Glucose transporter-1 gene expression is associated with pancreatic cancer invasiveness and MMP-2 activity

BACKGROUND: Overexpression of the facilitative glucose transporter-1 (GLUT-1) has been observed for a wide range of human cancers, with the degree of overexpression generally being inversely correlated with prognosis. We tested the effects of modulating GLUT-1 expression on pancreatic cancer cellular invasiveness. METHODS: GLUT-1 expression in MIAPaCa-2, PANC-1, BXPC-3, and CAPAN-2 cells was assayed using Western blotting. Cells were stably transfected with a GLUT-1 expression vector or a GLUT-1 RNA interference vector to alter GLUT-1 expression. Matrix metalloproteinase-2 (MMP-2) activity and expression were assayed using zymography and Western blotting, respectively. In vitro cellular invasiveness was assayed using Matrigel Boyden chambers, and in vivo metastatic potential was assessed using a nude mouse xenograft model. RESULTS: Variable baseline GLUT-1 expression levels were detected among the cell lines. Forced overexpression of GLUT-1 induced increases in MMP-2 expression and activity and in cellular invasiveness. GLUT-1 silencing induced reductions in MMP-2 expression and activity, cellular invasiveness, and metastatic potential in vivo. CONCLUSION: GLUT-1 promotes pancreatic cellular invasiveness. The therapeutic implications of this finding warrant further study.     

Presence of Chlamydia pneumoniae in abdominal aortic aneurysms is not associated with increased activity of matrix metalloproteinases.

OBJECTIVE: to test the hypothesis that the presence of Chlamydia pneumoniae (C. pneumoniae) in the wall of abdominal aortic aneurysms (AAA) is associated with increased activity of matrix metalloproteinase (MMP)-2 and/or MMP-9.DESIGN: case-control study. MATERIAL AND METHODS: in a series of 40 patients with AAA > or =5cm in maximal cross-sectional diameter, C. pneumoniae-DNA was identified in the aneurysm wall by nested PCR in 14 (35%) patients. Another 14 C. pneumoniae-DNA-negative AAA patients from the same series, matched for gender and aneurysm diameter, were used as controls. In each group there were 7 asymptomatic (aAAA) and 7 ruptured (rAAA) aneurysms. MMP-2 and -9 activity was estimated in AAA wall biopsies by gelatin zymography. RESULTS: patients with a C. pneumoniae-DNA-positive aneurysm wall specimen showed an over-all lower activity of MMP-2 and MMP-9 (pro- and active enzyme) compared to the C. pneumoniae-DNA negative patients. However, there were no statistically significant differences in MMP activity between the two groups of patients with aAAA. Among patients with rAAA both pro-MMP-9 (p=0,026) and active-MMP-9 (p=0.007) were significantly lower in C. pneumoniae-DNA-positive patients compared to C. pneumoniae-DNA-negative patients, whereas there were no significant differences in pro-MMP-2 or active-MMP-2. CONCLUSION: this preliminary study does not support the hypothesis that the presence of C. pneumoniae in the AAA wall is associated with increased activity of MMP-2 and MMP-9.     

Polycystin-2 expression is increased following experimental ischaemic renal injury.

BACKGROUND: Mutations in PKD2 account for 15% of patients with autosomal dominant polycystic kidney disease. Expression of the PKD2 protein, polycystin-2, is developmentally regulated, suggesting a major role for this protein during nephrogenesis. However, the regulation of polycystin-2 expression in the adult kidney has not been previously explored. METHODS: We have utilized an established model of renal ischaemic injury to study polycystin-2 expression in adult rat kidney for up to 120 h following ischaemia. RESULTS: Our results indicate that polycystin-2 expression is increased in the post-ischaemic kidney by up to 5-fold, with a peak in expression at 48 h reperfusion. This time course mirrored the increase in cell proliferation observed. In the non-ischaemic kidney, polycystin-2 expression was highest in distal nephron segments but faint proximal tubular staining was also observed. No expression was seen in glomeruli. In the ischaemic kidney, polycystin-2 expression was greatly increased but the increase in expression was not restricted to segments with the highest number of proliferating cells. Moreover, polycystin-2 was detectable mainly intracellularly following ischaemia. Consistent with this, polycystin-2 was completely sensitive to endoglycosidase H during renal recovery, suggesting that it remains largely retained within the endoplasmic reticulum under these conditions. CONCLUSIONS: Our results provide the first evidence that polycystin-2 is increased following renal ischaemia, but show that this increase is not restricted to actively proliferating cells. The increase in polycystin-2 may relate instead to the process of cellular repair or differentiation following injury.     

Tenascin expression is associated with a chronic inflammatory process in abdominal aortic aneurysms

Purpose: This study was performed to test whether tenascin, a large oligomeric glycoprotein of the extracellular matrix, is present in AAA disease and whether it could play a pathophysiologic role in the development of this disease.Methods: Tenascin immunoreactivity was investigated from samples of the aneurysmal walls of 17 patients with AAAs, and the results were compared with the results of those of six patients with aortoiliac occlusive disease (AOD) and one normal control patient. To study the source of tenascin mRNA synthesis, some tissue samples were also examined with a tenascin RNA probe by in situ hybridization.Results: The difference in immunoreactivity between the AODs and AAAs was especially prominent in the adventitial layer, where the specimens from AAAs displayed strong diffuse and reticular immunostaining. In AAAs the immunostaining was clearly associated with the degree of mononuclear inflammatory cell infiltrate and with the neovascularization of the adventitial layer.Conclusion: Tenascin expression is evident in AAA disease and is distinctly associated with mononuclear inflammatory cells. The adhesive properties of tenascin may offer a relevant explanation for the mechanism by which monocytes transmigrate into the aortic wall. The definitive role of tenascin in AAA process may be more complex, however, and will necessitate further investigation. (J Vasc Surg 1997;26:670-5.)     

Cyclooxygenase-2 expression and its association with increased angiogenesis in human abdominal aortic aneurysms

Although the mechanism whereby non-steroidal anti-inflammatory drugs may reduce abdominal aortic aneurysm (AAA) development is unknown, one potential route is via inhibition of the cyclooxygenase (COX) enzyme. Despite the fact that evidence from animal models suggests a role for the COX-2 isoform in promotion of AAA development, only very limited data exist on COX-2 expression in human AAAs. Semiquantitative immunohistochemistry for COX-2 was performed on a series of formalin-fixed, paraffin-embedded human AAAs (n = 49). Associated clinicopathological data, including the degree of inflammatory cell infiltration and neorevascularization, were obtained. COX-2 protein was detected in 46 of 49 (94%) human AAAs. Expression of COX-2 protein varied widely between AAAs. COX-2 protein localized to cells in the inflammatory infiltrate with a morphology characteristic of macrophages. COX-2 expression increased with the extent of inflammatory cell infiltration (P < 0.001) and with the degree of AAA neorevascularization (P < 0.001). Logistic regression analysis identified neorevascularization (P < 0.001) as the only significant independent predictor of COX-2 positivity in human AAAs. COX-2 protein is present at increased levels in the majority of human AAAs and is expressed by mononuclear cells in the inflammatory cell infiltrate. Promotion of angiogenesis by COX-2 may play a role in AAA development.     

Connective tissue growth factor expression is decreased in acute ascending aortic dissections and increased in degenerative ascending aortic aneurysms

Purpose. Both aortic aneurysms and aortic dissections exhibit abnormal extracellular matrix properties. Connective tissue growth factor (CTGF) can induce connective tissue cell proliferation and extracellular matrix synthesis. The role of CTGF in thoracic aortic disease has never been investigated. We sought to compare the expression of CTGF in degenerative ascending aortic aneurysms and ascending aortic dissection. Methods. Intraoperative samples of ascending aorta were obtained from 47 patients: 16 patients had ascending aortic aneurysms with medial degeneration, 10 had acute aortic dissection, 9 had aneurysms due to chronic dissection. Control ascending aorta was obtained from organ donors and heart transplant recipients (n = 10). Patients with Marfan syndrome were excluded from this study. CTGF mRNA expression within aortic wall was semiquantitatively determined by real-time RT-PCR using GAPDH as the internal standard. Results. There was a significant increase in CTGF mRNA in degenerative aneurysms compared to control tissue (P = 0.04). Conversely, patients with acute dissection had decreased CTGF mRNA expression compared with nondissection aneurysms (P = 0.019) and controls (P = 0.06). The increase in CTGF expression in chronic dissections compared to acute dissections approached statistical significance (P = 0.075). Conclusions. The altered tissue levels of CTGF in aneurysms and dissections suggest possibly different molecular pathology in these aortic disorders. Further investigation regarding the role of CTGF in thoracic aortic disease is warranted.     

Matrix metalloproteinase activity in thoracic aortic aneurysms associated with bicuspid and tricuspid aortic valves

OBJECTIVE: Matrix metalloproteinases are endopeptidases that function in cell matrix turnover. Abnormal matrix metalloproteinase activity has been implicated in the formation of atherosclerotic abdominal aortic aneurysms. Recent studies suggest that abnormal matrix metalloproteinase activity may also be associated with the formation of atherosclerotic and nonatherosclerotic thoracic aortic aneurysms. Bicuspid aortic valves are associated with an intrinsic aortic pathology that predisposes to formation of proximal thoracic aneurysms while tricuspid aortic valves are not. The objective of this study was to compare the activities of matrix metalloproteinases and levels of their inhibitors in thoracic aneurysms of patients with bicuspid and tricuspid aortic valves. METHODS: Endogenous and total activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 were measured in proximal nonatherosclerotic thoracic aortic aneurysms of 16 patients with bicuspid aortic valves and 12 patients with tricuspid aortic valves. Levels of tissue inhibitor metalloproteinase-1 and -2 were also measured. Results were standardized to total protein (mg). RESULTS: Total matrix metalloproteinase-2 activity was greater in aneurysms associated with bicuspid valves when compared with those from tricuspid valves (43 +/- 11 ng/mg vs 14 +/- 2 ng/mg, P =.02). Total matrix metalloproteinase-9 activity was also greater in aneurysms associated with bicuspid aortic valves (4.0 +/- 0.9 vs 1.5 +/- 0.3, P =.02). There was no meaningful difference between groups in levels of tissue inhibitor-1 and -2. CONCLUSION: The increased activity of matrix metalloproteinases in the walls of aneurysms associated with bicuspid aortic valves may partly explain the predilection to aneurysm formation in these patients.     

Differential expression of MMP-2/MMP-9 and potential benefit of an MMP inhibitor in experimental acute kidney allograft rejection

Acute cellular allograft rejection is characterized by leukocyte invasion and tissue destruction, associated with qualitative and quantitative alterations in the extracellular matrix (ECM) compartment. Metabolism of ECM proteins is mainly regulated by matrix metalloproteinases (MMP), that are zinc depended endoproteinases. MMP, especially basement membrane degrading MMP-2 and MMP-9, also facilitate tissue invasion of leukocytes. In addition, MMP-2 exerts a direct pro-inflammatory effect upon glomerular mesangial cells. Therefore, the investigation of the role of MMP in transplant rejection may lead to novel approaches in the therapy of rejection processes. To our knowledge, this is the first study of acute allograft rejection, formally addressing expression and activity of MMP, including the effect of a MMP inhibiting agent. For our studies, we used the orthotopic kidney allograft model in the stringent Dark Agouti-to-Lewis rat strain combination. Animals were divided into four groups: group A, healthy untreated Lewis rats (n=3); group B, sham operated Lewis rats (n=3); group C, transplanted Lewis rats treated with vehicle solution only (n=12); group D, transplanted Lewis rats treated with MMP inhibitor BB-94 (n=12). Respective animals were treated once daily intraperitonealy with BB-94 (30 mg/kg) or vehicle solution only. Treatment lasted from the third preoperative day until the end of the experiment, the time of severe rejection at day +7. Acute kidney allograft rejection led to alterations in the expression and activity of MMP. Overall MMP activity slightly increased despite severe destruction of kidney histology. The MMP inhibitor BB-94 successfully inhibited MMP activity to a high extent. MMP expression did not show uniform findings, since acute rejection led to differential expression of MMP-2 and MMP-9. During the rejection process, MMP-9 showed a small but significant increase, whereas MMP-2 production decreased substantially. Interestingly, BB-94 was able to keep proteinuria at a low level in transplanted animals. In conclusion, MMP-especially MMP-9-appear to represent new mediators involved in acute kidney transplant rejection.     

Enhanced invasive properties exhibited by smooth muscle cells are associated with elevated production of MMP-2 in patients with aortic aneurysms.

BACKGROUND: abdominal aortic aneurysms (AAA) are associated with excessive vascular matrix remodelling. Recent findings suggest a systemic overproduction of matrix metalloproteinases-2 (MMP-2) by vascular smooth muscle cells (SMC) may be pivotal aetiologically. SMC migration is facilitated by MMP mediated proteolysis of the basement membrane and extracellular matrix. Our aim was to see if enhanced MMP-2 production by these SMC exhibit increased invasion, in an in vitro model of migration. METHOD: SMC were derived from inferior mesenteric vein (IMV) harvested from patients undergoing aneurysm repair (n=6) or colectomy for diverticulosis (n=6, control). Using a modified Boyden chamber chemotaxis was measured towards platelet derived growth factor (PDGF) and foetal calf serum (FCS) and invasion through a Matrigel layer. MMP-2 production was quantified by ELISA and gelatin zymography. RESULTS: chemoattractant studies demonstrated no difference in the effect of PDGF or FCS between the two populations of SMC. However, invasive studies demonstrated a significant increase in the number of migrating SMC isolated from IMV of AAA patients. Analysis of culture media extracts revealed that this difference was associated with a significant increase in production of MMP-2. CONCLUSION: SMC derived from patients with AAA demonstrate increased invasive properties when compared to a control group. Increased migration appears to be due to overproduction of MMP-2. The enhanced migratory potential of these SMC may lead to extracellular matrix remodelling and subsequent medial disruption demonstrated in the aneurysmal aorta. These data further support evidence of the proteolytic role of MMP-2 in cell migration.     

High plasma microfibrillar-associated protein 4 is associated with reduced surgical repair in abdominal aortic aneurysms

ObjectiveIdentifying biomarkers for abdominal aortic aneurysms (AAA) could prove beneficial in prognosis of AAA and thus the selection for treatment. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein that is highly expressed in aorta. MFAP4 is involved in several tissue remodeling-related diseases. We aimed to investigate the potential role of plasma MFAP4 (pMFAP4) as a biomarker of AAA.MethodsPlasma samples and data were obtained for 504 male AAA patients and 188 controls in the Viborg Vascular (VIVA) screening trial. The pMFAP4 levels were measured by Alphalisa. The Mann-Whitney U test assessed differences in pMFAP4 levels between the presence and absence of different exposures of interest. The correlation between pMFAP4 and aorta growth rate were investigated through spearman's correlation analysis. Immunohistochemistry and multiple logistic regression adjusted for potential confounders assessed the association between pMFAP4 and AAA. Multiple linear regression assessed the correlation between pMFAP4 and aorta growth rate. Cox regression and competing risk regression were used to investigate the correlation between AAA patients with upper tertile pMFAP4 and the risk of undergoing later surgical repair.ResultsA significant negative correlation between pMFAP4 and aorta growth rate was observed using spearman's correlation analysis (ρ = −0.14; P = .0074). However, this finding did not reach significance when applying multiple linear regression. A tendency of decreased pMFAP4 was observed in AAA using immunohistochemistry. Competing risk regression adjusted for potential confounders indicated that patients with upper tertile pMFAP4 had a hazard ratio of 0.51 (P = .001) for risk of undergoing later surgical repair.ConclusionsHigh levels of pMFAP4 are associated with a decreased likelihood of receiving surgical repair in AAA. This observation warrants confirmation in an independent cohort.