Fabio F. di Mola

Nerve Growth Factor and Its High-Affinity Receptor in Chronic Pancreatitis

OBJECTIVE To study the mechanisms that are involved in nerve growth and contribute to pain generation in chronic pancreatitis (CP). SUMMARY BACKGROUND DATA Chronic pancreatitis is a painful disease associated with characteristic nerve changes, including an increase in nerve number and diameter. The mechanisms that influence nerve growth are not known. Nerve growth factor (NGF) and its high-affinity tyrosine kinase receptor A (TrkA) are involved in neural development and survival and growth of central and peripheral nerves. METHODS Nerve growth factor and TrkA were investigated by Northern blot analysis, in situ hybridization, and immunohistochemical staining in the pancreases of 24 patients with CP, and the findings were correlated with clinical parameters. RESULTS By Northern blot analysis, NGF and TrkA mRNA expression were increased in 42% (13.1-fold) and 54% (5.5-fold) of the CP samples (p < 0.01), respectively. In situ hybridization revealed that in CP, enhanced NGF mRNA expression was present in metaplastic ductal cells, in degenerating acinar cells, and in acinar cells dedifferentiating into tubular structures. TrkA mRNA was intensely present in the perineurium. Further, enhanced NGF and TrkA mRNA signals were also present in intrapancreatic ganglia cells in CP samples. Immunohistochemistry confirmed the in situ hybridization findings. Analysis of the molecular findings with clinical parameters revealed a significant relation (p < 0.05) between NGF mRNA levels and pancreatic fibrosis (r = 0.64) and acinar cell damage (r = 0.74) and between TrkA mRNA and pain intensity (r = 0.84). CONCLUSION Activation of the NGF/TrkA pathway occurs in CP. It might influence neural morphologic changes and the pain syndrome in this disorder.

Connective tissue growth factor is a regulator for fibrosis in human chronic pancreatitis

OBJECTIVE To evaluate the parameters that mediate fibrogenesis in chronic pancreatitis (CP). BACKGROUND Connective tissue growth factor (CTGF), which is regulated by transforming growth factor beta (TGF-beta), has recently been implicated in skin fibrosis and atherosclerosis. In the present study, the authors analyzed the concomitant presence of TGF-beta1 and its signaling receptors-TGF-beta receptor I, subtype ALK5 (TbetaR-I(ALK5)), and TGF-beta receptor II (TbetaR-II)-as well as CTGF and collagen type I in the pancreatic tissue of patients undergoing surgery for chronic pancreatitis. PATIENTS AND METHODS CP tissue samples were obtained from 40 patients (8 women, 32 men) undergoing pancreatic resection. Tissue samples of 25 previously healthy organ donors (12 women, 13 men) served as controls. The expression of TGF-beta1, TbetaR-I(ALK5), TbetaR-II, CTGF, and collagen type I was studied by Northern blot analysis. By in situ hybridization and immunohistochemistry, the respective mRNA moieties and proteins were localized in the tissue samples. RESULTS Northern blot analysis showed that CP tissue samples exhibited concomitant enhanced mRNA expression of TGF-beta1 (38-fold), TbetaR-II (5-fold), CTGF (25-fold), and collagen type I (24-fold) compared with normal controls. In addition, TbetaR-I(ALK5) mRNA was increased in 50% of CP tissue samples (1.8-fold). By in situ hybridization, TGF-beta1, TbetaR-I(ALK5), and TbetaR-II mRNA were often seen to be colocalized, especially in the ductal cells and in metaplastic areas where atrophic acinar cells appeared to dedifferentiate into ductal structures. In contrast, CTGF was located in degenerating acinar cells and principally in fibroblasts surrounding these areas. Moreover, CTGF mRNA expression levels correlated positively with the degree of fibrosis in CP tissues. CONCLUSION The concomitant overexpression of CTGF, collagen type I, TGF-beta1, and its signaling receptors in CP suggests that these proteins contribute to enhanced extracellular matrix synthesis and accumulation, resulting finally in the fibrogenesis observed in CP.

Desmoplastic Reaction Influences Pancreatic Cancer Growth Behavior

Connective tissue growth factor (CTGF), which is regulated by transforming growth factor-ß (TGFß), has recently been implicated in the pathogenesis of fibrotic diseases and tumor stroma. Inasmuch as generation of desmoplastic tissue is characteristic for pancreatic cancer, it is not known whether it gives pancreatic cancer cells a growth advantage or is a reaction of the body to inhibit cancer cell progression. In the present study we analyzed the expression and localization of CTGF and evaluated whether it influences the prognosis of pancreas cancer. Tissue samples were obtained from 25 individuals (6 women, 19 men) undergoing pancreatic resection for pancreatic cancer. Tissue samples from 13 previously healthy organ donors (5 women, 8 men) served as controls. Expression of CTGF was studied by Northern blot analysis. In situ hybridization and immunohistochemistry localized the respective mRNA moieties and proteins in the tissue samples. Northern blot analysis revealed that pancreatic cancer tissue samples exhibited a 46-fold increase in CTGF mRNA expression (p < 0.001) over that of normal controls. In vitro studies confirmed that pancreatic stellate cells are the major source of CTGF mRNA expression and revealed a large variance in basal and TGFß-induced CTGF expression in cultured pancreatic cancer cells. This could also be confirmed by in situ hybridization, indicating that CTGF mRNA signals were located principally in fibroblasts, with only weak signals in the cancer cells. High CTGF mRNA levels in the tissue samples correlated with better tumor differentiation (p < 0.03). In addition, patients whose tumors exhibited high CTGF mRNA levels (> onefold increase above normal controls) lived significantly longer than those whose tumors expressed low CTGF mRNA levels (none to onefold) (p < 0.04 multivariate analysis). Our present data indicate that CTGF, as a downstream mediator of TGFß, is overexpressed in connective tissue cells and to a lesser extent in pancreatic cancer cells. Because patients with high CTGF mRNA expression levels have a better prognosis, our findings indicate that the desmoplastic reaction provides a growth disadvantage for pancreatic cancer cells.

Connective tissue growth factor gene expression alters tumor progression in esophageal cancer

The ability of cancer cells to initiate specific fibroblast reactions may subsequently determine tumor evolution. In the present study we examined the coordinated expression of transforming growth factor-beta-1 (TGF-b1), its signaling receptors, and its downstream mediator—connective tissue growth factor (CTGF)—and their impact on tumor progression and fibrogenesis in esophageal carcinomas. Messenger ribonucleic acid (mRNA) expression of TGF-b1, CTGF, TGF-b receptor subtype I ALK5 (TbR-IALK5), and TGF-b receptor type II (TbR-II) was studied by Northern blot analysis in esophageal cancer and the normal esophagus. By means of immunohistochemistry and Western blot analysis, the respective proteins were localized in the tissue samples and the protein content was quantitated. Northern blot analysis revealed 3-fold and 4-fold increases (p <0.05) in TGF-b1 and CTGF mRNA levels, respectively, in esophageal cancer in comparison with normal controls, whereas TbR-I mRNA levels were significantly decreased and TbR-II mRNA levels were unchanged in the cancer samples. Immunostaining revealed results similar to those seen on the RNA level. TGF-b1 and CTGF immunoreactivity were increased, TbR-II was unchanged, and TbR-IALK5 immunoreactivity was decreased. CTGF immunoreactivity was mainly present in the stroma surrounding the cancer cells but was also present in the cancer cells. The degree of fibrosis was different in squamous and adenocarcinomas and was significantly related to CTGF mRNA expression levels. The presence of CTGF in squamous cell carcinomas was associated with longer survival, whereas in adenocarcinomas it influenced survival negatively. The findings indicate that TGF-b signaling is disturbed in esophageal cancer. CTGF, a downstream effector of TGF-b action, differentially influences the composition of tumor microenvironment and distinct cell-matrix interactions in the two histological types of esophageal carcinoma, resulting in differences in tumor progression and patient survival.

NK-1 receptor gene expression is related to pain in chronic pancreatitis

&NA; Recent theories of pathogenesis of pain in chronic pancreatitis (CP) are neuroimmune interactions of intrapancreatic nerves and inflammatory cells and increase in levels of pain neurotransmitters such as substance P (SP). This study analyzed the expression and localization of neurokinin 1 receptor (NK‐1R), which binds SP, and its association with pain and inflammation in CP. Pancreatic tissues from 31 patients (22 males, nine females; mean age 45.9±9.4 years) with CP were evaluated. Nine normal pancreases (five males, four females; mean age 42.9±9.5 years) served as controls. Quantitative PCR was used to determine the NK‐1R mRNA expression levels and in situ hybridization and immunohistochemistry were used to localize expression sites of NK‐1R mRNA and protein, respectively. We also analyzed whether an association exists between NK‐1R mRNA expression and pain and inflammation. In CP samples, in situ hybridization and immunohistochemistry localized NK‐1R mRNA expression and protein mainly in the nerves, ganglia, blood vessels, inflammatory cells and occasionally in fibroblasts. In patients with mild to moderate and strong intensity of pain, NK‐1R mRNA levels were increased 14‐ and 30‐fold over controls, respectively. There was a significant relationship between NK‐1R mRNA levels and intensity of pain (r=0.46, P=0.03), NK‐1R mRNA and the frequency of pain (r=0.51, P=0.04), and NK‐1 mRNA and duration of pain (r=0.46, P=0.01) in CP patients, but not with the degree of tissue inflammation. NK‐1R signaling may be involved in the pain syndrome of CP. The expression of NK‐1R in inflammatory cells and blood vessels also points to an interaction of immunoreactive substance P nerves, inflammatory cells and blood vessels, and further supports the existence of a neuroimmune interaction that probably influences the pain syndrome and chronic inflammatory changes so characteristic of CP.

Beneficial Effects of Batimastat (BB-94), a Matrix Metalloproteinase Inhibitor, in Rat Experimental Colitis

Background and Aims: Matrix metalloproteinases (MMPs) represent a group of enzymes that regulate cell-matrix composition playing a major role in the inflammatory response. In the present study we evaluated the ability of the MMP inhibitor Batimastat (BB-94) to modify the course of experimental colitis induced in the rat by trinitrobenzensulfonic acid (TNB). Methods: Colitis was induced in 40 rats by intracolonic administration of TNB. Animals were divided into four groups of ten rats each: group 1 received only intracolonic TNB, group 2 received TNB+5 mg/kg intraperitoneal BB-94, group 3 TNB+10 mg/kg BB-94 and group 4 TNB+20 mg/kg BB-94. The MMP inhibitor was administered 30 min before induction of colitis and twice daily until death. Ten rats receiving only intracolonic 0.9% saline served as controls. Animals were killed after seven days; segments of colon were removed and used for histological score of inflammation and myeloperoxidase (MPO) activity. Results: Rats receiving only intracolonic 0.9% saline showed no evidence of colitis. The inflammation score was 0.9, MPO activity 0.235 U/mg. Group 1 (TNB-treated rats) exhibited a high inflammation score (12.4) and MPO activity (0.715 U/mg). Conversely, BB-94-treated rats showed, compared to the TNB group, a significantly lower inflammation score and MPO activity in a dose-dependent fashion. Group 2: inflammatory score 10.1, MPO activity 0.474 (p < 0.05 vs. TNB); group 3: inflammatory score 8.3, MPO activity 0.287 (p < 0.01 vs. TNB); group 4: inflammatory score 5.0, MPO activity 0.256 (p < 0.01 vs. TNB). Conclusions: Treatment with BB-94 has dose-dependent beneficial effects on the inflammatory alterations in rat experimental colitis. Thus, the inhibition of MMPs may represent a novel therapeutic approach for treatment of intestinal inflammation.

Overexpressed Decorin in Pancreatic Cancer Potential Tumor Growth Inhibition and Attenuation of Chemotherapeutic Action

Purpose: The aim of this study was to investigate the expression and significance of decorin in pancreatic cancer. Experimental Design: Decorin expression in normal pancreas and excised tumors was examined by real-time quantitative PCR, Western blot analysis, and immunohistochemistry. Reverse transcription-PCR was used to analyze cultures of pancreatic cancer and stellate cells. Growth-inhibitory effects of decorin in vitro were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test, Western blot, and fluorescence-activated cell-sorting analysis. Results: Pancreatic cancer was characterized by striking overexpression of decorin mRNA in tumor tissues (9-fold by real-time quantitative PCR; 44 patients versus 18 healthy donors; P < 0.01). Strong decorin immunostaining was observed in the extracellular matrix of pancreatic cancer tissue, whereas tumor cells were devoid of decorin. Double staining for anti-smooth muscle actin and decorin and reverse transcription-PCR analysis of primary cultures revealed pancreatic stellate cells as the putative source of decorin. Human recombinant decorin was able to suppress growth of pancreatic cancer cells in vitro through p21mediated G1-S block of the cell cycle. However, in contrast to the previously described chemotherapy-potentiating capacity of decorin, this proteoglycan attenuated the cytostatic action of carboplatin and gemcitabine toward pancreatic cancer cells. Conclusions: Decorin might exert an antiproliferative effect toward pancreatic cancer cells, thus playing a role in a host stromal reaction aimed at sequestering and inhibiting growing malignant cells. However, in clinical settings, the importance of collagen-associated decorin as a moderate antitumor modality would be undermined by its ability to attenuate the efficiency of chemotherapeutics. Considering the general failure of adjuvant therapies in pancreatic cancer, the role of decorin in this process warrants further investigation.

Pathogenesis of Pain in Chronic Pancreatitis

The pathophysiology of pain in chronic pancreatitis (CP) is incompletely understood. Several hypotheses have been advanced, including pancreatic and extrapancreatic causes. The existence of different hypotheses to explain the genesis of pain in CP also reflects the different therapeutic approaches to pain in these patients. Increased intraductal pressure as a result of single or multiple strictures and/or calculi is believed to be a common cause of pain in CP patients with a dilated main pancreatic duct. Other suggested causes include pancreatic fibrosis, interstitial hypertension and pancreatic ischemia. Additionally, extrapancreatic causes like duodenal and common bile duct stenosis with scarring due to pancreatic inflammation are suggested as factors causing pain in CP. The ‘neurogenic inflammation’ hypothesis is a fascinating theory which is supported by different studies. Immunohistological reports have shown that the amount of neurotransmitters, such as substance P and its receptor, calcitonin gene-related peptide and other neurotransmitters, are increased in afferent pancreatic nerves and a correlation between pain and immune cell infiltration of the nerves has been reported in CP. In this review we will discuss the different pain hypotheses and will present the perspective that neuroimmune interaction is an important factor for pain generation in CP.

Expression of the Antiapoptotic Protein BAG3 Is a Feature of Pancreatic Adenocarcinoma and Its Overexpression Is Associated With Poorer Survival

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers, being the fourth leading cause of cancer-related deaths. Long-term survival reaching 15% is achieved in less than 5% of patients who undergo surgery, and median survival is only 6 months in those with inoperable lesions. A deeper understanding of PDAC biologic characteristics as well as novel prognostic markers are therefore required to improve outcomes. Herein we report that BAG3, a protein with recognized anti-apoptotic activity, was expressed in 346 PDACs analyzed, but was not expressed in the surrounding nonneoplastic tissue. In a cohort of 66 patients who underwent radical resection (R0), survival was significantly shorter in patients with high BAG3 expression (median, 12 months) than in those with low BAG3 expression (median, 23 months) (P = 0.001). Furthermore, we report that BAG3 expression in PDAC-derived cell lines protects from apoptosis and confers resistance to gemcitabine, offering a partial explanation for the survival data. Our results indicate that BAG3 has a relevant role in PDAC biology, and suggest that BAG3 expression level might be a potential marker for prediction of patient outcome.

Pain and pain generation in pancreatic cancer

BackgroundPain can be a frequent symptom during the natural history of a patient with pancreatic cancer. An increase in incidence with disease progression and the presence of unbearable pain may preclude a curative resection.Materials and methodsEven in those patients with resectable pancreatic cancer, the presence of pain has an impact on prognosis. To date, we do not really know why some patients develop pain.ResultsPerineural cancer cell invasion is one of the most intriguing characteristics of this neoplasia and may in some cases explain the pain sensation. In addition, so-called “neurogenic inflammation” might also play a role in pain generation in pancreatic cancer, just like in chronic pancreatitis.ConclusionIn conclusion, understanding the mechanisms of pain in pancreatic cancer could help patients because what counts is not only 5-year survival but also median survival with good quality of life.