Gilles Renault

Ultrasound and microbubble-assisted gene delivery in Achilles tendons: Long lasting gene expression and restoration of fibromodulin KO phenotype

The aim of this study is to deliver genes in Achilles tendons using ultrasound and microbubbles. The rationale is to combine ultrasound-assisted delivery and the stimulation of protein expression induced by US. We found that mice tendons injected with 10 μg of plasmid encoding luciferase gene in the presence of 5×10⁵ BR14 microbubbles, exposed to US at 1 MHz, 200 kPa, 40% duty cycle for 10 min were efficiently transfected without toxicity. The rate of luciferase expression was 100-fold higher than that obtained when plasmid alone was injected. Remarkably, the luciferase transgene was stably expressed for up to 108 days. DNA extracted from these sonoporated tendons was efficient in transforming competent E. coli bacteria, indicating that persistent intact pDNA was responsible for this long lasting gene expression. We used this approach to restore expression of the fibromodulin gene in fibromodulin KO mice. A significant fibromodulin expression was detected by quantitative PCR one week post-injection. Interestingly, ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar to that of wild type mice. Our results suggest that this gene delivery method is promising for clinical applications aimed at modulating healing or restoring a degenerative tendon while offering great promise for gene therapy due its safety compared to viral methods.

Identification of Quantitative Trait Loci responsible for embryonic lethality in mice assessed by ultrasonography.

Recurrent Spontaneous Abortion (RSA) is a frequent pathology affecting 1 to 5% of couples. In approximately 50 % of cases, the aetiology is unknown suggesting a subtle interaction between genetic and environmental factors. Previous attempts to describe genetic factors using the candidate gene approach have been relatively unsuccessful due to the physiological, cellular and genetic complexity of mammalian reproduction. Indeed, fertility can be considered as a quantitative feature resulting from the interaction of genetic, epigenetic and environmental factors. Herein, we identified Quantitative Trait Loci (QTL) associated with diverse embryonic lethality phenotypes and the subsequent embryonic resorption in 39 inter-specific recombinant congenic mice strains, using in vivo ultrasound bio-microscopy. The short chromosomal intervals related to the phenotypes will facilitate the study of a restricted number of candidate genes which are potentially dysregulated in patients affected by RSA.

Refined Mapping of a Quantitative Trait Locus on Chromosome 1 Responsible for Mouse Embryonic Death

Recurrent spontaneous abortion (RSA) is defined as the loss of three or more consecutive pregnancies during the first trimester of embryonic intrauterine development. This kind of human infertility is frequent among the general population since it affects 1 to 5% of women. In half of the cases the etiology remains unelucidated. In the present study, we used interspecific recombinant congenic mouse strains (IRCS) in the aim to identify genes responsible for embryonic lethality. Applying a cartographic approach using a genotype/phenotype association, we identified a minimal QTL region, of about 6 Mb on chromosome 1, responsible for a high rate of embryonic death (∼30%). Genetic analysis suggests that the observed phenotype is linked to uterine dysfunction. Transcriptomic analysis of the uterine tissue revealed a preferential deregulation of genes of this region compared to the rest of the genome. Some genes from the QTL region are associated with VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathways. This work may contribute to elucidate the molecular basis of a multifactorial and complex human disorder as RSA.

Tumor Stiffening, a Key Determinant of Tumor Progression, is Reversed by Nanomaterial-Induced Photothermal Therapy

Tumor stiffening, stemming from aberrant production and organization of extracellular matrix (ECM), has been considered a predictive marker of tumor malignancy, non-invasively assessed by ultrasound shear wave elastography (SWE). Being more than a passive marker, tumor stiffening restricts the delivery of diagnostic and therapeutic agents to the tumor and per se could modulate cellular mechano-signaling, tissue inflammation and tumor progression. Current strategies to modify the tumor extracellular matrix are based on ECM-targeting chemical agents but also showed deleterious systemic effects. On-demand excitable nanomaterials have shown their ability to perturb the tumor microenvironment in a spatiotemporal-controlled manner and synergistically with chemotherapy. Here, we investigated the evolution of tumor stiffness as well as tumor integrity and progression, under the effect of mild hyperthermia and thermal ablation generated by light-exposed multi-walled carbon nanotubes (MWCNTs) in an epidermoid carcinoma mouse xenograft. SWE was used for real-time mapping of the tumor stiffness, both during the two near infrared irradiation sessions and over the days after the treatment. We observed a transient and reversible stiffening of the tumor tissue during laser irradiation, which was lowered at the second session of mild hyperthermia or photoablation. In contrast, over the days following photothermal treatment, the treated tumors exhibited a significant softening together with volume reduction, whereas non-treated growing tumors showed an increase of tumor rigidity. The organization of the collagen matrix and the distribution of CNTs revealed a spatio-temporal correlation between the presence of nanoheaters and the damages on collagen and cells. This study highlights nanohyperthermia as a promising adjuvant strategy to reverse tumor stiffening and normalize the mechanical tumor environment.

Wnt proteins contribute to neuromuscular junction formation through distinct signaling pathways

Understanding the developmental steps that shape formation of the neuromuscular junction (NMJ) connecting motoneurons to skeletal muscle fibers is crucial. Wnt morphogens are key players in the formation of this specialized peripheral synapse, but their individual and collaborative functions and downstream pathways remain poorly understood at the NMJ. Here, we demonstrate through Wnt4 and Wnt11 gain-of-function studies in cell culture or in mice that Wnts enhance acetylcholine receptor (AChR) clustering and motor axon outgrowth. By contrast, loss of Wnt11 or Wnt-dependent signaling in vivo decreases AChR clustering and motor nerve terminal branching. Both Wnt4 and Wnt11 stimulate AChR mRNA levels and AChR clustering downstream of activation of the β-catenin pathway. Strikingly, Wnt4 and Wnt11 co-immunoprecipitate with Vangl2, a core component of the planar cell polarity (PCP) pathway, which accumulates at embryonic NMJs. Moreover, mice bearing a Vangl2 loss-of-function mutation (loop-tail) exhibit fewer AChR clusters and overgrowth of motor axons bypassing AChR clusters. Together, our results provide genetic and biochemical evidence that Wnt4 and Wnt11 cooperatively contribute to mammalian NMJ formation through activation of both the canonical and Vangl2-dependent core PCP pathways. Summary: Wnt4 and Wnt11 cooperatively contribute to NMJ formation in mice through activation of both the canonical and Vangl2-dependent core planar cell polarity pathways.

Cardiac Characterization of mdx Mice Using High-Resolution Doppler Echocardiography

OBJECTIVES Duchenne muscular dystrophy is an X-linked neuromuscular disorder. The heart is traditionally involved, leading to heart failure. The mdx mouse is a natural animal model of the disease. We conducted a prospective study to analyze left ventricular (LV) function in mdx mice at different ages using high-resolution Doppler echocardiography. METHODS Echocardiography was performed with a 30-MHz cardiac probe. Wild-type and mdx mice were scanned at 10 and 12 months. We measured the interventricular septal wall thickness, posterior wall thickness, and LV diameter in systole and diastole. The LV shortening fraction, LV ejection fraction, and LV mass were then calculated. RESULTS At 10 months, the shortening fractions in mdx and wild-type mice were relatively close (29% ± 5% versus 25% ± 4%). We found a significant difference in the posterior wall thickness change (40% ± 12% in mdx versus 28% ± 10% in wild-type; P = .048). The LV mass/body weight ratio was higher in mdx than wild-type mice (3.67 ± 0.25 versus 3.39 ± 0.26; P = .05). At 12 months, the LV mass was elevated in mdx mice compared to wild-type (152 ±16 versus 135 ± 3.7 mg; P = .04). The diastolic posterior wall thickness change was decreased in mdx mice at 12 months compared to wild-type (21% ± 7% versus 33% ± 4%; P = .01). The LV ejection fraction was not statistically different between mdx and wild-type mice (50% ± 6% versus 54% ± 2%). CONCLUSIONS Ten-month-old mdx mice had a significantly higher posterior wall thickness than wild-type mice, but it was not significant at 12 months. In 12-month-old mdx mice, the posterior wall thickness change was significantly lower, and the LV mass was significantly higher. These findings indicate the role of LV function in the early stages of Duchenne muscular dystrophy.

Targeting CD226/DNAX accessory molecule-1 (DNAM-1) in collagen-induced arthritis mouse models

BackgroundGenetic studies have pointed out that CD226 variants, encoding DNAM-1, could be associated with susceptibility to rheumatoid arthritis. Therefore, we aimed to determine the influence of DNAM-1 on the development of arthritis using the collagen-induced arthritis (CIA) mouse model.MethodsCIA was induced in mice on a DBA/1 background, treated in parallel with a DNAM-1 neutralizing monoclonal antibody, a control IgG and PBS, respectively. CIA was also induced in mice deficient for DNAM-1(dnam1−/−) and control dnam-1+/+ mice on a C57/BL6 background. Mice were monitored for clinical and ultrasound signs of arthritis. Histological analysis was performed to search for inflammatory infiltrates and erosions. The Mann–Whitney U test for non-related samples was used for statistical analysis.ResultsThere was a non-significant trend for a less arthritic phenotype in mice receiving anti-DNAM-1 mAb at both clinical, ultrasound and histological assessments. But, we did not observe any difference between dnam1+/+ and dnam1−/− mice for incidence nor severity of clinical arthritis. Histological analysis revealed inflammatory scores similar in both groups, without evidence of erosion. Collagen antibodies levels were similar in all mice, confirming immunization with collagen.ConclusionDespite some clues suggesting a role of DNAM-1 in arthritis, these complementary approaches demonstrate no contribution of CD226/DNAM-1 in the arthritic phenotype. These results contrast with previous studies showing a role in vivo of DNAM-1 in some autoimmune disorders.

Association of FOXD1 variants with adverse pregnancy outcomes in mice and humans

Recurrent spontaneous abortion (RSA) is a common cause of infertility, but previous attempts at identifying RSA causative genes have been relatively unsuccessful. Such failure to describe RSA aetiological genes might be explained by the fact that reproductive phenotypes should be considered as quantitative traits resulting from the intricate interaction of numerous genetic, epigenetic and environmental factors. Here, we studied an interspecific recombinant congenic strain (IRCS) of Mus musculus from the C57BL6/J strain of mice harbouring an approximate 5 Mb DNA fragment from chromosome 13 from Mus spretus mice (66H-MMU13 strain), with a high rate of embryonic resorption (ER). Transcriptome analyses of endometrial and placental tissues from these mice showed a deregulation of many genes associated with the coagulation and inflammatory response pathways. Bioinformatics approaches led us to select Foxd1 as a candidate gene potentially related to ER and RSA. Sequencing analysis of Foxd1 in the 66H-MMU13 strain, and in 556 women affected by RSA and 271 controls revealed non-synonymous sequence variants. In vitro assays revealed that some led to perturbations in FOXD1 transactivation properties on promoters of genes having key roles during implantation/placentation, suggesting a role of this gene in mammalian implantation processes.

Cardiac Metabolic Deregulation Induced by the Tyrosine Kinase Receptor Inhibitor Sunitinib is rescued by Endothelin Receptor Antagonism

The growing field of cardio-oncology addresses the side effects of cancer treatment on the cardiovascular system. Here, we explored the cardiotoxicity of the antiangiogenic therapy, sunitinib, in the mouse heart from a diagnostic and therapeutic perspective. We showed that sunitinib induces an anaerobic switch of cellular metabolism within the myocardium which is associated with the development of myocardial fibrosis and reduced left ventricular ejection fraction as demonstrated by echocardiography. The capacity of positron emission tomography with [18F]fluorodeoxyglucose to detect the changes in cardiac metabolism caused by sunitinib was dependent on fasting status and duration of treatment. Pan proteomic analysis in the myocardium showed that sunitinib induced (i) an early metabolic switch with enhanced glycolysis and reduced oxidative phosphorylation, and (ii) a metabolic failure to use glucose as energy substrate, similar to the insulin resistance found in type 2 diabetes. Co-administration of the endothelin receptor antagonist, macitentan, to sunitinib-treated animals prevented both metabolic defects, restored glucose uptake and cardiac function, and prevented myocardial fibrosis. These results support the endothelin system in mediating the cardiotoxic effects of sunitinib and endothelin receptor antagonism as a potential therapeutic approach to prevent cardiotoxicity. Furthermore, metabolic and functional imaging can monitor the cardiotoxic effects and the benefits of endothelin antagonism in a theranostic approach.

Cationic microbubbles and antibiotic-free miniplasmid for sustained ultrasound–mediated transgene expression in liver

Despite the increasing number of clinical trials in gene therapy, no ideal methods still allow non-viral gene transfer in deep tissues such as the liver. We were interested in ultrasound (US)-mediated gene delivery to provide long term liver expression. For this purpose, new positively charged microbubbles were designed and complexed with pFAR4, a highly efficient small length miniplasmid DNA devoid of antibiotic resistance sequence. Sonoporation parameters, such as insonation time, acoustic pressure and duration of plasmid injection were controlled under ultrasound imaging guidance. The optimization of these various parameters was performed by bioluminescence optical imaging of luciferase reporter gene expression in the liver. Mice were injected with 50μg pFAR4-LUC either alone, or complexed with positively charged microbubbles, or co-injected with neutral MicroMarker™ microbubbles, followed by low ultrasound energy application to the liver. Injection of the pFAR4 encoding luciferase alone led to a transient transgene expression that lasted only for two days. The significant luciferase signal obtained with neutral microbubbles decreased over 2days and reached a plateau with a level around 1 log above the signal obtained with pFAR4 alone. With the newly designed positively charged microbubbles, we obtained a much stronger bioluminescence signal which increased over 2days. The 12-fold difference (p<0.05) between MicroMarker™ and our positively charged microbubbles was maintained over a period of 6months. Noteworthy, the positively charged microbubbles led to an improvement of 180-fold (p<0.001) as regard to free pDNA using unfocused ultrasound performed at clinically tolerated ultrasound amplitude. Transient liver damage was observed when using the cationic microbubble-pFAR4 complexes and the optimized sonoporation parameters. Immunohistochemistry analyses were performed to determine the nature of cells transfected. The pFAR4 miniplasmid complexed with cationic microbubbles allowed to transfect mostly hepatocytes compared to its co-injection with MicroMarker™ which transfected more preferentially endothelial cells.