Effect of evolocumab on the progression of intraplaque neovascularization of the carotid based on contrast-enhanced ultrasonography (EPIC study): A prospective single-arm, open-label study.

Background and Purpose: The aim of this study was to explore the effect of half a year of evolocumab plus moderate-intensity statin treatment on carotid intraplaque neovascularization (IPN) and blood lipid levels. Methods: A total of 31 patients with 33 carotid plaques who received evolocumab plus statin treatment were included. Blood lipid levels, B-mode ultrasound and contrast-enhanced ultrasonography (CEUS) at baseline and after half a year of evolocumab plus statin therapy were collected. The area under the curve (AUC) reflected the total amount of acoustic developer entering the plaque or lumen within the 180 s measurement period. The enhanced intensity reflected the peak blood flow intensity during the monitoring period, and the contrast agent area reflected the area of vessels in the plaques. Results: Except for high-density lipoprotein cholesterol (HDL-c), all other lipid indices decreased. Compared with baseline, low-density lipoprotein cholesterol (LDL-c) decreased by approximately 57% (p < 0.001); total cholesterol (TC) decreased by approximately 34% (p < 0.001); small dense low-density lipoprotein (sd-LDL) decreased by approximately 52% (p < 0.001); and HDL-c increased by approximately 20% (p < 0.001). B-mode ultrasonography showed that the length and thickness of the plaque and the hypoechoic area ratio were reduced (p < 0.05). The plaque area, calcified area ratio, and lumen cross-sectional area changed little (p > 0.05). CEUS revealed that the area under the curve of plaque/lumen [AUC (P/L)] decreased from 0.27 ± 0.13 to 0.19 ± 0.11 (p < 0.001). The enhanced intensity ratio of plaque/lumen [intensity ratio (P/L)] decreased from 0.37 ± 0.16 to 0.31 ± 0.14 (p = 0.009). The contrast agent area in plaque/area of plaque decreased from 19.20 ± 13.23 to 12.66 ± 9.59 (p = 0.003). The neovascularization score decreased from 2.64 ± 0.54 to 2.06 ± 0.86 (p < 0.001). Subgroup analysis based on statin duration (<6 months and ≥6 months) showed that there was no significant difference in the AUC (P/L) or intensity ratio (P/L) at baseline or after half a year of evolocumab treatment. Conclusion: This study found that evolocumab combined with moderate-intensity statins significantly improved the blood lipid profile and reduced carotid IPN. Clinical Trial Registration: https://www.clinicaltrials.gov; identifier: NCT04423406.

Low-Intensity Focused Ultrasound-Responsive Ferrite-Encapsulated Nanoparticles for Atherosclerotic Plaque Neovascularization Theranostics.

Pathological angiogenesis is a crucial factor that causes atherosclerotic plaque rupture. Sinoporphyrin sodium-mediated sonodynamic therapy (DVDMS-SDT) induces regression of plaque neovascularization in humans without causing obvious side effects. However, a clinical noninvasive theranostic strategy for atherosclerotic plaque neovascularization is urgently needed. A nanoplatform designed for multimodality imaging-guided SDT in plaque angiogenesis theranostics, termed PFP-HMME@PLGA/MnFe2 O4 -ramucirumab nanoparticles (PHPMR NPs), is fabricated. It encapsulates manganese ferrite (MnFe2 O4 ), hematoporphyrin monomethyl ether (HMME), and perfluoropentane (PFP) stabilized by polylactic acid-glycolic acid (PLGA) shells and is conjugated to an anti-VEGFR-2 antibody. With excellent magnetic resonance imaging (MRI)/photoacoustic/ultrasound imaging ability, the distribution of PHPMR NPs in plaque can be observed in real time. Additionally, they actively accumulate in the mitochondria of rabbit aortic endothelial cells (RAECs), and the PHPMR NP-mediated SDT promotes mitochondrial-caspase apoptosis via the production of reactive oxygen species and inhibits the proliferation, migration, and tubulogenesis of RAECs. On day 3, PHPMR NP-mediated SDT induces apoptosis in neovessel endothelial cells and improves hypoxia in the rabbit advanced plaque. On day 28, PHPMR NP-mediated SDT reduces the density of neovessels, subsequently inhibiting intraplaque hemorrhage and inflammation and eventually stabilizing the plaque. Collectively, PHPMR NP-mediated SDT presents a safe and effective theranostic strategy for inhibiting plaque angiogenesis.

Advanced ultrasound methods in assessment of carotid plaque instability: a prospective multimodal study.

BACKGROUND: A significant proportion of ischemic strokes are caused by emboli from atherosclerotic, unstable carotid artery plaques. The selection of patients for endarterectomy in current clinical practice is primarily based on the degree of carotid artery stenosis and clinical symptoms. However, the content of the plaque is known to be more important for stroke risk. Intraplaque neovascularization (IPN) has recently emerged as a possible surrogate marker for plaque instability. Neo-microvessels from the adventitial vasa vasorum grow into the full thickness of the vessel wall in an adaptive response to hypoxia, causing subsequent intraplaque haemorrhage and plaque rupture. Conventional ultrasound cannot detect IPN. Contrast-enhanced ultrasound and Superb Microvascular Imaging (SMI), have, however, shown promise in IPN assessment. Recent research using Shear Wave Elastography (SWE) has also reported reduced tissue stiffness in the artery wall (reduced mean Young's modulus) in unstable compared to stable plaques. The purpose of this study is to identify unstable carotid artery plaques at risk of rupture and future ischemic stroke risk using multimodal assessments. METHODS: Forty five symptomatic and 45 asymptomatic patients > 18 years, with > 50% carotid stenosis referred to Oslo University Hospital ultrasound lab will be included in this on-going project. Patients will undergo contrast enhanced ultrasound, SMI, carotid-MRI and PET-(18F-FDG). Contrast enhanced ultrasound will be analyzed semi-quantitatively (5-levels visual classification) and quantitatively by plotting time-intensity curve analyses to obtain plaque peak contrast enhancement intensity. Plaques removed at carotid endarterectomy will be assessed histologically and the number of microvessels, areas of inflammation, granulation, calcification, lipid and fibrosis will be measured. DISCUSSION: This multimodality study will primarily provide information on the clinical value of advanced ultrasound methods (SMI, SWE) for the detection of unstable carotid artery plaque in comparison with other methods including contrast-enhanced ultrasound, carotid-MRI and PET-(18F-FDG) using histology as the gold standard. Secondly, findings from the methods mentioned above will be related to cerebrovascular symptoms, blood tests (leukocytes, CRP, ESR, lipoproteins and inflammatory markers) and cardiovascular risk factors at inclusion and at 1-year follow-up. The overall aim is to optimize detection of plaque instability which can lead to better preventive decisions and reduced stroke rate.

Rapid improvement in carotid adventitial angiogenesis and plaque neovascularization after rosuvastatin therapy in statin treatment-naïve subjects.

BACKGROUND: Statin therapy can improve plaque stability. However, the time course of effects of statin on adventitial angiogenesis and plaque neovascularization has not been studied. OBJECTIVE: The objective of the study was to investigate whether statin therapy reduces plaque neovascularization, associated with adventitial angiogenesis, over 24 months as assessed by using carotid dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). METHODS: Forty-three lipid treatment-naïve subjects with asymptomatic carotid atherosclerosis received rosuvastatin (5-20 mg/d) to lower low-density lipoprotein cholesterol to <80 mg/dL for 24 months. Carotid DCE-MRI was performed at baseline, 3, 12 and 24 months. Vascularity (Vp = fractional plasma volume) and vascular permeability (Ktrans = transfer constant) derived from kinetic modeling of DCE-MRI were measured in both adventitia and plaque. RESULTS: Adventitia Vp and adventitia Ktrans were significantly correlated with plaque Vp and plaque Ktrans at baseline. Rosuvastatin significantly reduced both adventitial and plaque Vp significantly at 3 months from 0.121 ± 0.064 to 0.085 ± 0.049 (P = .008) and from 0.096 ± 0.052 to 0.067 ± 0.043 (P = .013). Adventitial and plaque Vp continued to decrease by 43% and 34% at 12 months and by 49% and 45% at 24 months. However, the continued reductions from 3 to 12 months and from 12 to 24 months were not statistically significant. Adventitial and plaque Ktrans showed similar trends, but nonstatistically significant decreases during the 24 months of treatment. CONCLUSIONS: Rosuvastatin therapy rapidly and significantly decreased adventitial and plaque neovascularization at 3 months followed by continued, but nonstatistically significant, decreases at 12 and 24 months.

Assessing carotid plaque neovascularity and calcifications in patients prior to endarterectomy.

OBJECTIVE: The objective was to use two ultrasound image and signal processing techniques (MicroPure and superb microvascular imaging [SMI]; Toshiba Medical, Tokyo, Japan) to investigate carotid plaque calcification and intraplaque neovascularity flow as biomarkers for plaque vulnerability in patients before endarterectomy. METHODS: Thirty patients, with preoperative computed tomography angiography and scheduled for carotid endarterectomy, were enrolled in an institutional review board-approved study. Bilateral grayscale, power Doppler, SMI and MicroPure imaging of the carotids were performed using an Aplio 500 Platinum scanner (Toshiba). MicroPure combines nonlinear imaging and speckle suppression to mark calcifications as white spots in a blue overlay, and SMI uses clutter suppression to extract microvascular flow signals. Readers counted calcifications and scored them as present or absent; intraplaque neovascularity was scored on a 4-point scale by ultrasound imaging as well as by pathology (as the reference). MicroPure and SMI assessments were compared with conventional ultrasound examination and computed tomography angiography with pathology as the reference standard. RESULTS: Owing to technical difficulties and cancelled operations, 57 carotids were studied; endarterectomies yielded 28 specimens. Intraplaque neovascularization was detected by SMI in significantly more plagues than by power Doppler (41 vs 22 out 57 examined plaques or 72% vs 39%; P < .0001). There was no statistical difference between either reader compared with pathology (P > .37). Sensitivity specificity and accuracy for detecting intraplaque neovascularity based on color SMI and PDI were 84% (95% confidence interval [CI], 64%-96%), 33% (95% CI, 1%-91%), 79% (95% CI, 59%-92%), and 52% (95% CI, 31%-72%), 100% (95% CI, 23%-100%), and 57% (95% CI, 37%-76%), respectively. MicroPure did not correlate with any measures of intraplaque flow (P > .13). CONCLUSIONS: SMI may have potential for providing evidence of plaque vulnerability. MicroPure appears less useful in carotid applications.

Impact of Cardiovascular Risk Factors and Pharmacologic Treatments on Carotid Intraplaque Neovascularization Detected by Contrast-Enhanced Ultrasound.

BACKGROUND: Neovascularization is a marker of plaque vulnerability that can be assessed noninvasively using contrast-enhanced ultrasound (CEUS). The presence and extent of plaque neovascularization and their relation to cardiovascular risk factors and treatments were assessed in asymptomatic patients with carotid stenosis of intermediate severity and no indication for revascularization. METHODS: Sixty-six patients aged 69 ± 8 years (59% men) were prospectively enrolled. Plaque neovascularization was assessed using CEUS with sulfur hexafluoride contrast in each of the four carotid segments bilaterally (a total of 528 segments). In each plaque, the presence or absence of contrast enhancement was assessed semiquantitatively as CEUS grade 1 (no signal or signal confined to the adventitia and/or shoulder of the plaque) or CEUS grade 2 (signal within the plaque). RESULTS: Plaques were detectable in 289 of 528 carotid segments (54.7%). CEUS grade 2 was present in at least one plaque in 48 of 66 patients (72.7%) and was not influenced by stenosis severity or morphology. The highest CEUS grade 2 prevalence was observed in patients with diabetes and the lowest in those treated with angiotensin-converting enzyme inhibitors and statins, especially when low-density lipoprotein cholesterol was <100 mg/dL. Patients with multiple CEUS grade 2 plaques (20 of 66 [30%]) had both higher low-density lipoprotein and higher C-reactive protein. CONCLUSION: Intraplaque neovascularization is frequent in asymptomatic patients with intermediate carotid stenosis and is more prevalent in those with diabetes. Low-density lipoprotein cholesterol < 100 mg/dL and treatment with angiotensin-converting enzyme inhibitors seem to confer protection from neovascularization, although larger interventional studies are necessary to confirm these data.

Neovascularization of Carotid Atherosclerotic Plaque and Quantitative Methods of Its Dynamic Assessment in Vivo.

The study demonstrates significant variety of neovascularization degree and vessel diameter in the carotid atherosclerotic plaque. It is suggested that the increase in the number of vessels with a diameter <20 µ can be indicative of increased atherosclerosis activity, while the increase in the number of vessels with a diameter ≥40 µ indicates "reparative potential" of plaques. Duplex contrast-enhanced ultrasound scanning allows characterization of the localization and number of vessels with a diameter of ≥30 µ in the plaque, while even slight elevation of plasma concentration of basic fibroblast growth factor attests, first of all, to increased content of small vessels <30 µ in the plaque. The level of fibroblast growth factor >1.5 pg/ml is a reliable marker of increased number of both small and large vessels in the plaque.

Head-to-Head Comparison of Inflammation and Neovascularization in Human Carotid Plaques: Implications for the Imaging of Vulnerable Plaques.

BACKGROUND: Inflammation and intraplaque neovascularization are acknowledged to be 2 features of plaque vulnerability, although their temporal expression and their respective value in predicting clinical events are poorly understood. To determine their respective temporal associations, we conducted a comprehensive assessment of inflammation and intraplaque neovascularization in the carotid plaque of symptomatic and asymptomatic patients. METHODS AND RESULTS: Thirty patients with severe carotid stenosis underwent 18F-fluorodeoxyglucose-positron emission tomography/computed tomographic imaging. Plaque 18F-fluorodeoxyglucose-uptake, indicative of inflammation, was measured by calculating the target:background ratio. The presence of intraplaque neovascularization during contrast-enhanced ultrasound was judged semiquantitatively; low-grade contrast enhancement (CE) suggested its absence, and high-grade CE, the presence of neovascularization. Carotid surgery was performed 1.6±1.8 days after completing both imaging modalities in all patients, and the presence of macrophages and neovessels was quantified by immunohistochemistry. We identified a significant correlation between the target:background ratio and macrophage quantification (R=0.78; P<0.001). The number of vessels was also significantly higher in carotid plaque with high-CE (P<0.001). Surprisingly, immunohistochemistry showed that high-CE and vessel number were neither associated with an elevated target:background ratio (P=0.28 and P=0.60, respectively) nor macrophage infiltration (P=0.59 and P=0.40, respectively). Finally, macrophage infiltration and target:background ratio were higher in the carotid plaque of symptomatic patients (P=0.021 and P=0.05, respectively), whereas CE grade and the presence of neovessels were not. CONCLUSIONS: Inflammation and intraplaque neovascularization are not systematically associated in carotid plaques, suggesting a temporal separation between the 2 processes. Inflammation seems more pronounced when symptoms are present. These data highlight the challenges that face any imaging strategy designed to assess plaque vulnerability.

Circulating CD14+ and CD14highCD16- classical monocytes are reduced in patients with signs of plaque neovascularization in the carotid artery.

BACKGROUND AND AIMS: Monocytes are known to play a key role in the initiation and progression of atherosclerosis and contribute to plaque destabilization through the generation of signals that promote inflammation and neoangiogenesis. In humans, studies investigating the features of circulating monocytes in advanced atherosclerotic lesions are lacking. METHODS: Patients (mean age 69 years, 56% males) with intermediate asymptomatic carotid stenosis (40-70% in diameter) were evaluated for maximal stenosis in common carotid artery, carotid bulb and internal carotid artery, overall disease burden as estimated with total plaque area (TPA), greyscale and neovascularization in 244 advanced carotid plaques. Absolute counts of circulating CD14+ monocytes, of classical (CD14highCD16-), intermediate (CD14highCD16+) and non-classical (CD14lowCD16+) monocytes and HLA-DR+ median fluorescence intensity for each subset were evaluated with flow cytometry. RESULTS: No correlation was found between monocytes and overall atherosclerotic burden, nor with high sensitivity C-reactive protein (hsCRP) or interleukin-6 (IL-6). In contrast, plaque signs of neovascularization were associated with significantly lower counts of circulating CD14+ monocytes (297 versus 350 cells/mm3, p = 0.039) and of classical monocytes (255 versus 310 cells/mm3, p = 0.029). CONCLUSIONS: Neovascularized atherosclerotic lesions selectively associate with lower blood levels of CD14+ and CD14highCD16- monocytes independently of systemic inflammatory activity, as indicated by normal hsCRP levels. Whether the reduction of circulating CD14+ and CD14highCD16- monocytes is due to a potential redistribution of these cell types into active lesions remains to be explored.

Plaque Neovascularization Is Increased in Human Carotid Atherosclerosis Related to Prior Neck Radiotherapy: A Contrast-Enhanced Ultrasound Study.

OBJECTIVES: The aim of this study was to determine the effect of radiotherapy (RT) on intraplaque neovascularization (IPN) in human carotid arteries. BACKGROUND: Exposure of the carotid arteries to RT during treatment for head and neck cancer is associated with increased risk for stroke. However, the effect of RT on IPN, a precursor to intraplaque hemorrhage and thus associated with plaque vulnerability, is unknown. METHODS: In this cross-sectional study, patients who had undergone unilateral RT for head and neck cancer ≥2 years previously underwent B-mode and contrast-enhanced ultrasound of both RT-side and non-RT-side carotid arteries. Presence of IPN during contrast-enhanced ultrasound was judged semiquantitatively as grade 0 (absent), grade 1 (present but limited to plaque base), or grade 2 (extensive and noted within plaque body). RESULTS: Of 49 patients studied, 38 (78%) had plaques. The number of plaques was significantly greater in the RT than the non-RT arteries. Overall, 48 of 64 RT-side plaques (75%) had IPN compared with 9 of 23 non-RT-side (39%) plaques (p = 0.002). Among patients with plaques, IPN was present in 81% of patients with RT-side plaques and 41% of patients with non-RT-side plaques (p = 0.004). Grade 0 IPN was significantly more common in patients with non-RT-side plaques (25% vs. 61%; p = 0.002), whereas grade 2 plaques were more common on the RT side (31% vs. 9%; p = 0.03). The only clinical variable that predicted the presence or absence of IPN was RT laterality. CONCLUSIONS: This is the first study in humans to reveal a significant association between RT and the presence and extent of IPN. This may provide insights into the mechanisms underlying the increased stroke risk among survivors of head and neck cancer treated by RT.

The Use of Contrast-enhanced Ultrasonography for Imaging of Carotid Atherosclerotic Plaques: Current Evidence, Future Directions.

Contrast-enhanced ultrasonography (CEUS) is a rapidly evolving modality for imaging carotid artery disease and systemic atherosclerosis. CEUS coupled with diagnostic ultrasonography predicts the degree of carotid artery stenosis and is comparable with computed tomography and magnetic resonance angiography. This article reviews the literature on the evolving role of CEUS for the identification and characterization of carotid plaques with an emphasis on detection of intra-plaque neovascularization and related high-risk morphologic features notably present in symptomatic patients. CEUS carotid imaging may play a prominent additive role in risk stratifying patients and serve as a powerful tool for monitoring therapeutic interventions.

Expression of monocyte subsets and angiogenic markers in relation to carotid plaque neovascularization in patients with pre-existing coronary artery disease and carotid stenosis.

AIM: To characterize blood monocyte subsets in patients with different degrees of carotid atherosclerosis and pathological carotid plaque neovascularization. METHODS: Assessment of carotid plaque neovascularization using contrast ultrasonography and flow cytometric quantification of monocyte subsets and their receptors involved in inflammation, angiogenesis, and tissue repair was done in 40 patients with carotid stenosis ≥ 50% and CAD (CS > 50), 40 patients with carotid stenosis < 50% and documented CAD (CS < 50), 40 hypercholesterolaemic controls (HC group), and 40 normocholesterolaemic controls (NC). RESULTS: CS > 50 and CS < 50 groups had increased counts of Mon1 ('classical' CD14++ CD16-CCR2 + cells) compared to HCs (P = 0.03, and P = 0.009). Mon3 ('non-classical' CD14 + CD16++ CCR2- cells) were only increased in CS < 50 compared with HCs (P < 0.01). Both CS>50 and CS < 50 groups showed increased expression of proinflammatory interleukin-6 receptor on Mon1 and Mon2 ('intermediate' CD14++ CD16 + CCR2+ cells); TLR4, proangiogenic Tie2 on all subsets (P < 0.01 for all). In multivariate regression analysis only high Mon1 count was a significant predictor of carotid stenosis (P = 0.04) and intima-media thickness (P = 0.02). In multivariate regression analysis only the Mon1 subset was significantly associated with severe, grade 2 neovascularization (P = 0.034). CONCLUSION: In this pilot study classical monocytes (Mon1) represent the only monocyte subset predictive of the severity of carotid and systemic atherosclerosis, such as carotid intima-media thickness, degree of carotid stenosis, and presence of carotid intraplaque neovascularization.

New quantification methods for carotid intra-plaque neovascularization using contrast-enhanced ultrasound.

As carotid intra-plaque neovascularization (IPN) is linked to progressive atherosclerotic disease and plaque vulnerability, its accurate quantification might allow early detection of plaque vulnerability. We therefore developed several new quantitative methods for analyzing IPN perfusion and structure. From our analyses, we derived six quantitative parameters-IPN surface area (IPNSA), IPN surface ratio (IPNSR), plaque mean intensity, plaque-to-lumen enhancement ratio, mean plaque contrast percentage and number of micro-vessels (MVN)-and compared these with visual grading of IPN by two independent physicians. A total of 45 carotid arteries with symptomatic stenosis in 23 patients were analyzed. IPNSA (correlation r = 0.719), IPNSR (r = 0.538) and MVN (r = 0.484) were found to be significantly correlated with visual scoring (p < 0.01). IPNSA was the best match to visual scoring. These results indicate that IPNSA, IPNSR and MVN may have the potential to replace qualitative visual scoring and to measure the degree of carotid IPN.