Roger M. L. M. Lomme

Tissue levels of active matrix metalloproteinase-2 and -9 in colorectal cancer.

The bioactivity of matrix metalloproteinases was studied in tissues from colorectal cancer patients by means of both quantitative gelatin zymography and a fluorometric activity assay. Next to paired samples of tumour tissue and distant normal mucosa (n=73), transitional tissue was analysed from a limited (n=33) number of patients. Broad-spectrum matrix metalloproteinase activity and both the active and latent forms of the gelatinases matrix metalloproteinase-2 and -9 were higher in tumour than in normal mucosa. The ratios between active and latent forms of matrix metalloproteinase-2 and -9 were highest in tumour tissue and normal mucosa, respectively. Matrix metalloproteinase-2 levels, both active and latent forms, correlated inversely with stage of disease, the tumours without synchronous distant metastases containing significantly (P=0.005) more active matrix metalloproteinase-2 than the others. At much lower levels of activity, the same trend was observed in distant normal mucosa. The level of latent form of matrix metalloproteinase-9 in tumour depended on tumour location. Neither the active form of matrix metalloproteinase-9 nor broad-spectrum matrix metalloproteinase activity in tumour tissue did correlate with any of the clinicopathological parameters investigated. The results demonstrate explicit differences between the activity of matrix metalloproteinase-2 and -9, indicating different roles for both gelatinases in tumour progression. Such data are necessary in order to develop rational anti-cancer therapies based on inhibition of specific matrix metalloproteinases.

Plasma Levels of Matrix Metalloproteinase-2 and Tissue Inhibitor of Metalloproteinase-1 Correlate With Disease Stage and Survival in Colorectal Cancer Patients

PURPOSEThe matrix metalloproteinases and their inhibitors are known to be involved in the process of tumor invasion and progression. Our objective was to investigate the potential diagnostic and prognostic value of plasma matrix metalloproteinase-2 and -9 and tissue inhibitor of metalloproteinase-1 in colorectal cancer.METHODSGelatinase bioactivity and immunoreactivity of pro-matrix metalloproteinase-2 and -9, tissue inhibitor of metalloproteinase-1, and carcinoembryonic antigen were determined simultaneously in preoperative plasma and serum of colorectal cancer patients (n = 94) and in healthy controls (n = 51).RESULTSPlasma pro-matrix metalloproteinase-2 levels were lower in colorectal cancer patients (P < 0.0001) than in controls, and its gelatinolytic activity revealed an inverse correlation with adverse clinicopathologic parameters, such as lymph node involvement (P = 0.017), stage (0, I, II vs. III, IV; P = 0.012), and the carcinoembryonic antigen level (P = 0.016). Pro-matrix metalloproteinase-9 levels did not differ between patients and controls. Pro-matrix metalloproteinase-2 gelatinolytic activity showed potential value in colorectal cancer diagnosis, identifying patients with 70 percent sensitivity at 95 percent specificity. Pro-matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and carcinoembryonic antigen all showed lower sensitivities. Combining pro-matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 measurements increased the sensitivity significantly to 84 percent. With respect to prognosis, tissue inhibitor of metalloproteinase-1 showed value in predicting disease outcome in our patient group, whereas pro-matrix metalloproteinase-2 and -9 did not. The combination of tissue inhibitor of metalloproteinase-1 and carcinoembryonic antigen was better in predicting three-year survival than tissue inhibitor of metalloproteinase-1 alone, but it remains to be determined if the combination would be a better marker for survival than carcinoembryonic antigen alone.CONCLUSIONSLow pro-matrix metalloproteinase-2 levels and high tissue inhibitor of metalloproteinase-1 levels correlate with parameters of colorectal cancer disease. These correlations may be used in the search for new markers in colorectal cancer diagnosis and prognosis.

Persistent effects of everolimus on strength of experimental wounds in intestine and fascia.

The introduction of mTOR‐inhibitors in transplantation surgery has been associated with an increase in wound complications. We have previously reported a massive negative effect of everolimus on anastomotic strength in rat intestine at 7 days postoperatively. Because it is clinically important to know if this effect persists and occurs generally, repair in both intestine and abdominal wall has been investigated over a period of 4 weeks. Wistar rats received a daily dose of 1 or 2 mg/kg everolimus orally, from the operation day onwards. Controls received saline. In each rat a resection of ileum and colon was performed, and end‐to‐end anastomoses were constructed. On day 7, 14, and 28 the animals were killed and anastomoses and abdominal wall wounds were analyzed, wound strength being the primary parameter. Breaking strength of ileum, colon, and fascia was consistently and significantly reduced in the experimental groups at all time points. Anastomotic bursting pressures followed the same pattern. Loss of strength was accompanied by a decrease in hydroxyproline content after 7 days. Thus, the negative effect of everolimus on wound repair persists for at least 4 weeks after operation in this rodent model. This protracted effect may have clinical consequences and cause surgical morbidity.

Intestinal Anastomoses from Diabetic Rats Contain Supranormal Levels of Gelatinase Activity

AbstractPURPOSE: Early postoperative strength in intestinal anastomoses is reduced in diabetic rats, whereas collagen deposition is essentially unchanged, suggesting that increased matrix degradation may be the cause of diminished wound strength. The aim of this study was to investigate whether (gelatin-degrading) matrix metalloproteinase activity is enhanced in intestinal anastomoses from diabetic rats. METHODS: Sixty male young adult Wistar rats underwent resection and anastomosis of both ileum and colon. In half the animals diabetes was induced seven days before operation by streptozotocin injection (50 mg/kg intravenously). Gelatinase activities in extracts from uninjured intestine and anastomoses at one, three, or seven days after surgery were measured by quantitative gelatin zymography. RESULTS: After surgery, profound changes were observed with time for gelatinase activities with molecular weights of 50 and 60 kDa, thought to represent matrix metalloproteinase-2, and of 66, 80, 105, 140, 220, and 260 kDa, thought to represent various forms of matrix metalloproteinase-9. In many cases, specific activities were significantly (P < 0.05) higher in the anastomotic extracts from diabetic rats. Total anastomotic activities present at Day 7 were strongly elevated for most matrix metalloproteinase forms in ileum and colon from diabetic animals. CONCLUSION: Experimental diabetes leads to a sustained and elevated presence of gelatinase activity in intestinal anastomoses. Increased local matrix degradation may contribute significantly to impaired anastomotic strength in the intestine observed under this condition.

Perioperative Pain Relief by a COX-2 Inhibitor Affects Ileal Repair and Provides a Model for Anastomotic Leakage in the Intestine

The authors examined the potential of the cyclooxygenase 2 (COX-2) inhibitor carprofen to reproducibly induce anastomotic leakage. In experiment 1, an anastomosis was constructed in both ileum and colon of 20 rats, and they were given carprofen (5 mg/kg subcutaneously every 24 hours) or buprenorphine (0.02 mg/kg subcutaneously every 12 hours). In another 20 rats an anastomosis was constructed in either ileum or colon, and all received carprofen (experiment 2). Animals were sacrificed after 3 days. In experiment 1, the ileal dehiscence rate was 60% in the carprofen group and 0% in the buprenorphine group (P = .0108). Colonic anastomoses in both groups remained patent. In experiment 2, the anastomotic leakage rate was 80% in ileum and 0% in colon. Thus, COX-2 inhibitors can severely interfere with intestinal healing, particularly in the ileum. Perioperative administration of carprofen yields a unique model for anastomotic leakage, which allows translational research on the effectiveness of perisuture line reinforcement.

Reduction of oxygenation and blood flow in pedicled bowel segments in the rat and its consequences for anastomotic healing.

PURPOSE: Experimental studies indicate that perioperative hypoperfusion impairs anastomotic healing. In bowel surgery, the part of bowel that will be anastomosed is often pedicled, leaving the blood supply dependent on the marginal artery only. Little is known about the blood supply in such a segment, and whether anastomotic strength is affected when flow would be reduced. This study describes oxygenation and blood flow in pedicled bowel segments in the rat and investigates whether early anastomotic strength changes with variations in blood flow. METHODS: In rats, pedicled segments were created in ileum and colon by successive ligation of the feeding arteries. Oxygenation and blood flow were measured in the distal part of this segment by use of near-infrared spectroscopy with indocyanine green as an intravascular tracer. In a second experiment, a short pedicled colonic segment was created and, after flow measurements, an anastomosis was constructed. Wound strength and hydroxyproline content were analyzed 2 and 5 days after operation. RESULTS: After creation of a pedicled segment, the concentration of oxygenated hemoglobin decreased significantly. Blood flow also significantly decreased to even less than 10% of baseline. A very large variation was observed between animals, in particular, after ligation of the first arteries. The strength of colonic anastomoses was not significantly correlated with the blood flow in the pedicled segment before anastomotic construction. CONCLUSIONS: The creation of a pedicled bowel segment greatly reduces tissue oxygenation and blood flow to its distal part. Such impaired perioperative flow does not significantly affect early wound strength after anastomotic construction.

Increased expression of matrix metalloproteinases in the murine zymosan-induced multiple organ dysfunction syndrome

Matrix metalloproteinases (MMPs) have been implicated as mediators of tissue damage in several inflammatory diseases. Since the multiple organ dysfunction syndrome (MODS) is thought to result from systemic inflammation, overactivation of MMPs could contribute to the organ damage observed. The expression and activity of several MMPs were studied in a murine model for MODS. Sixty mice were given an aseptic intraperitoneal injection of lipopolysaccharide, followed, after 6 days, by zymosan. At days 2, 5, 8, 12, and 16 after the injection of zymosan, the liver, lungs, spleen, and kidneys were collected from groups of mice for either RNA extraction, gelatinase zymography and collagenase (MMP‐1 and ‐13) assays (six mice per time point), or immunohistochemistry (three mice per time point). A group of nine mice did not receive zymosan and acted as controls. The expression of MMP‐2 mRNA in zymosan‐treated mice was strongly up‐regulated in liver tissue only. For MMP‐9, this was the case in all organs examined. Quantitative gelatin zymography demonstrated the near complete absence of any gelatinase activity in tissues from control mice. However, in the liver, lungs, and especially the spleen of zymosan‐treated animals, significantly increased activity of proform and active MMP‐2 and ‐9 was observed with time. Overall, MMP‐1 and ‐13 activities were very low in all samples from the liver and lungs. In the spleen, however, high levels of MMP‐1 and ‐13 were observed in zymosan‐treated animals. Immunohistochemical staining for MMP‐2 was detected in the liver and spleen, but not in lung and kidney tissue of zymosan‐treated animals. Staining for MMP‐9 could be detected in liver, lung, and spleen tissues of zymosan‐treated mice. For both MMPs, staining appeared to be limited to phagocytes. In conclusion, the data suggest a role for MMPs, especially MMP‐9, in the pathogenesis of MODS. Copyright

Repeated remote ischemic preconditioning and isoflurane anesthesia in an experimental model of renal ischemia-reperfusion injury

BackgroundIn animal studies, remote ischemic preconditioning (RIPC) and anesthetic preconditioning are successful in reducing renal ischemia reperfusion injury (IRI), however the protective effect of RIPC may be improved by repeating the RIPC stimulus.MethodsSprague-Dawley rats underwent unilateral nephrectomy followed by 30 min of renal pedicle clamping. Animals were allocated into six groups: sham, control (IRI), RepISO (daily isoflurane anesthesia), RIPC (single dose isoflurane anesthesia and single dose RIPC), RepISO + RIPC (7-day isoflurane anesthesia and single dose RIPC) and RepISO + RepRIPC (7-day isoflurane anesthesia with 7-day RIPC). RIPC was applied by 3×5 min of cuff inflation on both thighs. Serum creatinine and urea levels were measured and histology was obtained at day two.ResultsRepISO diminished renal IRI, as reflected by a significant reduction in serum creatinine levels as compared to the control group, 170 ± 74 resp. 107 ± 29 μmol/L. The other preconditioning protocols showed similar reduction in serum creatinine levels as compared to the control group. No significant differences were observed between the different preconditioning protocols. For urea levels, only RepISO + RIPC resulted in significantly lower levels as compared to the control group, 14 ± 4 resp. 22 ± 7 mmol/L (p = 0.010). In the preconditioning groups only RepISO showed less histological damage as compared to controls 1.73 ± 1.19 resp. 2.91 ± 1.22 (p = 0.032).ConclusionsIn this study no additional protective effect of repeated ischemic preconditioning was observed as compared to single dose RIPC. Repeated administration of isoflurane provided stronger protection against renal IRI as compared to single dose isoflurane.

Tubular collagen scaffolds with radial elasticity for hollow organ regeneration

Tubular collagen scaffolds have been used for the repair of damaged hollow organs in regenerative medicine, but they generally lack the ability to reversibly expand in radial direction, a physiological characteristic seen in many native tubular organs. In this study, tubular collagen scaffolds were prepared that display a shape recovery effect and therefore exhibit radial elasticity. Scaffolds were constructed by compression of fibrillar collagen around a star-shaped mandrel, mimicking folds in a lumen, a typical characteristic of empty tubular hollow organs, such as ureter or urethra. Shape recovery effect was introduced by in situ fixation using a star-shaped mandrel, 3D-printed clamps and cytocompatible carbodiimide crosslinking. Prepared scaffolds expanded upon increase of luminal pressure and closed to the star-shaped conformation after removal of pressure. In this study, we applied this method to construct a scaffold mimicking the dynamics of human urethra. Radial expansion and closure of the scaffold could be iteratively performed for at least 1000 cycles, burst pressure being 132±22mmHg. Scaffolds were seeded with human epithelial cells and cultured in a bioreactor under dynamic conditions mimicking urination (pulse flow of 21s every 2h). Cells adhered and formed a closed luminal layer that resisted flow conditions. In conclusion, a new type of a tubular collagen scaffold has been constructed with radial elastic-like characteristics based on the shape of the scaffold, and enabling the scaffold to reversibly expand upon increase in luminal pressure. These scaffolds may be useful for regenerative medicine of tubular organs. STATEMENT OF SIGNIFICANCE In this paper, a new type I collagen-based tubular scaffold is presented that possesses intrinsic radial elasticity. This characteristic is key to the functioning of a number of tubular organs including blood vessels and organs of the gastrointestinal and urogenital tract. The scaffold was given a star-shaped lumen by physical compression and chemical crosslinking, mimicking the folding pattern observed in many tubular organs. In rest, the lumen is closed but it opens upon increase of luminal pressure, e.g. when fluids pass. Human epithelial cells seeded on the luminal side adhered well and were compatible with voiding dynamics in a bioreactor. Collagen scaffolds with radial elasticity may be useful in the regeneration of dynamic tubular organs.

Bioinspired coupled helical coils for soft tissue engineering of tubular structures - Improved mechanical behavior of tubular collagen type I templates

The design of constructs for tubular tissue engineering is challenging. Most biomaterials need to be reinforced with supporting structures such as knittings, meshes or electrospun material to comply with the mechanical demands of native tissues. In this study, coupled helical coils (CHCs) were manufactured to mimic collagen fiber orientation as found in nature. Monofilaments of different commercially available biodegradable polymers were wound and subsequently fused, resulting in right-handed and left-handed polymer helices fused together in joints where the filaments cross. CHCs of different polymer composition were tested to determine the tensile strength, strain recovery, hysteresis, compressive strength and degradation of CHCs of different composition. Subsequently, seamless and stable hybrid constructs consisting of PDSII® USP 2-0 CHCs embedded in porous collagen type I were produced. Compared to collagen alone, this hybrid showed superior strain recovery (93.5±0.9% vs 71.1±12.6% in longitudinal direction; 87.1±6.6% vs 57.2±4.6% in circumferential direction) and hysteresis (18.9±2.7% vs 51.1±12.0% in longitudinal direction; 11.5±4.6% vs 46.3±6.3% in circumferential direction). Furthermore, this hybrid construct showed an improved Youngs modulus in both longitudinal (0.5±0.1MPavs 0.2±0.1MPa; 2.5-fold) and circumferential (1.65±0.07MPavs (2.9±0.3)×10-2MPa; 57-fold) direction, respectively, compared to templates created from collagen alone. Moreover, hybrid template characteristics could be modified by changing the CHC composition and CHCs were produced showing a mechanical behavior similar to the native ureter. CHC-enforced templates, which are easily tunable to meet different demands may be promising for tubular tissue engineering. STATEMENT OF SIGNIFICANCE Most tubular constructs lack sufficient strength and tunability to comply with the mechanical demands of native tissues. Therefore, we embedded coupled helical coils (CHCs) produced from biodegradable polymers - to mimic collagen fiber orientation as found in nature - in collagen type I sponges. We show that the mechanical behavior of CHCs is very similar to native tissue and strengths structurally weak tubular constructs. The production procedure is relatively easy, reproducible and mechanical features can be controlled to meet different mechanical demands. This is promising in template manufacture, hence offering new opportunities in tissue engineering of tubular organs and preventing graft failure.