Susan Abramson

Connective tissue growth factor mediates transforming growth factor β-induced collagen synthesis: down-regulation by cAMP

Connective tissue growth factor (CTGF) is a cysteine‐rich peptide synthesized and secreted by fibroblastic cells after activation with transforming growth factor beta (TGF‐β) that acts as a downstream mediator of TGF‐β‐induced fibroblast proliferation. We performed in vitro and in vivo studies to determine whether CTGF is also essential for TGF‐β‐induced fibroblast collagen synthesis. In vitro studies with normal rat kidney (NRK) fibroblasts demonstrated CTGF potently induces collagen synthesis and transfection with an antisense CTGF gene blocked TGF‐β stimulated collagen synthesis. Moreover, TGF‐β‐induced collagen synthesis in both NRK and human foreskin fibroblasts was effectively blocked with specific anti‐CTGF antibodies and by suppressing TGF‐β‐induced CTGF gene expression by elevating intracellular cAMP levels with either membrane‐permeable 8‐Br‐cAMP or an adenylyl cyclase activator, cholera toxin (CTX). cAMP also inhibited collagen synthesis induced by CTGF itself, in contrast to its previously reported lack of effect on CTGF‐induced DNA synthesis. In animal assays, CTX injected intradermally in transgenic mice suppressed TGF‐β activation of a human CTGF promoter/lacZ reporter transgene. Both 8‐Br‐cAMP and CTX blocked TGF‐β‐induced collagen deposition in a wound chamber model of fibrosis in rats. CTX also reduced dermal granulation tissue fibroblast population increases induced by TGF‐β in neonatal mice, but not increases induced by CTGF or TGF‐β combined with CTGF. Our data indicate that CTGF mediates TGF‐β‐induced fibroblast collagen synthesis and that in vivo blockade of CTGF synthesis or action reduces TGF‐β‐induced granulation tissue formation by inhibiting both collagen synthesis and fibroblast accumulation.—Duncan, M. R., Frazier, K. S. Abramson, S., Williams, S., Klapper, H., Huang, X., Grotendorst, G. R. Connective tissue growth factor mediates transforming growth factor β‐induced collagen synthesis: down‐regulation by cAMP. FASEB J. 13, 1774–1786 (1999)

Hoxb13 knockout adult skin exhibits high levels of hyaluronan and enhanced wound healing

In contrast to adult cutaneous wound repair, early gestational fetal cutaneous wounds heal by a process of regeneration, resulting in little or no scarring. Previous studies indicate that down‐regulation of HoxB13, a member of the highly conserved family of Hox transcription factors, occurs during fetal scarless wound healing. No down‐regulation was noted in adult wounds. Here, we evaluate healing of adult cutaneous wounds in Hoxb13 knockout (KO) mice, hypothesizing that loss of Hoxb13 in adult skin should result in enhanced wound healing. Tensiometry was used to measure the tensile strength of incisional wounds over a 60‐day time course; overall, Hoxb13 KO wounds are significantly stronger than wild‐type (WT). Histological evaluation of incisional wounds shows that 7‐day‐old Hoxb13 KO wounds are significantly smaller and that 60‐day‐old Hoxb13 KO wounds exhibit a more normal collagen architecture compared with WT wounds. We also find that excisional wounds close at a faster rate in Hoxb13 KO mice. Biochemical and histochemcial analyses show that Hoxb13 KO skin contains significantly elevated levels of hyaluronan. Because higher levels of hyaluronan and enhanced wound healing are characteristics of fetal skin, we conclude that loss of Hoxb13 produces a more “fetal‐like” state in adult skin.

Membrane-type 1 matrix metalloproteinase mRNA expression in colorectal cancer

PURPOSE: Membrane-type matrix metalloproteinases are recently described proteolytic enzymes belonging to the matrix metalloproteinase family. Initial studies have indicated that membrane-type matrix metalloproteinases are involved in tumor invasion and metastasis. Membrane-type 1 matrix metalloproteinase is the first membrane-type matrix metalloproteinase to be described. The aim of this study was to investigate the expression of membrane-type 1 matrix metalloproteinase mRNA in colorectal cancer. METHODS: Samples were collected from surgical specimens of patients with colorectal adenocarcinoma and were immediately frozen in liquid nitrogen and stored at −80°C until processed. Both normal and cancer tissue was taken from each patient. TNM stage, tumor differentiation, mucin production, and vascular invasion were assessed. Northern blotting was used to quantify membrane-type 1 matrix metalloproteinase mRNA levels in the samples using a membrane-type 1 matrix metalloproteinase cDNA clone. X-ray film images were digitized and densitometry was used to quantify bands. All samples were normalized against 18S rRNA levels. Results are expressed as the ratio of cancer to normal tissue levels. Statistical analysis was performed using analysis of variance, withP<0.05 accepted as the level of significance. RESULTS: A total of 32 samples were prospectively analyzed. The correlation between TNM stage and increased expression of membrane-type 1 matrix metalloproteinase mRNA in cancer tissue over normal tissue is expressed in the mean ratio of cancer to normal tissue expression for Stages I through IV, respectively: 1.4 ± 0.2 (12 patients); 4.1 ± 2.6 (8 patients); 3.4 ± 3 (7 patients); and 4.5 ± 5 (5 patients). Stage I is significantly different from Stages II and IV (P<0.05). These preliminary results show an overall increasing trend in membrane-type 1 matrix metalloproteinase expression with increasing tumor stage. However, there was no correlation between membrane-type 1 matrix metalloproteinase expression and mucin production, degree of tumor differentiation, or vascular invasion. CONCLUSION: Preliminary results indicate that membrane-type 1 matrix metalloproteinase levels correlate with increasing tumor stage.

Altered serotonin immunoreactivities in the left colon of patients with colonic inertia

Serotonin is an important positive regulator of colonic motility and transit. Its quantity and distribution in the left colon could be abnormal in patients with colonic inertia (CI) and contribute to the disease.

Coincidence of Connective Tissue Growth Factor Expression with Fibrosis and Angiogenesis in Postoperative Peritoneal Adhesion Formation

Purpose: To investigate the relationship between connective tissue growth factor (CTGF) and fibrosis and angiogenesis in postoperative peritoneal adhesion formation. Methods: Adhesions were performed in 35 rats by creation of a peritoneal patch. Animals were sacrificed at 7 different time-points over 3 weeks. Adhesions and uninjured peritoneum from all animals were assessed by Northern blotting for CTGF and collagen-I mRNA and by immunohistochemistry for CTGF localization, degree of fibrosis and angiogenesis. Results: Persistent adhesions formed in all animals. CTGF and collagen-I mRNA were increased in adhesions compared to uninjured peritoneum (p < 0.05 for both). The temporal expression pattern depicted delayed peak levels of collagen-I mRNA with increasing tendency for both transcripts at the end of the observation period. Fibrosis within adhesions correlated positively with time after surgery (r = 0.85; p < 0.001) and showed typical signs of chronic tissue fibrosis at later time points. Angiogenesis was detected in adhesions but not in uninjured peritoneum (p = 0.001) and coincided with the spatial and temporal expression of CTGF protein in fibroblasts and vascular endothelial cells. Conclusions: The co-expression of CTGF with increasing fibrosis and angiogenesis in postoperative peritoneal adhesions suggests a role for CTGF as critical molecule in fibrous adhesive disease and target for future adhesion prevention.

Reduced expression of serotonin receptor(s) in the left colon of patients with colonic inertia

AbstractPURPOSE: Serotonin regulates colonic motility via receptors expressed on neural fibers and smooth muscle. Colonic inertia is characterized by delayed colonic transit. Abnormalities in serotonin receptor protein, as judged by immunoreactivity levels, could contribute to the origin of colonic inertia. The aim of this study was to investigate the expression of serotonin receptor(s) immunoreactivity in the left colon of patients with colonic inertia compared with controls. METHODS: Sixteen patients who underwent subtotal colectomy for colonic inertia were assessed. Colonic transit time was measured with the radiopaque marker technique and presented as the number of retained markers in the colon on Day 5. The control group consisted of 18 patients who underwent left hemicolectomy for colonic carcinoma; histologically normal tissues from the left colon were used. Immunohistochemical staining for serotonin receptor was performed with a rabbit anti-idiotypic antibody. The average positive area (square pixels) in the mucosa, muscularis mucosa, submucosa, and circular and longitudinal muscles per microscopic field (63×) was calculated based on measurement of the positively stained area in 20 randomly chosen microscopic fields in each related structure. The Scion Image computer analysis system was used. RESULTS: Serotonin receptor(s) immunoreactivity was mainly detected in the muscular mucosa, circular muscles, and longitudinal muscles and rarely in the mucosa and submucosa. In muscularis mucosa and circular muscle, the positive areas were significantly less in the colonic inertia group than in controls (muscularis mucosa: 29.1 ± 10.8 vs. 109.7 ± 28.2, P < 0.05; circular muscle: 25.6 ± 6.2 vs.90.2 ± 19.1, P < 0.01). There were significantly positive correlations in the control group in serotonin receptor(s) immunoreactivity levels between circular muscle and longitudinal muscle (r = 0.54, P < 0.05) and between muscular mucosa and longitudinal muscle (r = 0.57, P < 0.05) but not in colonic inertia patients. In addition, the positive areas in the circular muscle were positively correlated to the colonic transit time (Spearman’s rank correlation, 0.83; P < 0.01). CONCLUSION: In colonic inertia patients, the serotonin receptor(s) immunoreactivity level is lower in muscular mucosa and circular muscle. The absence of a correlation of serotonin receptor(s) immunoreactivity in the muscular mucosa and muscularis propria in the patient group implies that an uncoordinated expression of serotonin receptors may also contribute to colonic inertia. However, the positive correlation between serotonin receptor(s) immunoreactivity levels in the circular muscle and the transit time observed in colonic inertia patients suggests a decrease in stimulatory subtypes and at the same time an increase in inhibitory subtypes of serotonin receptors in this tissue.

Variability in serotonin and enterochromaffin cells in patients with colonic inertia and idiopathic diarrhoea as compared to normal controls

Aim To evaluate differences in distribution, density and staining intensity of enterochromaffin cells (EC) and serotonin cells (SC) in the colonic mucosa of patients with colonic inertia (CI), idiopathic diarrhoea (ID) and a control group.

Can the procedure for prolapsing hemorrhoids (PPH) be done twice? Results of a porcine model

AbstractBackground: The procedure for prolapsing hemorrhoids (PPH) is a new surgical method for the treatment of symptomatic hemorrhoids. In cases of recurrent prolapse, the performance of a second PPH may result in a ring of mucosa and submucosa between the two circular staple lines. In this study, we used a porcine model to assess whether PPH can be safely performed twice. Methods: Five adult pigs underwent two PPH procedures in one session, leaving a ring of ~1 cm of mucosa between the two staple lines. One month later, the pigs were examined under anesthesia. The anal canal was assessed using the following four methods: (a) clinical examination, (b) evaluation of mucosal blood perfusion at different levels of the anal canal via a laser Doppler flow detector, (c) measurement of concentrations of hydroxyproline and collagen to check for fibrosis, and (d) histopathological examination. Results: At the completion of the study period, all five pigs showed no clinical evidence of anorectal dysfunction. On examination under anesthesia 1 month after surgery, there was no evidence of anal stenosis in any of the pigs. The mean mucosal blood flow between the two staple lines did not differ significantly from the flow measured proximally and distally (394 vs 363 and 339 flow units, respectively; p = NS). The collagen levels, based on hydroxyproline concentration, were 81 mcg/mg between the staple lines, compared to 82 and 79 proximally and distally, respectively (p = NS). There was no significant difference in degree of fibrosis, as assessed histopathologically, between specimens taken from the ring between the staple lines and specimens taken from the area external to the staple lines. Conclusions: The results of this porcine model suggest that a second synchronous PPH is feasible. A controlled experience involving human subjects is required to determine the safety and usefulness of this technique in cases of metachronous application for recurrent or residual hemorrhoids.

Reduced expression of serotonin receptor (SR) in the left colon of patients with colonic inertia (CI)

Reduced expression of serotonin receptor (SR) in the left colon of patients with colonic inertia (CI)