John Bingley

Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo

PURPOSE The aim of this study was to determine whether heparan sulfate proteoglycans (HSPGs) from the normal arterial wall inhibit neointimal formation after injury in vivo and smooth muscle cell (SMC) phenotype change and proliferation in vitro. METHODS Arterial HSPGs were extracted from rabbit aortae and separated by anion-exchange chromatography. The effect of HSPGs, applied in a periadventitial gel, on neointimal formation was assessed 14 days after balloon catheter injury of rabbit carotid arteries. Their effect on SMC phenotype and proliferation was measured by point-counting morphometry of the cytoplasmic volume fraction of myofilaments (Vvmyo) and 3H-thymidine incorporation in SMCs in culture. RESULTS Arterial HSPGs (680 microg) reduced neointimal formation by 35% at 14 days after injury (P=.029), whereas 2000 microg of the low-molecular-weight heparin Enoxaparin was ineffective. HSPGs at 34 microg/mL maintained subconfluent primary cultured SMCs with the same high Vvmyo (52.1%+/-13.8%) after 5 days in culture as did cells freshly isolated from the arterial wall (52.1%+/-15.1%). In contrast, 100 microg/mL Enoxaparin was ineffective in preventing phenotypic change over this time period (Vvmyo 38.9%+/-14.6%, controls 35.9%+/-12.8%). HSPGs also inhibited 3H-thymidine incorporation into primary cultured SMCs with an ID50 value of 0.4 microg/mL compared with a value of 14 microg/mL for Enoxaparin (P< .01). CONCLUSION When used periadventitially in the rabbit arterial injury model, natural arterial HSPGs are effective inhibitors of neointimal formation. In vitro, the HSPGs maintain SMCs in a quiescent state by inhibiting phenotypic change and DNA synthesis. This study suggests that HSPGs may be a natural agent for the treatment of clinical restenosis.

Blockade of vascular smooth muscle cell proliferation and intimal thickening after balloon injury by the sulfated oligosaccharide PI-88: phosphomannopentaose sulfate directly binds FGF-2, blocks cellular signaling, and inhibits proliferation.

Abstract— Percutaneous transluminal coronary angioplasty is a frequently used interventional technique to reopen arteries that have narrowed because of atherosclerosis. Restenosis, or renarrowing of the artery shortly after angioplasty, is a major limitation to the success of the procedure and is due mainly to smooth muscle cell accumulation in the artery wall at the site of balloon injury. In the present study, we demonstrate that the antiangiogenic sulfated oligosaccharide, PI-88, inhibits primary vascular smooth muscle cell proliferation and reduces intimal thickening 14 days after balloon angioplasty of rat and rabbit arteries. PI-88 reduced heparan sulfate content in the injured artery wall and prevented change in smooth muscle phenotype. However, the mechanism of PI-88 inhibition was not merely confined to the antiheparanase activity of this compound. PI-88 blocked extracellular signal-regulated kinase-1/2 (ERK1/2) activity within minutes of smooth muscle cell injury. It facilitated FGF-2 release from uninjured smooth muscle cells in vitro, and super-released FGF-2 after injury while inhibiting ERK1/2 activation. PI-88 inhibited the decrease in levels of FGF-2 protein in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering the clotting time in these animals. Optical biosensor studies revealed that PI-88 potently inhibited (Ki 10.3 nmol/L) the interaction of FGF-2 with heparan sulfate. These findings show for the first time the capacity of this sulfated oligosaccharide to directly bind FGF-2, block cellular signaling and proliferation in vitro, and inhibit injury-induced smooth muscle cell hyperplasia in two animal models. As such, this study demonstrates a new role for PI-88 as an inhibitor of intimal thickening after balloon angioplasty. The full text of this article is available online at http://www.circresaha.org.

Up regulation of AT4 receptor levels in carotid arteries following balloon injury

Angiotensin IV, (V-Y-I-H-P-F), binds to AT4 receptors in blood vessels to induce vasodilatation and proliferation of cultured bovine endothelial cells. This latter effect may be important not only in developing tissues but also in injured vessels undergoing remodelling. In the present study, using normal rabbit carotid arteries, we detected AT4 receptors in vascular smooth muscle cells and in the vasa vasorum of the adventitia. Very low receptor levels were observed in the endothelial cells. In keeping with the described binding specificity of AT4 receptors, unlabelled angiotensin IV competed for [125I]angiotensin IV binding in the arteries, with an IC50 of 1.4 nM, whereas angiotensin II and angiotensin III were weaker competitors. Within the first week following endothelial denudation of the carotid artery by balloon catheter, AT4 receptor binding in the media increased to approximately 150% of control tissue. AT4 receptor binding further increased in the media, large neointima and re-endothelialized cell layer to 223% at 20 weeks after injury. In view of the known trophic effects of angiotensin IV, the elevated expression of AT4 receptors, in both the neointima and media of arteries, following balloon injury to the endothelium, suggests a role for the peptide in the adaptive response and remodelling of the vascular wall following damage.

Leukaemia inhibitory factor retards the progression of atherosclerosis

OBJECTIVE Our previous studies showed that the pleiotropic cytokine leukaemia inhibitory factor (LIF) inhibits the de novo formation of experimental atherosclerotic lesions. The present study examined whether LIF also inhibits progression of pre-existing atheroma. METHODS Balloon angioplasty was performed on the right carotid arteries of 18 rabbits immediately before placing animals on a cholesterol-enriched diet. After 4 weeks, at which time the intima:media ratio (I:M) was 0.99+/-0.12 (n=6), osmotic minipumps containing LIF (n=6) or saline control (n=6) were inserted into the peritoneal cavity of each of the remaining rabbits for a further 4 weeks. Arteries were then harvested for analysis. RESULTS Continuous administration of LIF for the final 4 weeks of an 8-week cholesterol-enriched diet completely inhibited lesion progression in injured carotid arteries (I:M 1.05+/-0.16) compared with the saline-treated group at 8 weeks (1.62+/-0.13; P<0.05). Similarly in contralateral uninjured carotid arteries, LIF treatment prevented an increase in I:M from a baseline of 0.11+/-0.01 at 4 weeks to 0.15+/-0.02 at 8 weeks compared with 0.40+/-0.04 for the saline-treated group at 8 weeks (P<0.05). LIF reduced the number of macrophages in the neointima of uninjured arteries, but had no effect on the cellular composition of injured arteries. LIF treatment normalised smooth muscle-dependent vasoreactivity to phenylephrine and sodium nitroprusside in both injured and uninjured arteries. Expression and activity of inducible nitric oxide synthase (iNOS) were up-regulated in response to hypercholesterolemia with levels further increased following endothelial denudation. With LIF treatment, iNOS expression was increased in uninjured arteries but marginally reduced in injured arteries. LIF receptors were expressed in both uninjured and injured arteries, with LIF treatment having no significant effect on expression levels. CONCLUSION LIF prevents progression of pre-formed atherosclerotic plaques, affecting lesion size and vascular reactivity. LIF treatment has differential effects within the artery wall, depending on the presence or absence of de-endothelialisation injury.

Inhibition of neointimal formation by natural heparan sulfate proteoglycans of the arterial wall.

Coronary angioplasty has been used clinically for more than a decade to treat primary atherosclerotic plaque. The initial promise of angioplasty as an alternative to coronary bypass surgery has only partially been filled because of a high rate of post-operative restenosis within the first six months.’,2 Post-mortem studies on patients dying several months after apparently successful angioplasty show clear evidence of plaque fracturing and medial dissection with neointimal proliferative tissue filling the crevices overlying the plaque and obstructing the coronary l~men.3 .~ It is believed that exposure of subintimal components of the vessel wall result in deposition of platelets and thrombus formation with release of enzymes and cytokines from platelets, circulating monocytes, endothelial cells, and perhaps vascular smooth muscle cells (SMC) within the vessel waL5 The resulting intimal hyperplasia leads to luminal restenosis consisting predominantly of SMC in a loose connective tissue matrix of sparse collagen fibrils and proteoglycans. While injury is accompanied by the liberation of cytokines, many of which are mitogenic for SMC, there may be more subtle control over SMC migration and proliferation than simply the presence or absence of cytokines. In the uninjured artery, SMC may be actively maintained in a quiescent state by inhibitory molecules. In such a “balance” model for control of cell proliferation, injury would not only present numerous cytokines to the SMC, but also remove the natural inhibitors. Maintaining these natural inhibitors could prove an effective therapeutic tool to prevent restenosis in the clinical setting. Heparan sulfate proteoglycans (HSPG) are a diverse family of molecules present both pericellularly and extracellularly around all c e k 6 The heparan sulfate sidechains from both endothelid’ and SMC8 proteoglycans have been shown to be similarly or more potently inhibitory of SMC proliferation in vitro as the closely related glycosaminoglycan heparin, and are thus good candidates to be naturat inhibitors of SMC proliferation in the normal artery wall. To examine this possibility, proteoglycans were extracted from the aortas of healthy rabbits by a two-step digestion process. The elution profile of the proteoglycans from DEAE-cellulose anion exchange chromatography showed two distinct

Barriers to screening and diagnosis of peripheral artery disease by general practitioners

Peripheral artery disease (PAD) is a strong predictor of cardiovascular morbidity and mortality yet it is under-recognised and undertreated. General practitioners (GPs) are best positioned to detect patients with PAD. This article investigates awareness of PAD by GPs; the prevalence of screening for PAD and tools used for screening and diagnosis, in particular the ankle–brachial index (ABI); and the barriers to PAD screening and measurement of the ABI in the general practice setting. A cross-sectional survey of primary care practitioners was conducted between September 2011 and March 2012. A mail-out survey was distributed to 1120 GPs practising in Queensland, Australia: 287 (26%) responded; 61% of GPs reported screening for PAD; 58% of GPs reported ‘never’ measuring the ABI; and 70% reported using arterial duplex ultrasound as their first-line diagnostic tool. Equipment availability, time constraints and lack of training and skills were identified as the most significant barriers to screening and ABI testing. In conclusion, there are deficits in the utilisation of guideline recommendations relating to PAD screening and diagnosis by Australian GPs. Our data suggest that earlier detection of PAD may be achieved through GP education combined with increased access to ABI equipment or the availability of a more time-efficient test.

Epigenetics and Peripheral Artery Disease

The term epigenetics is usually used to describe inheritable changes in gene function which do not involve changes in the DNA sequence. These typically include non-coding RNAs, DNA methylation and histone modifications. Smoking and older age are recognised risk factors for peripheral artery diseases, such as occlusive lower limb artery disease and abdominal aortic aneurysm, and have been implicated in promoting epigenetic changes. This brief review describes studies that have associated epigenetic factors with peripheral artery diseases and investigations which have examined the effect of epigenetic modifications on the outcome of peripheral artery diseases in mouse models. Investigations have largely focused on microRNAs and have identified a number of circulating microRNAs associated with human peripheral artery diseases. Upregulating or antagonising a number of microRNAs has also been reported to limit aortic aneurysm development and hind limb ischemia in mouse models. The importance of DNA methylation and histone modifications in peripheral artery disease has been relatively little studied. Whether circulating microRNAs can be used to assist identification of patients with peripheral artery diseases and be modified in order to improve the outcome of peripheral artery disease will require further investigation.

Expression of Heparan Sulphate N-deacetylase/N-sulphotransferase by Vascular Smooth Muscle Cells

Heparan sulphate is an important mediator in determining vascular smooth muscle cell (SMC) phenotype. The sulphation pattern of the heparan sulphate chains is critical to their function. We have examined the initial step in the biosynthesis of the sulphated domains mediated by the enzyme heparan sulphate N-deacetylase/N-sulphotransferase (NDST). Rabbit aortic SMC in primary culture exhibited NDST enzyme activity and expressed NDST-1 in their Golgi apparatus, with maximal expression in SMC 2 days after dispersal in primary culture confirmed by Western blot analysis. Endothelial cells, macrophages and fibroblasts expressed NDST-1 but had generally less intense staining than SMC, although SMC expression decreased with culture. The uninjured rat aorta also showed widespread expression of NDST-1. After balloon de-endothelialisation, NDST-1 could not be detected in SMC of the neointima in the early stages of neointimal formation, but was re-expressed at later time points (after 12 weeks). In human coronary arteries, SMC of the media and the diffuse intimal thickening expressed NDST-1, while SMC in the atherosclerotic plaque were negative for NDST-1. We conclude that SMC may regulate their heparan sulphate sulphation at the level of expression of the enzyme heparan sulphate NDST in a manner related to their phenotypic state.

A systematic review investigating the association of microRNAs with human abdominal aortic aneurysms

BACKGROUND AND AIMS There is increasing interest in identifying novel methods for abdominal aortic aneurysm (AAA) diagnosis. Non-coding RNA molecules such as microRNAs (miRNAs) are stable within the circulation and may serve as biomarkers for AAA. This systematic review aimed to identify miRNAs associated with a diagnosis of human AAA based on currently published original research. METHODS A systematic search of the MEDLINE and EMBASE databases identified studies assessing miRNA expression in abdominal aortic tissue or circulating blood from human AAA cases compared to non-aneurysmal controls. Data from included studies were extracted to assess methods and results after independent quality assessment by two reviewers. RESULTS 15 studies were included in this review. 11 studies obtained aortic tissue samples from 195 AAA cases and 104 controls with normal aortas. Nine studies obtained circulating blood samples from 526 AAA cases and 441 controls. miR-21 was differentially expressed in AAA tissue in five separate studies, with four studies reporting upregulation and one reporting downregulation. Seven other miRNAs were differentially expressed in AAA tissue in two separate studies. 15 circulating miRNAs were differentially expressed in two or more separate studies. miR-155 and miR-29b were the only miRNAs differentially expressed in two separate tissue- and blood-based studies. 11 studies offered mechanistic explanations of the role of miRNAs in AAA pathology through exploration of gene targets. Three studies assessed the diagnostic potential of circulating miRNAs with receiver operating characteristic curves. Only one study assessed the prognostic potential of circulating miRNAs in predicting AAA growth. CONCLUSIONS Several miRNAs have been found to be associated with human AAA. Their utility as AAA biomarkers requires further investigation.

Complete femoral nerve palsy secondary to a traumatic iliacus haematoma: Images for surgeons

Management of femoral nerve palsy secondary to traumatic iliacus haematomas is contentious, as owing to the rarity of the presentation, no evidence beyond a few case studies exists to inform clinical decision-making. We present such a case and advocate for prompt surgical decompression over conservative management in patients with progressive neurological deficits. Whereas femoral nerve palsies are widely reported in patients on oral anticoagulants or with blood dyscrasias, very few cases are reported secondary to traumatic iliacus haematomas. A recent systematic review identified only 16 cases of femoral neuropathy secondary to traumatic iliopsoas haematomas over a 40-year period to 2012. A reading of the cases reveals a trend towards conservative management of partial palsies and surgical decompression of complete palsies; however, it remains controversial when surgical intervention should be pursued over a conservative approach. The current authors freely admit that they were faced with a management uncertainty on initial presentation. Our patient, a 14-year-old boy, fell off a rope swing from a 2-m height landing on his back and left side. He presented later that day to a peripheral hospital complaining of left hip pain, and after a normal X-ray, was discharged home on oxycodone. The next day he was mobilizing with crutches but began experiencing altered sensation over the left anterior thigh. On the second day post-fall he represented with worsening hip pain and was admitted for analgesia and further imaging. A magnetic resonance imaging (MRI) study of the pelvis, performed on the third day post-fall, was discontinued by the patient due to hip pain on lying flat; however, the limited study completed identified a large iliacus haematoma measuring 6.2 cm × 3.9 cm × 10 cm (Fig. 1), prompting transfer to the Lady Cilento Children’s Hospital (LCCH). On arrival at LCCH, late in the evening on the fourth day post-fall, examination elicited left knee extension power 2/5, an absent knee jerk and altered L2 sensation. A computed tomography angiogram of the pelvis showed arterial phase contrast extravasation into the haematoma, unchanged in size since the MRI, from a branch of the left iliolumbar artery (Fig. 2). The palsy mechanism appears to be nerve stretching, with a possible compressive contribution. After emerging from lateral border of the psoas muscle, the femoral nerve descends under the iliac fascia in the intermuscular groove between psoas and iliacus, entering the thigh by passing deep to the inguinal ligament through the lacuna musculorum. As Figures 1 and 2 show, the left femoral nerve is displaced anteriorly and necessarily stretched by the iliacus haematoma. Goodfellow et al. suggest the nerve is also compressed by raised intra-compartmental pressure. Consultant discussions between paediatric and adult orthopaedics, vascular surgery and interventional radiology identified three potential management options: (i) conservative; (ii) radiological embolization and percutaneous drainage; and (iii) surgical decompression. The embolization was unable to be performed at LCCH and would require patient transfer to the Princess Alexandra Hospital the following day for the procedure and then back to LCCH for on-going management. With the above discussions in mind, the patient was managed conservatively overnight with a view to embolization and some form of decompression, either percutaneous or surgical, the following day. The patient was placed into slings and springs with the hip in flexion to decrease stretch on the femoral nerve and a dose of tranexamic acid was given to promote haemostasis. Throughout the next day his partial femoral nerve palsy progressed to a dense palsy with 0/5 quadriceps power, an absent knee jerk and L2 anaesthesia. Confronted with the overnight development of dense femoral nerve palsy and the unavailability of on-site radiological embolization, the decision was made to immediately surgically decompress the haematoma. The operation, performed by the last and penultimate authors, consultant orthopaedic and vascular surgeons, respectively, proceeded uneventfully via an iliac apophyseal splitting approach, evacuating 200 mL of clotted blood without encountering any active arterial bleeding. The patient experienced a rapid post-operative recovery in femoral nerve function. The day after surgery, quadriceps power was 3/5 and altered sensation supervened upon numbness; 3 months after surgery his only neurological deficit was an absent knee jerk. While the outcome of other management routes can never be known, prompt surgical intervention in this case produced a rapid and near-complete reversal of femoral neuropathy without any surgical complication. We advocate that acute development of a dense femoral nerve palsy