Major gaps in human evidence for structure and function of the vasa vasora limit our understanding of the link with atherosclerosis.

Atherosclerosis is the major pathology causing death in the developed world and, although risk factor modification has improved outcomes over the last decade, there is no cure. The role of the vasa vasora (VV) in the pathogenesis of atherosclerotic plaque is unclear but must relate to the predictability of diseased sites in the arterial tree. VV are small vessels found on major arteries and veins which supply nutrients and oxygen to the vessel wall itself while removing waste. Numerous studies have been carried out to investigate the anatomy and function of the VV as well as their significance in vascular disease. There is convincing evidence that VV are related to atherosclerotic plaque progression and vessel thrombosis, however, their link to the pathology of plaque initiation remains an interesting but neglected topic. We aim to present the evidence on the anatomy and functional behaviour of VV as well as their relationship to the initiation of atherosclerosis. At the same time, we wish to highlight inconsistencies in, and limitations of, the evidence available.

Arterial smooth muscle dynamics in development and repair.

Arterial vasculature distributes blood from early embryonic development and provides a nutrient highway to maintain tissue viability. Atherosclerosis, peripheral artery diseases, stroke and aortic aneurysm represent the most frequent causes of death and are all directly related to abnormalities in the function of arteries. Vascular intervention techniques have been established for the treatment of all of these pathologies, yet arterial surgery can itself lead to biological changes in which uncontrolled arterial wall cell proliferation leads to restricted blood flow. In this review we describe the intricate cellular composition of arteries, demonstrating how a variety of distinct cell types in the vascular walls regulate the function of arteries. We provide an overview of the developmental origin of arteries and perivascular cells and focus on cellular dynamics in arterial repair. We summarize the current knowledge of the molecular signaling pathways that regulate vascular smooth muscle differentiation in the embryo and in arterial injury response. Our review aims to highlight the similarities as well as differences between cellular and molecular mechanisms that control arterial development and repair.

Glycolysis and oxidative phosphorylation are essential for purinergic receptor-mediated angiogenic responses in vasa vasorum endothelial cells.

Angiogenesis is an energy-demanding process; however, the role of cellular energy pathways and their regulation by extracellular stimuli, especially extracellular nucleotides, remain largely unexplored. Using metabolic inhibitors of glycolysis (2-deoxyglucose) and oxidative phosphorylation (OXPHOS) (oligomycin, rotenone, and FCCP), we demonstrate that glycolysis and OXPHOS are both essential for angiogenic responses of vasa vasorum endothelial cell (VVEC). Treatment with P2R agonists, ATP, and 2-methylthioadenosine diphosphate trisodium salt (MeSADP), but not P1 receptor agonist, adenosine, increased glycolytic activity in VVEC (measured by extracellular acidification rate and lactate production). Stimulation of glycolysis was accompanied by increased levels of phospho-phosphofructokinase B3, hexokinase (HK), and GLUT-1, but not lactate dehydrogenase. Moreover, extracellular ATP and MeSADP, and to a lesser extent adenosine, increased basal and maximal oxygen consumption rates in VVEC. These effects were potentiated when the cells were cultured in 20 mM galactose and 5 mM glucose compared with 25 mM glucose. Treatment with P2R agonists decreased phosphorylation of pyruvate dehydrogenase (PDH)-E1α and increased succinate dehydrogenase (SDH), cytochrome oxidase IV, and ß-subunit of F1F0 ATP synthase expression. In addition, P2R stimulation transiently elevated mitochondrial Ca2+ concentration, implying involvement of mitochondria in VVEC angiogenic activation. We also demonstrated a critical role of phosphatidylinositol 3-kinase and Akt pathways in lactate production, PDH-E1α phosphorylation, and the expression of HK, SDH, and GLUT-1 in ATP-stimulated VVEC. Together, our findings suggest that purinergic and metabolic regulation of VVEC energy pathways is essential for VV angiogenesis and may contribute to pathologic vascular remodeling in pulmonary hypertension.

The adventitia: essential regulator of vascular wall structure and function.

The vascular adventitia acts as a biological processing center for the retrieval, integration, storage, and release of key regulators of vessel wall function. It is the most complex compartment of the vessel wall and is composed of a variety of cells, including fibroblasts, immunomodulatory cells (dendritic cells and macrophages), progenitor cells, vasa vasorum endothelial cells and pericytes, and adrenergic nerves. In response to vascular stress or injury, resident adventitial cells are often the first to be activated and reprogrammed to influence the tone and structure of the vessel wall; to initiate and perpetuate chronic vascular inflammation; and to stimulate expansion of the vasa vasorum, which can act as a conduit for continued inflammatory and progenitor cell delivery to the vessel wall. This review presents the current evidence demonstrating that the adventitia acts as a key regulator of vascular wall function and structure from the outside in.

Ascending aorta of hooded seals with particular emphasis on its vasa vasorum.

The pressure-volume relationship in the ascending aorta ("windkessel") of the hooded seal was determined and the morphology of its vasa vasorum described in some detail. We found that the ascending aorta has a high compliance and can easily accommodate the entire stroke volume when the peripheral vascular resistance becomes much increased and maintain perfusion pressure during the much extended diastole and thereby reduce cardiac stroke work during diving. We also found that the 3- to 5-mm thick wall of the ascending aorta had a very elaborate vasa vasorum interna with a hitherto undescribed vascular structure that penetrates the entire vascular wall. If similar structures with similar importance for the nutrition of the wall of the vessel are found in humans, important implications for the understanding of pathological conditions, such as aneurisms, may be indicated.

Nerve growth factor stimulates regeneration of perivascular nerve, and induces the maturation of microvessels around the injured artery.

An immature vasa vasorum in the adventitia of arteries has been implicated in induction of the formation of unstable atherosclerotic plaques. Normalization/maturation of the vasa vasorum may be an attractive therapeutic approach for arteriosclerotic diseases. Nerve growth factor (NGF) is a pleotropic molecule with angiogenic activity in addition to neural growth effects. However, whether NGF affects the formation of microvessels in addition to innervation during pathological angiogenesis is unclear. In the present study, we show a new role for NGF in neovessels around injured arterial walls using a novel in vivo angiogenesis assay. The vasa vasorum around arterial walls was induced to grow using wire-mediated mouse femoral arterial injury. When collagen-coated tube (CCT) was placed beside the injured artery for 7-14 days, microvessels grew two-dimensionally in a thin layer on the CCT (CCT-membrane) in accordance with the development of the vasa vasorum. The perivascular nerve was found at not only arterioles but also capillaries in the CCT-membrane. Biodegradable hydrogels containing VEGF and NGF were applied around the injured artery/CCT. VEGF significantly increased the total length and instability of microvessels within the CCT-membrane. In contrast, NGF induced regeneration of the peripheral nerve around the microvessels and induced the maturation and stabilization of microvessels. In an ex vivo nerve-free angiogenesis assay, although NGF potentially stimulated vascular sprouting from aorta tissues, no effects of NGF on vascular maturation were observed. These data demonstrated that NGF had potent angiogenic effects on the microvessels around the injured artery, and especially induced the maturation/stabilization of microvessels in accordance with the regeneration of perivascular nerves.

P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells.

Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca(2+) signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of [Ca(2+)](i) via Ca(2+) influx through plasma membrane channels as well as Ca(2+) mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca(2+) responses in both cytosolic and nuclear compartments. An increase in [Ca(2+)](i) was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPßS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αßMeATP, and ßγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca(2+) responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion.

Endothelial cell-selective adhesion molecule modulates atherosclerosis through plaque angiogenesis and monocyte-endothelial interaction.

Endothelial cell-selective adhesion molecule (ESAM) is a new member of the immunoglobulin superfamily, which is expressed in vascular endothelial cells. Previous studies have demonstrated that ESAM regulates angiogenesis, endothelial permeability, and leukocyte transmigration. However, little is known concerning the role of ESAM in atherosclerosis. In this study, we assessed the effects of ESAM inactivation on atherosclerosis in mice. ESAM-/- mice were bred with apoE-/- mice to generate double knockout mice, and the aortic lesion size of apoE-/- and ESAM-/-apoE-/- mice was compared histologically. Although plasma cholesterol levels were higher in ESAM-/-apoE-/- mice, the lesion size was markedly smaller than in apoE-/- mice. ESAM-/-apoE-/- mice exhibited a decrease in the number of vasa vasorum and macrophages in the vessel wall. In vitro adhesion assays showed that THP-1 cells, which did not express ESAM, bound to the ESAM-coated culture plates, suggesting that ESAM may interact with heterophilic ligand(s) on monocytes. Moreover, downregulation of ESAM by siRNA in the endothelial monolayer diminished transendothelial migration of THP-1 cells. In conclusion, ESAM inactivation can reduce susceptibility to atherosclerosis by inhibiting plaque neovascularization and macrophage infiltration into the atheroma.

Molecular imaging of inflammation and intraplaque vasa vasorum: a step forward to identification of vulnerable plaques?

Current developments in cardiovascular biology and imaging enable the noninvasive molecular evaluation of atherosclerotic vascular disease. Intraplaque neovascularization sprouting from the adventitial vasa vasorum has been identified as an independent predictor of intraplaque hemorrhage and plaque rupture. These intraplaque vasa vasorum result from angiogenesis, most likely under influence of hypoxic and inflammatory stimuli. Several molecular imaging techniques are currently available. Most experience has been obtained with molecular imaging using positron emission tomography and single photon emission computed tomography. Recently, the development of targeted contrast agents has allowed molecular imaging with magnetic resonance imaging, ultrasound and computed tomography. The present review discusses the use of these molecular imaging techniques to identify inflammation and intraplaque vasa vasorum to identify vulnerable atherosclerotic plaques at risk of rupture and thrombosis. The available literature on molecular imaging techniques and molecular targets associated with inflammation and angiogenesis is discussed, and the clinical applications of molecular cardiovascular imaging and the use of molecular techniques for local drug delivery are addressed.

The absence of lymphatics in normal and atherosclerotic coronary arteries in man: a morphologic study.

It has been suggested by various investigators that the impairment of lymphatic drainage from the coronary arteries may play a role in predisposition to coronary atherosclerosis, the pathogenesis of which is certainly multifactorial. In our study, no lymphatic vessels were found in the walls of the coronary arteries (adventitia, media and intima) in 51 human hearts from patients ranging in ages from 3 months to 83 years with normal coronary arteries, coronary atherosclerosis, and cardiomyopathy. Visualized lymphatics were located solely in the periadventitial area, and these lymphatics were more irregular in hearts from older persons. With injection, histology, and electronmicroscopy methods we could not detect penetration of lymphatics into the wall of coronary trunks in normal as well atherosclerotic arteries. In all coronary arteries studied, and particularly in the atherosclerotic lesions, blood vasa vasorum could be visualized. In the atherosclerotic areas, vasa vasorum (angiogenesis) could be seen penetrating into the media and intima. Many of the thin-walled vasa vasorum could easily be mistaken for lymphatics. The absence of lymphatics draining the epicardial coronary arteries may be a predisposing factor to coronary atherosclerosis.

Medial necrosis and acute alterations in aortic distensibility following removal of the vasa vasorum of canine ascending aorta.

OBJECTIVE: It is known that the outer layers of the thoracic aorta receive substantial blood flow through vasa vasorum. This study was undertaken to test the hypothesis that removal of vasa vasorum flow will alter the elastic properties of the ascending aorta. METHODS: Distensibility of the ascending aorta was determined before and 30 min after careful removal of the periaortic fat network which contains the vasa vasorum in 10 acutely instrumented dogs (experimental group) and the results were compared with those obtained from six weight matched sham operated control dogs. Aortic distensibility was measured using the formula: distensibility = 2 x pulsatile changes in aortic diameter divided by (diastolic aortic diameter x pulse pressure). Aortic pressures were measured directly from the ascending aorta by a pressure gauge. Aortic diameters were simultaneously determined by an elastic air filled ring connected to a transducer. The efficacy of the technique for the interruption of vasa vasorum blood supply to the aortic wall was proved in six additional animals by histology of transverse blocks of aortic wall from the area of interest. Histology was performed before vasa vasorum removal in two animals, 30 min after vasa vasorum removal in another two, and 15 d after vasa vasorum removal in the remainder. RESULTS: At baseline, there was no difference in the measured variables between the two groups. Aortic distensibility decreased significantly in the experimental group after vasa vasorum removal by 0.90(SEM 0.17) 10(-6).cm2.dyn-1 (p < 0.001), while it remained unchanged in the control group during the experiment. Complete removal of vasa vasorum of the ascending aorta was found in experimental group animals which were killed 30 min after operation, while ischaemic medial necrosis was observed in those killed 15 d after operation. CONCLUSIONS: Vasa vasorum removal led to an acute decrease in the distensibility of the ascending aorta. Lack of blood supply to the outer part of the aortic wall is most likely to have accounted for these findings.

Quantification of Adventitial Vasa Vasorum Vascularization in Double-injury Restenotic Arteries.

BACKGROUND: Accumulating evidence indicates a potential role of adventitial vasa vasorum (VV) dysfunction in the pathophysiology of restenosis. However, characterization of VV vascularization in restenotic arteries with primary lesions is still missing. In this study, we quantitatively evaluated the response of adventitial VV to vascular injury resulting from balloon angioplasty in diseased arteries. METHODS: Primary atherosclerotic-like lesions were induced by the placement of an absorbable thread surrounding the carotid artery of New Zealand rabbits. Four weeks following double-injury induced that was induced by secondary balloon dilation, three-dimensional patterns of adventitial VV were reconstructed; the number, density, and endothelial surface of VV were quantified using micro-computed tomography. Histology and immunohistochemistry were performed in order to examine the development of intimal hyperplasia. RESULTS: Results from our study suggest that double injured arteries have a greater number of VV, increased luminal surface, and an elevation in the intima/media ratio (I/M), along with an accumulation of macrophages and smooth muscle cells in the intima, as compared to sham or single injury arteries. I/M and the number of VV were positively correlated (R2 = 0.82, P < 0.001). CONCLUSIONS: Extensive adventitial VV neovascularization occurs in injured arteries after balloon angioplasty, which is associated with intimal hyperplasia. Quantitative assessment of adventitial VV response may provide insight into the basic biological process of postangioplasty restenosis.

The adventitia and atherogenesis: removal initiates intimal proliferation in the rabbit which regresses on generation of a 'neoadventitia'.

Removal of the carotid artery adventitia from rabbits induced the formation of an intimal hyperplastic lesion. In rabbits fed a normal diet, the lesion (measured as the intimal:medial ratio) was maximal by day 14 (0.456 +/- 0.079, n = 5, P < 0.01) and thereafter, regressed towards control dimensions (0.037 +/- 0.003, n = 14) by day 28 (0.080 +/- 0.025, n = 7, P = 0.14). In rabbits fed a high cholesterol diet, the lesion was again maximal by day 14 (0.376 +/- 0.056, n = 8, P < 0.01). Although some regression was seen, the lesion persisted to day 42 (0.272 +/- 0.052, n = 8, P < 0.01). Electron microscopy and immunocytochemistry showed two types of lesion, (a) smooth muscle cell predominant on normal diet and, (b) macrophage predominant on high cholesterol diet. Smooth muscle cell predominant lesions underwent almost complete regression, whereas macrophage predominant lesions persisted. We propose that lesion formation may be initiated following the development of arterial wall hypoxia, secondary to excision of the adventitial vasa vasorum. Furthermore, we have devised a novel method to restore a highly vascular 'neoadventitia' to an artery whose adventitia has previously been removed, using loosely placed PVC tubing. We suggest this 'neoadventitia' was able to inhibit the formation of an intimal hyperplastic lesion and to promote regression of an already established lesion by restoring arterial wall oxygenation.

Endothelin alters the reactivity of vasa vasorum: mechanisms and implications for conduit vessel physiology and pathophysiology.

1 The walls of certain large blood vessels are nourished by the vasa vasorum, a network of microvessels that penetrate the adventitia and media of the vessel wall. The purpose of this study was to characterize endothelin-1 (ET-1)-mediated contraction of vasa and to investigate whether threshold concentrations of ET-1 alters the sensitivity to constrictors. Arterial vasa were dissected from the walls of porcine thoracic aorta and mounted in a tension myograph. 2 ET-1 and ETB-selective agonist, sarafotoxin 6c (S6c), produced concentration-dependent contraction. ETA receptor antagonist, BQ123 (10 microM), caused a biphasic rightward shift of ET-1 response curves. ETB receptor antagonist, BQ788 (1 microM), produced a rightward shift of response curves to ET-1 and S6c of 5- and 80 fold respectively. 3 ET-1 responses were abolished in Ca2+-free PSS but unaffected by selective depletion of intracellular Ca2+ stores. Nifedipine (10 microM), an L-type Ca2+ channel blocker, attenuated ET-1 responses by 44%. Inhibition of receptor-operated Ca2+ channels or non-selective cation entry using SKF 96365 (30 microM) and Ni2+ (1 mM) respectively, attenuated ET-1 contractions by 60%. 4 ET-1 (1-3 nM) enhanced responses to noradrenaline (NA) (4 fold) but not to thromboxane A2-mimetic, whilst K+ (10-20 mM) sensitized vasa to both types of constrictor. 5 Therefore, ET-1-induced contraction of isolated vasa is mediated by ETA and ETB receptors and involves Ca2+ influx through L-type and non-L-type Ca2+ channels. Furthermore elevation of basal tone of vasa vasorum alters the profile of contractile reactivity. These results suggest that ET-1 may be an important regulator of vasa vasorum reactivity.

Human vascular to cardiac tissue selectivity of L- and T-type calcium channel antagonists.

1. Voltage-operated calcium channel (VOCC) antagonists are effective antihypertensive and antianginal agents but they also depress myocardial contractility. 2. We compared four L-type calcium channel antagonists, felodipine, nifedipine, amlodipine and verapamil and a relatively T-type selective calcium channel antagonist, mibefradil, on human and rat isolated tissue assays to determine their functional vascular to cardiac tissue selectivity (V/C) ratio. 3. The V/C ratio was calculated as the ratio of the IC50 value of the antagonist that reduced (by 50%) submaximally contracted (K+ 62 mM) human small arteries from the aortic vasa vasorum (vascular, V) mounted in a myograph and the IC50 value of the antagonist that reduced (-)-isoprenaline (6 nM) submaximally stimulated human right atrial trabeculae muscle (cardiac, C) mounted in organ chambers. 4. The average pIC50 values (-log IC50 M) for the human vascular preparations were felodipine 8.30, nifedipine 7.78, amlodipine 6.64, verapamil 6.26 and mibefradil 6.22. The average pIC50 values for the cardiac muscle were felodipine 7.21, nifedipine 6.95, verapamil 6.91, amlodipine 5.94, and mibefradil 4.61. 5. The V/C ratio calculated as antilog [pIC50V-pIC50C] is thus mibefradil 41, felodipine 12, nifedipine 7, amlodipine 5 and verapamil 0.2. 6. In rat small mesenteric arteries the pIC50 values for the five drugs were similar to the values for human vasa vasorum arteries contracted by K+ 62 mM. However for methoxamine (10 microM) contraction in the rat arteries the pIC50 values were lower for felodipine 7.24 and nifedipine 6.23, but similar for verapamil 6.13, amlodipine 6.28 and mibefradil 5.91. 7. In conclusion. in the human tissue assays, the putative T-channel antagonist mibefradil shows the highest vascular to cardiac selectivity ratio; some 3 fold higher than the dihydropyridine, felodipine, and some 200 fold more vascular selective than the phenylalkylamine, verapamil. This favourable vascular to cardiac selectivity for mibefradil, from a new chemical class of VOCC antagonist, may be explained by its putative T-channel selectivity.

Relationship between vasa vasorum and blood flow to vein bypass endothelial morphology.

In order to define the respective roles of the vasa vasorum blood supply and intraluminal arterial blood flow in maintaining the endothelial integrity of in situ vein bypasses, we have carried out two separate but interrelated experiments in a canine model. In vivo studies on eight dogs demonstrated that even in the absence of intraluminal blood flow the vasa vasorum maintained endothelial integrity and also showed that the endothelium was very sensitive to the loss of the vasa vasorum blood supply. In a second group of experiments on 14 in situ vein bypasses we studied the effect of division of the vasa vasorum immediately after arterialization of the bypass. This experiment showed that arterialization of the vein maintained endothelial integrity despite division of the vasa vasorum.

Why do radial artery grafts for aortocoronary bypass fail? A reappraisal.

The reasons for the high failure rate of radial artery grafts for aortocoronary bypass (ACB) are appraised. Each of 11 dogs received four types of grafts: femoral arteries were grafted to carotid arteries, and jugular veins were grafted to femoral arteries bilaterally. One of each pair of grafts was wrapped in a plastic sheet in order to interfere with the regeneration of vasa vasorum. The grafts were examined 2 months later. The results suggest that although operative trauma and intraluminal pressure change play important roles, the thick-walled artery appears to depend more on vasa vasorum for its integrity than does the thin-walled vein. A free radical artery graft, with vasa vasorum disrupted at both ends, cannot regenerate readily from the adjacent tissue, as is the case at the ACB position, and is therefore more vulnerable to subintimal hyperplasia and occlusion than either the vein graft or the internal mammary artery graft; both of these grafts are thinner, and the internal mammary artery graft remains undisrupted at one end.

Functional anatomy and hemodynamic characteristics of vasa vasorum in the walls of porcine coronary arteries.

In this study vasa vasorum in the walls of porcine coronary arteries were examined, using three-dimensional (3D) micro-CT scanning techniques. These techniques leave the 3D structure of the vasa vasorum tree intact and thus provide a much more direct view of this structure than is possible from conventional histological sections. The study demonstrates-for the first time, we believe-both the different types and the fine architecture of these vasa vasorum. Furthermore, with the use of automated tree analysis software, it was possible to obtain quantitative geometrical data on the 3D structure of vasa vasorum trees that have not previously been available. The results indicate that despite the restrictive topology of the space in which they are present, the branching architecture of the vasa vasorum trees, which we surveyed, is surprisingly similar to that of vasculature in general. The volume of vessel wall tissue perfused or drained by a vasa vasorum tree was found to correlate well with the cross-sectional area of the root segment of the vasa vasorum tree, and the luminal surface area corresponding to this volume was found to be comparable with the surface area of an early atherosclerotic lesion. This is consistent with earlier findings that the ligation or removal of vasa vasorum leads to atherogenesis.