Inhibitory Effect of Gamma Interferon on Cultured Human Keratinocyte Thrombospondin Production, Distribution, and Biologic Activities

Rapidly proliferating keratinocytes (KCs) maintained in low calcium, serum-free medium produce and utilize thrombospondin (TSP) as an attachment and spreading factor. To begin to understand the modulation of KC TSP metabolism, gamma interferon (IFN-γ), a product of activated T lymphocytes, was added to KC cultures. IFN-γ; was chosen because activated T cells appear at sites of cutaneous injury. Two additional cytokines including tumor necrosis factor (TNF) and IFN-β were also examined. IFN-γ (600 U/ml), but not TNF (500 U/ml) or IFN-β (103 U/ml), as single agents decreased KC TSP biosynthesis, secretion, and utilization as an attachment factor. IFN-γ alone did not detectably decrease TSP mRNA levels suggesting a post-transcriptional effect in KCs. However, the combination of IFN-γ (600 U/ml) and TNF (500 U/ml) inhibited TSP mRNA production. These results demonstrate the modulation of KC TSP metabolism and biologic activity

Cyclic stretching of mesangial cells up-regulates intercellular adhesion molecule-1 and leukocyte adherence: A possible new mechanism for glomerulosclerosis

Intraglomerular hypertension is a primacy causal factor in the progressive glomerulosclerosis that characterizes diabetic nephropathy or severe renal ablation. However, inflammation of the glomerular mesangium also participates in at least the early phase of these diseases. In glomerulonephritis, where inflammation is thought to be the predominant causal factor, intraglomerular hypertension is also often present. Mesangial cells (MCs) are critical in orchestrating key functions of the glomerulus including extracellular matrix metabolism, cytokine production, and interaction with leukocytes. Because MCs are subject to increased stretching when intraglomerular hypertension is present, and in glomerulonephritis MC/leukocyte interactions seem to be mediated primarily via the up-regulation of intercellular adhesion molecule-1 (ICAM-1), we examine the possibility that cyclic stretching is a stimulus for increased MC ICAM-1 activity. We demonstrate that the normal low levels of MC ICAM-1 mRNA and protein are dramatically up-regulated by even short intervals of cyclic stretch. This effect is dose- and time-dependent, and requires little amplitude and a brief period of elongation for significant induction. Stretch-induced MC ICAM-1 also leads to a marked elevation in phagocytic leukocyte adherence. This stimulated adherence is equal or greater than that induced by the inflammatory cytokine tumor necrosis factor-α, whereas an additive effect occurs when both are applied in combination. Our results indicate that stretch-induced ICAM-1 may provide a direct link between hypertension and inflammation in the progression of injury and glomerulosclerosis in diabetes, renal ablation, and other forms of glomerulonephritis. © 2001 American Society for Investigative Pathology

Putative subunits of the rat mesangial Katp: A type 2B sulfonylurea receptor and an inwardly rectifying K+ channel

Background. Sulfonylurea agents exert their physiological effects in many cell types via binding to specific sulfonylurea receptors (SUR). SUR couple to inwardly-rectifying K+ channel (Kir6.x) to form tetradimeric ATP-sensitive K+ channels (KATP). The SUR subunits confer ATP-sensitivity on KATP and also provide the binding sites for sulfonylureas and other pharmacological agents. Our previous work demonstrated that the exposure of mesangial cells (MC) to sulfonylureas generated profound effects on MC glucose uptake and matrix metabolism and induced heightened cell contractility in association with Ca2+ transients. Because these responses likely resulted from the binding of sulfonylurea to a mesangial SUR2, we subsequently documented [3H]-glibenclamide binding to MC and the gene expression of several mesangial SUR2 transcripts. From these data, we inferred that MC expressed the components of a mesangial KATP and sought to establish their presence in primary MC. Methods. To obtain mesangial SUR2 cDNA sequences, rapid amplification of cDNA ends (RACE) was utilized. DNA sequences were established by the fluorescent dye termination method. Gene expression of mesangial SUR2 and Kir6.1/2 was examined by reverse transcription polymerase chain reaction (RT-PCR) and Northern analysis. SUR2 proteins were identified by immunoblotting of mesangial proteins from membrane-enriched fractions with polyclonal antiserum directed against SUR2. Results. RACE cloning yielded two mesangial SUR2 cDNAs of 4.8 and 6.7 kbp whose open reading frames translated proteins of 964 and 1535 aa, respectively. Using probes specific to each cDNA, the presence of a unique, 5.5 kbp serum-regulated mesangial SUR2 splice variant was established. The sequence of this mesangial SUR2 (mcSUR2B) shares identity with the recently cloned rat SUR2B (rSUR2B), but, in comparison to rSUR2B, is truncated by 12 exons at the N-terminus where it contains a unique insert of 16 aa. Immunoblotting studies with anti-SUR2 antiserum demonstrated SUR2 proteins of 108 and 170 kD in membrane-enriched fractions of MC protein extracts. Complementary studies showed abundant gene expression of Kir6.1, thereby establishing gene expression of both compo- nents of KATP. Conclusions. Based upon analogy to vascular smooth muscle cells (VSMC), there are at least two putative mesangial KATP that most likely represent hetero-octamers, comprised of either rSUR2B or mcSUR2 in complex with Kir6.1. Our results define the mesangial SUR2B as the possible first link in a chain of cellular events that culminates in MC contraction and altered extracellular matrix metabolism following exposure to sulfonylureas. In addition, our results serve as the basis for the future elucidation of the electrophysiologic characteristics of the mesangial KATP and the study of endogenous regulators of mesangial cell contractility

Treatment with pyrophosphate inhibits uremic vascular calcification

Pyrophosphate, which may be deficient in advanced renal failure, is a potent inhibitor of vascular calcification. To explore its use as a potential therapeutic, we injected exogenous pyrophosphate subcutaneously or intraperitoneally in normal rats and found that their plasma pyrophosphate concentrations peaked within 15 min. There was a single exponential decay with a half-life of 33 min. The kinetics were indistinguishable between the two routes of administration or in anephric rats. The effect of daily intraperitoneal pyrophosphate injections on uremic vascular calcification was then tested in rats fed a high-phosphate diet containing adenine for 28 days to induce uremia. Although the incidence of aortic calcification varied and was not altered by pyrophosphate, the calcium content of calcified aortas was significantly reduced by 70%. Studies were repeated in uremic rats given calcitriol to produce more consistent aortic calcification and treated with sodium pyrophosphate delivered intraperitoneally in a larger volume of glucose-containing solution to prolong plasma pyrophosphate levels. This maneuver significantly reduced both the incidence and amount of calcification. Quantitative histomorphometry of bone samples after double-labeling with calcein indicated that there was no effect of pyrophosphate on the rates of bone formation or mineralization. Thus, exogenous pyrophosphate can inhibit uremic vascular calcification without producing adverse effects on bone

Tissue Destruction Resulting from the Interaction of Cytotoxic T Cells and Their Targets a

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73703/1/j.1749-6632.1988.tb36330.x.pd

Phorbol ester binding and phorol ester-induced arachidonic acid metabolism in a highly responsive murine fibrosarcoma cell line and in a less-responsive variant

Phorbol ester binding was examined in two lines of murine fibrosarcoma cells. The two cell lines were isolated from the same parent tumor but respond differentially to stimulation with phorbol esters. In one of the lines, these agents stimulate a rapid attachment and spreading response and induce directional migration. The other cell line does not migrate in response to stimulation with phorbol esters and the attachment and spreading response is slow. The cell line which responds actively to phorbol ester stimulation is highly malignant when injected into syngeneic animals while the other line is of low tumorigenicity and is virtually non-metastatic. In spite of these differences, both lines were found in the present study to bind [ 3 H]4β-phorbol-12β, 13α-dibutyrate in a receptor-mediated fashion. The characteristics of binding were virtually identical between the two cell lines. In additional studies, arachidonic acid metabolism was examined in the same two lines. In the highly responsive line, PMA stimulated a rapid release of [ 3 H]arachidonic acid and its conversion into cyclooxygenase and lipoxygenase products. In the less-responsive line, PMA stimulated a slower release of [ 3 H]arachidonic acid from prelabeled cells. The quantity of arachidonic acid metabolites produced was also much less. These studies suggest that the disparity between the two cell lines in their response to phorbol ester stimulation is not the result of differences in the initial interaction between the cells and ligand but may result from alterations in their signal transductance mechanism. This may be the result of inherent differences in capacity for arachidonic acid metabolism.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42593/1/10585_2004_Article_BF00053473.pd

TGF-β receptor expression and binding in rat mesangial cells: Modulation by glucose and cyclic mechanical strain

TGF-β receptor expression and binding in rat mesangial cells: Modulation by glucose and cyclic mechanical strain.BackgroundTransforming growth factor-β (TGF-β) is a causal factor in experimental glomerulosclerosis, and it mediates the increased extracellular matrix (ECM) accumulation that occurs in cultured mesangial cells (MCs) exposed to high glucose concentrations and cyclic mechanical strain. This change is associated with increased levels of TGF-β, but may also involve alterations in receptor expression and binding.MethodsRat MCs cultured in media containing either 8 or 35 mM glucose were seeded into culture plates with elastin-coated flexible bottoms. Thereafter, they were subjected to cyclic stretch or static conditions and then examined for125I-TGF-β1 binding and expression of TGF-β receptors at the gene and protein levels.ResultsKinetic studies showed that MCs bound TGF-β1 in a time- and concentration-dependent manner, expressing 6800 high-affinity receptors per cell, with an apparent dissociation constant (Kd) of 15.4 pM, while cross-linking analysis identified three TGF-β receptors (βR) corresponding to βRI, βRII, and βRIII of 54, 73, and 200 kDa, respectively. Immunocytochemical studies of βRI and βRII protein revealed MC expression in a homogeneous, punctate distribution, whereas Northern analysis demonstrated the presence of the corresponding mRNAs. Exposure to cyclic stretching significantly increased (10%) the overall number of TGF-β receptors, whereas ligands associated with βRs I, II, and III also increased (25 to 50%). The finding of increased (30 to 40%) βRI and βRII transcript levels and immunoreactive protein (163 and 59%, respectively) in the absence of significant changes in the apparent Kd indicated that stretch-induced binding was the result of increased receptor synthesis and expression and not due to a change in binding affinity. In a similar, but more dramatic fashion, exposure to high glucose also elevated (50%) the receptor number, as well as the amount of ligands associated with βRs I, II, and III (100 to 250%). This same treatment also increased the levels of βRI and βRII mRNA (30 to 40%) and the immunoreactive protein (82 and 82%, respectively), without significantly altering the binding affinity of the receptor. A concerted or synergistic effect of both stimuli was not evidenced.ConclusionThese results suggest that the modulation of TGF-β receptors may be an additional control point in mediating the glucose- and mechanical force-induced increase in ECM deposition by MCs

Patients with encapsulating peritoneal sclerosis have increased peritoneal expression of connective tissue growth factor (CCN2), transforming growth factor-beta 1, and vascular endothelial growth factor

Introduction: Encapsulating peritoneal sclerosis (EPS) is a devastating complication of peritoneal dialysis (PD). The pathogenesis is not exactly known and no preventive strategy or targeted medical therapy is available. CCN2 has both pro-fibrotic and pro-angiogenic actions and appears an attractive target. Therefore, we studied peritoneal expression of CCN2, as well as TGFb1 and VEGF, in different stages of peritoneal fibrosis. Materials and methods: Sixteen PD patients were investigated and compared to 12 hemodialysis patients and four pre-emptively transplanted patients. Furthermore, expression was investigated in 12 EPS patients in comparison with 13 PD and 12 non-PD patients without EPS. Peritoneal tissue was taken during kidney transplantation procedure or during EPS surgery. In a subset of patients, CCN2 protein levels in peritoneal effluent and plasma were determined. Samples were examined by qPCR, histology, immunohistochemistry, and ELISA. Results: Peritoneal CCN2 expression was 5-fold higher in PD patients compared to pre-emptively transplanted patients (P<0.05), but did not differ from hemodialysis patients. Peritoneal expression of TGF beta 1 and VEGF were not different between the three groups; neither was peritoneal thickness. Peritoneum of EPS patients exhibited increased expression of CCN2 (35-fold, P<0.001), TGF beta 1 (24-fold, P<0.05), and VEGF (77-fold, P<0.001) compared to PD patients without EPS. In EPS patients, CCN2 protein was mainly localized in peritoneal endothelial cells and fibroblasts. CCN2 protein levels were significantly higher in peritoneal effluent of EPS patients compared to levels in dialysate of PD patients (12.0 +/- 4.5 vs. 0.91 +/- 0.92 ng/ml, P<0.01), while plasma CCN2 levels were not increased. Conclusions: Peritoneal expression of CCN2, TGF beta 1, and VEGF are significantly increased in EPS patients. In early stages of peritoneal fibrosis, only CCN2 expression is slightly increased. Peritoneal CCN2 overexpression in EPS patients is a locally driven response. The potential of CCN2 as biomarker and target for CCN2-inhibiting agents to prevent or treat EPS warrants further study

Patients with encapsulating peritoneal sclerosis have increased peritoneal expression of connective tissue growth factor (CCN2), transforming growth factor- β1, and vascular endothelial growth factor

Introduction: Encapsulating peritoneal sclerosis (EPS) is a devastating complication of peritoneal dialysis (PD). The pathogenesis is not exactly known and no preventive strategy or targeted medical therapy is available. CCN2 has both profibrotic and pro-angiogenic actions and appears an attractive target. Therefore, we studied peritoneal expression of CCN2, as well as TGFb1 and VEGF, in different stages of peritoneal fibrosis.Materials and methods: Sixteen PD patients were investigated and compared to 12 hemodialysis patients and four preemptively transplanted patients. Furthermore, expression was investigated in 12 EPS patients in comparison with 13 PD and 12 non-PD patients without EPS. Peritoneal tissue was taken during kidney transplantation procedure or during EPS surgery. In a subset of patients, CCN2 protein levels in peritoneal effluent and plasma were determined. Samples were examined by qPCR, histology, immunohistochemistry, and ELISA.Results: Peritoneal CCN2 expression was 5-fold higher in PD patients compared to pre-emptively transplanted patients (P< 0.05), but did not differ from hemodialysis patients. Peritoneal expression of TGFβ1 and VEGF were not different between the three groups; neither was peritoneal thickness. Peritoneum of EPS patients exhibited increased expression of CCN2 (35- fold, P<0.001), TGFβ1 (24-fold, P< 0.05), and VEGF (77-fold, P<0.001) compared to PD patients without EPS. In EPS patients, CCN2 protein was mainly localized in peritoneal endothelial cells and fibroblasts. CCN2 protein levels were significantly higher in peritoneal effluent of EPS patients compared to levels in dialysate of PD patients (12.0±4.5 vs. 0.91±0.92 ng/ml, P<0.01), while plasma CCN2 levels were not increased.Conclusions: Peritoneal expression of CCN2, TGFβ1, and VEGF are significantly increased in EPS patients. In early stages of peritoneal fibrosis, only CCN2 expression is slightly increased. Peritoneal CCN2 overexpression in EPS patients is a locally driven response. The potential of CCN2 as biomarker and target for CCN2-inhibiting agents to prevent or treat EPS warrants further study

Characterization of thrombospondin synthesis, secretion and cell surface expression by human tumor cells

Previous studies have shown that thrombospondin (TSP) is an adhesion factor for some human tumor cells. The previous studies have shown further that tumor cells which utilize TSP as an adhesion factor also synthesize it. This study continues the effort to understand how TSP production and expression are regulated in human tumor cells and the consequences of this for the cells. It is shown that differences among cell lines in their capacity to biosynthesize TSP are associated with differences in TSP specific mRNA levels. This indicates that biosynthesis is regulated at the transcriptional level. There is also a direct relationship between TSP biosynthesis and secretion into the culture medium and expression at the cell surface. The cells which are the most biosynthetically active secrete amounts of TSP into the culture medium that are sufficient to elicit a detectable response in the cell-substrate adhesion assay. The kinetics of TSP secretion by these cells are in accord with the kinetics of attachment and spreading of the same cells in the absence of exogenous adhesion factors. These data are consistent with the idea that endogenously produced TSP promotes the adhesion of the cells which synthesize it in an autocrine manner.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42595/1/10585_2005_Article_BF01753679.pd