Nicholas S. Greenhill

Mast cells and type VIII collagen in human diabetic nephropathy

SummaryRenal injury in diabetes mellitus is associated with progressive interstitial fibrosis and extracellular matrix accumulation. However, the phenotypes of cells forming the interstitial infiltrate in diabetic nephropathy have not been precisely defined. There is increasing evidence for the association of mast cells with angiogenesis, chronic inflammatory conditions and fibrosis. We have recently shown that human mast cells can produce the non-fibrillar short chain type VIII collagen in vivo. Using immunohistochemistry, in situ hybridisation and reverse transcriptase-polymerase chain reaction, we examined the contribution of mast cells and type VIII collagen to the fibrotic changes occurring in biopsy-proven diabetic nephropathy. We observed that the number of interstitial mast cells was significantly increased in diabetic nephropathy compared with normal kidney tissue. In specimens from diabetic subjects, intense immunohistochemical staining for type VIII collagen was detected in mast cells, on periglomerular fibres and in perivascular and interstitial sites. The expression of type VIII collagen in periglomerular and interstitial sites coincided with that of alpha smooth muscle actin, a marker for myofibroblastic differentiation. mRNA for type VIII collagen was detected by reverse transcriptase-polymerase chain reaction in diabetic nephropathy and in a human mast cell line. By in situ hybridisation the transcripts for type VIII collagen were localised to renal mast cells. The increased number of mast cells and the elevated type VIII collagen deposition in human diabetic nephropathy provides a potential link between the extracellular matrix accumulation and the fibrosis observed in this condition.

The α1(VIII) and α2(VIII) collagen chains form two distinct homotrimeric proteins in vivo

The short chain collagen variant, type VIII, is considered to be comprised of two distinct gene products, the alpha1 and alpha2 polypeptide chains. However, recent in vitro translation studies suggest that these chains can form homotrimers. We report here data from biochemical, immunohistochemical and molecular biological experiments, which together provide evidence that alpha1 and alpha2 polypeptides of type VIII collagen exist as homotrimers in cells and tissues. High-performance liquid chromatographic separation of type VIII collagen isolated from Descemets membrane consistently demonstrated equimolar quantities of the two chains (alpha1:alpha2 1. 03+/-0.02 (S.E.M.); n=41). The availability of highly specific antibodies for the two polypeptides has assisted the in vivo characterisation of type VIII collagen. Immunoprecipitation of trimeric type VIII collagen from Descemets membrane with purified anti-alpha1(VIII) and anti-alpha2(VIII) yielded fractions that contained only the alpha1(VIII) and alpha2(VIII) chains, respectively. Cultured human mesangial cells synthesised both polypeptides, but the alpha1(VIII) chain was found exclusively in the cell pellet, while the media contained only the alpha2(VIII) chain. The RNA from human mesangial cells and cornea showed message for both chains. However, in peritoneal fibroblast and mesothelial cell RNA, only alpha1(VIII) mRNA was detectable, demonstrating that the transcription of these two genes was not always co-ordinated. Immunohistochemistry showed that both polypeptides were present in cornea, optic nerve, aorta and umbilical cord but did not always co-localise. These results indicate the alpha1(VIII) and alpha2(VIII) chains preferentially form pepsin-resistant, homotrimeric molecules and so can exist as two distinct proteins.

Changes in the localization of collagens IV and VIII in corneas obtained from patients with posterior polymorphous corneal dystrophy

Posterior polymorphous corneal dystrophy (PPCD) is a hereditary bilateral disorder affecting primarily the endothelium and Descemets membrane (DM). The aim of this study was to determine the changes in the presence and localization of the alpha1-alpha6 collagen IV chains and alpha1, alpha2 collagen VIII chains in Czech patients with PPCD. Twelve corneal buttons from ten PPCD patients who underwent corneal grafting, as well as eight unaffected corneas, were used. Enzymatic indirect immunohistochemistry was performed on cryosections using antibodies against the alpha1-alpha6 collagen IV chains and alpha1, alpha2 collagen VIII chains. The intensity of the signal was examined separately in the basal membrane of the epithelium (BME), stroma and DM. More than 50% of PPCD specimens exhibited positivity for alpha1 and alpha2 collagen IV chains in the BME and in the posterior stroma, while no staining was detected in these areas in control specimens. The signal for the alpha1 and alpha2 collagen IV chains was more intense in DM of PPCD corneas compared to controls and it was shifted from the stromal side (in control tissue) to the endothelial side of DM (in the patients). A less intensive signal in PPCD corneas for the alpha3 and alpha5 chains in DM and an accumulation of alpha3-alpha5 in the posterior stroma in diseased corneas were the only differences in staining for the alpha3-alpha6 collagen IV chains. The alpha1 collagen VIII chain was detected on both the endothelial and the stromal sides of DM in 90% of patients with PPCD, compared with the prevailing localization on the stromal side of DM in control corneas. A change in the localization of the alpha2 collagen VIII chain in DM from vertically striated features in control specimens to double line positivity in the DM of PPCD corneas and positive staining in the posterior collagenous layer of four patients were also detected. In three PPCD patients a fibrous pannus located under the BME, positive for alpha1-alpha3, alpha5 collagen IV chains and alpha1 collagen VIII chain, was observed. The increased expression of the alpha1, alpha2 collagen IV and alpha1 collagen VIII chains and the change in their localization in DM may contribute to the increased endothelial proliferative capacity observed in PPCD patients.

Cleavage of type VIII collagen by human neutrophil elastase

In this report, the susceptibility of type VIII collagen to human neutrophil elastase is compared to other extracellular matrix components. Type X collagen is degraded to specific fragments at a substrate to enzyme ratio of 5:1 after 20 h at room temperature, but type VIII collagen is almost completely degraded after only 4 h incubation at a substrate to enzyme ratio of 50:1 and partly degraded after only 15 min. Laminin, merosin and types I, III, IV and V collagen exhibit no susceptibility to neutrophil elastase under the latter conditions, while fibronectin is degraded.

Influence of tail versus cardiac sampling on blood glucose and lipid profiles in mice

Blood is collected during animal experimentation to measure haematological and metabolic parameters. It cannot be assumed that circulating blood has the same composition irrespective of its location, and indeed, differences in the composition of blood sampled from the arterial and venous compartments have been reported. Here we investigated whether blood collected by cardiac puncture (CP) versus that collected following removal of the distal 1 mm of the tail tip (TT) differs with respect to glucose and lipid profiles in male C57BL/6J mice at 4, 7, 20 and 28 weeks of age. Blood was first collected from the TT of unanaesthetized mice, which were then immediately anaesthetized using ketamine/xylazine, and a second blood sample was collected by CP. The CP glucose concentration was significantly higher than TT glucose by a positive bias averaging +80% (P < 0.01), irrespective of the age of the mice. Conversely, the concentrations of the CP lipids, including total cholesterol, high-density lipoprotein cholesterol and triglyceride were lower than TT lipids by a negative bias averaging −25% (P < 0.05). These observations highlight the difficulty in measuring and comparing metabolic parameters such as glucose and lipid between one blood compartment and another. They illustrate the need to standardize sampling sites, especially when repeated blood sampling is required.

an extract of the medicinal plant Artemisia annua modulates production of inflammatory markers in activated neutrophils

Purpose To investigate the ability of a commercial extract from the medicinal plant Artemisia annua to modulate production of the cytokine, tumor necrosis factor-alpha (TNF-α), and the cyclooxygenase (COX) inflammatory marker, prostaglandin E2 (PGE2) in activated neutrophils. Methods Neutrophils were harvested from rat whole blood and cultured in the presence of plant extract or control samples. Neutrophils, except unactivated control cells, were activated with 10 μg/mL lipopolysaccharide (LPS). The cells were cultured with a range of different concentrations of the A. annua extracts (400–1 μg/mL) and artemisinin (200 and 100 μg/mL) and the supernatants were then tested by enzyme-linked immunosorbent assay (ELISA) for the concentrations of TNF-α and PGE2. Each sample was assayed in triplicate. Positive controls with an inhibitor were assayed in triplicate: chloroquine 2.58 and 5.16 μg/mL for TNF-α, and ibuprofen 400 μg/mL for PGE2. An unsupplemented group was also assessed in triplicate as a baseline control. Results Neutrophils were stimulated to an inflammatory state by the addition of LPS. A. annua extract significantly inhibited TNF-α production by activated neutrophils in a dose-dependent manner. There was complete inhibition by the A. annua extract at 200, 100, and 50 μg/mL (all P≤0.0003). At A. annua extract concentrations of 25, 10, and 5 μg/mL, TNF-α production was inhibited by 89% (P<0.0001), 54% (P=0.0002), and 38% (P=0.0014), respectively. A. annua 1 μg/mL did not significantly inhibit TNF-α production (8.8%; P>0.05). Concentrations of 400, 200, and 100 μg/mL A. annua extract significantly inhibited PGE2 production by 87% (P=0.0128), 91% (P=0.0017), and 93% (P=0.0114), respectively. Conclusion An extract of A. annua was shown to be a potent inhibitor of TNF-α and a strong inhibitor of PGE2 production in activated neutrophils at the concentrations tested. Further studies are warranted with this promising plant extract.

Fuchs Endothelial Corneal Dystrophy: A Systematic Immunofluorescence Study of Collagen Type VIII Suggests Heterogeneous Pathophysiology.

Purpose: The purpose of this study was to investigate the pathophysiologic heterogeneity of Fuchs endothelial corneal dystrophy (FECD). Methods: We conducted a systematic immunofluorescence study on 39 Descemet membrane samples from FECD patients and compared these with 10 Descemet membrane samples from patients with pseudophakic bullous keratopathy (PBK) and 7 normal corneas. Samples were analyzed with immunofluorescence using antibodies to the &agr;1-chain [collagen VIII &agr;1-chain (COL8A1)] and &agr;2-chain (COL8A2). Intensity of staining was assessed using a subjective grading scale from 0 to 3. The presence of specific staining patterns was noted. Results: The overall distribution of COL8A1 staining intensity between groups was significantly different (P = 0.002). There was marked/intense staining in 85% (33/39) of the FECD samples, 40% (4/10) of the PBK samples (P = 0.034), and 29% (2/7) of normal samples (P = 0.004). The overall distribution of COL8A2 staining intensity was not significantly different between groups (P = 0.39). There was marked/intense staining in 33% (13/39) of the FECD samples, 10% (1/10) of PBK samples, and 14% (1/7) of the normal samples. There was substantial variation in staining intensity in the FECD group, a phenomenon that was especially pronounced for the COL8A2 antibody. Conclusions: We found increased staining for COL8A1, but not COL8A2 in FECD samples. Further, there was striking variation of staining intensity in FECD patients, indicating pathophysiological heterogeneity.

SALT SOLUBLE CROSS-LINKED ELASTIN: FORMATION AND COMPOSITION OF FIBERS

Cross-linked elastin has been isolated from the salt extract of sheep vascular tissue by means of hydrophobic interaction chromatography on a column of decyl-agarose. Dialysis of the dimethylformamide and sodium dodecyl sulphate column eluates against distilled water produced a precipitate that was fibrous and that resembled insoluble elastin fibers. As judged by amino acid analyses and SDS-polyacrylamide gel electrophoresis, this precipitation resulted in further purification of the soluble cross-linked elastin. Similar chromatography and precipitation of oxalic acid solubilized cross-linked elastin (alpha-elastin) produced identical results.