Long-term Intravital Investigation of an Orthotopic Glioma Mouse Model via Optical Coherence Tomography Angiography.

BACKGROUND/AIM: Probing brain tumor microvasculature holds significant importance in both basic cancer research and medical practice for tracking tumor development and assessing treatment outcomes. However, few imaging methods commonly used in clinics can noninvasively monitor the brain microvascular network at high precision and without exogenous contrast agents in vivo. The present study aimed to investigate the characteristics of microvasculature during brain tumor development in an orthotopic glioma mouse model. MATERIALS AND METHODS: An orthotopic glioma mouse model was established by surgical orthotopic implantation of U87-MG-luc cells into the mouse brain. Then, optical coherence tomography angiography (OCTA) was utilized to characterize the microvasculature progression within 14 days. RESULTS: The orthotopic glioma mouse model evaluated by bioluminescence imaging and MRI was successfully generated. As the tumor grew, the microvessels within the tumor area slowly decreased, progressing from the center to the periphery for 14 days. CONCLUSION: This study highlights the potential of OCTA as a useful tool to noninvasively visualize the brain microvascular network at high precision and without any exogenous contrast agents in vivo.

[18F]FDG PET/CT Signal Correlates with Neoangiogenesis Markers in Patients with Fibrotic Interstitial Lung Disease Who Underwent Lung Biopsy: Implication for the Use of PET/CT in Diffuse Lung Diseases.

The use of [18F]FDG PET/CT as a biomarker in diffuse lung diseases is increasingly recognized. We investigated the correlation between [18F]FDG uptake with histologic markers on lung biopsy of patients with fibrotic interstitial lung disease (fILD). Methods: We recruited 18 patients with fILD awaiting lung biopsy for [18F]FDG PET/CT. We derived a target-to-background ratio (TBR) of maximum pulmonary uptake of [18F]FDG (SUVmax) divided by the lung background (SUVmin). Consecutive paraffin-embedded lung biopsy sections were immunostained for alveolar and interstitial macrophages (CD68), microvessel density (MVD) (CD31 and CD105/endoglin), and glucose transporter 1. MVD was expressed as vessel area percentage per high-power field (Va%/hpf). Differences in imaging and angiogenesis markers between histologic usual interstitial pneumonia (UIP) and non-UIP were assessed using a nonparametric Mann-Whitney test. Correlation of imaging with angiogenesis markers was assessed using the nonparametric Spearman rank correlation. Univariate Kaplan-Meier survival analysis assessed the difference in the survival curves for each of the angiogenesis markers (separated by their respective optimal cutoff) using the log-rank test. Statistical analysis was performed using SPSS. Results: In total, 18 patients were followed for an average of 41.36 mo (range, 5.69-132.46 mo; median, 30.07 mo). Only CD105 MVD showed a significantly positive correlation with [18F]FDG TBR (Spearman rank correlation, 0.556; P < 0.05, n = 13). There was no correlation between [18F]FDG uptake and macrophage expression of glucose transporter 1. CD105 and CD31 were higher for UIP than for non-UIP, with CD105 reaching statistical significance (P = 0.011). In all patients, MVD assessed with either CD105 or CD31 quantification on biopsy predicted overall survival. Patients with CD105 MVD of less than 12 Va%/hpf or CD31 MVD of less than 35 Va%/hpf had a significantly better prognosis (no deaths during follow-up in the case of CD105) than did patients with higher scores of CD105 MVD (median survival, 35 mo; P = 0.041, n = 13) or CD31 MVD (median survival, 28 mo; P = 0.014, n = 13). Conclusion: Previous work has used [18F]FDG uptake in PET/CT as a biomarker in fILD. Here, we highlight a correlation between angiogenesis and [18F]FDG TBR. We show that MVD is higher for UIP than for non-UIP and is associated with mortality in patients with fILD. These data set the scene to investigate the potential role of vasculature and angiogenesis in fibrosis.

Effects of Glutamine Synthetase on Neovascularization in Glioma: In Vivo MR Vessel Size Imaging and Histology.

BACKGROUND: Glutamine Synthetase (GS) could induce vascular sprouting through the improvement of endothelial cell migration in inflammatory diseases. MR vessel-size imaging has been proposed as a valuable approach for visualizing the underlying angiogenic processes in the brain. OBJECTIVE: This study aims to investigate the role of GS in the neovascularization of gliomas through the utilization of MR vessel-size imaging and histopathological techniques. METHODS: In this exploratory animal study, we randomly divided the C6 glioma rat models into a control group and an L-methionine sulfoximine (MSO) treatment group. Daily intraperitoneal injections were administered for three consecutive days, starting from day 10 following the implantation of C6 glioma cells in rats. Subsequently, MR vessel size imaging was conducted using a BRUKER 7 T/200 mm MRI scanner, and the MRI results were validated through histopathological examination. RESULTS: A significant decrease in microvessel density was observed in both the tumor periphery and center areas in the MSO treatment group compared to that in the control group. The mean vessel diameter (mVD) and vessel size index (VSI) did not exhibit significant changes compared to the control group. Moreover, the staining intensity of platelet endothelial cell adhesion molecule-1 (CD31) and GS in the tumor periphery was significantly decreased in the MSO treatment group. Additionally, the MSO treatment demonstrated a substantial inhibition of tumor growth. CONCLUSION: GS inhibitors significantly reduced angiogenesis in the periphery area of C6 glioma, exerting an inhibitory effect on tumor progression. Thus, GS inhibitors could be potential therapeutic agents for treating glioma. Additionally, in vivo MR vessel size imaging detects changes in vascularrelated parameters after tumor treatment, making it a promising method for detecting neovascularization in glioma.

Dynamic contrast-enhanced magnetic resonance imaging assessment of residual tumor angiogenesis after insufficient microwave ablation and donafenib adjuvant therapy.

To analyze the correlation between dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) permeability parameters and serum vascular endothelial growth factor (VEGF) levels in a rabbit VX2 liver cancer model with insufficient microwave ablation (MWA), to observe the dynamic changes in residual tumor angiogenesis in the short term after MWA, and to assess the effectiveness of donafenib as adjuvant therapy. Forty rabbits with VX2 liver tumors were randomly divided into three groups: an insufficient MWA group (n = 15), a combined treatment group (n = 15) and a control group (n = 10). The dynamic changes in VEGF expression after MWA and the effectiveness of donafenib as adjuvant therapy were evaluated by DCE-MRI and serum VEGF levels before surgery and 1, 3, 7, and 14 days after surgery. The correlation between the volume translate constant (Ktrans) of DCE-MRI parameters and serum VEGF levels fluctuated after ablation, but the coefficient was always positive (all p < 0.001). Repeated-measures ANOVA revealed significant changes in the serum VEGF concentration (F = 40.905, p < 0.001; partial η2 = 0.689), Ktrans (F = 13.388, p < 0.001; partial η2 = 0.420), and tumor diameter in each group (F = 34.065, p < 0.001; partial η2 = 0.648) at all five time points. Pairwise comparisons showed that the serum VEGF level, Ktrans value and tumor diameter in the insufficient MWA group and combined treatment group were significantly lower at 1 d than in the control group, but these values gradually increased over time (all p < 0.05). Ktrans and tumor diameter were significantly greater in the insufficient MWA group than in the control group at 14 days (all p < 0.05). The serum VEGF concentration, Ktrans, and tumor diameter were significantly lower in the combined treatment group than in the other two groups at 3, 7, and 14 days (all p < 0.05). Ktrans is positively correlated with the serum VEGF concentration. Ktrans and the serum VEGF concentration changed significantly after treatment with insufficient ablation or in combination with donafenib, and Ktrans may change faster. Insufficient MWA promotes the progression of residual tumors. Adjuvant treatment with donafenib is effective.

Study on the Consistency of Angiogenesis in Carotid Plaque Evaluated by Contrast-Enhanced Ultrasound and Superb Microvascular Imaging and Its Correlation With Stroke Occurrence.

OBJECTIVES: This study aimed to investigate the value of contrast-enhanced ultrasound (CEUS) and superb microvascular imaging (SMI) in evaluating angiogenesis in carotid artery plaques and prognosis in stroke patients. METHODS: Sixty-one patients with carotid atherosclerotic plaques were selected. All patients received conventional ultrasound, CEUS, and SMI examination, including 32 patients with cerebral infarction and 29 patients without cerebral infarction. The results of CEUS and SMI neovascularization of patients were graded 0, 1, and 2 points according to the image characteristics. The consistency between SMI results and CEUS results was evaluated, and the differences in neovascularization in carotid plaques between patients with cerebral infarction and those without cerebral infarction were compared. RESULTS: SMI showed that the neovascularization score in plaque was 0 point in 13 cases, 1 point in 24 cases, and 2 points in 24 cases. There were no significant differences in age, sex, plaque size, or echo between the two groups. There was no significant difference between the SMI and CEUS results, P > .05. The CEUS neovascularization grade of patients with cerebral infarction had a higher score, which was significantly different from that of patients without cerebral infarction, P < .05. The SMI neovascularization grade of patients with cerebral infarction had a higher score, which was significantly different from that of patients without cerebral infarction, P < .05. CONCLUSION: SMI can show neovascularization in plaques, with a significantly higher grade of neovascularization in those of patients with cerebral infarction than in those without cerebral infarction.

In vivo MR imaging for tumor-associated initial neovascularization by supramolecular contrast agents.

Microvascular imaging is required to understand tumor angiogenesis development; however, an appropriate whole-body imaging method has not yet been established. Here, we successfully developed a supramolecular magnetic resonance (MR) contrast agent for long-term whole-tissue observation in a single individual. Fluorescein- and Gd-chelate-conjugated polyethylene glycols (PEGs) were synthesized, and their structures were optimized. Spectroscopic and pharmacokinetic analyses suggested that the fluorescein-conjugated linear and 8-arm PEGs with a molecular weight of approximately 10 kDa were suitable to form a supramolecular structure to visualize the microvessel structure and blood circulation. Microvascular formation was evaluated in a glioma cell transplantation model, and neovascularization around the glioma tissue at 5 days was observed, with the contrast agent leaking out into the cancer tissue. In contrast, after 12 days, microvessel structures were formed inside the glioma tissue, but the agents did not leak out. These imaging data for the first time proved that the microvessels formed inside cancer tissues at the early stage are very leaky, but that they form continuous microvessels after 12 days.

Liver metastases in renal cell carcinoma: The vascularization pattern and the wash out phenomenon.

The wash out behavior of focal liver lesions in contrast-enhanced ultrasound plays a central role in tumor classification. Besides hepatocellular carcinomas, other hypervascular tumor entities like renal cell carcinomas may also show a very late wash out, possibly caused by portal-venous tumor vessels. There is need to observe long enough in the late phase in order to classify them correctly.

Neovascularization From the Carotid Artery Lumen Into the Carotid Plaque Confirmed by Contrast-Enhanced Ultrasound and Histology.

OBJECTIVE: This study was aimed at assessing intraplaque neovessels, focusing on neovascularization from the vascular luminal side using contrast-enhanced ultrasound (CEUS) and determining that this contrast effect indicates that the neovessel is connected to the vessel lumen histopathologically. Whether plaque vulnerability can be assessed more accurately was also investigated. METHODS: We enrolled consecutive patients with internal carotid artery stenosis who underwent carotid endarterectomy (CEA) and pre-operative CEUS with perflubutane of the carotid arteries. We graded the contrast effect semi-quantitatively from the vascular luminal and adventitial sides. We compared the contrast effect with the pathological findings, especially the neovascularization of the CEA specimens. RESULTS: In total, 68 carotid arterial atheromatous plaques (47 symptomatic) were analyzed. Symptomatic plaques were significantly correlated with stronger contrast effects from the luminal side than from the adventitial side (p = 0.0095). Microbubbles from the luminal side appeared to flow mainly into the plaque shoulder. The contrast effect value for the plaque shoulder and neovessel density were significantly correlated (ρ = 0.35, p = 0.031). Neovessel density was significantly higher in symptomatic than in asymptomatic plaques (56.2 ± 43.7/mm2 and 18.1 ± 15.2/mm2, respectively, p < 0.0001). Serial histological sections of CEA specimens in a symptomatic plaque with a strong contrast effect from the luminal side revealed multiple neovessels fenestrated to the vessel lumen with endothelial cells, consistent with the CEUS findings. CONCLUSION: Contrast-enhanced ultrasound can be used to evaluate neovessels originating from the luminal side, histopathologically confirmed in serial sections. Symptomatic vulnerable plaque is correlated more significantly with intraplaque neovascularization from the luminal side than with neovascularization from the adventitia.

Multi-layered adaptive neoangiogenesis Intra-Operative quantification (MANIOQ).

Quantification of vascularization volume can provide valuable information for diagnosis and prognosis in vascular pathologies. It can be adapted to inform the surgical management of gliomas, aggressive brain tumors characterized by exuberant sprouting of new blood vessels (neoangiogenesis). Filtered ultrafast Doppler data can provide two main parameters: vascularization index (VI) and fractional moving blood volume (FMBV) that clinically reflect tumor micro vascularization. Current protocols lack robust, automatic, and repeatable filtering methods. We present a filtrating method called Multi-layered Adaptive Neoangiogenesis Intra-Operative Quantification (MANIOQ). First, an adaptive clutter filtering is implemented, based on singular value decomposition (SVD) and hierarchical clustering. Second a method for noise equalization is applied, based on the subtraction of a weighted noise profile. Finally, an in vivo analysis of the periphery of the B-mode hyper signal area allows to measure the vascular infiltration extent of the brain tumors. Ninety ultrasound acquisitions were processed from 23 patients. Compared to reference methods in the literature, MANIOQ provides a more robust tissue filtering, and noise equalization allows for the first time to keep axial and lateral gain compensation (TGC and LGC). MANIOQ opens the way to an intra-operative clinical analysis of gliomas micro vascularization.

Molecular Ultrasound Imaging Depicts the Modulation of Tumor Angiogenesis by Acetylsalicylic Acid.

Acetylsalicylic acid (ASA) is a well-established drug for heart attack and stroke prophylaxis. Furthermore, numerous studies have reported an anti-carcinogenic effect, but its exact mechanism is still unknown. Here, we applied VEGFR-2-targeted molecular ultrasound to explore a potential inhibitory effect of ASA on tumor angiogenesis in vivo. Daily ASA or placebo therapy was performed in a 4T1 tumor mouse model. During therapy, ultrasound scans were performed using nonspecific microbubbles (CEUS) to determine the relative intratumoral blood volume (rBV) and VEGFR-2-targeted microbubbles to assess angiogenesis. Finally, vessel density and VEGFR-2 expression were assessed histologically. CEUS indicated a decreasing rBV in both groups over time. VEGFR-2 expression increased in both groups up to Day 7. Towards Day 11, the binding of VEGFR-2-specific microbubbles further increased in controls, but significantly (p = 0.0015) decreased under ASA therapy (2.24 ± 0.46 au vs. 0.54 ± 0.55 au). Immunofluorescence showed a tendency towards lower vessel density under ASA and confirmed the result of molecular ultrasound. Molecular US demonstrated an inhibitory effect of ASA on VEGFR-2 expression accompanied by a tendency towards lower vessel density. Thus, this study suggests the inhibition of angiogenesis via VEGFR-2 downregulation as one of the anti-tumor effects of ASA.

Scandium-44: Diagnostic Feasibility in Tumor-Related Angiogenesis.

Angiogenesis-related cell-surface molecules, including integrins, aminopeptidase N, vascular endothelial growth factor, and gastrin-releasing peptide receptor (GRPR), play a crucial role in tumour formation. Radiolabelled imaging probes targeting angiogenic biomarkers serve as valuable vectors in tumour identification. Nowadays, there is a growing interest in novel radionuclides other than gallium-68 (68Ga) or copper-64 (64Cu) to establish selective radiotracers for the imaging of tumour-associated neo-angiogenesis. Given its ideal decay characteristics (Eß+average: 632 KeV) and a half-life (T1/2 = 3.97 h) that is well matched to the pharmacokinetic profile of small molecules targeting angiogenesis, scandium-44 (44Sc) has gained meaningful attention as a promising radiometal for positron emission tomography (PET) imaging. More recently, intensive research has been centered around the investigation of 44Sc-labelled angiogenesis-directed radiopharmaceuticals. Previous studies dealt with the evaluation of 44Sc-appended avb3 integrin-affine Arg-Gly-Asp (RGD) tripeptides, GRPR-selective aminobenzoyl-bombesin analogue (AMBA), and hypoxia-associated nitroimidazole derivatives in the identification of various cancers using experimental tumour models. Given the tumour-related hypoxia- and angiogenesis-targeting capability of these PET probes, 44Sc seems to be a strong competitor of the currently used positron emitters in radiotracer development. In this review, we summarize the preliminary preclinical achievements with 44Sc-labelled angiogenesis-specific molecular probes.

Neovascularization, vascular mimicry and molecular exchange: The imaging of tumorous tissue aggressiveness based on tissue perfusion.

Angiogenesis in healthy tissue and within malignant tumors differs on many levels, which may partly be explained by vascular mimicry formation resulting in altered contrast material or different radiopharmaceuticals distributions. Failed remodulation results in changes in the molecular exchange through the capillary wall and those consequences affect the behavior of contrast agents and radiopharmaceuticals. One of the most indicative signs of malignant tissue is the increased permeability and the faster molecular exchange that occurs between the extracellular and intravascular spaces. Dynamic imaging can help to assess the changed microenvironment. The fast-distribution of molecules reflects newly developed conditions in blood-flow redistribution inside a tumor and within the affected organ during the early stages of tumor formation. Tumor development, as well as aggressiveness, can be assessed based on the change to the vascular bed development, the level of molecular exchange within the tissue, and/or indicative distribution within the organ. The study of the vascular network organization and its impact on the distribution of molecules is important to our understanding of the image pattern in several imaging methods, which in turn influences our interpretation of the findings. A hybrid imaging approach (including PET/MRI) allows the quantification of vascularization and/or its pathophysiological impressions in structural and metabolic images. It might optimize the evaluation of the pretreatment imaging, as well as help assess the effect of therapy targeting neovascularization; antiVEGF drugs and embolization-based therapies, for example.

Utility of ultrasound Angio-PLUS imaging for detecting blood flow in breast masses and comparison with color Doppler for differentiating benign from malignant masses.

BACKGROUND: Tumor neo-angiogenesis plays an important role in the development and growth of breast cancers, but its detection by imaging is challenging. A novel microvascular imaging (MVI) technique, Angio-PLUS, promises to overcome the limitations of color Doppler (CD) in detecting low-velocity flow and small diameter vessels. PURPOSE: To determine the utility of the Angio-PLUS technique for detecting blood flow in breast masses and compare it with CD for differentiating benign from malignant masses. MATERIAL AND METHODS: A total of 79 consecutive women with breast masses were prospectively evaluated using CD and Angio-PLUS techniques, and biopsied as per BI-RADS recommendations. Vascular imaging scores were assigned using three factors (number, morphology, and distribution) and vascular patterns were divided into five groups: internal-dot-spot, external-dot-spot, marginal, radial, and mesh patterns. The independent samples t-test, Mann-Whitney U test, Wilcoxon signed rank test, or Fisher's exact test were used to compare the two groups as appropriate. Area under the receiver operating characteristic (ROC) curve (AUC) methods were used to assess diagnostic accuracy. RESULTS: Vascular scores were significantly higher on Angio-PLUS than CD (median=11, [IQR=9-13] vs. 5 [IQR=3-9], P < 0.001). Malignant masses had higher vascular scores than benign masses on Angio-PLUS (P < 0.001). AUC was 80% (95% CI=70.3-89.7; P < 0.001) for Angio-PLUS and 51.9% for CD. Using Angio-PLUS at a cutoff value of ≥9.5, sensitivity was 80% and specificity was 66.7%. Vascular pattern descriptors on AP showed good correlation with histopathological results (PPV mesh 95.5%, radial 96.9%, and NPV of marginal orientation 90.5%). CONCLUSION: Angio-PLUS was more sensitive in detecting vascularity and superior in differentiating benign from malignant masses compared to CD. Vascular pattern descriptors on Angio-PLUS were useful.

Value of Intraplaque Neovascularization on Contrast-Enhanced Ultrasonography in Predicting Ischemic Stroke Recurrence in Patients With Carotid Atherosclerotic Plaque.

OBJECTIVE: Patients with a history of ischemic stroke are at risk for a second ischemic stroke. This study aimed to investigate the relationship between carotid plaque enhancement on perfluorobutane microbubble contrast-enhanced ultrasonography (CEUS) and future recurrent stroke, and to determine whether plaque enhancement can contribute to risk assessment for recurrent stroke compared with the Essen Stroke Risk Score (ESRS). MATERIALS AND METHODS: This prospective study screened 151 patients with recent ischemic stroke and carotid atherosclerotic plaques at our hospital between August 2020 and December 2020. A total of 149 eligible patients underwent carotid CEUS, and 130 patients who were followed up for 15-27 months or until stroke recurrence were analyzed. Plaque enhancement on CEUS was investigated as a possible risk factor for stroke recurrence and as a possible adjunct to ESRS. RESULTS: During follow-up, 25 patients (19.2%) experienced recurrent stroke. Patients with plaque enhancement on CEUS had an increased risk of stroke recurrence events (22/73, 30.1%) compared to those without plaque enhancement (3/57, 5.3%), with an adjusted hazard ratio (HR) of 38.264 (95% confidence interval [CI]:14.975-97.767; P < 0.001) according to a multivariable Cox proportional hazards model analysis, indicating that the presence of carotid plaque enhancement was a significant independent predictor of recurrent stroke. When plaque enhancement was added to the ESRS, the HR for stroke recurrence in the high-risk group compared to that in the low-risk group (2.188; 95% CI, 0.025-3.388) was greater than that of the ESRS alone (1.706; 95% CI, 0.810-9.014). A net of 32.0% of the recurrence group was reclassified upward appropriately by the addition of plaque enhancement to the ESRS. CONCLUSION: Carotid plaque enhancement was a significant and independent predictor of stroke recurrence in patients with ischemic stroke. Furthermore, the addition of plaque enhancement improved the risk stratification capability of the ESRS.

Microvascular damage evaluation based on nailfold videocapillarosopy in sarcoidosis.

OBJECTIVES: Microvascular damage is thought to play a role in the pathogenesis of sarcoidosis. We aimed to evaluate the nailfold capillaroscopy (NVC) pattern to detect microvascular changes in patients with sarcoidosis and the relationship of capillaroscopic parameters with clinical variables and disease-related measurements. PATIENTS AND METHODS: Forty-two patients with sarcoidosis and 42 age- and sex-matched patients with systemic sclerosis (SSc) and healthy individuals were included in this cross-sectional case-control study. Patients aged 18-80 years who met the current American Thoracic Society criteria for sarcoidosis were included. NVC was performed by a digital microscope under a magnification of × 200. Capillary density, number of dilated, giant and neoangiogenic capillaries, capillary loop diameter, capillary shape, micro-hemorrhages, and number of avascular areas, were evaluated by an assessor who was blind to the groups allocation. RESULTS: Among the capillaroscopic parameters, neoangiogenesis and dilated capillaries, which can be seen in the pattern of scleroderma, were detected in patients with sarcoidosis but not significantly different from the control group (p = 0.055; p = 0.433, respectively). The rate of tortuosity and crossing capillaries of 50% and above was significantly higher in the sarcoidosis group than in SSc and healthy controls (p < 0.05). In patients with sarcoidosis, the only statistically significant finding was; forced expiratory volume (FEV1) in one second was lower in patients with a crossed capillary rate > 50% than in patients with a crossed capillary rate of less than 50% (FEV1; 87.21 ± 19.3, 102.5 ± 14.8, p = 0.04; respectively). CONCLUSION: Patients with a diagnosis of sarcoidosis have different capillaroscopic patterns. The presence of these nonspecific abnormal patterns may reflect microvascular damage in the pathophysiology of sarcoidosis. Key Points • Microvascular damage may play a role in the pathogenesis of sarcoidosis. • There may be some nonspecific abnormal findings in capillaroscopy findings in sarcoidosis. • Capillaroscopy may be valuable in reflecting sarcoidosis lung injury.

Image-based modeling of vascular organization to evaluate anti-angiogenic therapy.

In tumor therapy anti-angiogenic approaches have the potential to increase the efficacy of a wide variety of subsequently or co-administered agents, possibly by improving or normalizing the defective tumor vasculature. Successful implementation of the concept of vascular normalization under anti-angiogenic therapy, however, mandates a detailed understanding of key characteristics and a respective scoring metric that defines an improved vasculature and thus a successful attempt. Here, we show that beyond commonly used parameters such as vessel patency and maturation, anti-angiogenic approaches largely benefit if the complex vascular network with its vessel interconnections is both qualitatively and quantitatively assessed. To gain such deeper insight the organization of vascular networks, we introduce a multi-parametric evaluation of high-resolution angiographic images based on light-sheet fluorescence microscopy images of tumors. We first could pinpoint key correlations between vessel length, straightness and diameter to describe the regular, functional and organized structure observed under physiological conditions. We found that vascular networks from experimental tumors diverted from those in healthy organs, demonstrating the dysfunctionality of the tumor vasculature not only on the level of the individual vessel but also in terms of inadequate organization into larger structures. These parameters proofed effective in scoring the degree of disorganization in different tumor entities, and more importantly in grading a potential reversal under treatment with therapeutic agents. The presented vascular network analysis will support vascular normalization assessment and future optimization of anti-angiogenic therapy.

Optical imaging technology realizes early tumor diagnosis by detecting angiogenesis.

The occurrence and development of blood vessels play a key role in different stages of tumor growth, while current imaging techniques are difficult to detect early tumor angiogenesis because of their low sensitivity. Therefore, this article introduces high-sensitivity optical imaging technology to achieve early tumor diagnosis by detecting tumor angiogenesis. Liver and pancreatic tumor models in nude mice were respectively established to represent tumors with a rich or poor blood supply. The two optical imaging methods, in vivo confocal fluorescence imaging and photoacoustic imaging, were used to detect tumor angiogenesis at different stages. Finally, the changes in blood vessels were verified by immunostaining. Both autoluminescence imaging and pathological staining confirmed that these two tumor models were successfully established. In vivo confocal fluorescence imaging found that the early tumor blood vessel structure had obvious characteristics: disorder, tortuous deformation, thin diameter, which were significantly different from the normal tissues. Photoacoustic imaging could effectively identify blood vessels inside early tumors, which were small and disordered and might be used as one of the predictors of early tumor development. CD31 immunostaining was used to evaluate the vascular status of tumors at different stages and under different blood supply conditions. The vascular structures observed under the microscope in the two tumor models were consistent with the results observed by optical imaging methods. The optical imaging methods could monitor the characteristics of angiogenesis in the rich or poor blood supply tumors, especially the early diagnosis of tumors.

Evaluation of carotid plaque vulnerability with different echoes by shear wave elastography and CEUS.

OBJECTIVE: Using shear wave elastography (SWE) and contrast enhanced ultrasound (CEUS)to examine carotid plaques with different echoes, and explore a reliable method to quantify characteristics associated with vulnerable carotid plaques. METHODS: 2D ultrasound, SWE and CEUS were performed on 244 carotid plaques, and the echoes were evaluated according to the Gray-Weale classification scale and gray-scale median (GSM), and the mean Young's modulus (YM) of the plaque was measured and the intraplaque neovascularization was observed to investigate the relationship between carotid plaque types with different echo characteristics, GSM and the values of each parameter of YM and CEUS. The relationship between GSM and YM and CEUS values was investigated. RESULTS: The differences between GSM values (F = 49.742, P < 0.001), with the maximum, mean, and minimum YM values of ultrasound elastography (P < 0.001), and with the number (P < 0.001) and density (P = 0.047) of neovascularization on CEUS were statistically significant for the different echogenic types of plaques, and the lower the echogenicity of the plaque, the lower the GSM values (r = 0.632, P < 0.001), the smaller the YM values (all r > 0, P < 0.001), and the higher the neovascularization number and density values (r < 0, P < 0.001); and there were also statistically significant differences between the above indicators in the vulnerable and stable plaque groups (all P < 0.05). CONCLUSION: GSM, SWE, and CEUS techniques can quantitatively evaluate the vulnerability of different echo carotid plaques in a more comprehensive and objective manner, which may help clinical identification of vulnerable plaques, and provide important reference values for early diagnosis and treatment in clinical practice.

Early Enhancement with Contrast-Enhanced Ultrasonography Relates to the Number of Small-Diameter Neovessels in the Carotid Plaque.

BACKGROUND AND PURPOSE: Intraplaque neovessels (INVs) have been recognized as a major cause of intraplaque hemorrhage and subsequent vulnerability of the carotid plaque. However, the exact mechanisms by which INVs cause intraplaque hemorrhage remain unclear. Various sizes of INVs coexist in carotid plaques pathologically, and we hypothesized that the size of INVs would be associated with carotid plaque histology, particularly in terms of intraplaque hemorrhage. Detection method of INV is important when determining whether carotid plaques are vulnerable, and contrast-enhanced ultrasonography (CEUS) is one of the most useful methods to detect them. The purpose of this study was to examine the relationship between findings from CEUS and vascular pathology obtained by carotid endarterectomy (CEA). We focused on associations between small and large INVs evaluated by CEUS and histologically defined intraplaque hemorrhage. METHODS: Participants comprised 115 patients (mean age, 73.0 ± 7.2 years; 96 men) who underwent preoperative CEUS and underwent CEA. CEUS findings were evaluated as vascular grade at 0 min (Vas-G0) and 10 min (Vas-G10) after contrast injection. Plaques were histologically evaluated quantitatively for the total area of intraplaque hemorrhage, cholesterol, and calcification and the thinnest fibrous cap. Immunohistochemical studies were conducted using anti-CD-34 antibody as a marker for endothelial cells. INVs were divided into two groups depending on diameter: small INVs, <50 µm; and large INVs, ≥50 µm. The numbers of small and large blood vessels in the plaque were quantified histologically. Associations of small and large INVs with CEUS, plaque histology, and clinical findings were assessed by uni- and multivariable analyses. RESULTS: Multivariable analyses indicated that CEUS Vas-G0 was associated with the 4th quartile of the number of small INVs compared with other quartiles, and Vas-G10 was associated with the 4th quartile of the number of large INVs. Histologically, the presence and area of intraplaque hemorrhage were associated with the number of small INVs, while the increased number of large INVs was associated with infrequent plaque disruption and thicker fibrous cap. CONCLUSIONS: Our study showed that early phase enhancement in the CEUS can help identify plaque vulnerability by predicting a larger number of small INVs. This information can also help determine treatment strategies for carotid plaque.

The Value of Superb Microvascular Imaging and Contrast-enhanced Ultrasound for the Evaluation of Neovascularization in Carotid Artery Plaques.

OBJECTIVE: To compare the consistency between superb microvascular imaging (SMI) and contrast-enhanced ultrasound (CEUS) for the detection of neovascularization in carotid plaques of different thicknesses and to evaluate the applied value of these two methods for detecting neovascularization in carotid plaques in the clinic. METHODS: A total of 45 patients with carotid artery plaques who were diagnosed in our hospital involving 76 hypoechoic plaques with a thickness ≥2.0 mm were selected. According to thickness, the plaques were divided into three groups: 2.0-2.5 mm, 2.5-3.0 mm and ≥3.0 mm. Each group underwent both SMI and CEUS, and two experienced sonographers (A and B) analyzed the ultrasound images to evaluate the neovascularization of carotid plaques. The amount of the neovascular signal was assessed using a semi-quantitative grading scale (vascularity grade: grade 0-3). SMI and CEUS were graded respectively according to the visual methods as follows: grade 0: no blood flow signal/enhancement within plaques; grade 1: a few blood flow signals/enhancement within plaques; grade 2: medium blood flow signals/enhancement within plaques; and grade 3: extensive blood flow signals/enhancement within plaques. Kappa consistency test was used to analyze the consistency of the grade of neovascularization in plaques between SMI with CEUS. Gamma rank correlation analysis was used to examine the correlation between neovascularization grade by SMI and CEUS in plaque and plaque thickness. RESULTS: Of these patients, 14 had unilateral plaques and 31 had bilateral plaques. The two sonographers were highly consistent in terms of applying SMI and CEUS methods for diagnosing neovascularization in carotid plaques (Kappa values were 0.736 and 0.680>0). Consistency was found between SMI and CEUS by sonographers (sonographer A: Kappa=0.823; sonographer B: Kappa=0.842) in evaluating the neovascular grade in the carotid plaques. SMI and CEUS grades were positively correlated with plaque thickness (sonographer A: γ = 0.735 and 0.772; sonographer B: γ = 0.805 and 0.798). CONCLUSION: Neovascularization in carotid plaques was successfully detected by SMI in a manner that concurred well with CEUS results. Our data indicate that both CEUS and SMI have high diagnostic value for assessing the neovascularization of plaques.