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Intraplaque neovascularization contributes to the progression of atherosclerosis. Our aim is to understand the mobilization of cells and factors involved in this process. We investigated the localization of hepatocyte growth factor (HGF) and its receptor, c-Met, in human atherosclerotic plaques, together with the effects of HGF on pericyte migration in vitro. Atherosclerotic femoral arterial segments were collected and analysed from 13 subjects who were undergoing lower limb amputation. Pericytes were identified in human lesions using a 3G5 antibody. Immunohistochemical analysis localized HGF mainly around microvessels, in association with some, but not all, CD31-positive endothelial cells. c-Met expression was mainly associated with smooth muscle cells and pericytes, around some, but not all, microvessels within the atherosclerotic lesions; no detection was apparent in normal internal mammary arteries. Using RT-PCR, we demonstrated expression of HGF and c-Met in a rat pericyte cell-line, TR-PCT1, and in primary pericytes. HGF treatment of TR-PCT1 cells induced their migration, but not their proliferation, in a dose-dependent manner (10-100 ng/ml, p<0.01), an effect mediated by activation of the serine/threonine kinase Akt, shown by western blot analysis. Treating the cells with the PI3K inhibitors Wortmannin (0.1 microM) or LY294002 (10 microM) abolished these effects. This work demonstrates the expression of c-Met and HGF in human atherosclerotic arteries, in association with SM-actin-positive cells and CD-31-positive cells, respectively. HGF induces pericyte migration via PI3-kinase and Akt activation in vitro. HGF and c-Met may be involved in neovascularization during plaque development, and may recruit pericytes to neovessels. Since pericytes are thought to mechanically stabilize new blood vessels, these factors may function to protect against haemorrhage., (Copyright (c) 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Calcification of the vessel wall is a regulated process with many similarities to osteogenesis. Progenitor cells may play a role in this process. Previously, we identified a novel gene, Vascular Calcification Associated Factor (VCAF), which was shown to be important in pericyte osteogenic differentiation. The aim of this study was to determine the localization and expression pattern of VCAF in human cells and tissues. Immunohistochemical analysis of seven atherosclerotic arteries confirmed VCAF protein expression within calcified lesions. In addition, individual VCAF-positive cells were detected within the intima and adventitia in areas where sporadic 3G5-positive pericytes were localized. Furthermore, VCAF-positive cells were identified in newly formed microvessels in association with CD34-positive/CD146-positive/c-kit-positive cells as well as in intact CD31-positive endothelium in internal mammary arteries. Western blot analysis confirmed the presence of VCAF (18 kD) in protein lysates extracted from human smooth muscle cells, endothelial cells, macrophages, and osteoblasts. In fracture callus samples from three patients, VCAF was detected in osteoblasts and microvessels. This study demonstrates the presence of VCAF in neovessels and raises the possibility that VCAF could be a new marker for vascular progenitor cells involved in a number of differentiation pathways. These data may have implications for the prevention or treatment of vascular disease., (Copyright 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

The pathophysiology of chronic low back pain is poorly understood, mainly because it is difficult to study experimentally or objectively. Recently it has been found that there is a relationship between neovascularization and innervation of the usually avascular and aneural intervertebral disc at the sites of discogenic pain. These data, together with the recognized involvement of mast cells in tissue repair, in the induction of angiogenesis, and in the production of and response to neurotrophic stimuli such as nerve growth factor, has suggested the hypothesis that mast cells may have a causative role in chronic low back pain. If so, the mast cell may represent an attractive therapeutic target., (Copyright 2002 John Wiley & Sons, Ltd.)

Following a previous description of nociceptive nerve fibre growth into usually aneural inner parts of painful intervertebral disc (IVD), this study has investigated whether nociceptive nerve ingrowth into painful IVD is stimulated by local production of neurotrophins. Immunohistochemistry and in situ hybridization have been used to investigate expression of the candidate neurotrophin, nerve growth factor (NGF), and its high- and low-affinity receptors trk-A and p75, respectively, in painful IVD excised for the management of low back pain. IVD from patients with back pain were of two types: those that when examined by discography reproduced the patient symptoms (pain level IVD) and those that did not (non-pain level IVD). Microvascular blood vessels accompanied nerve fibres growing into pain level IVD and these expressed NGF. The adjacent nerves expressed the high-affinity NGF receptor trk-A. These vessels entered the normally avascular IVD through the discal end plates. NGF expression was not identified in non-pain level or control IVD. Some non-pain level IVD had vessels within them, which entered through the annulus fibrosus. These did not express NGF nor did nerves accompany them. These findings show that nociceptive nerve ingrowth into painful IVD is causally linked with NGF production by blood vessels growing into the IVD, from adjacent vertebral bodies., (Copyright 2002 John Wiley & Sons, Ltd.)

Until recently, material removed from the intervertebral disc (IVD) at surgery consisted either of 'loose bodies' from the centre of the IVD or discal tissue displaced (prolapsed) into the intervertebral root or spinal canals. This material is best regarded as a by-product of disc degeneration and therefore not representative of the disease process itself. Recent advances in surgical techniques, particularly anterior fusion, in which large segments of the anterior part of the IVD are excised with the anatomical relationships between different components intact, have generated material that can be investigated with modern molecular and cell biological techniques. This is an important area of study because degeneration of the lumbar IVDs is associated, perhaps causally, with low back pain, one of the most common and debilitating conditions in the West. 'Degeneration' carries implications of inevitable progression of wear-and-tear associated conditions. Modern research on human IVD tissue has shown that this is far from the case and that disruption of the micro-anatomy described as degeneration is an active process, regulated by locally produced molecules. The exciting consequence of this observation is the possibility of being able to inhibit or even reverse the processes of degeneration using targeted therapy., (Copyright 2002 John Wiley & Sons, Ltd.)

Eosinophils are a recognized feature of inflammatory bowel disease (IBD), but their tissue distribution and functional importance in Crohn's disease (CD) and ulcerative colitis (UC) remain obscure. This study describes an improved immunohistochemical protocol to identify eosinophils in full thickness bowel wall specimens of IBD (n=40) and their in situ relationships with the chemoattractants eotaxin and RANTES. Eosinophils were identified using immunohistochemistry with a combination of monoclonal antibodies (EG1+EG2+MBP), an ultrasensitive technique superior to other methodologies, and their tissue distributions were related to those for eotaxin, RANTES, mast cells and neutrophils. Increased numbers of eosinophils (up to 400 cells/mm(2)) were observed in active, fulminant inflammation in both CD and UC, this being related to the severity of inflammation and not the diagnosis of the two disorders. The chemoattractants eotaxin (CCL11) and RANTES (CCL5) were upregulated in IBD tissues showing eosinophilia. Neutrophils and mast cells were commonly associated with eosinophil accumulations. Eosinophil numbers and their in situ activation are increased in active rather than chronic IBD. The observations strongly suggest a pivotal role for the eosinophil and its potent mediators in many pathophysiological symptoms of CD and UC, where it represents the major proportion of all granulocytic cells in active inflammatory bowel disease., (Copyright 2001 John Wiley & Sons, Ltd.)

An improved immunohistochemical method has been used to assess neovascularization within the vulnerable 'shoulder' regions of atherosclerotic plaques from carotid arteries. A combination of monoclonal antibodies (CD31, CD34, +/- von Willebrand factor) was shown to be far more effective than conventional techniques in demonstrating extensive vascularizations within the 'shoulder' and cap regions of late-stage plaques. Such sites were shown to be microfocal, often appearing as a plexus of both large and small vessels which occupied a significant proportion of the 'shoulder' area. These regions of marked neovascularization were commonly associated with accumulations of macrophages, mast cells, and T-cells, indicative of local inflammatory reactions. The matrix components elastin and collagen type VI showed variable distributions which suggested extensive tissue remodelling, whereas collagen type IV was recognized as a basement membrane protein of most blood vessels, as well as being associated with 'stellate' smooth muscle cells. Evidence of local microvascular damage within the shoulder regions of some specimens was demonstrated by extravascular red blood cells, macrophages containing haemosiderin, and perivascular fibrin deposition. These local haemorrhages derived from microvessels beneath the lining of the arterial lumen are a further indication of how the microfocal vascularization of the plaque 'shoulder' might contribute to further complications of inflammation and plaque destabilization in late-stage disease., (Copyright 1999 John Wiley & Sons, Ltd.)

Calcification has been examined in 250 samples of atherosclerotic lesions (types II to VI) of human carotid arteries using von Kossa and haematoxylin staining. Early calcification described as 'stippling' was first noted in stage III specimens, with intermediate and solid calcifications becoming increasingly prominent within advanced plaques, especially stages Vb and VI. Although the relative frequencies of stippling, intermediate and large calcified deposits varied between plaques of the same stage, the prevalent sites of calcification were recognized as the deeper regions of the intima and the atheroma. Immunolocalization and histochemical techniques were used to identify the associations of mast cells (MCs), macrophages, smooth muscle cells (SMCs), and elastin with the different stages of calcification. Early, dispersed stippling was commonly associated with local accumulations of macrophages (HAM56 and CD68-positive), MCs and extracellular MC tryptase, the presence of immunoreactive elastin, but the relative absence of SMCs. Intermediate stages of calcification described as 'morula' deposits were also associated with local increases in the numbers of macrophages and MCs. Larger calcified deposits, even within the same plaque specimen, showed no regular pattern of cellular or elastin associations. However, in the vast majority of specimens, macrophages represented the predominant cell type associated with different phases of calcification. By contrast, the calcification less frequently observed in the media beneath advanced plaques was commonly associated with SMCs and elastin; only rarely were macrophages or MCs present. These studies are the first to demonstrate that macrophages, MCs, and extracellular tryptase frequently occupy micro-environmental loci showing the first stages of calcification within the atherosclerotic plaque; similar associations with more advanced mineral deposits are discussed in relation to plaque rupture.

Immunohistochemical staining for mast cell tryptase and chymase was used to examine the distribution, activation, and tryptase/chymase phenotype of mast cells (MCs) in 250 samples of atherosclerotic lesions (type I to VI) of human carotid arteries. Dual immunolocalization and histochemical techniques were used to identify the associations of MCs with macrophages, smooth muscle cells, and extracellular matrix components. Whereas normal carotid arteries contained very few MCs within the intima, atherosclerotic lesions showed increased MC numbers with variable focal accumulations. MCs were identifiable from the earliest stages of atherosclerosis, and especially at the shoulder regions of the fully formed atheroma. They were observed in close association with macrophages (HAM56 positive) and extracellular lipid, as well as at sites of foam cell formation. MCs and diffuse tryptase staining were also evident within sites of new calcification and around small calcified deposits. Extensive MC activation/degranulation, as judged by diffuse extracellular tryptase staining, was a common feature of the advanced atherosclerotic plaques complicated by fissure, haemorrhage, and thrombus formation. Moreover, such sites of extracellular MC tryptase were often associated with localized oedema and disruption of the stromal matrix. MCs which contained both tryptase and chymase (the MCTC phenotype) represented approximately 80-95 per cent of all MCs. These studies are the first to demonstrate significant numbers and focal accumulations of MCs in all developmental stages of atherosclerotic carotid arteries. Since MCs contain or express a variety of potent mediators, their release could profoundly influence the development and pathological complications of atherosclerotic plaques.