Mario Pescatori

An Integrated Approach for Experimental Target Identification of Hypoxia-induced miR-210

miR-210 is a key player of cell response to hypoxia, modulating cell survival, VEGF-driven endothelial cell migration, and the ability of endothelial cells to form capillary-like structures. A crucial step in understanding microRNA (miRNA) function is the identification of their targets. However, only few miR-210 targets have been identified to date. Here, we describe an integrated strategy for large-scale identification of new miR-210 targets by combining transcriptomics and proteomics with bioinformatic approaches. To experimentally validate candidate targets, the RNA-induced silencing complex (RISC) loaded with miR-210 was purified by immunoprecipitation along with its mRNA targets. The complex was significantly enriched in mRNAs of 31 candidate targets, such as BDNF, GPD1L, ISCU, NCAM, and the non-coding RNA Xist. A subset of the newly identified targets was further confirmed by 3′-untranslated region (UTR) reporter assays, and hypoxia induced down-modulation of their expression was rescued blocking miR-210, providing support for the approach validity. In the case of 9 targets, such as PTPN1 and P4HB, miR-210 seed-pairing sequences localized in the coding sequence or in the 5′-UTR, in line with recent data extending miRNA targeting beyond the “classic” 3′-UTR recognition. Finally, Gene Ontology analysis of the targets highlights known miR-210 impact on cell cycle regulation and differentiation, and predicts a new role of this miRNA in RNA processing, DNA binding, development, membrane trafficking, and amino acid catabolism. Given the complexity of miRNA actions, we view such a multiprong approach as useful to adequately describe the multiple pathways regulated by miR-210 during physiopathological processes.

Common micro-RNA signature in skeletal muscle damage and regeneration induced by Duchenne muscular dystrophy and acute ischemia

The aim of this work was to identify micro‐RNAs (miRNAs) involved in the pathological pathways activated in skeletal muscle damage and regeneration by both dystrophin absence and acute ischemia. Eleven miRNAs were deregulated both in MDX mice and in Duchenne muscular dystrophy patients (DMD signature). Therapeutic interventions ameliorating the mdx‐phenotype rescued DMD‐signature alterations. The significance of DMD‐signature changes was characterized using a damage/regeneration mouse model of hind‐limb ischemia and newborn mice. According to their expression, DMD‐signature miRNAs were divided into 3 classes. 1) Regeneration miRNAs, miR‐31, miR‐34c, miR‐206, miR‐335, miR‐449, and miR‐494, which were induced in MDX mice and in DMD patients, but also in newborn mice and in newly formed myofibers during postischemic regeneration. Notably, miR‐206, miR‐34c, and miR‐335 were up‐regulated following myoblast differentiation in vitro. 2) Degenerative‐miRNAs, miR‐1, miR‐29c, and miR‐135a, that were down‐modulated in MDX mice, in DMD patients, in the degenerative phase of the ischemia response, and in newborn mice. Their down‐modulation was linked to myofiber loss and fibrosis. 3) Inflammatory miRNAs, miR‐222 and miR‐223, which were expressed in damaged muscle areas, and their expression correlated with the presence of infiltrating inflammatory cells. These findings show an important role of miRNAs in physiopathological pathways regulating muscle response to damage and regeneration.—Greco, S., De Simone, M., Colussi, C., Zaccagnini, G., Fasanaro, P., Pescatori, M., Cardani, R., Perbellini, R., Isaia, E., Sale, P., Meola, G., Capogrossi, M. C., Gaetano, C., Martelli, F. Common micro‐RNA signature in skeletal muscle damage and regeneration induced by Duchenne muscular dystrophy and acute ischemia. FASEB J. 23, 3335–3346 (2009). www.fasebj.org

HDAC2 blockade by nitric oxide and histone deacetylase inhibitors reveals a common target in Duchenne muscular dystrophy treatment

The overlapping histological and biochemical features underlying the beneficial effect of deacetylase inhibitors and NO donors in dystrophic muscles suggest an unanticipated molecular link among dystrophin, NO signaling, and the histone deacetylases (HDACs). Higher global deacetylase activity and selective increased expression of the class I histone deacetylase HDAC2 were detected in muscles of dystrophin-deficient MDX mice. In vitro and in vivo siRNA-mediated down-regulation of HDAC2 in dystrophic muscles was sufficient to replicate the morphological and functional benefits observed with deacetylase inhibitors and NO donors. We found that restoration of NO signaling in vivo, by adenoviral-mediated expression of a constitutively active endothelial NOS mutant in MDX muscles, and in vitro, by exposing MDX-derived satellite cells to NO donors, resulted in HDAC2 blockade by cysteine S-nitrosylation. These data reveal a special contribution of HDAC2 in the pathogenesis of Duchenne muscular dystrophy and indicate that HDAC2 inhibition by NO-dependent S-nitrosylation is important for the therapeutic response to NO donors in MDX mice. They also define a common target for independent pharmacological interventions in the treatment of Duchenne muscular dystrophy.

Glyceryl trinitrate for chronic anal fissure - Healing or headache? Results of a multicenter, randomized, placebo-controlled, double-blind trial

PURPOSE: Internal anal sphincterotomy for treating chronic anal fissure can irreversibly damage anal continence. Reversible chemical sphincterotomy may be achieved by anal application of glyceryl trinitrate ointment (nitric oxide donor), which has been reported to heal the majority of patients with anal fissure by inducing sphincter relaxation and improving anodermal blood flow. This trial aimed to further clarify the role of glyceryl trinitrate in the treatment of chronic anal fissure. METHODS: A total of 132 consecutive patients from nine centers were randomly assigned to receive 0.2 percent glyceryl trinitrate ointment or placebo twice daily for at least four weeks. The severity of pain and maximum anal resting pressure were measured before and after one week of treatment. Anodermal blood flow was measured before and after application of glyceryl trinitrate or placebo in ten patients. RESULTS: The study was completed by 119 patients (59 glyceryl trinitrate and 60 placebo), matched for gender, age, duration of symptoms, duration of treatment, site of fissure, previous attempts to treat, pain score, and maximum anal resting pressure. Twenty-nine patients (49.2 percent) healed after glyceryl trinitrate and 31 patients (51.7 percent) healed after placebo (P= not significant). Pain score fell significantly in both groups, in addition to maximum anal resting pressure. Anodermal blood flow improved significantly in seven patients receiving glyceryl trinitrate, but not in the three receiving placebo. Twenty-three patients (33.8 percent) experienced headache and 4 (5.9 percent), orthostatic hypotension after glyceryl trinitrate. CONCLUSION: This trial fails to demonstrate any superiority of topical 0.2 percent glyceryl trinitrate treatmentvs. a placebo, although the effects of glyceryl trinitrate on anodermal blood flow and sphincter pressure are confirmed. This finding, together with the high incidence of side-effects, should discourage the use of this treatment as a substitute for surgery in chronic anal fissure.

Bleeding, incontinence, pain and constipation after STARR transanal double stapling rectotomy for obstructed defecation

Abstract.Background:The STARR double stapling procedure (DSP), i. e. transanal anteroposterior rectotomy, has been recently reported as a low-morbidity and effective operation for the treatment of rectocele and internal rectal mucosal prolapse (R-IMP) causing obstructed defecation. We report the postoperative complications and recurrence of symptoms following this novel operation.Patients and methods:Fourteen chronically constipated women with RIMP, aged 36–72 years, presented with either severe complications or recurrence of symptoms following DSP performed by means of two circular staplers. All were followed for a median period of 12 months (range, 2–24) after DPS.Results:Severe rectal bleeding occurred in two cases postoperatively. Persistent severe anal pain was reported by seven patients, all presenting with anxiety. Four of them were multiparous. Three patients had fecal incontinence, both had vaginal deliveries. R-IMP recurred in six, obstructed defecation in seven cases. Four patients needed reintervention, one for suturing the bleeding area, one excising the recurrent prolapse, one for colpocele and one for rectal stricture. Four patients required biofeedback training for non-relaxing puborectalis and two needed psychotherapy.Conclusion:Parity, spastic floor syndrome and psychoneurosis seem to be the risk factors predisposing to failure of DSP, which may be followed by severe complications and early recurrence of symptoms requiring reoperation.

Results, Outcome Predictors, and Complications after Stapled Transanal Rectal Resection for Obstructed Defecation

PurposeObstructed defecation may be treated by stapled transanal rectal resection, but different complications and recurrence rates have been reported. The present study was designed to evaluate stapled transanal rectal resection results, outcome predictive factors, and nature of complications.MethodsClinical and functional data of 123 patients were retrospectively analyzed. All patients had symptoms of obstructed defecation before surgery and had rectocele and/or intussusception. Of them, 85 were operated on by the authors and 38 were referred after stapled transanal rectal resection had been performed elsewhere.ResultsAt a median follow-up of 17 (range, 3–44) months, 65 percent of the patients operated on by the authors had subjective improvement. Recurrent rectocele was present in 29 percent and recurrent intussusception was present in 28 percent of patients. At univariate analysis, results were worse in those with preoperative digitation (P < 0.01), puborectalis dyssynergia (P < 0.05), enterocele (P < 0.05), larger size rectocele (P < 0.05), lower bowel frequency (P < 0.05), and sense of incomplete evacuation (P < 0.05). Bleeding was the most common perioperative complication occurring in 12 percent of cases. Reoperations were needed in 16 patients (19 percent): 9 for recurrent disease. In the 38 patients referred after stapled transanal rectal resection, the most common problems were perineal pain (53 percent), constipation with recurrent rectocele and/or intussusception (50 percent), and incontinence (28 percent). Of these patients, 14 (37 percent) underwent reoperations: 7 for recurrence. Three patients presented with a rectovaginal fistula. One other patient died for necrotizing pelvic fasciitis.ConclusionsStapled transanal rectal resection achieved acceptable results at the cost of a high reoperation rate. Patients with puborectalis dyssynergia and lower bowel frequency may do worse because surgery does not address the causes of their constipation. Patients with large rectoceles, enteroceles, digitation, and a sense of incomplete evacuation may have more advanced pelvic floor disease for which stapled transanal rectal resection, which simply removes redundant tissue, may not be adequate. This, together with the complications observed in patients referred after stapled transanal rectal resection, suggests that this procedure should be performed by colorectal surgeons and in carefully selected patients.

Inflammatory conditions affect gene expression and function of human adipose tissue-derived mesenchymal stem cells

There is emerging interest in the application of mesenchymal stem cells (MSC) for the prevention and treatment of autoimmune diseases, graft‐versus‐host disease and allograft rejection. It is, however, unknown how inflammatory conditions affect phenotype and function of MSC. Adipose tissue‐derived mesenchymal stem cells (ASC) were cultured with alloactivated peripheral blood mononuclear cells (PBMC) (mixed lymphocyte reaction: MLR), with proinflammatory cytokines [interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and interleukin (IL)‐6] or under control conditions, and their full genome expression and function examined. Proinflammatory cytokines mainly increased indoleamine‐2,3‐dioxygenase expression, whereas ASC cultured with MLR showed increased expression of COX‐2, involved in prostaglandin E2 production. Both conditions had a stimulatory, but differential, effect on the expression of proinflammatory cytokines and chemokines, while the expression of fibrotic factors was decreased only in response to proinflammatory cytokines. Functional analysis demonstrated that inflammatory conditions affected morphology and proliferation of ASC, while their differentiation capacity and production of trophic factors was unaffected. The immunosuppressive capacity of ASC was enhanced strongly under inflammatory conditions. In conclusion, ASC showed enhanced immunosuppressive capacity under inflammatory conditions, while their differentiation capacity was preserved. Therefore, in vitro preconditioning provides ASC with improved properties for immediate clinical immune therapy.

Effect of perioperative blood transfusions on recurrence of colorectal cancer

PURPOSE: This study was undertaken to evaluate the influence of perioperative blood transfusions on colorectal cancer recurrence. METHODS: All articles published up to December 1996 in English (or with an English abstract) were retrieved, both using MEDLINE and scanning their references, to be considered for this meta-analysis. RESULTS: One hundred thirty-one articles were identified, and 99 of them were excluded because they analyzed survival or mortality, were repetitive publications, or were reviews or letters. Thirty-two original studies (9 were prospective) on 11,071 patients were included for further analysis; 20 showed a detrimental effect of perioperative blood transfusions. Nineteen articles used also multivariable techniques, and 11 found perioperative blood transfusions to be an independent prognostic factor. Pooled estimates of the effect of perioperative blood transfusions on colorectal cancer recurrence yielded an overall odds ratio of 1.68 (95 percent confidence interval, 1.54–1.83) and a rate difference of 0.13 (95 percent confidence interval, 0.09–0.17) against patients who received transfusions. Stratified meta-analyses also confirmed these findings when stratifying patients by site and stage of disease. The effect of perioperative blood transfusion was observed in a dose-related fashion, regardless of timing and type, although some heterogeneity was detected. Data on surgical techniques were not available for further analysis. CONCLUSIONS: A consistently detrimental association was discovered between the use of perioperative blood transfusion and colorectal cancer recurrence. Further studies are needed to confirm that blood transfusion has a causal association.

Anal continence after rectocele repair.

INTRODUCTION: Rectocele may be associated with both chronic constipation and anal incontinence. Several different surgical procedures have been advocated for rectocele repair. The aim of the present study was to evaluate anorectal function and clinical outcome in a consecutive series of patients who underwent selected endorectal or transperineal surgery for rectocele for whom operative treatment was determined by clinical and proctographic features. Attention was paid to the cohort of rectocele patients presenting with incontinence as a leading symptom. METHODS: Sixty consecutive patients with symptomatic rectocele underwent surgical treatment at our institution. Fifty-eight of the patients were female (mean age 56; range, 21–70 years). Incontinence was graded according to a previously reported scoring system that accounts for the type and frequency of incontinence episodes. Preoperative anorectal manometry was performed using an open perfused polyethylene probe. Rectal sensation was recorded by balloon distention. Endoanal ultrasonography was performed with a 7.5-MHz probe. Preoperative defecography was performed at rest and on maximal squeeze and straining. Patients with obstructed defecation as their principal symptom, with associated mucosal rectal prolapse, underwent an endorectal procedure. For patients with associated anal incontinence (Grade B2 or greater), and without a rectal mucosal prolapse, a transperineal approach was performed with either an anterior external overlapping sphincteroplasty or levatorplasty. The median follow-up was 48 (range, 9–122) months. RESULTS: There was no operative mortality. Postoperative complications occurred in 18 patients (30 percent). Of 43 patients with incontinence, 34 (79 percent) were available for postoperative evaluation. None were fully continent. However, in 25 patients (73.5 percent), continence improved after surgery; half had only mucus soiling or loss of gas. Incontinence scores decreased (i.e., improved) from 4.8 ± 0.9 to 3.9 ± 0.9 (P = 0.002). A significant improvement was found both after transanal and perineal procedures. Only ten initially continent patients were available for postoperative assessment. All patients stated that they had clinical improvement in constipation. Their preoperative mean anal resting pressure was 62.5 ± 3.9 (standard error of the mean) mmHg, with a postoperative mean of 75.5 ± 7 mmHg. The preoperative mean squeeze pressure was 83.1 ± 8.5 mmHg, with a mean postoperative squeeze pressure of 88.5 ± 7.9 mmHg (P = not significant). The maximal tolerable volumes were all within normal limits, confirming the proctographic evidence that there were no cases of megarectum in our patient series. The pudendal nerve terminal motor latency was abnormal in all but two patients with incontinence (mean pudendal nerve terminal motor latency = 3.1; range, 1.2–4 milliseconds). Rectoceles recurred in six patients (10 percent): five after a Block procedure and one after a Sarles-type operation. The postoperative endosonographic appearance varied according to the nature of the procedure performed. CONCLUSION: There are few data concerning patients with rectocele who have associated anal incontinence, however, surgical decision analysis resulted in improvement in both constipation and incontinence in the majority of our patients with rectocele. Nevertheless, because none of the patients gained full continence postoperatively, pelvic floor rehabilitation might be also needed to achieve better sphincter function in patients with incontinence.

Functionalized carbon nanotubes as immunomodulator systems

In view of the broad potential biomedical applications of carbon nanotubes (CNTs) different studies were performed to assess their effect on the immune system. However, the work performed to date was able to give a restricted view looking only at some activation markers and cytokine expression. The immune system is rarely limited to few molecule interactions being instead always a balance of switching several genes on and off. Whole genome expression (microarray) is a technology able to give the full picture on genome expression. Here we describe a microarray genome-wide study on Jurkat cells, a T lymphocyte cell line, and THP1, a monocytic cell line, representative of both types of immune response, the adaptive and innate, respectively. Since any structure or molecule modification may lead to very different immune reactions, we treated the two cell lines with four types of functionalized multi-walled CNTs that differ in terms of functionalization and diameter. After having assessed the internalization and the lack of toxicity of CNTs in both cell types, we used the Affymetrix technology to analyze the expression of about 32,000 transcripts. Three of the tested nanotubes (i.e., ox-MWCNT-1, ox-MWCNT-NH3(+)-1, and ox-MWCNT-NH3(+)-2) activated immune-related pathways in monocytes but not in T cells. In view of these charateristics they were named as monocyte activating CNTs (MA-CNTs). Molecular pathways upregulated by MA-CNTs included IL6, CD40, dendritic cell maturation, tumor necrosis factor-(TNF)-α/TNFR1-2, NFKB signaling and T helper 1 chemokine pathways (CXCR3 and CCR5 ligand pathways). These pathways are commonly activated during acute inflammatory processes as those associated with immune-mediated tumor rejection and pathogen clearance. One of them (i.e., ox-MWCNT-2) downregulated genes associated with ribosomal proteins in both monocytes and T cells. We validated our findings at gene expression level by performing real-time PCR assessing the most highly modulated genes in monocytes. To confirm the results at protein level, the secretion of IL1β, TNFα, IL6 and IL10 by THP1 and primary monocytes was assessed by ELISA, corroborating gene-expression data. Our results provide new insights into the whole gene expression modulation by different CNTs on immune cells. Considering the well known drug carrier ability of CNTs, our findings demonstrate that MA-CNTs here behave as cell specific immunostimulatory systems, giving very interesting future perspectives for their application also as immunotherapeutic agents and/or vaccine adjuvants.