Intracellular cyclic nucleotide analogues inhibit in vitro mitogenesis and activation of fibroblasts derived from obstructed rat kidneys.

As several studies indirectly suggest that inhibiting the intracellular breakdown of cyclic nucleotides may inhibit fibrogenesis, this study used membrane permeable cyclic nucleotide analogues to examine the role of cAMP and cGMP signaling pathways in the regulation of renal fibroblast function. Fibroblasts were isolated by explant outgrowth culture of rat kidneys post unilateral ureteric obstruction. Subcultured cells were exposed to 10- 1,000 microM of the cyclic nucleotide analogues 8-bromo-cAMP (8br-cAMP) and 8-bromo-cGMP (8br-cGMP). Functional parameters examined included mitogenesis (thymidine incorporation), collagen synthesis (proline incorporation), myofibroblast differentiation (Western blotting for alpha-smooth muscle actin; alpha-SMA) and expression of CTGF (Northern blotting), a TGF-beta(1)-driven immediate early response gene. Serum-stimulated mitogenesis was decreased 27 +/- 4% by 100 microM 8br-cAMP (p < 0.01), 49 +/- 6% by 1,000 microM 8br-cAMP (p < 0.001) and 43 +/- 7% by 1,000 microM 8br-cGMP (p < 0.01). 1,000 microM 8br-cAMP and 8br-cGMP reduced basal collagen synthesis by 80 +/- 5 and 60 +/- 21% respectively (both p < 0.05). Maximum dose of 8br-cAMP but not 8br-cGMP inhibited basal expression of the differentiation marker alpha-SMA by 43 +/- 33 (p < 0.05), resulted in a more rounded cell morphology and reduced expression of CTGF by 39 +/- 24% (p < 0.05). Measurement of mitochondrial activity confirmed that effects were independent of cell toxicity. In conclusion, cyclic nucleotides inhibit fibrogenesis in vitro. Strategies which elevate intracellular cyclic nucleotide concentrations may therefore be therapeutically valuable in preventing the proliferation and activation of fibroblasts in progressive renal disease.

Sox18 is transiently expressed during angiogenesis in granulation tissue of skin wounds with an identical expression pattern to Flk-1 mRNA.

Sox18 encodes a member of the Sry-related high mobility group box (SOX) family of developmental transcription factors. Examination of Sox18 expression during embryogenesis has shown that Sox18 is expressed transiently in endothelial cells of developing blood vessels, and mutations in Sox18 have been found to underlie the mouse vascular and hair follicle mutant ragged. In this study we have examined the expression of Sox18 in angiogenesis during wound healing. Full-thickness skin wounds were created in mice, and subsequent expression of vascular endothelial growth factor (VEGF), the VEGF receptor Flk-1, alpha1 (iv) collagen (Col4a1), and Sox18 were studied using in situ hybridization. As has been previously reported, VEGF was expressed predominantly in the keratinocytes at the wound margins. Sox18 expression was found five days after wounding during capillary sprouting in granulation tissue and persisted through the proliferative phase of healing, but was not detected in fully epithelialized wounds 21 days after wounding. Sox18 mRNA expression was detected in capillaries within the granulation tissue and showed an identical pattern of distribution to Flk-1 and Col4a1 mRNA expression in endothelial cells. Immunostaining with a polyclonal anti-Sox18 antibody showed SOX18 protein localized in capillary endothelial cells within the granulation tissue. Capillaries in the subcutaneous tissue of unwounded skin showed no Sox18 expression. Sox18 may therefore represent a transcription factor involved in the induction of angiogenesis during wound healing and tissue repair, but not in the maintenance of endothelial cells in undamaged tissue.

Evolution of tubulointerstitial fibrosis in experimental renal infection and scarring.

Renal tubulointerstitial fibrosis may result from a loss of tubulointerstitial volume, which produces a disproportionate increase in the density of matrix. This study examines the relationship between fibrogenesis and collapse in scar formation after experimental renal infection. Escherichia coli were inoculated into the renal cortex of Sprague Dawley rats, with saline substituted in a control group. Glomerular, tubular, and interstitial profile areas were determined. Density of glomerular profiles was used as a measure of tubulointerstitial collapse. Collagen type I, III, and IV expression was examined by in situ hybridization and immunohistochemistry. Myofibroblasts were identified by alpha smooth muscle actin immunohistochemistry, and matrix metalloproteinase-1 (MMP-1) and MMP-2 were localized with appropriate antisera. Acute interstitial edema was followed by increasing density of glomerular profiles, paralleled by loss of interstitial volume and progressive tubular atrophy. Glomerular profile area remained unchanged. Density of glomerular profiles was not temporally related to myofibroblast accumulation. Procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) transcription was focal, spatially related but temporally ordered. Collagen I, III, and IV immunostaining was increased from days 3, 24, and 100, respectively (P < 0.05 versus day 0 and day 100 saline). However, when corrected for glomerular density, collagen I immunostaining decreased between days 24 and 100, whereas collagen III and IV no longer differed from day 0. MMP staining within the lesion was confined to occasional interstitial and epithelial cells throughout. It is concluded that in this model, contraction and collapse of the tubulointerstitial parenchyma has a greater influence than new collagen production on final fibrotic density.

Apoptosis is increased in a model of diabetes-impaired wound healing in genetically diabetic mice.

Impaired wound healing is a common complication of diabetes mellitus. The underlying pathophysiology of diabetes-impaired healing is poorly understood. In the present study we have compared cell proliferation rates, apoptosis (programmed cell death), the myofibroblast marker alpha-smooth muscle actin and procollagen I mRNA expression, between diabetic and control mice. Full-thickness skin wounds were made in non-obese diabetic (NOD) mice and C57B6 controls. NOD mice showed a marked retardation of wound healing at both 7 and 14 days after wounding. Comparison of cell proliferation rates 7 days after wounding, using 5-bromo-2'-deoxy-Uridine incorporation, showed higher rates of cell proliferation in controls (88.1 +/- 12.8) than in NOD wounds (52.1 +/- 9.9, p < 0.02, n = 4). Immunohistochemical detection of alpha-smooth muscle actin, showed a later onset in diabetic wounds, suggesting that wound contraction may be delayed in the diabetic animals. In situ hybridisation for alpha 1 (I) procollagen mRNA expression, showed reduced procollagen I expression in the diabetic wounds when compared with controls. Lastly, there appeared to be higher levels of apoptosis in diabetic wounds, shown by the terminal transferase mediated UTP nick end-labelling technique. Apoptotic cells were rare in control wounds confirming previous studies, which showed that apoptosis occurs late in normal wound healing as the wound matures into scar tissue. In conclusion, we hypothesize that reduced cell proliferation, retarded onset of the myofibroblast phenotype, reduced procollagen I mRNA expression and aberrant control of apoptotic cell death may contribute to impaired wound healing seen in this diabetic model.

DNA polymorphisms of the cholesteryl ester transfer protein (CETP) gene in Italian and Greek migrants to Australia.

The distribution of two common TaqI restriction fragment length polymorphisms (RFLPs) of the cholesteryl ester transfer protein (CETP) gene were determined in 271 Italian-born and 170 Greek-born migrants to Melbourne, Australia. A much smaller number were examined for the EcoNI RFLP of the same gene. Allele frequencies of the TaqI A RFLP exhibited the least variation in both ethnic groups, and no significant regional heterogeneity in allele or genotype frequencies of either TaqI RFLP was detected for Greece or Italy. There was no difference between Italians and Greeks for the TaqI A polymorphism and the variability at the B RFLP was of borderline significance. Comparisons with other Caucasian populations revealed that allele frequencies of all three CETP RFLPs are remarkably uniform within Caucasians, with the TaqI B polymorphism being the most variable.

Polymorphisms of the gene coding for the cholesteryl ester transfer protein and plasma lipid levels in Italian and Greek migrants to Australia.

The relation between TaqI restriction fragment length polymorphisms (RFLPs) of the cholesteryl ester transfer protein (CETP) gene and plasma lipid and lipoprotein phenotypes was investigated in a sample of Italian and Greek migrants of both sexes, age 40-69 years. Italians display significantly higher mean triglyceride and lower mean high-density lipoprotein (HDL) cholesterol levels than Greeks. Greek females have significantly higher HDL cholesterol than Greek males, and Italian females have significantly higher low-density lipoprotein (LDL), HDL, and total cholesterol than Italian males. The differences in RFLP allele frequencies between the two ethnic groups and sexes are insignificant. Multivariate analyses show that in the Greek sample the TaqI B RFLP of the CETP gene has a highly significant effect on HDL cholesterol levels regardless of sex and that the TaqI A polymorphism has a significant effect on HDL levels in females but modulates LDL cholesterol concentrations in males. Among Italians, with the sexes considered separately or combined, no such effects of the CETP TaqI polymorphisms are detected. Kruskal-Wallis tests detected associations between the TaqI B polymorphism in all Greek samples but not in the Italian samples. Genotype CETP*B2 exhibits significantly higher HDL cholesterol concentrations than either of the other two TaqI B genotypes, but there is no evidence of a dosage effect of the *B2 allele. These data suggest that associations between the CETP gene and lipid phenotypes can be population specific. Further, they suggest that such associations are mediated in some way by gender.