A type IV collagenase inhibitor, N-hydroxy-3-phenyl-2-(4-phenylbenzenesulfonamido) propanamide (BiPS), suppresses skin injury induced by sulfur mustard.

Sulfur mustard (SM) is a highly toxic blistering agent thought to mediate its action, in part, by activating matrix metalloproteinases (MMPs) in the skin and disrupting components of the basement membrane zone (BMZ). Type IV collagenases (MMP-9) degrade type IV collagen in the skin, a major component of the BMZ at the dermal-epidermal junction. In the present studies, a type IV collagenase inhibitor, N-hydroxy-3-phenyl-2-(4-phenylbenzenesulfonamido) propanamide (BiPS), was tested for its ability to protect the skin against injury induced by SM in the mouse ear vesicant model. SM induced inflammation, epidermal hyperplasia and microblistering at the dermal/epidermal junction of mouse ears 24-168 h post-exposure. This was associated with upregulation of MMP-9 mRNA and protein in the skin. Dual immunofluorescence labeling showed increases in MMP-9 in the epidermis and in the adjacent dermal matrix of the SM injured skin, as well as breakdown of type IV collagen in the basement membrane. Pretreatment of the skin with BiPS reduced signs of SM-induced cutaneous toxicity; expression of MMP-9 mRNA and protein was also downregulated in the skin by BiPS. Following BiPS pretreatment, type IV collagen appeared intact and was similar to control skin. These results demonstrate that inhibiting type IV collagenases in the skin improves basement membrane integrity after exposure to SM. BiPS may hold promise as a potential protective agent to mitigate SM induced skin injury.

Bioactive compounds from Cornus officinalis fruits and their effects on diabetic nephropathy.

ETHNOPARMACOLOGICAL RELEVANCE: The fruit of Cornus officinalis, called "Shanzhuyu", a traditional medicine in China, is used for the treatment of kidney diseases, including diabetic nephropathy. The aim of this study is to investigate the anti-diabetic nephropathy activity of Shanzhuyu and the active compounds in the fruit. MATERIALS AND METHODS: The air dried fruit of Cornus officinalis was extracted in 80% EtOH, the obtained residue was fractioned on D101 resin column eluted with H2O/EtOH solution to get five crude fractions (fr. A-E). The anti-diabetic nephropathy activity of fractions (fr. A-E) was evaluated in vitro by inhibiting the expression of collagen IV (Col V), fibronectin (FN) and IL-6 in high-glucose-induced mesangial cells. By preliminary bio-assay screenings, repeated column chromatography on fraction B-D led the isolation of 22 compounds, whose structures were determined by extensive spectroscopic analysis, and the anti-diabetic nephropathy activity of the isolated compounds was also evaluated. RESULTS: Two new iridoid glucosides, logmalicids A and B (1 and 2), together with 20 known compounds (3-22) were isolated from the extract of Shanzhuyu under the bioassay-guided screenings. The anti-diabetic nephropathy activity assay displayed that fractions A, D and E could significantly inhibit the production of Col IV; fractions A and C could significantly inhibit the expression of FN and IL-6 in the high-glucose-stimulated mesangial cells at concentration of 50 µg/mL; and loganin (3) and its derivatives (1 and 2) could significantly inhibit the expression of FN and IL-6 at concentration of 10 µM, respectively. CONCLUSIONS: The results suggested that loganin and its derivatives were the active compounds in Cornus officinalis fruit (Shanzhuyu) on diabetic nephropathy. This study further supported the traditional use of Shanzhuyu to treat diabetic nephropathy and related kidney diseases.

Exenatide reduces urinary transforming growth factor-ß1 and type IV collagen excretion in patients with type 2 diabetes and microalbuminuria.

AIMS: It was reported that exenatide ameliorated renal injury in diabetic rats. The present study was carried out to evaluate the effect of exenatide on 24-hour urinary albumin, urinary transforming growth factor-ß(1) (TGF-ß(1)) and type IV collagen excretion in patients with type 2 diabetes and microalbuminuria. METHODS: 31 type 2 diabetic patients with microalbuminuria were randomly allocated to receive exenatide (group Exe, n = 13) or glimepiride treatment (group Glm, n = 18) for 16 weeks. Body mass index (BMI), fasting plasma glucose, 2-hour postprandial plasma glucose, glycated hemoglobin A(1c), systolic blood pressure, diastolic blood pressure, 24-hour urinary albumin, urinary TGF-ß(1) and type IV collagen concentration were analyzed between the two treatment groups. 20 age- and BMI-matched healthy subjects were chosen as the normal control group (group NC, n = 20). RESULTS: After 16 weeks of treatment, 24-hour urinary albumin, urinary TGF-ß(1) and type IV collagen in group Exe were significantly lower than those of group Glm (p < 0.01), while glycemic control had no statistical difference between the two groups. CONCLUSIONS: Our results indicate that exenatide reduces urinary TGF-ß(1) and type IV collagen excretion in patients with type 2 diabetes and microalbuminuria, which may be partly contributory to its directly renoprotective role.

Role of reactive oxygen species in transforming growth factor beta1-induced alpha smooth-muscle actin and collagen production in nasal polyp-derived fibroblasts.

BACKGROUND: Myofibroblasts are detected in nasal polyps and are involved in nasal polyp formation by inducing extracellular matrix accumulation. Reactive oxygen species (ROS) are released during the differentiation of fibroblasts to myofibroblasts. The purpose of this study was to investigate ROS production and nicotinamide adenine dinucleotide phosphate oxidase (NOX) expression in nasal polyp-derived fibroblasts (NPDFs) and to evaluate whether ROS from NOX mediates transforming growth factor (TGF)-ß1-induced production of alpha smooth-muscle actin (α-SMA) and collagen production. METHODS: NPDFs were incubated and treated with TGF-ß1. The mRNA expression of NOXs, α-SMA, and collagen type I and IV was determined by reverse transcription-polymerase chain reaction, and the expression of α-SMA protein was determined by immunofluorescence microscopy. The amount of total soluble collagen production was analyzed by the SirCol assay. The ROS generation of cells was investigated using the 2',7'-dichlorfluorescein-diacetate. The fluorescence was captured by fluorescent microscope and measured using a fluorometer. RESULTS: Stimulation with TGF-ß1 increased ROS production by NPDFs compared with NPDFs not treated with TGF-ß1. Stimulation with TGF-ß1 increased the expression of NOX4 mRNA most potently among various Nox enzymes. siNOX4 was able to decrease the level of ROS production. Myofibroblast differentiation and the production of collagen in NPDFs were prevented by inhibition of ROS generation with diphenyliodonium, N-acetylcysteine, ebselen, and siNox4. CONCLUSIONS: This study showed that NOX4 and ROS have a role in myofibroblast differentiation and collagen production of TGF-ß1-induced NPDFs and that these processes are inhibited by the elimination of ROS.

Bradykinin inhibits high glucose- and growth factor-induced collagen synthesis in mesangial cells through the B2-kinin receptor.

Mesangial matrix expansion is an early lesion leading to glomeruloclerosis and chronic renal diseases. A beneficial effect is achieved with angiotensin I-converting enzyme inhibitors (ACEI), which also favor bradykinin (BK) B2 receptor (B2R) activation. To define the underlying mechanism, we hypothesized that B2R activation could be a negative regulator of collagen synthesis in mesangial cells (MC). We investigated the effect of BK on collagen synthesis and signaling in MC. Inflammation was evaluated by intercellular adhesion molecule-1 (ICAM-1) expression. BK inhibited collagen I and IV synthesis stimulated by high glucose, epithelial growth factor (EGF), and transforming growth factor-ß (TGF-ß) but did not alter ICAM-1. Inhibition of collagen synthesis was B2R but not B1R mediated. PKC or phosphatidylinositol 3-kinase (PI3K) inhibitors mimicked the BK effect. B2R activation inhibited TGF-ß- and EGF-induced Erk1/2, Smad2/3, Akt S473, and EGFR phosphorylation. A phosphatase inhibitor prevented BK effects. The in vivo impact of B2R on mesangial matrix expansion was assessed in streptozotocin-diabetic rodents. Deletion of B2R increased mesangial matrix expansion and albuminuria in diabetic mice. In diabetic rats, matrix expansion and albuminuria were prevented by ACEI but not by ACEI and B2R antagonist cotreatment. Consistently, the lowered BK content of diabetic glomeruli was restored by ACEI. In conclusion, deficient B2R activation aggravated mesangial matrix expansion in diabetic rodents whereas B2R activation reduced MC collagen synthesis by a mechanism targeting Erk1/2 and Akt, common pathways activated by EGF and TGF-ß. Taken together, the data support the hypothesis of an antifibrosing effect of B2R activation.

Glucose autoxidation induces functional damage to proteins via modification of critical arginine residues.

Nonenzymatic modification of proteins in hyperglycemia is a major mechanism causing diabetic complications. These modifications can have pathogenic consequences when they target active site residues, thus affecting protein function. In the present study, we examined the role of glucose autoxidation in functional protein damage using lysozyme and RGD-α3NC1 domain of collagen IV as model proteins in vitro. We demonstrated that glucose autoxidation induced inhibition of lysozyme activity as well as NC1 domain binding to α(V)ß(3) integrin receptor via modification of critical arginine residues by reactive carbonyl species (RCS) glyoxal (GO) and methylglyoxal while nonoxidative glucose adduction to the protein did not affect protein function. The role of RCS in protein damage was confirmed using pyridoxamine which blocked glucose autoxidation and RCS production, thus protecting protein function, even in the presence of high concentrations of glucose. Glucose autoxidation may cause protein damage in vivo since increased levels of GO-derived modifications of arginine residues were detected within the assembly interface of collagen IV NC1 domains isolated from renal ECM of diabetic rats. Since arginine residues are frequently present within protein active sites, glucose autoxidation may be a common mechanism contributing to ECM protein functional damage in hyperglycemia and oxidative environment. Our data also point out the pitfalls in functional studies, particularly in cell culture experiments, that involve glucose treatment but do not take into account toxic effects of RCS derived from glucose autoxidation.

Cation-independent mannose 6-phosphate receptor inhibitor (PXS25) inhibits fibrosis in human proximal tubular cells by inhibiting conversion of latent to active TGF-beta1.

Hyperglycemia and hypoxia have independent and convergent roles in the development of renal disease. Transforming growth factor-ß(1) (TGF-ß(1)) is a key cytokine promoting the production of extracellular matrix proteins. The cationic-independent mannose 6-phosphate receptor (CI-M6PR) is a membrane protein that binds M6P-containing proteins. A key role is to activate latent TGF-ß(1). PXS25, a novel CI-MPR inhibitor, has antifibrotic properties in skin fibroblasts, but its role in renal fibrosis is unclear. The aim was to study the role of PXS25 in matrix protein production under high glucose ± hypoxic conditions in human proximal tubule (HK-2) cells. HK-2 cells were exposed to high glucose (30 mM) ± 100 µM PXS25 in both normoxic (20% O(2)) and hypoxic (1% O(2)) conditions for 72 h. Cellular fibronectin, collagen IV, and matrix metalloproteinase-2 (MMP-2) and MMP-9 were assessed. Total and active TGF-ß(1) were measured by ELISA. High glucose and hypoxia independently induced TGF-ß(1) production. Active TGF-ß(1), but not total TGF-ß(1) was reduced with concurrent PXS25 in the presence of high glucose, but not in hyperglycemia+hypoxia conditions. Hyperglycemia induced fibronectin and collagen IV production (P < 0.05), as did hypoxia, but only hyperglycemia-induced increases in matrix proteins were suppressed by concurrent PXS25 exposure. High glucose induced MMP-2 and -9 in normoxic and hypoxic conditions, which was not modified in the presence of PXS25. High glucose and hypoxia can independently induce endogenous active TGF-ß(1) production in human proximal tubular cells. PXS25 inhibits conversion of high glucose-induced release of active TGF-ß(1), only in the absence of hypoxia.

Collagen IV contributes to nitric oxide-induced angiogenesis of lung endothelial cells.

Nitric oxide (NO) mediates endothelial angiogenesis via inducing the expression of integrin α(v)ß(3). During angiogenesis, endothelial cells adhere to and migrate into the extracellular matrix through integrins. Collagen IV binds to integrin α(v)ß(3), leading to integrin activation, which affects a number of signaling processes in endothelial cells. In the present study, we evaluated the role of collagen IV in NO-induced angiogenesis. We found that NO donor 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (NOC-18) causes increases in collagen IV mRNA and protein in lung endothelial cells and collagen IV release into the medium. Addition of collagen IV into the coating of endothelial culture increases endothelial monolayer wound repair, proliferation, and tube formation. Inhibition of collagen IV synthesis using gene silencing attenuates NOC-18-induced increases in monolayer wound repair, cell proliferation, and tube formation as well as in the phosphorylation of focal adhesion kinase (FAK). Integrin blocking antibody LM609 prevents NOC-18-induced increase in endothelial monolayer wound repair. Inhibition of protein kinase G (PKG) using the specific PKG inhibitor KT5823 or PKG small interfering RNA prevents NOC-18-induced increases in collagen IV protein and mRNA and endothelial angiogenesis. Together, these results indicate that NO promotes collagen IV synthesis via a PKG signaling pathway and that the increase in collagen IV synthesis contributes to NO-induced angiogenesis of lung endothelial cells through integrin-FAK signaling. Manipulation of collagen IV could be a novel approach for the prevention and treatment of diseases such as alveolar capillary dysplasia, severe pulmonary arterial hypertension, and tumor invasion.

Effects of aminoguanidine and vitamin C on collagen type IV in diabetic nephropathy rats.

The aim of this article is to investigate the effects of Aminoguanidine and vitamin C (VitC) on type IV collagen in diabetic nephropathy rats. Diabetic nephropathy rats were induced by intraperitoneal injection of STZ. Rats were randomly divided into five groups: normal control group (n = 10), diabetes group (n = 10), aminoguanidine group (n = 10), VitC group (n = 10), aminoguanidine and VitC group (n = 10). After 16 weeks, the general conditions, blood gloucose, glycosylated hemoglobin, blood urea nitrogen, serum creatinine, serum type IV collagen, urinary albumin excretion rate, and creatinine clearance rate were detected, type IV collagen protein was determined by immunohistochemical analysis as well as the expression of collagen type IVα1 mRNA were determined by in situ hybridization analysis in the kidneys of each group. The results were (1) diabetes mellitus and renal lesions occurred in the diabetes group, aminoguanidine group, VitC group, VitC and aminoguanidine group; (2) aminoguanidine and VitC improved the general conditions of diabetic nephropathy rats, decreased blood urea nitrogen, serum creatinine, and urinary albumin excretion rate as well as increased creatinine clearance rate. The expressions of collagen type IV were significantly down-regulated in treatment groups in contrast to the diabetes group. Aminoguanidine and VitC protect renal lesions in diabetic nephropathy, respectively, by inhibiting expression of type IV collagen, while aminoguanidine and VitC have a synergistic effect on them.

[Effect of compound Puerarin on the collage IV in streptozotocin-induced diabetic nephropathy rats].

OBJECTIVE: To observe the effect of compound Puerarin on collagen IV of streptozotocin-induced diabetic rats. METHODS: Diabetic nephropathy rats were induced by intraperitoneal injection of streptozotocin (STZ). Rats were allocated randomly to control group (10), diabetes model group (10), Vitamin C group (10), Puerarin group (10), vitamin C plus Puerarin group (10). The study period lasted for 12 weeks. During and after the treatment, the general state, blood glucose levels, glycosylated hemoglobin, blood urea nitrogen, serum collagen IV, blood urea nitrogen, serum creatinine, urinary albumin excretion rate of the 24-hour, and clearance rate of creatinine collagen IV protein were determined by immunohistochemistoche analysis as well as type the gene expression of collagen IV alpha 1 mRNA were determined by in situ hybridization analysis in the kidney tissue of different groups. RESULTS: (1) Diabetes mellitus and renal function lesion occurred in the four groups. (2) Vitamin C and Puerarin could improve the general conditions of diabetic Rats, decrease blood urea nitrogen [(8.68 +/- 0.43), (7.98 +/- 0.47) and (5.76 +/- 0.82) micromol/L, serum creatinine [(74.68 +/- 8.20), (75.52 +/- 7.98) and (58.66 +/- 6.65) mmol/L], and urinary albumin excretion rate of the 24-hour [(18.40 +/- 0.37), (17.24 +/- 0.30) and (9.97 +/- 1.27) mg/24 h x 10(-3)]; increase clearance rate of creatinine [(0.59 +/- 0.21), (0.61 +/- 0.14) and (0.69 +/- 0.32) ml/min], the expression of collage IV absorbance [(111.56 +/- 14.61), (110.78 +/- 9.69) and (95.44 +/- 9.97) ] in the diabetic Rats were significantly inhibited at the same time. CONCLUSION: The compound Puerarin might have some functions on preventing ren by inhibiting expression of type IV collagen.

Dexamethasone induces the expression of metalloproteinase inhibitor TIMP-1 in the murine cerebral vascular endothelial cell line cEND.

In many neuroinflammatory conditions, including multiple sclerosis (MS), encephalitis, meningitis, brain tumours and cerebral ischaemia, the matrix metalloproteinases (MMPs) play an important role in disrupting the blood-brain barrier (BBB). Normally under tight regulation, increased MMP-9 cerebrospinal fluid levels and excessive proteolytic activity is detected in the blood and cerebrospinal fluid in patients with acute MS. MMP-9 is a member of the type IV collagenases, which attack components of the endothelial basal lamina, including type IV collagen. The disruption of the BBB and clinical symptoms can be reduced with different inhibitors to MMPs including activators of tissue inhibitor of metalloproteinases-1 (TIMP-1), the cognate tissue inhibitor of MMP-9. Since intravenous glucocorticoid (GC) treatment reduces the levels of MMP-9 markedly in patients, we hypothesized that GC effects might be mediated by transcriptional activation of the TIMP-1 gene in addition to reported repressive effects on MMP-9 transcription. Our results provide direct evidence that GCs increase TIMP-1 in the brain endothelial cell line cEND, prevent alterations in microvascular integrin alpha1 subunit expression and help maintain endothelial barrier function in response to pro-inflammatory stimuli (TNFalpha administration). GC-induced up-regulation of TIMP-1 expression by the CNS vascular endo-thelium may thus play a role in preservation of the endothelial basal lamina and maintain integrin alpha1 and tight junction protein expression important for vessel wall integrity.

Spironolactone ameliorates renal injury and connective tissue growth factor expression in type II diabetic rats.

Administration of spironolactone provides a beneficial effect in various animal models of renal injury. In this study, we investigated whether spironolactone prevents the progression of diabetic nephropathy through reduction of connective tissue growth factor (CTGF) synthesis in type II diabetic rats. In addition, we evaluated the effect of aldosterone and spironolactone on CTGF and collagen production in cultured cells. Renal functional and morphologic changes were examined in Otsuka Long-Evans Tokushima Fatty rats with or without spironolactone treatment (20 mg/kg/day) for 8 months, as well as in non-diabetic age-matched Long-Evans Tokushima Otsuka rats. Spironolactone treatment did not induce any significant differences in body weight, kidney/body weight ratio, serum creatinine concentration, blood glucose levels, or systolic blood pressure. However, urinary protein and albumin excretion were significantly decreased in the spironolactone treatment group, which was associated with amelioration of glomerulosclerosis. In addition, renal CTGF, collagen synthesis demonstrated marked decreases in the spironolactone treatment group. In cultured MC and PTC, aldosterone induced significant increases in CTGF gene expression and protein synthesis associated with increased collagen synthesis, which was abolished by prior treatment with spironolactone. However, aldosterone treatment did not induce transforming growth factor (TGF)-beta1 overproduction, and inhibition of TGF-beta1 by neutralization of TGF-beta1 protein did not significantly prevent aldosterone-induced CTGF production. These results suggest that the antifibrotic effects of spironolactone may be mediated by CTGF through a TGF-beta1-independent pathway in this animal model of diabetic nephropathy.

Expression, purification, and bioactivity of human tumstatin from Escherichia coli.

Tumstatin is a M(r) 28,000 C-terminal NC1 fragment of type alpha3 (IV) collagen that inhibits pathological angiogenesis and suppresses proliferation of endothelial cells and growth of tumors. We report here high cytoplasmic expression of recombinant human tumstatin in Escherichia coli and its purification, in vitro refolding, and inhibitory activity analysis. Human tumstatin was expressed in the bacterial cytoplasm as an insoluble N-terminal polyhistidine tagged protein, which accounted for more than 30% of total bacterial protein in BL21 (DE3) cells. After extraction and solubilization in guanidine-HCl, recombinant protein was purified to homogeneity using a simple one-step Ni(2+)-chelate affinity chromatography and then refolded by dialysis against acidic pH buffers with gradually decreasing concentrations of denaturant. The renatured recombinant tumstatin could specifically inhibit endothelial cell proliferation in a dose-dependent manner, and suppress bFGF-induced angiogenesis in chick embryo chorioallantoic membrane and tumor growth in mouse B16 melanoma xenograft models.

Antihypertensive treatment and renal damage: amlodipine exerts protective effect through the polyol pathway.

Besides generating renal damage, hypertension plays an important role in the progression of diabetic nephropathy. The fructose-fed rat is a well-established model both of high blood pressure and renal impairment, which is similar to diabetic nephropathy. To clarify the relationship between hypertension, glucose metabolism, and kidney remodeling, we investigated the renal level of Glut 1 and Glut 5, their relation to fibrosis and the effects of an antihypertensive drug on renal damage. Twenty-four male WK rats were divided into three groups: 8 animals received a fructose-enriched diet, 8 a control diet, and 8 animals a high-fructose diet plus amlodipine (5 mg/Kg). After six weeks of treatment, we observed a significant increase in Glut 5, fibronectin, and sorbitol in fructose-fed rats compared with control and amlodipine-treated animals; there was a positive correlation between Glut 5 and fibronectin levels (r = 0.63). Glut 1 levels were similar in all three groups, whereas collagen IV was higher in fructose-fed rats; amlodipine prevented the increase of collagen IV and sorbitol. Collagen I was statistically higher in the fructose group than in the other two groups. Therefore, prolonged fructose feeding results in renal fibrosis via polyol pathway overactivity that can be prevented by means of an antihypertensive drug.

BMP7 antagonizes TGF-beta -dependent fibrogenesis in mesangial cells.

Exogenous administration of recombinant human bone morphogenetic protein (BMP)-7 was recently shown to ameliorate renal glomerular and interstitial fibrosis in rodents with experimental renal diseases. We tested the hypothesis that BMP7 functions by antagonizing profibrogenic events that are induced by transforming growth factor (TGF)-beta in cultured mesangial cells. Incubation of murine mesangial cells with TGF-beta (50-200 pM) increased cell-associated collagen type IV and fibronectin, soluble collagen type IV, thrombospondin, and connective tissue growth factor (CTGF). Coincubation with recombinant human BMP7 (200 pM) reduced the increase of these ECM proteins and CTGF. The changes in collagen type IV and fibronectin proteins occurred without concomitant changes in collagen type alpha(1)IV and fibronectin mRNA levels, suggesting that TGF-beta and BMP7 act primarily by affecting ECM protein degradation. Indeed, TGF-beta decreases the levels and activity of matrix metalloprotease (MMP)-2, the major metalloprotease that is secreted by mesangial cells. Moreover, BMP7 inhibits TGF-beta-induced activation of MMP2. Because TGF-beta reduces the activity of MMPs through increasing plasminogen activator inhibitor (PAI)-1, we tested whether BMP7 interferes with this TGF-beta effect. BMP7 reduces, by about two-thirds, the activation of a PAI-1 promoter/luciferase reporter in cells stably transfected with this construct. The findings from these studies indicate that BMP7 reduces TGF-beta-induced ECM protein accumulation in cultured mesangial cells primarily by maintaining levels and activity of MMP2 partially through prevention of TGF-beta-dependent upregulation of PAI-1.

Renal fibrosis: collagen composition and assembly regulates epithelial-mesenchymal transdifferentiation.

Type IV collagen is a major component of basement membranes and it provides structural and functional support to various cell types. Type IV collagen exists in a highly complex suprastructure form and recent studies implicate that protomer (the trimeric building unit of type IV collagen) assembly is mediated by the NC1 domain present in the C-terminus of each collagen alpha-chain polypeptide. Here we show that type IV collagen contributes to the maintenance of the epithelial phenotype of proximal tubular epithelial cells, whereas type I collagen promotes epithelial-to-mesenchymal transdifferentiation (EMT). In addition, the recombinant human alpha1NC1 domain inhibits assembly of type IV collagen NC1 hexamers and potentially disrupts the deposition of type IV collagen, facilitating EMT in vitro. Inhibition of type IV collagen assembly by the alpha1NC1 domain up-regulates the production of transforming growth factor-beta1 in proximal tubular epithelial cells, an inducer of EMT. These results strongly suggest that basement membrane architecture is pivotal for the maintenance of epithelial phenotype and that changes in basement membrane architecture potentially lead to up-regulation of transforming growth factor-beta1, which contributes to EMT during renal fibrosis.

A reliable method to reduce collagen scar formation in the lesioned rat spinal cord.

Following traumatic injury, the formation of a glial scar and deposition of extracellular matrix (ECM) contributes to the regeneration failure in the adult mammalian central nervous system (CNS). Using a postcommissural fornix transection as a brain lesion model in rat, we have previously shown that the collagenous basement membrane (BM) at the lesion site is a major impediment for axon regeneration. Deposition of BM in this lesion model can be delayed by administration of the iron chelator 2,2'-bipyridine (BPY), an inhibitor of prolyl 4-hydroxylase (PH), a key enzyme of collagen biosynthesis. To examine whether this potential therapeutic approach is transferable to other CNS regions, we have chosen the mechanically lesioned rat spinal cord to investigate the effects of BPY administration on BM formation. Due to the close proximity of the lesion zone to meningeal fibroblasts, a cell-type secreting large amounts of collagen IV, BM deposition was much more extensive in the spinal cord than in the brain lesion. Neither immediate injections nor continuous application of BPY resulted in a detectable reduction of BM formation in the spinal cord. Only a combination of anti-scarring treatments including (i) injection of the more potent PH inhibitor [2,2'-bipyridine]-5,5'-dicarboxylic acid (BPY-DCA), (ii) selective inhibition of fibroblast proliferation and ECM production by 8-Br-cAMP, and (iii) continuous application of BPY-DCA, reduced the lesion-induced BM significantly. The present results clearly demonstrate, that the exclusive application of BPY according to a protocol designed for treatment of brain lesions is not sufficient to reduce BM formation in the lesioned adult rat spinal cord.