Edda Battaglia

Thrombopoietin Stimulates Endothelial Cell Motility and Neoangiogenesis by a Platelet-Activating Factor–Dependent Mechanism

In this study, we demonstrate that human umbilical cord vein-derived endothelial cells (HUVECs) expressed c-Mpl, the thrombopoietin (TPO) receptor, and that TPO activates HUVECs in vitro, as indicated by directional migration, synthesis of 1-alkyl-/1-acyl-platelet-activating factor (PAF) and interleukin-8 (IL-8), and phosphorylation of the signal transducers and activators of transcription (STAT) STAT1 and STAT5B. The observation that WEB 2170 and CV3988, 2 structurally unrelated PAF receptor antagonists, prevented the motility of HUVECs induced by TPO suggests a role of PAF as secondary mediator. Moreover, kinetic analysis of TPO-induced tyrosine phosphorylation of STAT demonstrated that STAT5B activation temporally correlated with the synthesis of PAF. PAF, in turn, induced a rapid tyrosine phosphorylation of STAT5B and PAF receptor blockade, by WEB 2170, preventing both TPO- and PAF-mediated STAT5B activation. The in vivo angiogenic effect of TPO, studied in a mouse model of Matrigel implantation, demonstrated that TPO induced a dose-dependent angiogenic response that required the presence of heparin. Moreover, the in vivo angiogenic effect of TPO was inhibited by the PAF receptor antagonist WEB 2170 but not by the anti-basic fibroblast growth factor neutralizing antibody. These results indicate that the effects of TPO are not restricted to cells of hematopoietic lineages, because TPO is able to activate endothelial cells and to induce an angiogenic response in which the recruitment of endothelial cells is mediated by the synthesis of PAF. Moreover, biochemical analysis supports the hypothesis that STAT5B may be involved in the signaling pathway leading to PAF-dependent angiogenesis.

long-term Study on the Effects of Visual Biofeedback and Muscle Training as a Therapeutic Modality in Pelvic Floor Dyssynergia and Slow-transit Constipation

PURPOSE: Biofeedback training has been shown as an effective therapeutic measure in patients with pelvic floor dyssynergia, at least in the short term. Long-term effects have received less attention. Moreover, its effects in patients with slow-transit constipation have been scarcely investigated. This study was designed to assess in an objective way the medium- and long-term effects of biofeedback and muscle training in patients with pelvic floor dyssynergia and slow-transit constipation. METHODS: Twenty-four patients (14 with pelvic floor dyssynergia and 10 with slow transit) meeting the Rome II criteria for constipation, and unresponsive to conventional treatments, entered the study. Clinical evaluation and anorectal manometry were performed basally and three months after a cycle of electromyographic biofeedback and muscle training; moreover, a clinical interview was obtained one year after biofeedback. Patients with slow-transit constipation also had colonic transit time reassessed at one year. RESULTS: Clinical variables (abdominal pain, straining, number of evacuations/week, use of laxatives) all significantly improved in both groups at three-month assessment; anorectal manometric variables remained unchanged, apart from a significant decrease of sensation threshold in the pelvic floor dyssynergia group and of the maximum rectal tolerable volume in the slow-transit constipation group. At one-year control, 50 percent of patients with pelvic floor dyssynergia still maintained a beneficial effect from biofeedback, whereas only 20 percent of those complaining of slow-transit constipation did so. Moreover, the latter displayed no improvement in colonic transit time. CONCLUSIONS: In our experience, patients with pelvic floor dyssynergia are likely to have continued benefit from biofeedback training in the time course, whereas its effects on slow-transit constipation seems to be maximal in the short-term course.

Manometric investigation of anorectal function in early and late stage Parkinson's disease

Abnormal gastrointestinal function is relatively frequent in Parkinsons disease, and constipation is a disturbing symptom in many patients. However, it remains to be established whether anorectal abnormalities are characteristic of the late stages of the disease. Clinical and anorectal manometric function were investigated in groups of early and late stage parkinsonian patients. Thirty one patients (19 men, 12 women, age range 22 to 89 years) entered the study. The disease severity was assessed by Hoehn and Yahr staging: there were four (12.9%) stage I, seven (22.6%) stage II, 10 (32.2%) stage III, and 10 (32.2%) stage IV patients. Anorectal variables were measured by standard manometric equipment and techniques. Values obtained in early stage patients (Hoehn and Yahr stage I and II) were compared with those obtained in late stage patients (Hoehn and Yahr stage III and IV). Overall, more than 70% of patients complained of chronic constipation, with chronic laxative use reported in more than 30%. Late stage patients were slightly older than their early stage counterparts. Pelvic floor dyssynergia was documented in more than 60% of patients. Manometric variables were not different in the two groups. In conclusion, defecatory dysfunction is frequent in Parkinsons disease, it is not confined to late stage patients, and it is found early in the course of the disease. This has potential implications for a targeted therapeutic approach.

Platelet-Activating Factor Enhances Vascular Endothelial Growth Factor–Induced Endothelial Cell Motility and Neoangiogenesis in a Murine Matrigel Model

We previously reported that platelet-activating factor (PAF) enhances the angiogenic activity of certain polypeptide mediators such as tumor necrosis factor and hepatocyte growth factor by promoting endothelial cell motility. The purpose of the present study was to evaluate whether the synthesis of PAF induced by vascular endothelial growth factor (VEGF) might affect endothelial cell motility, microvascular permeability, and angiogenesis. The neoangiogenesis and synthesis of PAF induced by VEGF were studied in vivo in a murine Matrigel model. Dermal permeability was studied in mice by injection of (125)I-albumin. The synthesis of PAF, cell motility, and the increased (125)I-albumin transfer across endothelial monolayers were studied in vitro by using cultures of human umbilical cord vein-derived endothelial cells (HUVECs). The results obtained demonstrate that the neoangiogenesis induced by VEGF in vivo was associated with a local synthesis of PAF and was inhibited by WEB2170 and CV3988, 2 chemically unrelated, specific PAF-receptor antagonists. In contrast, WEB2170 did not inhibit VEGF-enhanced dermal permeability, suggesting that the latter was independent of the synthesis of PAF. In vitro, it was found that VEGF induced the synthesis of PAF by HUVECs in a dose- and time-dependent manner. The cell motility induced by VEGF was inhibited by PAF-receptor antagonists. In contrast, VEGF-induced proliferation of HUVECs and albumin transfer through HUVEC monolayer were unaffected by PAF-receptor antagonists. These results suggest that the synthesis of PAF induced by VEGF enhances endothelial cell migration and contributes to the angiogenic effect of VEGF in the in vivo Matrigel model.

Expression of CD40 and its ligand, CD40L, in intestinal lesions of Crohn's disease

Objective:Selected mechanisms of the immune system participate in the development of inflammatory bowel disease. Recently, overexpression of the ligand for CD40 (CD40L), a lymphocyte costimulatory molecule, was shown to induce severe inflammatory bowel disease in transgenic mice. In the present study, we examined the expression of CD40 and CD40L on surgical specimens of ileum from 12 patients with Crohns disease and 10 patients with diverticulitis.Methods:Several CD40L+ cells were present in the affected tissue of patients with Crohns disease, whereas few scattered CD40L+ cells were detected in sections of histologically normal ileum, resected distantly from the affected tissue, in patients with diverticulitis and in normal ileum portions obtained from colorectal cancer undergoing extensive surgery. The phenotype of CD40L+ cells was mainly CD4+.Results:In patients with Crohns disease, several CD40+ cells were detectable in the same areas of lymphocytes expressing CD40L, whereas in patients with diverticulitis, the number of CD40+ cells was significantly lower. Most of the CD40+ cells costained with CD20, thus showing to be B-lymphocytes, and only a few were CD14+ macrophages. Several von Willebrand-positive vessels were also positive for CD40. In addition, several infiltrating macrophages were found to express B7-1 and B7-2 molecules, the ligands of CD28 and CTLA-4, which cooperate with the CD40-CD40L pathway in lymphocyte activation. Staining of ileal lesions with anti-CTLA-4 antibodies resulted in detection of none or very few positive cells. In contrast, in patients with diverticulitis, an enhanced number of B7-1 and B7-2 and CTLA-4 was observed.Conclusion:The local accumulation of CD40L+ together with CD40+ cells within intestinal lesions of Crohns disease suggests the involvement of this co-stimulatory pathway.

Alterations in Colonic Motility and Relationship to Pain in Colonic Diverticulosis

BACKGROUND AND AIMS Although the pathophysiologic basis of colonic diverticular disease is understood incompletely, there is agreement that abnormal colon motility probably plays a major role. However, several different abnormalities have been reported in such patients. The purpose of this study was to assess whether patients with diverticulosis display an abnormal duration of regular colonic contractile patterns, which has been observed in other conditions characterized by spasticity of the viscus, such as the irritable bowel syndrome. METHODS Twelve patients with symptomatic uncomplicated diverticular disease entered the study and underwent 24-hour colonic manometric recordings using a standard technique. The duration of regular contractile patterns was compared with that recorded in 20 healthy volunteers. RESULTS Patients with diverticulosis had a significant increase of the duration of regular patterns of phasic pressure activity compared with healthy controls (31% vs. 6.4%, P < .001). In both groups, the 2- or 3-cycles-per-minute activity represented more than 80% of such activity, especially in the sigmoid colon. More than 30% of patients, but none of the controls, reported episodes of abdominal pain (cramping lower abdominal pain with characteristics similar to those experienced at home) during the occurrence of a regular colonic contractile pattern. This was significant by symptom association probability criteria. CONCLUSIONS Patients with symptomatic uncomplicated colonic diverticulosis displayed increased duration of rhythmic, low-frequency, contractile activity, particularly in the segments bearing diverticula. These regular rhythms are associated significantly with reporting of abdominal pain.

Gluten-Free Diet Normalizes Mouth-to-Cecum Transit of a Caloric Meal in Adult Patients with Celiac Disease

The mechanisms responsible for boweldisturbances in celiac disease are still relativelyunknown. Recent reports suggested that small bowel motorabnormalities may be involved in this pathologicalcondition; however, there are no studies addressing smallbowel transit in celiac disease before and after agluten-free diet. We studied the mouth-to-cecum transittime of a caloric liquid meal in a homogeneous group of celiac patients presenting with clinical andbiochemical evidence of malabsorption and complaining ofdiarrhea. Sixteen patients were recruited andinvestigated by means of hydrogen breath test through ingestion of 20 g lactulose together with anenteral gluten-free diet formula. A urinary D-xylosetest was also done in each patient. Both breath testsand D-xylose tests were carried out basally and after a period of gluten-free diet. Twenty healthyvolunteers were recruited as a control group andunderwent the same breath testing. At the time of thediagnosis, mouth-to-cecum transit time was significantly prolonged in celiacs with respect to controls(243 ± 10 vs 117 ± 6 min, P = 0.0001). TheD-xylose test was also abnormal (average urinaryconcentration 2.8 ± 0.25 g, normal values>4.5). No correlation was found in patients between mouth-to-cecum transit timeand urinary D-xylose output (r = 0.22). After thegluten-free diet period, mouth-tocecum transit time inceliacs was significantly reduced compared to prediet transit (134 ± 8 vs 243 ± 10 min,P = 0.0001) and did not show statistical difference whencompared to that found in controls (P = 0.1). TheD-xylose test reverted to normal in all but twosubjects, who were found to be noncompliant with the diet.Mouth-to-cecum transit time is significantly prolongedin patients affected by untreated celiac disease whencompared to healthy controls. This alteration might notbe correlated to intestinal malabsorption, and theprolonged orocecal transit could be due to impairedsmall bowel function (deranged motility?). Sinceintestinal transit returned to normal values after an adequate gluten-free period, a link with severeactive mucosal lesions is suggestive.

Regulation of polymorphonuclear cell activation by thrombopoietin.

Thrombopoietin (TPO) regulates early and late stages of platelet formation as well as platelet activation. TPO exerts its effects by binding to the receptor, encoded by the protooncogene c-mpl, that is expressed in a large number of cells of hematopoietic origin. In this study, we evaluated the expression of c-Mpl and the effects of TPO on human polymorphonuclear cells (PMN). We demonstrate that PMN express the TPO receptor c-Mpl and that TPO induces STAT1 tyrosine phosphorylation and the formation of a serum inducible element complex containing STAT1. The analysis of biological effects of TPO on PMN demonstrated that TPO, at concentrations of 1-10 ng/ml, primes the response of PMN to n-formyl-met-leu-phe (FMLP) by inducing an early oxidative burst. TPO-induced priming on FMLP-stimulated PMN was also detected on the tyrosine phosphorylation of a protein with a molecular mass of approximately 28 kD. Moreover, we demonstrated that TPO by itself was able to stimulate, at doses ranging from 0.05 to 10 ng/ml, early release and delayed synthesis of interleukin 8 (IL-8). Thus, our data indicate that, in addition to sustaining megakaryocytopoiesis, TPO may have an important role in regulating PMN activation.

Ski/SnoN expression in the sequence metaplasia-dysplasia-adenocarcinoma of Barrett's esophagus☆

Barretts esophagus (BE) is a precancerous condition. However, the mechanisms underlying the transformation from metaplastic to dysplastic to adenocarcinomatous epithelium are still poorly understood. As loss of transforming growth factor-beta growth inhibition is considered a hallmark of several human neoplasms, we evaluated the expression of Ski and SnoN (proteins that antagonize transforming growth factor-beta signaling through physical interaction with Smad complex and by recruiting histone deacetylases), as markers of the transforming growth factor-beta signaling pathway, in BE with and without dysplasia. Biopsy samples from 37 patients (26 men, aged 60 +/- 8 years) with histologically proven BE were evaluated; 10 patients had concomitant low-grade dysplasia, 7 high-grade dysplasia (HGD), and 6 HGD associated with adenocarcinoma. Ski and SnoN expression was assessed immunohistochemically. Neither Ski nor SnoN was expressed in normal esophageal epithelium, but both were strongly expressed in BE tissue, with intense cytoplasmic positivity. Expression of these proteins decreased markedly in dysplastic areas in patients with low-grade dysplasia and was absent in those with HGD or HGD/adenocarcinoma. Ski and SnoN proteins are overexpressed in BE and may be involved in abnormal signaling elicited by transforming growth factor-beta in this epithelium, enhancing the tumorigenesis process. These observations might help to elucidate the molecular mechanisms involved in the BE tumorigenesis process.

Botulinum toxin treatment of oesophageal achalasia in the old old and oldest old: a 1-year follow-up study.

BackgroundIntrasphincteric injection of botulinum toxin (BTX) has become one of the most frequent therapeutic approaches for the treatment of oesophageal achalasia. This treatment seems particularly effective in elderly patients who are not candidates for more invasive procedures.AimsThere are few or no data on BTX treatment of achalasia in the old old and oldest old. Therefore, we evaluated BTX treatment in a group of patients with achalasia in the extreme age range who were too ill or frail to undergo surgery or pneumatic dilatation.Patients and MethodsTwelve elderly achalasic patients (age range 81–94 years, average age 86 years) with American Society of Anesthesiologists (ASA) class III–IV status were recruited for the study. After baseline clinical and instrumental evaluations, BTX 100U was injected at time 0 and 1 month later. Clinical follow-up was carried out after 3, 6 and 12 months.ResultsA significant improvement in symptom score was documented at each follow-up step. On the basis of improvements in scores, approximately 70% of patients were considered responders at the end of follow-up.ConclusionsBTX treatment is an effective treatment in a substantial proportion of achalasic patients >80 years of age, in whom benefits are still detectable after 12 months. BTX is a therapeutic option in patients unsuitable for surgery or pneumatic dilatation.