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.

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.

Safety, Tolerability, and EEG-Based Target Engagement of STP1 (PDE3,4 Inhibitor and NKCC1 Antagonist) in a Randomized Clinical Trial in a Subgroup of Patients with ASD.

This study aimed to evaluate the safety and tolerability of STP1, a combination of ibudilast and bumetanide, tailored for the treatment of a clinically and biologically defined subgroup of patients with Autism Spectrum Disorder (ASD), namely ASD Phenotype 1 (ASD-Phen1). We conducted a randomized, double-blind, placebo-controlled, parallel-group phase 1b study with two 14-day treatment phases (registered at clinicaltrials.gov as NCT04644003). Nine ASD-Phen1 patients were administered STP1, while three received a placebo. We assessed safety and tolerability, along with electrophysiological markers, such as EEG, Auditory Habituation, and Auditory Chirp Synchronization, to better understand STP1's mechanism of action. Additionally, we used several clinical scales to measure treatment outcomes. The results showed that STP1 was well-tolerated, with electrophysiological markers indicating a significant and dose-related reduction of gamma power in the whole brain and in brain areas associated with executive function and memory. Treatment with STP1 also increased alpha 2 power in frontal and occipital regions and improved habituation and neural synchronization to auditory chirps. Although numerical improvements were observed in several clinical scales, they did not reach statistical significance. Overall, this study suggests that STP1 is well-tolerated in ASD-Phen1 patients and shows indirect target engagement in ASD brain regions of interest.

Characterization of a Clinically and Biologically Defined Subgroup of Patients with Autism Spectrum Disorder and Identification of a Tailored Combination Treatment.

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental disorders (NDDs) with a high unmet medical need. The diagnosis of ASD is currently based on behavior criteria, which overlooks the diversity of genetic, neurophysiological, and clinical manifestations. Failure to acknowledge such heterogeneity has hindered the development of efficient drug treatments for ASD and other NDDs. DEPI® (Databased Endophenotyping Patient Identification) is a systems biology, multi-omics, and machine learning-driven platform enabling the identification of subgroups of patients with NDDs and the development of patient-tailored treatments. In this study, we provide evidence for the validation of a first clinically and biologically defined subgroup of patients with ASD identified by DEPI, ASD Phenotype 1 (ASD-Phen1). Among 313 screened patients with idiopathic ASD, the prevalence of ASD-Phen1 was observed to be ~24% in 84 patients who qualified to be enrolled in the study. Metabolic and transcriptomic alterations differentiating patients with ASD-Phen1 were consistent with an over-activation of NF-κB and NRF2 transcription factors, as predicted by DEPI. Finally, the suitability of STP1 combination treatment to revert such observed molecular alterations in patients with ASD-Phen1 was determined. Overall, our results support the development of precision medicine-based treatments for patients diagnosed with ASD.

The high angiogenic activity in very early breast cancer enables reliable imaging with VEGFR2-targeted microbubbles (BR55).

OBJECTIVES: Tumour xenografts of well-discernible sizes can be examined well by molecular ultrasound. Here, we investigated whether very early breast carcinomas express sufficient levels of VEGFR2 for reliable molecular ultrasound imaging with targeted microbubbles. METHODS: MCF-7 breast cancer xenografts were orthotopically implanted in nude mice (n = 26). Tumours measuring from 4 mm(3) (2 mm diameter) up to 65 mm(3) (5 mm diameter) were examined with automated 3D molecular ultrasound using clinically translatable VEGFR2-targeted microbubbles (BR55). Additionally, the relative tumour blood volume was assessed with non-targeted microbubbles (BR38). In vivo ultrasound data were validated by quantitative immunohistochemistry. RESULTS: Very small lesions 2 mm in diameter showed the highest binding of VEGFR2-specific microbubbles. In larger tumours significantly less BR55 accumulated (p = 0.023). Nonetheless, binding of VEGFR2-targeted microbubbles was still high enough for imaging. The relative blood volume was comparable at all tumour sizes. Both findings were confirmed by immunohistochemistry. Additionally, a significantly enhanced number of large and mature vessels were detected with increasing tumour size (p < 0.01), explaining the decrease in VEGFR2 expression during tumour growth. CONCLUSIONS: 3D molecular ultrasound using BR55 is very well suited to depicting the angiogenic activity in very small breast lesions, suggesting its potential for detecting and characterising these lesions.

Percutaneous removal of sentinel lymph nodes in a swine model using a breast lesion excision system and contrast-enhanced ultrasound.

OBJECTIVES: To investigate the feasibility of percutaneous removal of the entire sentinel lymph node (SLN) in an animal model using a breast lesion excision system after identifying these nodes using contrast-enhanced ultrasound (CEUS) and intradermal microbubbles. METHODS: Animal studies approval was obtained. SLNs were identified using CEUS and intradermal injection of microbubbles in two young pigs. Microbubbles were mixed with blue dye and injected around the mammary papillae to access lymphatic drainage to the superficial inguinal lymph nodes. When enhancing nodes were identified, the breast lesion excision system (BLES) was used to remove these nodes percutaneously. Both animals then underwent surgical lymph node dissection. Histopathological examination of all the samples was performed. RESULTS: Removal of the entire SLN was successful in three groins in the pigs. All three nodes were stained with blue dye. No other stained nodes were observed in the node dissection specimens. The nodal architecture of removed lymph nodes was well preserved on microscopy. There were no signs of excess trauma within the biopsy bed. CONCLUSION: The results obtained from the swine model demonstrated that it is feasible to remove the entire SLN percutaneously under the guidance of CEUS and microbubbles. KEY POINTS: Intradermal injection of microbubbles and CEUS can identify sentinel lymph nodes • Ultrasound could then guide percutaneous removal of intact and complete SLNs • We have shown this was feasible in pigs but not yet in humans • This technique may eventually have the potential to reduce futile SLN biopsies.

Heterogeneity in Fragile X Syndrome Highlights the Need for Precision Medicine-Based Treatments.

Fragile X syndrome (FXS) is the most frequent monogenic cause of autism or intellectual disability, and research on its pathogenetic mechanisms has provided important insights on this neurodevelopmental condition. Nevertheless, after 30 years of intense research, efforts to develop treatments have been mostly unsuccessful. The aim of this review is to compile evidence from existing research pointing to clinical, genetic, and therapeutic response heterogeneity in FXS and highlight the need of implementing precision medicine-based treatments. We comment on the high genetic and phenotypic heterogeneity present in FXS, as a contributing factor to the difficulties found during drug development. Given that several clinical trials have showed a non-negligeable fraction of positive responders to drugs targeting core FXS symptoms, we propose that success of clinical trials can be achieved by tackling the underlying heterogeneity in FXS by accurately stratifying patients into drug-responder subpopulations. These precision medicine-based approaches, which can be first applied to well-defined monogenic diseases such as FXS, can also serve to define drug responder profiles based on specific biomarkers or phenotypic features that can associate patients with different genetic backgrounds to a same candidate drug, thus repositioning a same drug for a larger number of patients with NDDs.

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.

Hemin prevents in-stent stenosis in rat and rabbit models by inducing heme-oxygenase-1.

OBJECTIVE: The introduction of drug-eluting stents (DES) has largely added benefit to the percutaneous coronary intervention. Questions about the long-term safety of DES have been raised, however, particularly with respect to late stent thrombosis. Research efforts are now being directed toward therapeutics that can impede smooth muscle proliferation and promote vascular healing. Emerging data suggest that heme oxygenase-1 (HO-1), an inducible oxidoreductase enzyme system, can exert cytoprotective effects on endothelial cells and limit smooth muscle cell proliferation. We assessed the ability of hemin, a potent HO-1 inducer, to reduce in-stent stenosis without compromising re-endothelialization. METHODS: Rat aorta and rabbit iliac arteries were stented. Animals received ongoing treated with intraperitoneal hemin (50 mg/kg) or vehicle. At 7 to 28 days after surgery, stented arterial segments were collected and processed for histologic, electron microscopy, or protein analysis. RESULTS: In both models, treatment with hemin reduced neointima growth without compromising re-endothelialization of the stented arteries. In the rat aorta, analysis of protein expression at 7 and 28 days after stenting revealed that hemin increased HO-1 expression and limited the early inflammatory, apoptotic, and proliferative cellular events that are common to in-stent stenosis. Hemin treatment decreased the expression of the Ki-67 protein and the activity of key regulators of smooth muscle cell proliferation, including p42/44, RhoA, and up-regulated the expression of cyclin-dependent kinase inhibitors. The beneficial effects of hemin were abolished in the presence of tin-protoporphyrin IX, an HO inhibitor. Finally, treatment with tricarbonylchloro(glycinato)ruthenium(II), a carbon monoxide donor, reduced in-stent stenosis in the rat aorta, suggesting that carbon monoxide, a by-product of heme degradation, might contribute to the protective effect of hemin. CONCLUSION: These results suggest that HO-1 is important in limiting in-stent stenosis and can be regarded as a new therapeutic target.

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.

Short- and long-term cardioprotective effect of darbepoetin-alpha: role of Bcl-2 family proteins.

The purpose of this study was to assess the short- and long-term cardioprotective effects of darbepoetin-alpha (DA) in a rat myocardial ischemia and reperfusion model and to investigate the signaling pathway through which DA limits cardiomyocytes apoptosis. Rats were subjected to 40 minutes of coronary artery ligation followed by 72 hours or 4 weeks reperfusion and received either DA (3 or 30 microg/kg, DA3 and D30 groups) or vehicle (control) prior to ischemia. In the DA groups reperfused for 72 hours, left ventricular shortening fraction and left ventricular ejection fraction were higher than that in the control rats (P < 0.05), in agreement with a smaller left ventricular (LV) infarct size. DA treatment activated the JAK2/Akt signaling pathway, lowered cleaved caspase-3, and increased both phosphorylated-Bad and phosphorylated-GSK-3beta proteins. This was consistent with the decrease of reactive oxygen species production and the lowered binding of Bad to Bcl-xL and Bcl-2 in a DA30 group of rats. Similarly, in the DA-4-week group, LV function was greater compared to the control. Histology alterations implicated lower LV cardiac fibrosis and greater capillary density; furthermore, both Bcl-xL and Bcl-2 were upregulated. In conclusion, DA afforded short- and long-term cardioprotective effects. Antiapoptotic effects, through the activation of Akt that regulates the Bcl-2 family proteins and activates GSK-3beta, are central in the DA cardioprotective mechanism.

Heme oxygenase-1 inducer hemin attenuates the progression of remnant kidney model.

BACKGROUNDS/AIMS: Heme oxygenase-1 (HO-1) has been shown to protect against fibrotic proliferation and apoptosis in several models of renal damage. The purpose of this study was to evaluate the impact of a treatment with the HO-1 inducer hemin on the progression of chronic kidney disease in nephrectomized rats versus the AT1 receptor antagonist losartan. METHODS: The rats underwent 5/6 renal ablation and after the procedure received either losartan (20 mg/kg/day; n = 9), hemin (50 mg/kg/twice a week; n = 8) or vehicle (n = 8) over a 12-week period. At week 12, blood pressure was measured, urine, blood and remnant kidney were collected for biochemical (proteinuria, urea, creatinine) and histopathological (degrees of glomerulosclerosis and tubular atrophy) analysis. The expressions of HO-1, bone morphogenic protein 7 (BMP-7) and TGF-beta were assessed by immunochemistry, and the level of the apoptosis marker protein caspase-3 by Western blot, on the remnant kidney. RESULTS: Hemin significantly reduced blood pressure, proteinuria, inhibited the expression of TGF-beta and caspase-3 protein and increased BMP-7 expression protein. Hemin-treated rats had lower glomerulosclerosis and tubular atrophy indexes than controls. CONCLUSION: Hemin treatment attenuates the progression of chronic kidney disease and appears more efficient than losartan in our rat model hypothetically because of the impact of hemin on the renal expression of BMP-7.

Echocardiography, a non-invasive method for the assessment of cardiac function and morphology in preclinical drug toxicology and safety pharmacology.

BACKGROUND: Echocardiography (EC) is a method used for the investigation of cardiac morphology and function. Two-dimensional EC gives a visualisation of the morphology of the heart. M-mode EC allows heart function to be monitored. Pulsed Doppler EC is the method of choice to measure blood flows. OBJECTIVE: To describe the information EC can provide for cardiovascular investigation in laboratory animals, with a special focus on the potential helpfulness of EC in preclinical toxicology and safety pharmacology. METHODS: This review includes publications describing the methodology of EC and its application to several animal species used in biological experimentation. RESULTS/CONCLUSION: EC has been established in dogs, monkeys, rodents, rabbits and pigs. As demonstrated by experiments in different species, EC can be particularly helpful in toxicology and safety pharmacology, based on the amount of information it can give on the causes and consequences of drug adverse effects on the cardiovascular system. Furthermore, EC does not require any surgery and is therefore a key refinement compared to invasive methods generally used for investigating the cardiovascular function in laboratory animals. Despite some limitations of the method (the need for trained people, time required for an accurate EC recording, lack of current validation), EC should be further developed in preclinical toxicology and safety pharmacology.

Microbubbles combined with ultrasound therapy in ischemic stroke: A systematic review of in-vivo preclinical studies.

BACKGROUND: Microbubbles (MBs) combined with ultrasound sonothrombolysis (STL) appears to be an alternative therapeutic strategy for acute ischemic stroke (IS), but clinical results remain controversial. OBJECTIVE: The aim of this systematic review is to identify the parameters tested; to assess evidence on the safety and efficacy on preclinical data on STL; and to assess the validity and publication bias. METHODS: Pubmed® and Web of ScienceTM databases were systematically searched from January 1995 to April 2017 in French and English. We included studies evaluating STL on animal stroke model. This systematic review was conducted in accordance with the PRISMA guidelines. Data were extracted following a pre-defined schedule by two of the authors. The CAMARADES criteria were used for quality assessment. A narrative synthesis was conducted. RESULTS: Sixteen studies met the inclusion criteria. The result showed that ultrasound parameters and types of MBs were heterogeneous among studies. Numerous positive outcomes on efficacy were found, but only four studies demonstrated superiority of STL versus recombinant tissue-type plasminogen activator on clinical criteria. Data available on safety are limited. LIMITATIONS: Quality assessment of the studies reviewed revealed a number of biases. CONCLUSION: Further in vivo studies are needed to demonstrate a better efficacy and safety of STL compared to currently approved therapeutic options. SYSTEMATIC REVIEW REGISTRATION: http://syrf.org.uk/protocols/.

Heme oxygenase-1 inducer hemin prevents vascular thrombosis.

Hemin is a heme oxygenase-1 (HO-1) inducer which provides endogenous carbon monoxide known for playing roles in cell proliferation, inflammation or aggregation process. The objective of the current study was to examine the effect of prophylactic treatment with hemin in a thrombosis vascular model. Three groups of Wistar rats, control (n = 6), hemin (n = 6) and hemin + HO-1 inhibitor (n = 6), were used for this study. Hemin-treated animals received hemin (50 mg/kg/d; I.P.) for seven days and HO-1 inhibitor group received hemin at the same dose and SnPP IX (60 mg/kg/d; I.P.). All animals were exposed to electric stimulation of the left carotid according to Kawasaki's procedure to induce reproducible thrombus formation. The hemin treatment did not induce blood pressure disturbance. Effects of hemin on vascular thrombosis were quantified by histopathology and its influence on haemostasis was assessed by measuring prothrombin time (PT), activated partial thromboplastin time (APTT) and blood parameters at the end of treatment. The HO-1 mRNA and protein level variation were also checked out. Results showed that chronic treatment with hemin significantly (p < 0.01) reduced the vascular occlusion degree when compared to control and hemin SnPP groups with 7.2 +/- 4.6 vs. 71.1 +/- 14.7 and 74.0 +/- 8.8%, respectively. Moreover, we observed significant (p < 0.05) perturbations of blood parameters in hemin-treated and hemin-SnPP treated rats. Interestingly, hemin treatment did not significantly increase both PT and APTT. Finally, the HO-1 mRNA and protein levels were increased in hemin-treated carotid artery. In conclusion, hemin by inducing HO-1 expression may be a preventive agent against clinical disorders associated to an increased risk of thrombosis events and may limit haemorrhagic risks.

Inhibition of Rho-kinase attenuates hypoxia-induced angiogenesis in the pulmonary circulation.

Pulmonary hypertension (PH) is a common complication of chronic hypoxic lung diseases, which increase morbidity and mortality. Hypoxic PH has previously been attributed to structural changes in the pulmonary vasculature including narrowing of the vascular lumen and loss of vessels, which produce a fixed increase in resistance. Using quantitative stereology, we now show that chronic hypoxia caused PH and remodeling of the blood vessel walls in rats but that this remodeling did not lead to structural narrowing of the vascular lumen. Sustained inhibition of the RhoA/Rho-kinase pathway throughout the period of hypoxic exposure attenuated PH and prevented remodeling in intra-acinar vessels without enlarging the structurally determined lumen diameter. In chronically hypoxic lungs, acute Rho kinase inhibition markedly decreased PVR but did not alter the alveolar to arterial oxygen gap. In addition to increased vascular resistance, chronic hypoxia induced Rho kinase-dependent capillary angiogenesis. Thus, hypoxic PH was not caused by fixed structural changes in the vasculature but by sustained vasoconstriction, which was largely Rho kinase dependent. Importantly, this vasoconstriction had no role in ventilation-perfusion matching and optimization of gas exchange. Rho kinase also mediated hypoxia-induced capillary angiogenesis, a previously unrecognized but potentially important adaptive response.

Dexfenfluramine does not worsen but moderates progression of chronic hypoxia-induced pulmonary hypertension.

This study shows for the first time, that dexfenfluramine, a 5-HT(2) receptor agonist, attenuates the development of chronic hypoxia-induced pulmonary hypertension. Chronic exposure to hypoxia, 4 weeks, induced hypoxic pulmonary hypertension in adult rat as haemodynamic and cardiac measurements showed significant modifications in right ventricle parameters (free wall right ventricle thickness; pulmonary acceleration time and velocity time integral) in chronic hypoxic control when compared to normoxic control animals. We observed that free wall right ventricle thickness and pulmonary velocity time integral were significantly less in chronic hypoxic rats treated with dexfenfluramine when compared to chronic hypoxic control rats. Similarly, rats exposed to chronic hypoxia exhibited an increase in both right ventricle pressure and weight by comparison to normoxic control animals but those variations were significantly diminished in dexfenfluramine-treated rats, indicating the moderating influence exerted by dexfenfluramine on chronic hypoxia-induced pulmonary hypertension and cardiac alterations. Thus, we report here the ability of dexfenfluramine to limit chronic hypoxia-induced pulmonary hypertension, emphasizing the importance of the time after the dexfenfluramine treatment discontinuation to assess the influence of this 5-HT receptor agonist on the development of chronic hypoxia-induced pulmonary hypertension.

Sonothrombolysis with BR38 Microbubbles Improves Microvascular Patency in a Rat Model of Stroke.

BACKGROUND: Early recanalization of large cerebral vessels in ischemic stroke is associated with improved clinical outcome, however persisting hypoperfusion leads to poor clinical recovery despite large vessel recanalization. Limited experimental sonothrombolysis studies have shown that addition of microbubbles during treatment can improve microvascular patency. We aimed to determine the effect of two different microbubble formulations on microvascular patency in a rat stroke model. METHODS: We tested BR38 and SonoVue® microbubble-enhanced sonothrombolysis in Wistar rats submitted to 90-minute filament occlusion of the middle cerebral artery. Rats were randomized to treatment (n = 6/group): control, rt-PA, or rt-PA+3-MHz ultrasound insonation with BR38 or SonoVue® at full or 1/3 dose. Treatment duration was 60 minutes, beginning after withdrawal of the filament, and sacrifice was immediately after treatment. Vascular volumes were evaluated with microcomputed tomography. RESULTS: Total vascular volume of the ipsilateral hemisphere was reduced in control and rt-PA groups (p<0.05), but was not significantly different from the contralateral hemisphere in all microbubble-treated groups (p>0.1). CONCLUSIONS: Microbubble-enhanced sonothrombolysis improves microvascular patency. This effect is not dose- or microbubble formulation-dependent suggesting a class effect of microbubbles promoting microvascular reopening. This study demonstrates that microbubble-enhanced sonothrombolysis may be a therapeutic strategy for patients with persistent hypoperfusion of the ischemic territory.

CO inhalation at dose corresponding to tobacco smoke worsens cardiac remodeling after experimental myocardial infarction in rats.

We hypothesized that inhalation of carbon monoxide (CO) (500 ppm), similar to that in tobacco smoke, disturbs the cardiovascular adaptation after myocardial infarction by increasing remodeling. Four groups of rats were assessed. Two groups had myocardial infarction induced by the ligation of the left coronary artery: the first group was exposed to air (infarcted air group, n = 12), and the second was exposed to CO (infarcted CO group, n = 11). They were compared to two sham-operated groups, a control air group (n = 10), and a control CO group (n = 7) exposed (3 weeks) to CO. Aerobic endurance capacity was assessed in both the infarct CO and infarct air group (endurance capacity = 0.043 +/- 0.006 m.min(-1).g(-1) vs. 0.042 +/- 0.005 m.min(-1).g(-1), not significant). In the infarcted CO group compared to the infarcted air group, the dilatation of the left ventricle observed 3 weeks after infarction was increased, (left ventricular diastolic (LVD) diameter (D) = 9 +/- 0.4 vs. 7 +/- 0.4 mm, p < 0.05; left ventricular systolic (LVS) diameter (D) = 6 +/- 0.6 vs. 4.1 +/- 0.4, p < 0.05), and the diastolic posterior wall thickness was augmented (posterior wall diastolic thickness = 1.7 +/- 0.1 vs. 1.3 +/- 0.1 mm, p < 0.05). Hemodynamic pressure measurements in both ventricles and pulmonary artery showed elevated diastolic pressure after CO exposure compared to air exposure (LVD pressure = 32 +/- 1.6 vs. 19 +/- 2.3 mm Hg, p < 0.05; right ventricular diastolic pressure = 16 +/- 1.6 vs. 8.6 +/- 1.6 mm Hg, p < 0.05; pulmonary arterial pressure in diastole (PAD) = 27 +/- 1.6 vs. 20 +/- 2.3 mm Hg, p < 0.05). In the infarcted CO group, the infarct size increased. Echocardiography and histology showed hypertrophy of the contralateral wall similar to that observed in the noninfarcted control CO group. In conclusion, chronic CO inhalation worsens heart failure in rats with myocardial infarction by an increase in the infarct size and hypertrophy remodeling.

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.