Neovessel Density Research Articles

Abstract 18348: Total Mast Cell Numbers in Human Carotid Atherosclerotic Plaques and Tryptase Plasma Levels are Predictive for Future Cardiovascular Events

Mast cells (MCs) are inflammatory cells known to contribute to the pathogenesis of atherosclerotic disease. Experimental studies have revealed that MCs might be involved in the progression of atherosclerosis by inducing intraplaque neovascularization. OBJECTIVE: This study was designed to assess total MC numbers and relate the score to neovascularization in human atherosclerotic plaques. In addition we related the presence of plaque MCs with the occurrence of adverse cardiovascular events during follow-up. METHODS AND RESULTS: In the current study atherosclerotic plaques of 270 patients suffering from carotid artery stenosis, were stained for the presence of MCs (MC tryptase), macrophages (CD68), neutrophils (CD66) and microvessels (CD34). Furthermore, during a follow-up of 3 years, cardiovascular related endpoints were assessed in all patients. Counting of mast cells revealed a surprising high number of MCs that were present in plaques (165 ± 10 MC per plaque). We equally divided the patient cohort into a group with low (n=126) and high (n=127) total MC numbers. High MC numbers were associated with high microvessel density 6,0 [4,3-9,0] versus 10,7 [6,5-14,2] vessels per hotspot (P<0.001). Patients with high plaque MC numbers showed significantly more cardiovascular events during follow up (35 [28%] versus 54 [43%] events (P<0.05)). Macrophage and neutrophil presence did not show this association with the occurrence of cardiovascular events. In addition, in 80 patients (1:1 event versus no event) MC tryptase and chymase have been measured in plasma. Patients with high plasma tryptase showed significantly more cardiovascular events (11 [28%] versus 24 [60%] events (P<0.01)). Plasma chymase levels did not show predictive value in our cohort. CONCLUSIONS: MCs are highly prevalent in carotid atherosclerotic lesions and associated with plaque neovessel density. Total intraplaque MC numbers and MC plasma tryptase levels are predictive for future cardiovascular events. These data support the view that MCs could play an important role in progression of atherosclerotic disease.

The Influence of Nerve Growth Factor (NGF) on Distribution of Perivascular Nerves in Tumor Neovasculatures of Mouse Corneal

Angiogenesis, or new blood vessel formation, is critical for the growth and spread of tumors. The vascular tone and tissue blood flow are maintained and regulated by perivascular nerves. However, many studies have reported that tumor neovascular vessels have no innervation of perivascular nerves. We have shown that nerve growth factor (NGF) facilitated perivascular innervation and suppressed the tumor growth. From these results, we hypothesized that the neuronal regulation of blood flow toward tumors via perivascular nerves may lead suppression of the tumor growth. Therefore, the aim of this study is to investigate effect of NGF on distribution of perivascular nerves and neovessel form in tumor tissues, which were generated by mouse corneal micropocket method. A gel, which contained DU145 prostate carcinoma cells, was implanted into the mouse corneal. NGF or saline was subcutaneously administered using an osmotic mini-pump. After 1 week, the distribution of perivascular nerves in mouse corneal were immunohistochemically studied. Also, the density of neovessels (immunocytochemically stained CD31) and smooth muscles (α-smooth muscle actin; SMA) in tumor tissues was quantified by the computer-assisted image processing. Four days after implantation of tumor cells in mouse corneal, many neovessels generated from corneal limbal vessels were observed in tumor tissues. Treatment of mouse with NGF resulted in innervation of perivascular nerves around tumor neovessels, but not observed in saline-treated group. NGF treatment increased SMA-, but not CD31-, immunopositive cells. These results suggest that NGF may facilitate innervations of perivascular nerve to regulate the blood flow in tumor neovessels.

Increased vascularization of shoulder regions of carotid atherosclerotic plaques from patients with diabetes

Increased vascularization is considered an important contributing factor for plaque vulnerability. Microvascular proliferative disease in patients with diabetes results in renal damage and visual loss. We assessed the hypothesis that vascularization in carotid atherosclerotic tissue is increased in diabetic patients, especially in the critical shoulder regions of the plaque. Carotid endarterectomy specimens, clinical data, and blood samples were collected from patients with symptomatic carotid artery stenosis (median 85 days after clinical event) and pharmacologic treatment for diabetes (n = 26) or no diabetes (n = 85). Plaques were fixed in formalin and transverse tissue sections prepared. Histopathology and immunohistochemistry were performed for detection of endothelial cells (anti-CD34), macrophages (anti-CD68), vascular endothelial growth factor (VEGF), and its receptor (VEGFR-2). Neovascularization was assessed as CD34(+) neovessel density in the entire section area and by the presence or absence of CD34(+) vessels in the shoulder and cap regions of the plaques. The patient groups did not differ significantly in neovascularization in the entire transverse sections (2.0 vs 2.1 vessels/mm(2); P = .61) or in the fibrous cap (52% of the patients in both groups; P = .95). Neovascularization of the plaque shoulder regions was observed in 52% of the diabetic patients and in 26% of the nondiabetic patients (P = .028). VEGF-stained areas were similar in the two patient groups (0.4% and 0.2% of shoulder area; P = .61). Patients with diabetes had more VEGFR-2 (1.0% vs 0.2% of shoulder area; P < .016) and less CD68 staining (0.4% vs 3.6% of shoulder area; P < .008). Time from clinical event to surgery was positively associated with neovascularization of the plaque shoulder regions (≤90 days, 18% of patients; >90 days, 50% of patients; P = .002), independently of diabetes status. Diabetes was associated with increased vascularization of the shoulder regions in patients with symptomatic carotid atherosclerotic plaques. This was accompanied by increased expression of VEGFR-2. The increased vascularization of the plaque shoulder regions may help explain why patients with diabetes are at increased risk of atherosclerotic complications.

Intramural coronary lipid injection induces atheromatous lesions expressing proinflammatory chemokines: implications for the development of a porcine model of atherosclerosis

Intramural delivery of lipids into the coronaries of pigs fed high-cholesterol diet results in the formation of localized atherosclerotic-like lesions within 12 weeks. These lesions are located in positively remodeled vessels and are associated to the development of abundant adventitial vasa vasorum and mononuclear cell infiltrate. In this study, we aimed to analyze the degree of expression of various inflammatory chemokines within the developed lesions compared with control segments injected with saline. Balloon injury was performed in 15 coronary arteries of pigs fed high-cholesterol diet for 12 weeks. Two weeks after procedure, 60 coronary segments were randomized to either intramural injections of complex lipids (n=30) or normal saline (n=30). Neovessel density in the lesions was analyzed by lectin stain. Segments were processed for RNA expression of inflammatory chemokines such as monocyte chemoattractant protein-1 and vascular endothelial growth factor. At 12 weeks, the percentage area of stenosis seen in histological sections was modest in both groups (lipids: 17.3±15 vs. saline: 32.4±22.8, P=.017). The lipid group showed higher vasa vasorum (VV) quantity (saline: 18.2±14.9 VV/section vs. lipids: 30.6±21.6 VV/section, P<.05) and vasa vasorum density (saline: 7.3±4.6 VV/mm(2) vs. lipids: 16.5±9 VV/mm(2), P<.001). In addition, monocyte chemoattractant protein-1 expression was higher in the lipid group (1.5±1.12) compared with saline control group (0.83±0.34, P<.01). Vascular endothelial growth factor expression was also higher in the lipid group (1.36±0.9) compared with saline group (0.87±0.33, P<.05). The intramural injection of complex lipids into the coronary arteries of pigs maintained in a high-cholesterol diet results in focal lesions located in positively remodeled vessels that have a high neovessel count and express proinflammatory chemokines.

Vascular Gene Transfer of SDF-1 Promotes Endothelial Progenitor Cell Engraftment and Enhances Angiogenesis in Ischemic Muscle

Gene therapy approaches to enhance endothelial progenitor cell (EPC) homing may augment cell engraftment to ischemic tissue and lead to a greater therapeutic response. Therefore, we assessed the effects of ultrasound-mediated (UM) transfection of the chemokine stromal cell-derived factor-1 (SDF-1) on homing and engraftment of intravenously administered EPCs and the subsequent angiogenic response in chronically ischemic skeletal muscle. Bone marrow-derived EPCs were isolated from donor Fisher 344 rats, cultured and labeled in preparation for injection into recipient animals via a jugular vein. Using a model of chronic hindlimb ischemia in rats, we demonstrated that UM destruction of intravenous carrier microbubbles loaded with SDF-1 plasmid DNA resulted in targeted transfection of the vascular endothelium within ischemic muscle and greater local engraftment of EPCs. The combination of SDF-1gene therapy and EPCs lead to the greatest increase in tissue perfusion and microvascular density within ischemic muscle, compared to no treatment or either monotherapy alone. Our results demonstrate that UM transfection of SDF-1 improves EPC targeting to chronically ischemic tissue, enhancing vascular engraftment and leading to a more robust neovascularization response.

Abstract 1517: Retinoblastoma tumor development: Role of tumor-associated macrophages and their sub-type in LHBETATAG retinal tumor progression

Abstract Purpose: The purpose of the current study is to establish the distribution of tumor associated macrophages (TAMs) during tumor development in a transgenic retinoblastoma mouse model, study the contribution of bone-marrow derived macrophages in these tumors, and assess the supportive role of TAMs in retinoblastoma tumor growth. Methods: Macrophage infiltration in transgenic retinoblastoma tumors was assessed by immunohistochemistry at different time points in tumorigenesis. To establish the origin of TAMs in transgenic retinoblastoma tumors, 107 bone marrow cells from green fluorescent protein (GFP) positive 16-week-old mice were transplanted into age-matched, irradiated LHBETATAG mice via tail vein injections. Macrophage depletion was performed by subconjuctival (SC) delivery of liposomal clodronate. Results: TAMs’ density increased from 4- to 12-weeks of age in mice with small to medium-sized tumors (p=0.037), and remained stable in the later stages of the disease (i.e., 16-week-old with large tumors; p=0.20). 38% of TAMs (2.5 ± 3.2 cells per 400X hpf) in 16-week old mice were GFP-positive, bone marrow-derived macrophages. Total TAM depletion was correlated with a significant decrease in the expression levels of MMP-9 (p=0.014) and mature vessels (p&amp;lt;0.001), and a nonsignificant decrease in the density of neovessels (p=0.94). The density of M2-polarized TAMs did not change significantly following TAM depletion (p=0.68). Following M1-polarized TAM depletion, the tumor burden increased (p=0.056). Conclusions: The current study expands our understanding of the complex role that macrophages play in retinoblastoma. Macrophage modulation in the tumor microenvironment is a critical factor in retinoblastoma tumor development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1517. doi:10.1158/1538-7445.AM2011-1517

Increased macrophage infiltration and neovascularization in congenital bicuspid aortic valve stenosis

Patients with congenital bicuspid aortic valves have aortic valve stenosis at a relatively young age compared with patients with tricuspid aortic valves. We hypothesize that aortic valve stenosis evolves from a more aggressive inflammatory process, with increased macrophage/T-cell and neovessel content in congenital bicuspid aortic valveswhen compared with that seen in tricuspid valves. Fifty-one severely stenotic aortic valves were obtained at the time of aortic valve replacement. A total of 17 bicuspid and 34 tricuspid aortic valves were evaluated. Macrophage/T-cell infiltration (CD68 plus CD3) and neovessel density (CD34) were evaluated with immunohistochemical staining. Leaflet calcification and ossification were also quantified. Real-time polymerase chain reaction was used to assess expression of chondromodulin 1 and vascular endothelial growth factor. The density of macrophages/T cells was greater in congenital bicuspid aortic valves than in tricuspid valves (51 ± 31 vs 23 ± 13 cells/mm(2), P = .002). Neovascularization was more frequently noted in congenital bicuspid aortic valves when compared with tricuspid valves (31 ± 10 vs 21 ± 9 vessels/mm(2), P = .0005), and calcification was more severe (P = .03). Expression of chondromodulin 1 demonstrated a 6-fold downregulation (P = .0003) and expression of vascular endothelial growth factor demonstrated a 2-fold increase (P = .02) in congenital bicuspid aortic valves compared with that seen in tricuspid valves. Multivariable analyses demonstrated significant associations between bicuspid aortic valve anatomy and increased inflammatory cell infiltration (β = 25.8, P = .0007) and neovascularization (β = 9.4, P = .001), despite adjusting for measured covariates. The pathogenesis of aortic valve stenosis in bicuspid aortic valves is associated with a more aggressive inflammatory process with increased macrophage infiltration and neovascularization when compared with that seen in tricuspid valves.

Angiotensin Type 1 Receptor Blocker Reduces Intimal Neovascularization and Plaque Growth in Apolipoprotein E–Deficient Mice

The interactions between the renin-angiotensin system and neovascularization in atherosclerotic plaque development are unclear. We investigated the effects of angiotensin II type 1 receptor antagonism in the pathogenesis of atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice with a special focus on plaque neovascularization. ApoE(-/-) mice fed a high-fat diet were randomly assigned to 1 of 2 groups and administered vehicle or olmesartan for 12 weeks. Quantification of plaque areas at the aortic root and in the thoracic and abdominal aorta revealed that, in all 3 of the regions, olmesartan reduced intimal neovessel density and the mRNA levels of toll-like receptor (TLR) 2 and TLR4. Olmesartan increased the levels of collagen and elastin, reduced the level of macrophages in the aortic root, and reduced the mRNA and the activity of matrix metalloproteinase (MMP) 2 in aortic roots and thoracic aortas. Aortic ring assay revealed that olmesartan-treated ApoE(-/-) mice had a markedly lower angiogenic response than that of untreated ApoE(-/-) mice. Bone marrow-derived endothelial progenitor cell-like c-Kit(+) cells from olmesartan-treated ApoE(-/-) mice showed marked impairment of cellular functions and lower expression of TLR2/TLR4 and MMP-2 compared with those of untreated controls. MMP-2 deficiency reduced intimal neovessel density and atherosclerotic lesion formation. Olmesartan and small-interfering RNA targeting TLR2 reduced the levels of TLR2, and MMP-2 mRNA induced angiotensin II in cultured endothelial cells. Angiotensin II type 1 receptor antagonism appears to inhibit intimal neovascularization in ApoE(-/-) mice, partly by reducing TLR2/TLR4-mediated inflammatory action and MMP activation, thus decreasing atherosclerotic plaque growth and increasing plaque instability.

Application of avastin in vitrectomy for proliferative diabetic retinopathy and its mechanism

Background Researches demonstrated that avastin-assisted vitrectomy for serious proliferative diabetic retinopathy can decrease intra-operation complication and bring down difficulty of surgery.It is speculated that this is associated with suppression of avastin on neovascularization.However,the evidence of its pathology is lack.Objective The aim of this study was to evaluate the application and mechanism of avastin in vitrectomy for severe proliferative diabetic retinopathy. Methods Twenty-four eyes from consecutive 24 patients with vitrectomy for severe proliferative diabetic retinopathy were enrolled in this study.Fourteen eyes received all intravitreal injection of 0.06 ml avastin(1.5 mg)14 days prior to vitrectomy。And the other 10 eyes underwent only vitrectomy without avastin injection as controls,Preretinal membranes were collected during vitrectomy for histopathologic examination by hemotoxylin and eosin staining.The differences in the density of the neovessels and micro-neovessels between the two groups were observed by detecting the expression of CD34 in vesse]endothelial cells using immunohistochemistry.Written informed consent was obtained from each patient before surgery. Results No statistically significant differences were found in the demography and eye manifestations between only vitreetomy group and avastin+vitrectomy group(P＞0.05).The neovessels with grade three was in 10 eyes in only vitrectomy group and 1 eye in avastin+vitrectomy group(P＜0.01).More capillary-like neovascularization with single vascular endothelial cells,obvious hemorrhage and inflammatory cells infiltration were observed on preretinal membranes in vitrectomy group.However,there were less hemorrhage,ceUular components and few capillary-like neovascularization but more hyaline degeneration of fibrous tissue were observed in avastin+vitrectomy group under the light microscope.Immunochemistry revealed that CD34 was positively expressed in vascular endothelial cells on preretinal membrane in both two groups.The neovessels density and miero-neovessels density were 15.40±7.42/field and 1.88±1.70/field in avastin+vitrectomy group and those in vitreetomy group were 18.00±3.80/field and 0.45±0.56/field respectively,showing significant differences between these two groups(neovessels density:Z=-4.102,P＜0.01;micro-neovessels density:Z=-4.137,P＜0.01).Conclusion As an adjunct drug during the vitrectomy for proliferative diabetic retinopathy, avastin can improve the successful rate of surgery by inhibiting the neovascular formation and alleviating retinal edema. Key words: Diabetic retinopathy; Avastin; Preretinal membrane; Neovascularization

Novel retinoblastoma treatment avoids chemotherapy: the effect of optimally timed combination therapy with angiogenic and glycolytic inhibitors on LHBETATAG retinoblastoma tumors

PurposeThe purpose of this study was to evaluate the effect of optimally timed combination treatment with angiogenic and glycolytic inhibitors on tumor burden, hypoxia, and angiogenesis in advanced retinoblastoma tumors.MethodsLHBETATAG mice (n =30) were evaluated. Mice were divided into 5 groups (n =6) and received injections at 16 weeks of age (advanced tumors) with a) saline, b) anecortave acetate (AA), c) 2-deoxyglucose (2-DG), d) AA +2-DG (1 day post-AA treatment), or e) AA +2-DG (1 week post-AA treatment). Eyes were enucleated at 21 weeks and tumor sections were analyzed for hypoxia, angiogenesis, and tumor burden.ResultsEyes treated with 2-DG 1 day post-AA injection showed a 23% (P =0.03) reduction in tumor burden compared with 2-DG alone and a 61% (P < 0.001) reduction compared with saline-treated eyes. Eyes treated with 2-DG 1 week post-AA injection showed no significant decrease in tumor burden compared with 2-DG alone (P = 0.21) and a 56% (P < 0.001) decrease in comparison with saline-treated eyes. 2-DG significantly reduced the total density of new blood vessels in tumors by 44% compared to saline controls (P < 0.001), but did not affect the density of mature vasculature.ConclusionsCombination therapy with angiogenic and glycolytic inhibitors significantly enhanced tumor control. Synergistic effects were shown to be dependent on the temporal course of treatment, emphasizing optimal timing. 2-DG was shown to reduce the density of neovessels, demonstrating an antiangiogenic effect in vivo. As a result, angiogenic and glycolytic inhibitors may have significant potential as alternative therapies for treating children with retinoblastoma.

Contrast-Enhanced Ultrasound Imaging Detects Intraplaque Neovascularization in an Experimental Model of Atherosclerosis

The aims of this study were to investigate the feasibility of contrast-enhanced ultrasound (CEU) imaging for in vivo visualization of intraplaque neovascularization and to correlate the in vivo observations with histological assessment of neovessel density and plaque composition in an experimental animal model of advanced atherosclerosis. Recent evidence has linked plaque angiogenesis with enhanced atherosclerotic plaque progression and vulnerability. Increased neovascularization has been detected in ruptured human lesions and is associated with clinical manifestations of plaque rupture. Advanced aortic atherosclerosis was induced in New Zealand white rabbits (n = 21; high cholesterol-rich diet/double-balloon aortic denudation). Animals underwent standard and CEU imaging at the end of the atherosclerosis induction period. Six age-matched animals served as control subjects. Within 24 h, animals were euthanized and aortas processed for histopathological evaluation of plaque composition and neovascularization. Imaged plaques were classified as contrast enhanced (CE) positive or CE negative, according to their contrast enhancement on CEU imaging. The lesions were also classified as class III (predominantly echogenic) or class II (predominantly echolucent), according to their echogenicity on non-CEU images. No contrast enhancement was observed in control animals. In atherosclerotic animals, class III lesions showed an increased contrast enhancement compared with class II lesions and CE-positive lesions showed greater neovascularization than CE-negative plaques. Macrophage density, but not smooth muscle cell density, was significantly higher in CE-positive than CE-negative lesions. As expected, class III lesions showed increased macrophage density compared with class II plaques. Intraplaque neovessel density at histology was significantly higher in CE-positive than in CE-negative lesions. Class III plaques showed a significantly higher neovessel density compared with class II lesions. A strong correlation between intraplaque neovessels and contrast enhancement was found. CEU imaging is a feasible noninvasive imaging modality to evaluate intraplaque neovascularization. A noninvasive imaging modality to assess lesion neovascularization could be of great importance to identify vascularized, "high-risk" lesions before rupture.

BAS/BSCR2 Analysis of microvessels within carotid plaques: comparison of symptomatic and asymptomatic patients to identify those at risk from plaque rupture

IntroductionStroke is the third most common cause of death world wide. Most cases of stroke are caused by atherosclerotic plaque rupture. A challenge facing clinicians is identifying asymptomatic patients at...

Impact of adventitial neovascularisation on atherosclerotic plaque composition and vascular remodelling in a porcine model of coronary atherosclerosis

There is little in vivo data in regards to the impact of adventitial neovascularisation on vascular remodelling and plaque composition. Using a porcine model of coronary atherosclerosis, we aimed to determine the impact of adventitial neovascularisation on plaque composition and vascular remodelling evaluated by IVUS.Coronary atherosclerosis was induced by adventitial delivery of lipids and a high cholesterol diet. At termination all vessels were analysed using IVUS to determine the degree of remodelling of each individual segment containing atherosclerotic lesions. Then, each segment was correlated with its correspondent histological frame for plaque composition and neovessel density. A total of 57 atherosclerotic lesions at different stages of development were analysed. The total neovessel count (TNC) correlated to the degree of plaque burden (15.6+/-7.2 TNC in <40% stenosis versus 35.7+/-14.0 TNC in >60% stenosis, p<0.01) and to the amount of intra-plaque collagen (32.4+/-14.1%, lower TNC tertile versus 47.5+/-8.9% upper TNC tertile, p< 0.01). The amount of intra-plaque SMC content inversely correlated with the TNC (49.7+/-18.9% versus 36.4+/-14.4%, lower versus upper tertiles, p<0.05). Plaques with the highest TNC showed higher remodelling indexes by IVUS (0.89+/-0.32 in lower TNC tertile versus 1.36+/-0.73 in upper TNC tertile, p<0.05) and higher macrophage cell content (161.42+/-157.6 in lower TNC tertile versus 340.6+/-127.2 in upper TNC tertile, p<0.05) compared to non-remodelled segments.Adventitial neovascularisation is more prominent in positively remodelled segments and appears to be associated to SMC loss, increase collagen deposition and localised macrophage infiltration.

Impact of adventitial neovascularisation on atherosclerotic plaque composition and vascular remodelling in a porcine model of coronary atherosclerosis.

There is little in vivo data in regards to the impact of adventitial neovascularisation on vascular remodelling and plaque composition. Using a porcine model of coronary atherosclerosis, we aimed to determine the impact of adventitial neovascularisation on plaque composition and vascular remodelling evaluated by IVUS. Coronary atherosclerosis was induced by adventitial delivery of lipids and a high cholesterol diet. At termination all vessels were analysed using IVUS to determine the degree of remodelling of each individual segment containing atherosclerotic lesions. Then, each segment was correlated with its correspondent histological frame for plaque composition and neovessel density. A total of 57 atherosclerotic lesions at different stages of development were analysed. The total neovessel count (TNC) correlated to the degree of plaque burden (15.6+/-7.2 TNC in <40% stenosis versus 35.7+/-14.0 TNC in >60% stenosis, p<0.01) and to the amount of intra-plaque collagen (32.4+/-14.1%, lower TNC tertile versus 47.5+/-8.9% upper TNC tertile, p< 0.01). The amount of intra-plaque SMC content inversely correlated with the TNC (49.7+/-18.9% versus 36.4+/-14.4%, lower versus upper tertiles, p<0.05). Plaques with the highest TNC showed higher remodelling indexes by IVUS (0.89+/-0.32 in lower TNC tertile versus 1.36+/-0.73 in upper TNC tertile, p<0.05) and higher macrophage cell content (161.42+/-157.6 in lower TNC tertile versus 340.6+/-127.2 in upper TNC tertile, p<0.05) compared to non-remodelled segments. Adventitial neovascularisation is more prominent in positively remodelled segments and appears to be associated to SMC loss, increase collagen deposition and localised macrophage infiltration.

A Comparative Study of Carotid Atherosclerotic Plaque Microvessel Density and Angiogenic Growth Factor Expression in Symptomatic Versus Asymptomatic Patients

Objective A challenge facing clinicians is identifying patients with asymptomatic carotid disease at risk of plaque instability. We hypothesise that locally released angiogenic growth factors contribute to plaque instability. Methods Carotid endarterectomy specimens from eight symptomatic and eight asymptomatic patients were interrogated for microvessel density and angiogenic growth factor expression histologically using immunofluorescence, and biochemically using quantitative real-time polymerase chain reaction (q-RT-PCR). Bio-Plex™ suspension array was used to assess circulating biomarkers in venous blood from the same patients and six healthy age-matched controls. Results Immunofluorescence demonstrated significantly greater neovessel density in symptomatic plaques ( P = 0.010) with elevated expression of hepatocyte growth factor ( HGF) ( P = 0.001) and its receptor MET ( P = 0.011) than in asymptomatic plaques. The q-RT-PCR demonstrated up-regulation of Endoglin (CD105), HGF ( P = 0.001) and MET ( P = 0.011) in the plaques of symptomatic versus asymptomatic patients. Bio-Plex™ suspension array demonstrated elevated HGF ( P = 0.002) serum levels in symptomatic versus asymptomatic patients and healthy controls, and decreased platelet-derived growth factor (PDGF) ( P = 0.036) serum levels in symptomatic versus asymptomatic patients. Conclusion Plaque instability may be mediated by HGF-induced formation of new microvessels, and decreased vessel stability resulting from decreased PDGF. Suspension array technology has the potential to identify circulating biomarkers that correlate with plaque rupture risk.

Impact of Tumor-Associated Macrophages in LHBETATAGMice on Retinal Tumor Progression: Relation to Macrophage Subtype

To determine the distribution of tumor-associated macrophages (TAMs) during retinoblastoma tumor development, examine the contribution of bone marrow-derived TAMs in retinoblastoma tumors, and evaluate the supportive role of TAMs in tumor growth in a transgenic retinoblastoma mouse model. The time course of macrophage infiltration in transgenic retinoblastoma tumors was assessed by immunohistochemistry at different time points in tumorigenesis. The origin of TAMs in transgenic retinoblastoma tumors was determined by transplanting 10(7) bone marrow cells from green fluorescent protein (GFP)-positive 16-week-old mice into age-matched, irradiated LH(BETA)T(AG) mice via tail vein injections. Macrophage depletion was performed by subconjunctival (SC) delivery of liposomal clodronate. The density of TAMs increased from 4 to 12 weeks of age in mice with small to medium tumors (P = 0.037) and remained stable in the later stages of disease (i.e., 16 weeks old with large tumors; P = 0.20). In 16-week-old mice, 38% (2.5 +/- 3.2 cells per 400x high-power field) of TAMs were GFP-positive, bone marrow-derived macrophages. Total TAM depletion was associated with a significant decrease in the expression levels of MMP-9 (P = 0.014) and mature vessels (P < 0.001) and a nonsignificant decrease in the density of neovessels (P = 0.94). The density of M2-polarized TAMs did not change significantly after TAM depletion (P = 0.68). After M1-polarized TAM depletion, the tumor burden increased (P = 0.056). This work extends understanding of the complex role that macrophages play in retinoblastoma. Macrophage modulation in the tumor microenvironment is a critical factor in retinoblastoma tumor progression.

Metronomic Dosing Enhances the Anti-Angiogenic Effect of Epothilone B

High doses (10 nM) of epothilone B, a microtubule stabilizer, will inhibit the development of human tumor-derived angiogenesis following short (14 d) drug exposure times. Metronomic dosing regimes use lower drug doses and prolonged drug exposure times in an attempt to decrease toxicity compared with standard dosing schedules. We hypothesized that epothilone B would be an effective anti-angiogenic agent when administered at very low doses over an extended period of time. Fragments of four fresh human tumors were cultured in a fibrin-thrombin matrix and maintained in nutrient media plus 20% fetal bovine serum (FBS) for 56 d. Tumor fragments (n=40-60 per group) were exposed to weekly doses of epothilone B at concentrations of 10, 5, 1, 0.5, or 0.1 nM. All of these concentrations are clinically achievable. Tumor angiogenesis was assessed weekly on d 14-56 using a validated visual grading system. This system rates neovessel growth, density, and length on a 0-16 scale [angiogenic index, (AI)]. The average change in AI between d 14 and 56 was calculated for all samples and used to evaluate the metronomic response. Epothilone B produced a dose-dependent anti-angiogenic response in all tumors. Two of the four tumors demonstrated a clear and significant metronomic anti-angiogenic effect over time. Epothilone B, when dosed by a metronomic schedule may have a significant anti-angiogenic effect on human solid tumors. This study provides evidence for the potential use of epothilone B on a metronomic dosing schedule.

Increased Neovascularization in Advanced Lipid-Rich Atherosclerotic Lesions Detected by Gadofluorine-M-Enhanced MRI

Background— Inflammation and neovascularization may play a significant role in atherosclerotic plaque progression and rupture. We evaluated gadofluorine-M-enhanced MRI for detection of plaque inflammation and neovascularization in an animal model of atherosclerosis. Methods and Results— Sixteen rabbits with aortic plaque and 6 normal control rabbits underwent gadofluorine-M-enhanced MRI. Eight rabbits had advanced atherosclerotic lesions, whereas the remaining 8 had early lesions. Magnetic resonance atherosclerotic plaque enhancement was meticulously compared with plaque inflammation and neovessel density as assessed by histopathology. Advanced plaques and early atheroma were enhanced after gadofluorine-M injection. Control animals displayed no enhancement. After accounting for the within-animal correlation of observations, mean contrast-to-noise ratio was significantly higher in advanced plaques than compared with early atheroma (4.29�0.21 versus 3.00�0.32; P =0.004). Macrophage density was higher in advanced plaques in comparison to early atheroma (geometric mean=0.50 [95% CI, 0.19 to 1.03] versus 0.25 [0.07 to 0.42]; P =0.05). Furthermore, higher neovessel density was observed in advanced plaques (1.83 [95% CI, 1.51 to 2.21] versus 1.29 [0.99 to 1.69]; P =0.05). The plaque accumulation of gadofluorine-M correlated with increased neovessel density as shown by linear regression analysis ( r =0.67; P &lt;0.001). Confocal and fluorescence microscopy revealed colocalization of gadofluorine-M with plaque areas containing a high density of neovessels. Conclusion— Gadofluorine-M-enhanced MRI is effective for in vivo detection of atherosclerotic plaque inflammation and neovascularization in an animal model of atherosclerosis. These findings suggest that gadofluorine-M enhancement reflects the presence of high-risk plaque features believed to be associated with plaque rupture. Gadofluorine-M plaque enhancement may therefore provide functional assessment of atherosclerotic plaque in vivo.

Blood Vessel Maturation in Human Uveal Melanoma: Spatial Distribution of Neovessels and Mature Vasculature

Purpose: The aims of this study are (1) to evaluate the spatial distribution of neovessels and mature vessels in human uveal melanoma tumors and (2) to determine whether vessel maturation is associated with the major indicators for poor prognosis. Methods: Immunohistochemical analyses were performed on human tissue specimens from enucleated eyes (n = 14) to assess total vessels, neovessels, mature vessels, and cell proliferation. Tumor morphology was analyzed by hematoxylin and eosin and modified periodic acid-Schiff (PAS) staining.The spatial distribution of neovessels and mature vessels was analyzed by immunohistochemistry, and correlated with major indicators of poor prognosis (i.e., aggressive PAS patterns, epithelioid cytology, mitotic figures, extraocular extension, anterior tumor location, ciliary body involvement, large tumor size, cell proliferation, and angiogenic activity). Results: Neovesseldensity was greater than mature vessel density in apical (p = 0.17), central (p = 0.036), and peripheral (p = 0.31) regions of the tumors, while mature vessel density was greater than neovessel density in basal areas of the tumor (p = 0.47). This pattern indicated that vessel maturation begins at the base of the tumor and later extends to the peripheral and apical regions. The difference between mature and neovessel densities for the apical (–0.8 ± 1.9) and central areas (–0.8 ± 1.3) of the tumor was significantly higher than the difference obtained for the basal area (0.3 ± 1.6; p = 0.014 and p = 0.012, respectively), indicating a higher density of mature vessels compared to neovessels at the base. Statistical correlations were found between mature vessel density and tumor size (r = 0.48, p = 0.084), cell proliferation (r = 0.62, p = 0.042), and mitotic figures (r = 0.76, p = 0.001). Conclusions: Significant differences exist in the spatial distribution of mature versus neovessels in human uveal melanoma. Vessel maturation is associated with known clinical and pathologic indicators of poor prognosis (e.g., cell proliferation). Antiangiogenic therapy should be considered for the treatment of ocular malignancies; however, the results of this study indicate that blood vessel maturation heterogeneity may limit the efficacy of vessel targeting agents.

Contrast-Enhanced Ultrasound Imaging of Intraplaque Neovascularization in Carotid Arteries: Correlation With Histology and Plaque Echogenicity

This study was designed to evaluate contrast-enhanced ultrasound imaging of carotid atherosclerosis as a clinical tool to study intraplaque neovascularization. Plaque neovascularization is associated with plaque vulnerability and symptomatic disease; therefore, imaging of neovascularization in carotid atherosclerosis may represent a useful tool for clinical risk stratification and monitoring the efficacy of antiatherosclerotic therapies. Thirty-two patients with 52 carotid plaques were studied by standard and contrast-enhanced ultrasound imaging. In 17 of these patients who underwent endarterectomy, the surgical specimen was available for histological determination of microvessel density by CD31/CD34 double staining. Plaque echogenicity and degree of stenosis at standard ultrasound imaging were evaluated for each lesion. Contrast-agent enhancement within the plaque was categorized as absent/peripheral (grade 1) and extensive/internal (grade 2). In the surgical subgroup, plaques with higher contrast-agent enhancement showed a greater neovascularization at histology (grade 2 vs. grade 1 contrast-agent enhancement: median vasa vasorum density: 3.24/mm(2) vs. 1.82/mm(2), respectively, p = 0.005). In the whole series of 52 lesions, echolucent plaques showed a higher degree of contrast-agent enhancement (p < 0.001). Stenosis degree was not associated with neovascularization at histology or with the grade of contrast-agent enhancement. Carotid plaque contrast-agent enhancement with sonographic agents correlates with histological density of neovessels and is associated with plaque echolucency, a well-accepted marker of high risk lesions, but it is unrelated to the degree of stenosis. Contrast-enhanced carotid ultrasound imaging may provide valuable information for plaque risk stratification and for assessing the response to antiatherosclerotic therapies, beyond that provided by standard ultrasound imaging.