Contrast Enhanced Ultrasound Molecular Imaging of Spontaneous Chronic Inflammatory Bowel Disease in an Interleukin-2 Receptor α-/- Transgenic Mouse Model Using Targeted Microbubbles.

Inflammatory bowel disease (IBD) is a lifelong inflammatory disorder with relapsing-remission cycles, which is currently diagnosed by clinical symptoms and signs, along with laboratory and imaging findings. However, such clinical findings are not parallel to the disease activity of IBD and are difficult to use in treatment monitoring. Therefore, non-invasive quantitative imaging tools are required for the multiple follow-up exams of IBD patients in order to monitor the disease activity and determine treatment regimens. In this study, we evaluated a dual P- and E-selectin-targeted microbubble (MBSelectin) in an interleukin-2 receptor α deficient (IL-2Rα-/-) spontaneous chronic IBD mouse model for assessing long-term anti-inflammatory effects with ultrasound molecular imaging (USMI). We used IL-2Rα-/- (male and female on a C57BL/6 genetic background; n = 39) and C57BL/6 wild-type (negative control; n = 6) mice for the study. USMI of the proximal, middle, and distal colon was performed with MBSelectin using a small animal scanner (Vevo 2100) up to six times in each IL-2Rα-/- mouse between 6-30 weeks of age. USMI signals were compared between IL-2Rα-/- vs. wild-type mice, and sexes in three colonic locations. Imaged colon segments were analyzed ex vivo for inflammatory changes on H&E-stained sections and for selectin expression by immunofluorescence staining. We successfully detected spontaneous chronic colitis in IL-2Rα-/- mice between 6-30 weeks (onset at 6-14 weeks) compared to wild-type mice. Both male and female IL-2Rα-/- mice were equally (p = 0.996) affected with the disease, and there was no significant (p > 0.05) difference in USMI signals of colitis between the proximal, middle, and distal colon. We observed the fluctuating USMI signals in IL-2Rα-/- mice between 6-30 weeks, which might suggest a resemblance of the remission-flare pattern of human IBD. The ex vivo H&E and immunostaining further confirmed the inflammatory changes, and the high expression of P- and E-selectin in the colon. The results of this study highlight the IL-2Rα-/- mice as a chronic colitis model and are suitable for the long-term assessment of treatment response using a dual P- and E-selectin-targeted USMI.

Ultrasound Molecular Imaging for the Guidance of Ultrasound-Triggered Release of Liposomal Doxorubicin and Its Treatment Monitoring in an Orthotopic Prostatic Tumor Model in Rat.

Liposome encapsulation of drugs is an interesting approach in cancer therapy to specifically release the encapsulated drug at the desired treatment site. In addition to thermo-, pH-, light-, enzyme- or redox-responsive liposomes, which have had promising results in (pre-) clinical studies, ultrasound-triggered sonosensitive liposomes represent an exciting alternative to locally trigger the release from these cargos. Localized drug release requires precise tumor visualization to produce a targeted and ultrasound stimulus. We used ultrasound molecular imaging (USMI) with BR55, a vascular endothelial growth factor receptor 2 (VEGFR2)-targeted ultrasound contrast agent, to guide ultrasound-triggered release of sonosensitive liposomes encapsulating doxorubicin (L-DXR) in an orthotopic prostatic rodent tumor model. Forty-eight hours after L-DXR injection, local release of doxorubicin was triggered with a confocal ultrasound device with two focused transducers, 1.1-MHz center frequency, and peak positive and negative pressures of 20.5 and 13 MPa at focus. Tumor size decreased by 20% in 2 wk with L-DXR alone (n = 9) and by 70% after treatment with L-DXR and confocal ultrasound (n = 7) (p < 0.01). The effect of doxorubicin on perfusion/vascularity and VEGFR2 expression was evaluated by USMI and immunohistochemistry of CD31 and VEGFR2 and did not reveal differences in perfusion or VEGFR2 expression in the absence or after the triggered release of liposomes. USMI can provide precise guidance for ultrasound-triggered release of liposomal doxorubicin mediated by a confocal ultrasound device; moreover, the combination of B-mode imaging and USMI can help to follow the response of the tumor to the therapy.

Microfluidic preparation of various perfluorocarbon nanodroplets: Characterization and determination of acoustic droplet vaporization (ADV) threshold.

Over the last two decades, liquid perfluorocarbon nanodroplets (PFC-NDs), also known as Phase Change Contrast Agents (PCCAs), that are capable of vaporizing into gaseous echogenic microbubbles via an external stimulus, have gained much attention for diagnostic and therapeutic applications. In the present work, a microfluidic platform is evaluated for the preparation of various size-controlled nanodroplets. Here, two major lines of investigations were carried out. The first was to define the microfluidic device settings for the preparation of nanodroplets depending on the nature of the encapsulating shell such as lipids, fluorinated surfactants and PLGA biopolymers as well as the liquid perfluorocarbon core (perfluoropentane, perfluorohexane). Specifically, the effect of the microfluidic system parameters, such as total flow rate and flow rate ratio on PFC-NDs attributes including size and uniformity was assessed. Secondly, a custom-made set-up, based on echogenicity signals from produced bubbles, was designed and successfully applied to determine the Acoustic Droplet Vaporization (ADV) threshold of PFC-NDs. Finally, the influence of various formulation parameters on the vaporization outcome was investigated depending on the PFC type and the encapsulating shell composition (soft versus hard shells). This study indicates the usefulness of this novel formulation platform enabling the rapid design and optimization of narrowly dispersed nanodroplets at a reliable yield and ultimately accelerate nanomedicines development.

Ultrasound Molecular Imaging With BR55, a Predictive Tool of Antiangiogenic Treatment Efficacy in a Chemo-Induced Mammary Tumor Model.

OBJECTIVES: The aim of this study was to evaluate the added value of ultrasound molecular imaging of the vascular growth factor receptor 2 (VEGFR2) expression, using the clinical grade contrast agent BR55, for the early evaluation of antiangiogenic treatment efficacy in a chemo-induced rat mammary tumor model. MATERIALS AND METHODS: In this preclinical study, chemo-induced rat mammary tumors were obtained after a single injection of N-nitroso-N-methylurea intraperitoneally in 46 prepubescent (age 38 ± 2 days) female rats. All experiments were performed under the authorization of the Direction Générale de la Santé, Geneva, Switzerland. Once tumor reached 0.8 cm in the largest cross-section, animals were enrolled in a sunitinib- or vehicle-treated group. Ultrasound molecular imaging was performed using BR55, a clinical grade targeted contrast agent against VEGFR2, before therapy and up to 72 hours. Anatomical changes of tumor over time, that is, area of the tumor largest cross-section and tumor volume, were measured in B-mode. Signal from microbubbles was detected in a nonlinear contrast mode (power modulation) using the iU22 diagnostic ultrasound system (Phillips, United States) equipped with a L12-5 linear transducer (transmit frequency 5 MHz). Peak enhancement and wash-in area under the curve were extracted from the time intensity curves generated by a dedicated quantification software for contrast ultrasound, so-called VueBox (Bracco Suisse SA, Switzerland). The signal of bound BR55 microbubbles in the tumor was quantified 10 minutes after injection. Altogether, these parameters were used to monitor tumoral response to treatment at the anatomical, functional, and molecular levels. At each time point, a cohort of tumors was harvested for the assessment of CD31 and VEGFR2 expression by immunohistochemistry staining. RESULTS: Under sunitinib therapy, assessment of the expression of VEGFR2 by ultrasound molecular imaging with BR55 reveals a significant difference as early as 12 hours after first dosing (-25%), whereas tumor size significant change occurs only after 24 hours. At the end of the therapeutic protocol, 72 hours after the onset of treatment, molecular changes are more marked with a 80% decrease compared with only ~40% for the anatomic parameters. Ultrasound molecular imaging observations suggesting a decrease in VEGFR2 expression in treated tumors were corroborated by semiquantitative grading of VEGFR2, showing a decrease expression over time. Functional parameters measured in the perfusion phase also show a decrease along treatment, significant for 24 hours and of 48% of peak enhancement at the end of protocol. CONCLUSIONS: Anatomical, functional, and molecular evaluations are feasible in a single examination using BR55 ultrasound targeted contrast agent. Ultrasound molecular imaging of VEGFR2 can depict an early response to antiangiogenic treatment in a rat mammary tumor model. This imaging modality has a potential for early assessment of each patient's response, which could be useful to take decisions on therapeutic protocol, providing as such an imaging tool for personalized medicine.

Microbubble Agents: New Directions.

Microbubble ultrasound contrast agents have now been in use for several decades and their safety and efficacy in a wide range of diagnostic applications have been well established. Recent progress in imaging technology is facilitating exciting developments in techniques such as molecular, 3-D and super resolution imaging and new agents are now being developed to meet their specific requirements. In parallel, there have been significant advances in the therapeutic applications of microbubbles, with recent clinical trials demonstrating drug delivery across the blood-brain barrier and into solid tumours. New agents are similarly being tailored toward these applications, including nanoscale microbubble precursors offering superior circulation times and tissue penetration. The development of novel agents does, however, present several challenges, particularly regarding the regulatory framework. This article reviews the developments in agents for diagnostic, therapeutic and "theranostic" applications; novel manufacturing techniques; and the opportunities and challenges for their commercial and clinical translation.

Chronic Model of Inflammatory Bowel Disease in IL-10-/- Transgenic Mice: Evaluation with Ultrasound Molecular Imaging.

Objective: Acute mouse models of inflammatory bowel disease (IBD) fail to mirror the chronic nature of IBD in patients. We sought to develop a chronic mouse IBD model for assessing long-term anti-inflammatory effects with ultrasound molecular imaging (USMI) by using dual P- and E-selectin targeted microbubbles (MBSelectin). Materials and Methods: Interleukin 10 deficient (IL-10-/- on a C57BL/6 genetic background; n=55) and FVB (n=16) mice were used. In IL-10-/-mice, various experimental regimens including piroxicam, 2,4,6-trinitrobenzenesulfonic acid (TNBS) or dextran sulfate sodium (DSS), respectively were used for promoting colitis; colitis was induced with DSS in FVB mice. Using clinical and small animal ultrasound scanners, evolution of inflammation in proximal, middle and distal colon, was monitored with USMI by using MBSelectin at multiple time points. Imaged colon segments were analyzed ex vivo for inflammatory changes on H&E staining and for P-selectin expression on immunofluorescence staining. Results: Sustained colitis was not detected with USMI in IL-10-/- or FVB mice with various experimental regimens. USMI signals either gradually decreased after the colitis enhancing/inducing drug/agents were discontinued, or the mortality rate of mice was high. Inflammation was observed on H&E staining in IL-10-/- mice with piroxicam promotion, while stable overexpression of P-selectin was not found on immunofluorescence staining in the same mice. Conclusion: Sustained colitis in IL-10-/- mice induced with piroxicam, TNBS or DSS, and in FVB mice induced with DSS, was not detected with USMI using MBSelectin, and this was verified by immunofluorescence staining for inflammation marker P-selectin. Thus, these models may not be appropriate for long-term monitoring of chronic colitis and subsequent treatment response with dual-selectin targeted USMI.

US Molecular Imaging of Acute Ileitis: Anti-Inflammatory Treatment Response Monitored with Targeted Microbubbles in a Preclinical Model.

Purpose To evaluate whether dual-selectin-targeted US molecular imaging allows longitudinal monitoring of anti-inflammatory treatment effects in an acute terminal ileitis model in swine. Materials and Methods The Institutional Animal Care and Use Committee approved all animal studies. Fourteen swine with chemically induced acute terminal ileitis (day 0) were randomized into the following groups: (a) an anti-inflammatory treatment group (n = 8; meloxicam, 0.25 mg per kilogram of body weight; prednisone, 0.5 mg/kg) and (b) a control group (n = 6; saline). US molecular imaging was performed with a clinical US machine after intravenous injection of clinically translatable dual P- and E-selectin-targeted microbubbles (5 × 108/kg). Three inflamed bowel segments per swine were imaged at baseline, as well as on days 1, 3, and 6 after treatment initiation. At day 6, bowel segments were analyzed ex vivo for selectin expression levels by using quantitative immunofluorescence. Results After induction of inflammation, US molecular imaging signal increased at day 1 in both animal groups (P < .001). At day 3, signal in the treatment group decreased (P < .001 vs day 1), while signal in control animals did not significantly change (P = .18 vs day 1) and was higher (P = .001) compared with that in the treatment group. At day 6, signal in the treatment group further decreased and remained lower (P = .02) compared with that in the control group. Immunofluorescence confirmed significant (P ≤ .04) downregulation of both P- and E-selectin expression levels in treated versus control bowel segments. Conclusion Dual-selectin-targeted US molecular imaging allows longitudinal monitoring of anti-inflammatory treatment effects in a large-animal model of acute ileitis. This supports further clinical development of this quantitative and radiation-free technique for monitoring inflammatory bowel disease. © RSNA, 2018 Online supplemental material is available for this article.

Anatomical Road Mapping Using CT and MR Enterography for Ultrasound Molecular Imaging of Small Bowel Inflammation in Swine.

OBJECTIVES: To evaluate the feasibility and time saving of fusing CT and MR enterography with ultrasound for ultrasound molecular imaging (USMI) of inflammation in an acute small bowel inflammation of swine. METHODS: Nine swine with ileitis were scanned with either CT (n = 3) or MR (n = 6) enterography. Imaging times to load CT/MR images onto a clinical ultrasound machine, fuse them to ultrasound with an anatomical landmark-based approach, and identify ileitis were compared to the imaging times without anatomical road mapping. Inflammation was then assessed by USMI using dual selectin-targeted (MBSelectin) and control (MBControl) contrast agents in diseased and healthy control bowel segments, followed by ex vivo histology. RESULTS: Cross-sectional image fusion with ultrasound was feasible with an alignment error of 13.9 ± 9.7 mm. Anatomical road mapping significantly reduced (P < 0.001) scanning times by 40%. Localising ileitis was achieved within 1.0 min. Subsequently performed USMI demonstrated significantly (P < 0.001) higher imaging signal using MBSelectin compared to MBControl and histology confirmed a significantly higher inflammation score (P = 0.006) and P- and E-selectin expression (P ≤ 0.02) in inflamed vs. healthy bowel. CONCLUSIONS: Fusion of CT and MR enterography data sets with ultrasound in real time is feasible and allows rapid anatomical localisation of ileitis for subsequent quantification of inflammation using USMI. KEY POINTS: • Real-time fusion of CT/MRI with ultrasound to localise ileitis is feasible. • Anatomical road mapping using CT/MRI significantly decreases the scanning time for USMI. • USMI allows quantification of inflammation in swine, verified with ex vivo histology.

Characteristics and Echogenicity of Clinical Ultrasound Contrast Agents: An In Vitro and In Vivo Comparison Study.

OBJECTIVES: To compare physicochemical characteristics and in vitro and in vivo contrast-enhanced ultrasound imaging performance of 3 commercially available ultrasound contrast agents: SonoVue (Bracco Imaging SpA, Colleretto Giacosa, Italy; also marketed as Lumason in the USA), Definity (Lantheus Medical Imaging, North Billerica, MA) and Optison (GE Healthcare AS, Oslo, Norway). METHODS: Physicochemical characteristics were measured with a Multisizer Coulter Counter (Beckman Coulter, Fullerton, CA). Two ultrasound systems (Aplio 500; Toshiba Medical Systems Corp, Tochigi-ken, Japan; and Logiq E9; GE Healthcare, Little Chalfont, England) were used with different transducers. Contrast enhancement was measured in vitro by dose-ranging measurements using a custom-built beaker setup; in vivo imaging performances were compared in pigs (heart and liver) and rabbits (liver). Quantitative analyses were performed with VueBox quantification software (Bracco Suisse SA, Plan-les-Ouates, Switzerland). RESULTS: Measured physicochemical characteristics were in agreement with those provided by the manufacturers. In vitro data demonstrated that the performance of SonoVue was similar to or better than that of Definity but superior to Optison (normalized scattered power 2- to 10-fold higher with SonoVue). Similar results were obtained in vivo, although the duration of enhancement in the pig heart was longer for SonoVue compared to Definity, and quantitative analysis revealed higher enhancement for SonoVue (1.5-fold increase). For liver imaging, SonoVue and Definity showed similar contrast enhancement and duration of enhancement, but compared to Optison, both peak enhancement and duration of enhancement were superior for SonoVue (up to 2-fold increase). CONCLUSIONS: Imaging performance of SonoVue was similar to or slightly better than that of Definity, but it was superior to Optison for the conditions used in this study.

Design of Microbubbles for Gene/Drug Delivery.

The role of ultrasound contrast agents (UCA) initially designed for diagnosis has evolved towards a therapeutic use. Ultrasound (US) for triggered drug delivery has many advantages. In particular, it enables a high spatial control of drug release, thus potentially allowing activation of drug delivery only in the targeted region, and not in surrounding healthy tissue. Moreover, UCA imaging can also be used firstly to precisely locate the target region to, and then used to monitor the drug delivery process by tracking the location of release occurrence. All these features make UCA and ultrasound attractive means to mediate drug delivery. The three main potential clinical indications for drug/gene US delivery are (i) the cardiovascular system, (ii) the central nervous system for small molecule delivery, and (iii) tumor therapy using cytotoxic drugs. Although promising results have been achieved in preclinical studies in various animal models, still very few examples of clinical use have been reported. In this chapter will be addressed the aspects pertaining to UCA formulation (chemical composition, mode of preparation, analytical methods…) and the requirement for a potential translation into the clinic following approval by regulatory authorities.

Quantitative Assessment of Inflammation in a Porcine Acute Terminal Ileitis Model: US with a Molecularly Targeted Contrast Agent.

PURPOSE: To evaluate the feasibility and reproducibility of ultrasonography (US) performed with dual-selectin-targeted contrast agent microbubbles (MBs) for assessment of inflammation in a porcine acute terminal ileitis model, with histologic findings as a reference standard. MATERIALS AND METHODS: The study had institutional Animal Care and Use Committee approval. Acute terminal ileitis was established in 19 pigs; four pigs served as control pigs. The ileum was imaged with clinical-grade dual P- and E-selectin-targeted MBs (MBSelectin) at increasing doses (0.5, 1.0, 2.5, 5.0, 10, and 20 × 10(8) MB per kilogram of body weight) and with control nontargeted MBs (MBControl). For reproducibility testing, examinations were repeated twice after the MBSelectin and MBControl injections. After imaging, scanned ileal segments were analyzed ex vivo both for inflammation grade (by using hematoxylin-eosin staining) and for expression of selectins (by using quantitative immunofluorescence analysis). Statistical analysis was performed by using the t test, intraclass correlation coefficients (ICCs), and Spearman correlation analysis. RESULTS: Imaging signal increased linearly (P < .001) between a dose of 0.5 and a dose of 5.0 × 10(8) MB/kg and plateaued between a dose of 10 and a dose of 20 × 10(8) MB/kg. Imaging signals were reproducible (ICC = 0.70), and administration of MBSelectin in acute ileitis resulted in a significantly higher (P < .001) imaging signal compared with that in control ileum and MBControl. Ex vivo histologic grades of inflammation correlated well with in vivo US signal (ρ = 0.79), and expression levels of both P-selectin (37.4% ± 14.7 [standard deviation] of vessels positive; P < .001) and E-selectin (31.2% ± 25.7) in vessels in the bowel wall of segments with ileitis were higher than in control ileum (5.1% ± 3.7 for P-selectin and 4.8% ± 2.3 for E-selectin). CONCLUSION: Quantitative measurements of inflammation obtained by using dual-selectin-targeted US are reproducible and correlate well with the extent of inflammation at histologic examination in a porcine acute ileitis model as a next step toward clinical translation.

Ultrasound molecular imaging of transient acute myocardial ischemia with a clinically translatable P- and E-selectin targeted contrast agent: correlation with the expression of selectins.

OBJECTIVE: The diagnosis of acute coronary syndrome remains challenging especially in patients without clear symptoms or electrocardiographic and/or biomarker features. A hallmark of ischemia/reperfusion is activation of endothelial cells leading to altered expression of molecular markers, including selectins. In this context, we aimed to validate the value of ultrasound molecular imaging for detecting transient myocardial ischemia by using a clinically translatable dual P- and E-selectin-targeted ultrasound contrast agent (UCA) and microbubble (MB(selectin)). MATERIAL AND METHODS: Transient (20 minutes) myocardial ischemia of rat heart was produced by ligation of the left anterior descending coronary artery ligation followed by 2-, 5-, or 24-hour reperfusion. Imaging of the transient ischemic event was achieved by the use of MB(selectin). Performance of this clinically translatable targeted UCA was compared with that of antibody-targeted streptavidin MBs. Finally, immunohistochemistry staining of rat myocardial ischemic tissue was performed to assess expression of selectins accessible to targeted UCA. RESULTS: In rats subjected to myocardial ischemia (20 minutes) followed by reperfusion (2 hours), injection of MB(selectin) produced high late phase (ie, 10-minute postinjection) ultrasound molecular imaging enhancement in the myocardium, which colocalized with the ischemic area. Late phase enhancement persisted 5 and 24 hours after reperfusion. Similarly, the use of MBP and MBE, comprising antibodies specific for P- and E-selectin, respectively, showed high late-phase enhancement within the ischemic area compared with remote myocardial tissue. Two and 5 hours after ischemia has resolved, a persistent expression of these 2 selectins was detected. After 24 hours of reperfusion, only MBE produced late phase enhancement within the ischemic myocardium. Immunohistochemical findings revealed that both P- and E-selectin were expressed and accessible on the surface of the activated endothelium 2 and 5 hours after the acute ischemic event, whereas only E-selectin remained accessible after 24 hours. CONCLUSIONS: Ultrasound molecular imaging of transient myocardial ischemia using dual selectin-targeted UCA is able to monitor the time course of expression of selectins after resolution of the ischemic event, paving the way for a large clinical diagnostic window.

Molecular imaging of inflammation in inflammatory bowel disease with a clinically translatable dual-selectin-targeted US contrast agent: comparison with FDG PET/CT in a mouse model.

PURPOSE: To develop and test a molecular imaging approach that uses ultrasonography (US) and a clinically translatable dual-targeted (P- and E-selectin) contrast agent (MBSelectin) in the quantification of inflammation at the molecular level and to quantitatively correlate selectin-targeted US with fluorodeoxyglucose (FDG) combined positron emission tomography (PET) and computed tomography (CT) in terms of visualization and quantification of different levels of inflammation in a murine acute colitis model. MATERIALS AND METHODS: Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care at Stanford University. MBSelectin was developed by covalently binding an analog of the naturally occurring binding ligand P-selectin glycoprotein ligand 1 fused to a human fragment crystallizable(or Fc) domain onto the lipid shell of perfluorobutane and nitrogen-containing MBs. Binding specificity of MBSelectin was assessed in vitro with a flow chamber assay and in vivo with a chemically induced acute colitis murine model. US signal was quantitatively correlated with FDG uptake at PET/CT and histologic grade. Statistical analysis was performed with the Student t test, analysis of variance, and Pearson correlation analysis. RESULTS: MBSelectin showed strong attachment to both human and mouse P- and E-selectin compared with MBControl in vitro (P ≤ .002). In vivo, US signal was significantly increased (P < .001) in mice with acute colitis (173.8 arbitrary units [au] ± 134.8 [standard deviation]) compared with control mice (5.0 au ± 4.5). US imaging signal strongly correlated with FDG uptake on PET/CT images (ρ = 0.89, P < .001). Ex vivo analysis enabled confirmation of inflammation in mice with acute colitis and high expression levels of P- and E-selectin in mucosal capillaries (P = .014). CONCLUSION: US with MBSelectin specifically enables detection and quantification of inflammation in a murine acute colitis model, leveraging the natural pathway of leukocyte recruitment in inflammatory tissue. US imaging with MBSelectin correlates well with FDG uptake at PET/CT imaging.

Ultrasound-mediated stimulation of microbubbles after acute myocardial infarction and reperfusion ameliorates left-ventricular remodelling in mice via improvement of borderzone vascularization.

AIMS: Post-infarction remodelling (PIR) determines left-ventricular (LV) function and prognosis after myocardial infarction. The aim of this study was to evaluate transthoracic ultrasound-mediated microbubble stimulation (UMS) as a novel gene- and cell-free therapeutic option after acute myocardial infarction and reperfusion (AMI/R) in mice. METHODS AND RESULTS: For myocardial delivery of UMS, a novel therapeutic ultrasound-system (TIPS, Philips Medical) and commercially available microbubbles (BR1, Bracco Suisse SA) were utilized in a closed-chest mouse model. UMS was performed as myocardial post-conditioning (PC) on day four after 30 minutes of coronary occlusion and reperfusion. LV-morphology, as well as global and regional function were measured repeatedly with reconstructive 3-dimensional echocardiography applying an additional low-dose dobutamine protocol after two weeks. Scar size was quantified by means of histomorphometry. A total of 41 mice were investigated; 17 received PC with UMS. Mean ejection fraction (EF) prior UMS was similar in both groups 53%±10 (w/o UMS) and 53%±14 (UMS, p = 0.5), reflecting comparable myocardial mass at risk 17%±8 (w/o UMS), 16%±13 (UMS, p = 0.5). Two weeks after AMI/R, mice undergoing UMS demonstrated significantly better global LV-function (EF = 53%±7) as compared to the group without PC (EF = 39%±11, p<0.01). The fraction of akinetic myocardial mass was significantly lower among mice undergoing UMS after AMI/R [27%±10 (w/o UMS), 13%±8 (UMS), p<0.001)]. Our experiments showed a fast onset of transient, UMS-induced upregulation of vascular-endothelial and insulin-like growth factor (VEGF-a, IGF-1), as well as caveolin-3 (Cav-3). The mice undergoing PC with UMS after AMI/R showed a significantly lower scar size. In addition, the microvascular density was significantly higher in the borderzone of UMS-treated animals. CONCLUSION: UMS following AMI/R ameliorates PIR in mice via up-regulation of VEGF-a, IGF-1 and Cav-3, and consecutive improvement of myocardial borderzone vascularization.

The increase of VEGF secretion from endothelial progenitor cells post ultrasonic VEGF gene delivery enhances the proliferation and migration of endothelial cells.

We investigated the feasibility of exogenous gene expression in endothelial progenitor cells (EPCs) through the use of ultrasonic microbubble transfection (UMT). EPCs originating from porcine peripheral blood were cultured in a medium containing constructed vascular endothelial growth factor (VEGF) pDNA followed by UMT. Simultaneously, comprehensive functional evaluations were conducted to investigate the effects of UMT of the VEGF gene on the EPCs. The results showed that UMT yielded significant VEGF protein expression. VEGF-containing supernatant originating from EPCs post UMT led to significantly enhanced activities of proliferation by more than 20% and migration by approximately 30% in human aortic endothelial cells. The duration of additional secretion of VEGF protein attributable to the exogenous VEGF gene in the EPCs post UMT lasted more than 96 hours. In conclusion, UMT successfully delivers the VEGF gene into porcine EPCs, and VEGF-containing supernatant derived from EPCs post UMT enhances the proliferation and migration of human aortic endothelial cells.

Ultrasound molecular imaging contrast agent binding to both E- and P-selectin in different species.

OBJECTIVES: Ultrasound molecular imaging is increasingly used in preclinical studies to measure the expression of vascular markers during inflammation process. In this context, a new ultrasound contrast agent functionalized with a recombinant P-selectin glycoprotein ligand-1 analogue (rPSGL-Ig) was developed (MBrPSGL-Ig). This agent was assayed in vitro and in vivo to evaluate its binding performance and potential to image expression of inflammatory markers E- and P-selectin. Performance of this newly developed agent was compared with that of antibody (MBAb) or sialyl Lewis X (MBsLe) containing microbubbles and with control microbubbles (MBC). MATERIALS AND METHODS: The targeted ultrasound contrast agents were prepared by coupling biotin-conjugated ligands onto streptavidin-functionalized microbubbles. First, in vitro experiments were performed to measure the adhesion efficiency of these microbubble constructs under static or flow conditions (114 sec), on cell monolayer (human umbilical vein endothelial cells and bEnd.5), or coatings of E- or P-selectin of various animal species, respectively. Second, molecular imaging studies were performed in a rat inflammatory model 24 hours after intramuscular injection of lipopolysaccharide in the hind limb. Finally, immunohistochemistry staining of rat inflamed muscle tissue was performed to assess expression of E- and P-selectin. RESULTS: Microbubbles functionalized with rPSGL-Ig (MBrPSGL-Ig) displayed firm in vitro binding on the coating of both recombinant E- or P-selectin, with an efficiency similar to microbubbles comprising antibody specific for E-selectin (MBE) or P-selectin (MBP). In contrast, lower binding capacity was measured with MBsLe. At the surface of inflamed endothelial cells, MBrPSGL-Ig were able to interact specifically with E- and P-selectin. Binding specificity was demonstrated by performing blocking experiments with target-specific antibodies, resulting in an 80% to 95% decrease in binding. Ten minutes after microbubble injection, echo signal measured with MBrPSGL-Ig in the inflamed muscles was 20-fold higher compared with MBC. Moreover, the in vivo adhesion of MBrPSGL-Ig was 2- and 7-fold higher compared with P-selectin or E-selectin-specific microbubbles, respectively. Immunohistochemistry revealed a temporal coexpression of E- and P-selectin in the vascular bed of inflamed rat muscle 24 hours after lipopolysaccharide injection. CONCLUSION: The molecular imaging study demonstrates that MBrPSGL-Ig provide imaging signal higher than those measured with antibody or sialyl Lewis X containing microbubbles. These results suggest that MBrPSGL-Ig is a powerful agent to image the expression of both E- and P-selectin in the context of an inflammatory process.

Use of intravital microscopy to study the microvascular behavior of microbubble-based ultrasound contrast agents.

PURPOSE: The study describes the use of intravital microscopy (IVM) to assess the behavior of ultrasound contrast agents (UCAs), including targeted UCAs, in the microcirculation of rodents. MATERIALS AND METHODS: IVM was performed on various exteriorized organs: hamster cheek pouch, rat mesentery, liver, spinotrapezius muscle, and mouse cremaster muscle. A dorsal skin-fold chamber with MatBIII tumor cells was also implanted in rats. Nontargeted UCAs (SonoVue(®) and BR14) and targeted UCAs (BR55 and P-selectin targeted microbubbles) were tested. IVM was used to measure microbubble size, determine their persistence, and observe their behavior in the blood circulation. RESULTS: Intravenous and intra-arterial injections of high doses of UCAs did not modify the local microvascular hemodynamics. No microbubble coalescence and no increased size were observed. Adhesion of some microbubbles to leukocytes was observed in various microcirculation models. Microbubbles are captured by Kupffer cells in the liver. Targeted microbubbles were shown to adhere specifically to endothelial receptors without compromising local blood flow. CONCLUSION: These results support the safety of both targeted and nontargeted UCAs as no microvascular flow alteration or plugging of microvessels were observed. They confirm that binding observed with targeted microbubbles are due to the binding of these microbubbles to specific endothelial receptors.

A minimally-invasive closed chest myocardial occlusion-reperfusion model in rhesus monkeys (Macaca mulatta): monitoring by contrast-enhanced ultrasound imaging.

Myocardial infarction is frequently developed in canine and porcine models but exceptionally in non-human primates. The aim of this study was to develop a minimally invasive myocardial ischemic/reperfusion model in the monkey intended to be combined with imaging techniques, in particular myocardial contrast echocardiography (MCE). A balloon-tipped catheter was advanced via the femoral artery into the left anterior descending artery (LAD) under fluoroscopic guidance in ten anaesthetized male rhesus monkeys (Macaca mulatta). The balloon was inflated to completely occlude the vessel. Coronary angiography (CA) was performed to control the reality of the LAD occlusion/reperfusion. The ischemia period was followed by 3-6 h of reperfusion. Myocardial perfusion was evaluated during ischemia and at reperfusion by MCE using a novel ultrasound contrast agent (BR38). Occlusion was successfully induced during 18-50 min in nine out of the ten evaluated monkeys. ST segment elevation indicated myocardial ischemia. MCE showed complete transmural arrest of myocardial blood flow during the ischemia period and no persistent microvascular perfusion defects during reperfusion. A minimally invasive closed-chest model was successfully developed for creating myocardial ischemia in the rhesus monkey (Macaca mulatta). This technique could have an important role in mimicking acute coronary syndrome under physiologically and ethically-acceptable conditions. MCE provides non-invasively information on myocardial perfusion status, information not available from CA.

Upregulation of coronary endothelial P-selectin in a monkey heart ischemia reperfusion model.

The design of targeted ultrasound contrast agents for molecular imaging of myocardial ischemia-reperfusion (IR) requires the availability of an adequate in vivo model in a species in which cross reactivity with the target occurs. P-selectin (Psel) is an activation-dependent endothelial receptor that supports rapid and reversible cell adhesion in a flowing system. Together with E- and L-selectins it constitutes the selectin family of adhesion molecules. We investigated the myocardial expression of selectins in a closed chest minimally invasive monkey myocardial IR model. Catheter-based occlusion (30-50 min) followed by reperfusion (3 or 5 h) of left anterior descending artery (LAD) was performed in anesthetised monkeys. At the end of each procedure animals were killed, and their hearts were excised. The tissues were analyzed immunohistochemically using an anti-human Psel antibody (AK-6 clone) that cross reacts with rhesus monkey. Histopathological features confirm the presence of IR injuries in myocardial tissues. There was significant increase in the Psel expression in vessels from the IR areas. However, significantly higher Psel immunoreactivity was also seen in areas which are distant from IR injuries.

Ultrasonic microbubble-mediated gene delivery causes phenotypic changes of human aortic endothelial cells.

Ultrasound, in combination with microbubbles, serves as a feasible nonviral method in vascular gene delivery. However, the effects of ultrasonic microbubble transfection (UMT) on vascular endothelial cells remained unclear. We therefore investigated whether UMT itself causes phenotypic changes of the human aortic endothelial cells (HAEC) in vitro. HAEC were cultured with solution containing luciferase reporter gene and microbubbles followed by exposure to ultrasound of selected parameters. Thereafter, the proliferation and migration activities of HAEC were investigated. Real-time RT-PCR and/or western blotting were performed to assess expression profile of HAEC, including growth-related factors (vascular endothelial growth factor, fins-like tyrosine kinase-1 [Flt-1] and kinase insert domain-containing receptor [KDR]), coagulatory factor (von Willebrand factor), vasodilatory enzyme (endothelial nitric oxide synthase), gap junctional protein connexin43 and adhesion molecules (P-selectin, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1). The results showed that in conditions where UMT lead to expression of luciferase, proliferation capacity is enhanced (p<0.001), partly attributable to the effect of ultrasound (p<0.05), after excluding the effect of contact inhibition. In addition, the expression of KDR and Flt-1 were found increased at either the mRNA level, protein level, or both (p<0.05). Other markers did not have significant changes (all p>0.2). Similarly, the migration capacity was minimally changed (p>0.3). In conclusion, UMT causes phenotypic changes of HAEC by enhancing proliferation and upregulating KDR and Flt-1, while possesses no obvious adverse effect on viable transfected cells. Further investigation is required to clarify the impact of these changes by UMT in vivo.