In this study, we have identified significantly increased CD105-positive microvessel density with increasing grade of atherosclerotic lesion in carotid arteries with stenosis <50% and in coronary arteries. Increased CD105-positive intimal neovessels were demonstrated previously by Li et al , whilst they also showed that CD105 staining was almost absent in normal arteries. Since we previously showed that cells (myoblasts) which do not normally express CD105, when transfected with this gene, exhibited increased cellular attachment, spreading, survival and growth concomitant with increased phosphorylation of JNK , and that CD105 expression is also up-regulated by hypoxia and TGF-β, also features of plaque development , the current observations suggest that CD105 expression is associated with active angiogenic neovascularization in these lesions. In selected cases we compared CD105 staining with CD34 positive vessels. There was less CD105 positive vessels, further suggesting that this marker is selective for active or new vessels.
There was a statistically significant increase in the maximum thickness of the neointima with increasing grade of lesion in coronary, carotid and MCA arteries. This increase correlated with an increase in the expression of CD105-positive microvessels. The early stage (grade I-III) coronary arteries generally contained stable uncomplicated plaques with few areas of inflammation. This was in contrast to the larger calibre carotid arteries which exhibited more complex heterogeneous features with more evidence of inflammation and the presence of CD105-positive vessels in grade I-III + lesions. This adds more weight to the evidence suggesting significantly different developmental profiles between carotid and coronary artery diseases with that of the carotid arteries demonstrating higher angiogenic activity, possibly in part due to the increased inflammatory infiltration and ultimately resulting in earlier production of unstable regions prone to haemorrhage and rupture [1, 12]. The prevalence of neovascularization in carotid arteries of symptomatic patients could help to explain the more rapid development of unstable lesion and subsequent thrombosis. Further, we know from the clinical experience that patients with low to moderate carotid stenosis suffer strokes or transient ischaemic attacks. These often are classified as of unknown origin if screening for cardiac source of emboli is negative and only non-significant intracranial or extracranial stenosis is detected [23, 24]. Quickly evolving carotid lesions without important lumen occlusion may be at least partially responsible for these events.
In more advanced plaques (grade IV-V) there remained a notable difference between coronary and carotid vessels with respect to the numbers of neointimal CD105-positive microvessels and this was associated with larger intimal thickness in the carotid arteries. There were no notable differences in plaque CD105-positive vessel density in coronary or carotid arteries from patients who had been undergoing treatment with antiplatelets and statins and the others.
Atherosclerosis in the intracranial arteries is less studied, although stenosis of MCA is a common cause of stroke in some populations . The exact frequency and role of intracranial artery plaques in living patients with stroke is still unknown in Caucassian population . Intracranial plaques and stenoses is highly prevalent in fatal stroke, however, some of the reported stenoses graded 30% to 75% may be causal and correspond to superimposed clot on ulcerated plaque. In our series MCA showed evidence of intimal angiogenesis. This is in contrast to some of the studies which reported higher prevelance of neovasculature in plaques associated with stroke . However, the authors did not use any marker to show neovessels and overall number of plaques with neovasculature was low. Plaques developing in the intracranial arteries can probably survive and obtain nutrients by diffusion from the existing adventitia. However the existence of CD105-positive vasa vasorum allows for the possibility that plaque development still activates these vessels and that they are affected by secretion of pro-angiogenic molecules from the intimal site. It would be interesting to compare and identify the mitogenic factors expressed in different vascular beds.
Our results indicate that intraplaque biomarkers, rather than conventional risk factors, may reveal clinical and radiological differences between patients with carotid and MCA atherosclerosis. Plaques associated with MCA atherosclerosis may be more stable than those associated with carotid atherosclerosis .
Perhaps surprisingly, there were no significant differences between the numbers of CD105-positive vessels in the adventitia adjacent to the plaque area compared with non-plaque containing regions in carotid arteries. Since the number of CD105-positive vessels was increased significantly in all areas of the adventitia, it is possible that a more general activation of the vasa vasorum occurs and neointimal infiltrating endothelial cells are derived from a more widespread region. This was demonstrated recently by Coli et al , who showed that the number of CD31/CD34 positive vasa vasorum microvessels correlated with plaque echolucency, although not with the degree of stenosis. In contrast, the numbers of vasa vasorum CD105-positive vessels correlated strongly with areas of plaque remodelling suggesting a more focussed process of remodelling.
In the neointima we observed abnormal morphology in many of the neovessels even in some of the early stage grade II-III carotid lesions. Some of the vessels demonstrated an elongated appearance but remained patent whilst others were more irregular, multilobular and often collapsed and non-functional. These immature vessels, similar to those identified in growing tumours  are more prone to rupture and haemorrhage. Dunmore et al  showed that dilated, highly irregular multilobular vessels were found almost exclusively in carotid plaques from symptomatic patients and not in asymptomatic ones. The vessels generally lacked smooth muscle cells, were immature and also existed in regions rich in VEGF-containing macrophages. We also showed that the majority of CD105-positive vessels were thin walled and immature suggesting they were undergoing angiogenesis and/or maturation associated with cellular activation.
Endoglin is a component of the TGFβ receptor complex expressed mainly on the surface of endothelial cells. TGFβ has notable effects on cell proliferation, differentiation, adhesion, migration, and extracellular matrix production, as well as other activities . Endoglin binds to members of the TGFβ superfamily and modulates cellular responses to TGF-β1. The growth factor TGF-β1 is involved in the pathogenesis of atherosclerosis through its pleiotropic effects . Some reports show anti-atherogenic properties to TGF-β1 [32, 33], whereas others show that it has atherogenic properties [34, 35]. Piao et al found an increased expression of TGF-β1 in atherosclerotic lesions and an especially strong over-expression and higher expression rate in the advanced lesions, suggesting that TGF-β1 participates in the formation of early lesions and the progression to advanced lesions . CD105 may initiate its effects in atherosclerosis at an early stage through interaction with TGFβ, but we do not know if the expression of these proteins is causative or a result of the atherogenesis.
It could be useful in a therapeutic setting to differentiate perhaps with specific markers and imaging, patent functional and stable vessels from mal-formed, leaky and unstable ones liable to haemorrhage. Future treatment aimed at stabilization of immature vessels could help in the control of developing lesions.