Development and Assessment of Herpes Simplex Virus Type 1 (HSV-1) Amplicon Vectors with Sensory Neuron-Selective Promoters.

BACKGROUND: Neurogenic detrusor overactivity (NDO) is a severe pathological condition characterized by involuntary detrusor contractions leading to urine leakage. This condition is frequent after spinal cord injury (SCI). Gene therapy for NDO requires the development of vectors that express therapeutic transgenes driven by sensory neuron-specific promoters. The aim of this study was to develop and assess tools for the characterization of sensory neuron-specific promoters in dorsal root ganglia (DRG) neurons after transduction with herpes simplex virus type 1 (HSV-1)-based amplicon defective vectors. METHODS: The HSV-1 vector genome encoded two independent transcription cassettes: one expressed firefly luciferase (FLuc) driven by different promoters' candidates (rTRPV1, rASIC3, rCGRP, or hCGRP), and the other expressed a reporter gene driven by an invariable promoter. The strength and selectivity of promoters was assessed in organotypic cultures of explanted adult DRG, or sympathetic and parasympathetic ganglia from control and SCI rats. RESULTS: The rCGRP promoter induced selective expression in the DRG of normal rats. The rTRPV-1 promoter, which did not display selective activity in control rats, induced selective expression in DRG explanted from SCI rats. CONCLUSIONS: This study provides a methodology to assess sensory neuron-specific promoters, opening new perspectives for future gene therapy for NDO.

Nanotubular crosstalk with distressed cardiomyocytes stimulates the paracrine repair function of mesenchymal stem cells.

Mesenchymal stem cells (MSC) are known to repair broken heart tissues primarily through a paracrine fashion while emerging evidence indicate that MSC can communicate with cardiomyocytes (CM) through tunneling nanotubes (TNT). Nevertheless, no link has been so far established between these two processes. Here, we addressed whether cell-to-cell communication processes between MSC and suffering cardiomyocytes and more particularly those involving TNT control the MSC paracrine regenerative function. In the attempt to mimic in vitro an injured heart microenvironment, we developed a species mismatch coculture system consisting of terminally differentiated CM from mouse in a distressed state and human multipotent adipose derived stem cells (hMADS). In this setting, we found that crosstalk between hMADS and CM through TNT altered the secretion by hMADS of cardioprotective soluble factors such as VEGF, HGF, SDF-1α, and MCP-3 and thereby maximized the capacity of stem cells to promote angiogenesis and chemotaxis of bone marrow multipotent cells. Additionally, engraftment experiments into mouse infarcted hearts revealed that in vitro preconditioning of hMADS with cardiomyocytes increased the cell therapy efficacy of naïve stem cells. In particular, in comparison with hearts treated with stem cells alone, those treated with cocultured ones exhibited greater cardiac function recovery associated with higher angiogenesis and homing of bone marrow progenitor cells at the infarction site. In conclusion, our findings established the first relationship between the paracrine regenerative action of MSC and the nanotubular crosstalk with CM and emphasize that ex vivo manipulation of these communication processes might be of interest for optimizing current cardiac cell therapies.

Partial restoration of brain dystrophin by tricyclo-DNA antisense oligonucleotides alleviates emotional deficits in mdx52 mice.

The mdx52 mouse model recapitulates a frequent mutation profile associated with brain involvement in Duchenne muscular dystrophy. Deletion of exon 52 impedes expression of two dystrophins (Dp427, Dp140) expressed in brain, and is eligible for therapeutic exon-skipping strategies. We previously showed that mdx52 mice display enhanced anxiety and fearfulness, and impaired associative fear learning. In this study, we examined the reversibility of these phenotypes using exon 51 skipping to restore exclusively Dp427 expression in the brain of mdx52 mice. We first show that a single intracerebroventricular administration of tricyclo-DNA antisense oligonucleotides targeting exon 51 restores 5%-15% of dystrophin protein expression in the hippocampus, cerebellum, and cortex, at stable levels between 7 and 11 week after injection. Anxiety and unconditioned fear were significantly reduced in treated mdx52 mice and acquisition of fear conditioning appeared fully rescued, while fear memory tested 24 h later was only partially improved. Additional restoration of Dp427 in skeletal and cardiac muscles by systemic treatment did not further improve the unconditioned fear response, confirming the central origin of this phenotype. These findings indicate that some emotional and cognitive deficits associated with dystrophin deficiency may be reversible or at least improved by partial postnatal dystrophin rescue.

Human mesenchymal stem cells reprogram adult cardiomyocytes toward a progenitor-like state through partial cell fusion and mitochondria transfer.

Because stem cells are often found to improve repair tissue including heart without evidence of engraftment or differentiation, mechanisms underlying wound healing are still elusive. Several studies have reported that stem cells can fuse with cardiomyocytes either by permanent or partial cell fusion processes. However, the respective physiological impact of these two processes remains unknown in part because of the lack of knowledge of the resulting hybrid cells. To further characterize cell fusion, we cocultured mouse fully differentiated cardiomyocytes with human multipotent adipose-derived stem (hMADS) cells as a model of adult stem cells. We found that heterologous cell fusion promoted cardiomyocyte reprogramming back to a progenitor-like state. The resulting hybrid cells expressed early cardiac commitment and proliferation markers such as GATA-4, myocyte enhancer factor 2C, Nkx2.5, and Ki67 and exhibited a mouse genotype. Interestingly, human bone marrow-derived stem cells shared similar reprogramming properties than hMADS cells but not human fibroblasts, which suggests that these features might be common to multipotent cells. Furthermore, cardiac hybrid cells were preferentially generated by partial rather than permanent cell fusion and that intercellular structures composed of f-actin and microtubule filaments were involved in the process. Finally, we showed that stem cell mitochondria were transferred into cardiomyocytes, persisted in hybrids and were required for somatic cell reprogramming. In conclusion, by providing new insights into previously reported cell fusion processes, our data might contribute to a better understanding of stem cell-mediated regenerative mechanisms and thus, the development of more efficient stem cell-based heart therapies.

Monitoring of erectile and urethral sphincter dysfunctions in a rat model mimicking radical prostatectomy damage.

INTRODUCTION: Animal models of urinary incontinence and erectile dysfunction following radical prostatectomy (RP) are lacking. AIMS: To develop an animal model of combined post-RP urethral sphincter and erectile dysfunctions, and noninvasive methods to assess erectile function (EF) and urinary sphincter function (USF) during prolonged follow-up. METHODS: In the main experiments, 60 male Sprague Dawley rats were randomized to a sham operation (N = 30) or electrocautery of both sides of the striated urethral sphincter (N = 30). EF and USF were evaluated preoperatively and on postoperative days 7, 15, 30, 60, and 90. Sphincter and penile tissue samples were evaluated histologically on days 7 (N = 10) and 30 (N = 10) to detect apoptosis (TUNEL assays) and fibrosis (Trichrome Masson staining). MAIN OUTCOME MEASURES: To assess EF, we measured systemic and penile blood flow using penile laser Doppler and penile rigidity using a durometer before and after apomorphine injection. USF was assessed based on the retrograde leak point pressure (LPPr). RESULTS: Apomorphine increased baseline Doppler flow by 180% (95% confidence interval, 156-202%) and penile hardness from 3.49 ± 0.5 to 7.16 ± 0.82 Shore A units but did not change systemic arterial flow. Mean LPPr was 76.8 ± 6.18 mm Hg at baseline and decreased by 50% after injury, with no response to apomorphine on day 7. EF and USF impairments persisted up to 90 days post injury. Histology showed penile apoptosis on day 7 and extensive urethral sphincter and penile fibrosis on day 30. Our data did not allow us to determine whether the impairment in erectile response to apomorphine preponderantly reflected arterial penile insufficiency or veno-occlusive dysfunction. CONCLUSION: Electrocautery of the striated urethral sphincter caused severe and lasting impairment of EF and USF that could be monitored repeatedly using minimally invasive methods. This new animal model may hold potential for developing new treatments designed to correct post-RP impairments.

How does chronic sildenafil prevent vascular oxidative stress in insulin-resistant rats?

INTRODUCTION: Insulin resistance features both endothelial dysfunction and increased oxidative stress. Both disorders are targeted by a chronic treatment with sildenafil. However, the mechanism of action by which chronic sildenafil exerts its effects on reactive oxygen species sources is still largely unknown. AIM: We therefore investigated how chronic sildenafil administration could impact vascular endothelial NO and superoxide release in a rat model of insulin resistance induced by fructose overload. METHODS: Adult male Wistar rats were fed a fructose-enriched diet (fructose-fed rats [FFR]) for 9 weeks. From weeks 6-8, sildenafil was administered subcutaneously twice daily (20 mg/kg), followed by a 1-week washout. MAIN OUTCOME MEASURES: Vascular endothelial NO and superoxide release were monitored in vitro in thoracic aortic segments using oxidative fluorescence. Specific inhibitors were used to distinguish the respective role of the main superoxide-producing systems within the vascular wall (i.e., mitochondrial respiratory chain and NADPH oxidases). The levels of expression of eNOS, Akt, and NADPH oxidase subunits were determined in the abdominal aorta. RESULTS: Chronic sildenafil administration corrected hyperglycemia, hyperinsulinemia, and hypertriglyceridemia in FFR. Moreover, after 9 weeks of diet, while global unstimulated aortic endothelial NO and superoxide release were unchanged in FFR, the relative contribution of the mitochondrial respiratory chain and NADPH oxidases was modified. Chronic sildenafil treatment, even after the 1-week washout period, was able to increase endothelial NO release independently of Akt-dependent phosphorylation by up-regulating eNOS expression, and restored the relative contribution of each superoxide-producing system examined, yielding endothelial superoxide release. Finally, in vitro incubation of aortic segments with sildenafil markedly decreased the endothelial aortic superoxide release. CONCLUSIONS: The present study showed that chronic sildenafil produced sustained vascular antioxidant effects in insulin-resistant rats by increasing NO release and regulating vascular superoxide release, supporting therefore further investigations using chronic sildenafil administration in preventing cardiovascular alterations associated with oxidative stress.

Botulinum Neurotoxin Light Chains Expressed by Defective Herpes Simplex Virus Type-1 Vectors Cleave SNARE Proteins and Inhibit CGRP Release in Rat Sensory Neurons.

A set of herpes simplex virus type 1 (HSV-1) amplicon vectors expressing the light chains (LC) of botulinum neurotoxins (BoNT) A, B, C, D, E and F was constructed. Their properties have been assessed in primary cultures of rat embryonic dorsal root ganglia (DRG) neurons, and in organotypic cultures of explanted DRG from adult rats. Following infection of primary cultures of rat embryonic DRG neurons, the different BoNT LC induced efficient cleavage of their corresponding target Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) protein (VAMP, SNAP25, syntaxin). A similar effect was observed following infection by BoNT-A LC of organotypic cultures of adult rat DRG. To quantify and compare the functional activities of the different BoNT LC, the inhibition of calcitonin gene-related protein (CGRP) secretion was assessed in DRG neurons following infection by the different vectors. All BoNT-LC were able to inhibit CGRP secretion although to different levels. Vectors expressing BoNT-F LC displayed the highest inhibitory activity, while those expressing BoNT-D and -E LC induced a significantly lower CGRP release inhibition. Cleavage of SNARE proteins and inhibition of CGRP release could be detected in neuron cultures infected at less than one transducing unit (TU) per neuron, showing the extreme efficacy of these vectors. To our knowledge this is the first study investigating the impact of vector-expressed transgenic BoNT LC in sensory neurons.

Low-affinity state beta1-adrenoceptor-induced vasodilation in SHR.

Low-affinity state beta1-adrenoceptor (beta1-AR) was functionally expressed in some blood vessels and was different from beta1, beta2 and beta3-AR. In rat aorta, low-affinity state beta1-AR activation produced an endothelium-independent relaxation which was impaired in spontaneously hypertensive rats (SHRs). In the present work, we investigated whether renin-angiotensin system was involved in this alteration by evaluating the effects of enalapril, an angiotensin converting enzyme (ACE) inhibitor or losartan, an AT1 angiotensin receptor antagonist. Cumulative concentration-response curves to low-affinity state beta1-AR agonists (CGP 12177, cyanopindolol or alprenolol) and to NS 1619, a large conductance Ca2+-activated K+ channels (BK) agonist were performed in denuded aortic rings isolated from control or treated Wistar Kyoto (WKY) rats or SHRs in different experimental conditions. The low-affinity state beta1-AR-mediated aortic vasodilation was impaired in 5 and 12 weeks old SHRs when compared to age-matched WKY. Twelve days enalapril (5 mg/kg/day) or losartan (15 mg/kg/day) treatments reduced systolic blood pressure (SBP) only in 12 weeks old SHRs whereas no significant change was observed in other groups. These treatments improved low-affinity state beta1-AR effect only in SHRs groups. In 12 weeks old WKY rats, CGP 12177-induced relaxation was insensitive to glibenclamide, a K(ATP)+ channel blocker, but was reduced by TEA or iberiotoxin, two large conductance Ca2+-activated K+ channel (BK) blockers. The impairment of NS 1619-induced vasodilation in both 5 and 12 weeks old SHRs was restored by enalapril or losartan. These results suggested that improvement of the low-affinity state beta1-AR-mediated vasodilation in 5 and 12 weeks old SHRs could be attributed to enhanced BK channels-induced hyperpolarization in SHRs independently of lowering of SBP.

Impact of a long-term sildenafil treatment on pressor response in conscious rats with insulin resistance and hypertriglyceridemia.

BACKGROUND: Insulin resistance constitutes a risk factor for endothelial dysfunction and subsequent cardiovascular diseases including hypertension. Daily treatment with phosphodiesterase type 5 (PDE5) inhibitors has beneficial effects on endothelial function in men with increased cardiovascular risk. Endothelium-dependent vasomotor function is ultimately linked to blood pressure (BP) regulation. We postulated that sildenafil would ameliorate BP and biological markers of endothelial function in fructose-fed rats (FFRs). METHODS: Wistar rats were fed a standard chow or a 60% fructose-enriched diet containing 12% fat for 8 weeks (FFR). From week 6 through 8, sildenafil (twice a day subcutaneously, 20 mg/kg) was administered followed by a 1-week washout period. At the end of the washout period, BP was recorded using radiotelemetry following cumulative infusion of norepinephrine (from 50 to 400 ng/kg/min). RESULTS: FFR displayed both an impaired glucose tolerance and elevated triglyceridemia. The latter was corrected by sildenafil treatment. Resting BP was similar in all rats, whereas pressor responses were significantly enhanced in FFR (maximal increase in mean BP to norepinephrine: 25.6 +/- 3.8 vs. 40.8 +/- 4.0 mm Hg, P < 0.05) and normalized by sildenafil treatment (24.9 +/- 5.3 mm Hg, not significant vs. control). Urinary levels of 8-isoprostanes and thromboxane B(2) were increased in FFR and corrected by sildenafil treatment. CONCLUSION: Thus, chronic treatment with sildenafil normalized BP regulation in an experimental model of insulin resistance and hypertriglyceridemia while restoring normal excretion of urinary biological markers of oxidative stress and cyclooxygenase-derived vasoconstrictors. The modulation of ROS and cyclooxygenase-derived vasoconstrictors generation by a chronic treatment with sildenafil may represent an added benefit beyond PDE5 inhibition.