk of cardiovascular disease in men. Previous studies have suggested that androgens directly inhibit atherosclerotic lesion formation although the underlying mechanisms for this remain unclear. This study addressed the hypothesis that endogenous androgens inhibit arterial remodelling by a direct action on the androgen receptor (AR) in the vascular wall. Methods and results We studied a series of novel mouse lines with cell-specific deletion of the AR in either the endothelium or in smooth muscle cells or both cell types. Findings were compared with a model of global androgen deficiency in wild-type mice (castrated). We characterized the cardiovascular phenotype, vascular pharmacology and histology, and assessed neointimal lesion formation following vascular injury to the femoral artery. Cell-specific AR deletion did not alter body weight, circulating testosterone levels or seminal vesicle weight, but caused limited alterations in arterial contractility and blood pressure. Neointimal lesion formation was unaltered by selective deletion of AR from the vascular endothelium, smooth muscle, or both cell types. Castration in wild-type mice increased neointimal lesion volume (Sham vs. Castration: 2.4 × 107 ± 4.5 × 106 vs. 3.9 × 107 ± 4.9 × 106 µm3, P = 0.04, n = 9–10). Conclusion Vascular cell-specific AR deletion had no effect on neointimal lesion formation, while low systemic androgen levels adversely affect neointimal lesion size. These findings suggest that the cardio-protective effects of androgens are mediated either by AR outside the vasculature or by AR-independent mechanisms.

Inhibition of Inducible Nitric Oxide Synthase Promotes Vein Graft Neoadventitial Inflammation and Remodelling

Background: Grafting veins into the arterial circulation causes endothelial damage and neointimal hyperplasia. However, the remodelling of vein grafts and the contribution of the endothelium is not well understood. Since nitric oxide (NO) has a crucial role in vascular function, we investigated the importance of NO synthases (NOSs) in vein graft re-endothelialization and remodelling in this study. Methods and Results: Mouse isogenic vena cava was grafted into the carotid artery. Progressive remodelling of the grafted veins was evidenced by re-endothelialization at 2 weeks and subsequent appearance of vasomotor function at 4 weeks. Pharmacological inhibition of inducible NOS (iNOS) with the specific inhibitor 1400W, administered between 2 and 4 weeks after grafting, when re-endothelialization was complete, resulted in neoadventitial inflammation, neoadventitial thickening and impaired functional remodelling. Conclusion: Completion of re-endothelialization is pivotal in vein graft remodelling in the mouse and is associated with a series of changes in inflammation, proliferation and initiation of vascular functional remodelling. After re-endothelialization, iNOS upregulation may be an important mechanism to prevent secondary neoadventitial inflammation and preserve ongoing functional remodelling. iNOS activity could therefore be beneficial for long-term patency of the vein graft.

Perivascular mast cells regulate vein graft neointimal formation and remodeling

Objective. Emerging evidence suggests an important role for mast cells in vein graft failure. This study addressed the hypothesis that perivascular mast cells regulate in situ vascular inflammatory and proliferative responses and subsequent vein graft neointimal lesion formation, using an optimized local mast cell reconstitution method. Methods and Results. Neointimal hyperplasia was induced by insertion of a vein graft into the right carotid artery in wild type and mast cell deficient KitW−sh/W−sh mice. In some experiments, mast cells were reconstituted systemically (tail vein injection of bone marrow-derived mast cells) or locally (directly into the right neck area) prior to vein grafting. Vein graft neointimal lesion formation was significantly (P < 0.05) reduced in KitW−sh/W−sh mice. Mast cell deficiency reduced the number of proliferating cells, and inhibited L-selectin, CCL2, M-CSF and MIP-3α expression in the vein grafts. Local but not systemic mast cell reconstitution restored a perivascular mast cell population that subsequently promoted neointimal formation in mast cell deficient mice. Conclusion. Our data demonstrate that perivascular mast cells play a key role in promoting neointima formation by inducing local acute inflammatory and proliferative responses. These results suggest that ex vivo intraoperative targeting of mast cells may have therapeutic potential for the prevention of pathological vein graft remodeling.

Optical projection tomography permits efficient assessment of infarct volume in the murine heart postmyocardial infarction.

Optical projection tomography permits rapid high-resolution imaging of intact murine heart in vitro and identification of tissue heterogeneity within individual optical slices of postmyocardial infarction hearts. Infarct volume derived from >400 slices correlates with in vivo magnetic resonance imaging and avoids the need for histological staining of multiple physical sections.

PET Cell Tracking Using 18 F-FLT is Not Limited by Local Reuptake of Free Radiotracer

Assessing the retention of cell therapies following implantation is vital and often achieved by labelling cells with 2′-[18F]-fluoro-2′-deoxy-D-glucose (18F-FDG). However, this approach is limited by local retention of cell-effluxed radiotracer. Here, in a preclinical model of critical limb ischemia, we assessed a novel method of cell tracking using 3′-deoxy-3′-L-[18F]-fluorothymidine (18F-FLT); a clinically available radiotracer which we hypothesise will result in minimal local radiotracer reuptake and allow a more accurate estimation of cell retention. Human endothelial cells (HUVECs) were incubated with 18F-FDG or 18F-FLT and cell characteristics were evaluated. Dynamic positron emission tomography (PET) images were acquired post-injection of free 18F-FDG/18F-FLT or 18F-FDG/18F-FLT-labelled HUVECs, following the surgical induction of mouse hind-limb ischemia. In vitro, radiotracer incorporation and efflux was similar with no effect on cell viability, function or proliferation under optimised conditions (5 MBq/mL, 60 min). Injection of free radiotracer demonstrated a faster clearance of 18F-FLT from the injection site vs. 18F-FDG (p ≤ 0.001), indicating local cellular uptake. Using 18F-FLT-labelling, estimation of HUVEC retention within the engraftment site 4 hr post-administration was 24.5 ± 3.2%. PET cell tracking using 18F-FLT labelling is an improved approach vs. 18F-FDG as it is not susceptible to local host cell reuptake, resulting in a more accurate estimation of cell retention.

The role of androgen receptors in atherosclerosis

Male disadvantage in cardiovascular health is well recognised. However, the influence of androgens on atherosclerosis, one of the major causes of many life-threatening cardiovascular events, is not well understood. With the dramatic increase in clinical prescription of testosterone in the past decade, concerns about the cardiovascular side-effects of androgen supplementation or androgen deprivation therapy are increasing. Potential atheroprotective effects of testosterone could be secondary to (aromatase-mediated) conversion into oestradiol or, alternatively, to direct activation of androgen receptors (AR). Recent development of animal models with cell-specific AR knockout has indicated a complex role for androgen action in atherosclerosis. Most studies suggest androgens are atheroprotective but the precise role of AR remains unclear. Increased use of AR knockout models should clarify the role of AR in atherogenesis and, thus, lead to exploitation of this pathway as a therapeutic target.