Tyrphostin AG490 reduces inflammation and fibrosis in neonatal obstructive nephropathy.

BACKGROUND: Congenital obstructive nephropathy is the main cause of end-stage renal disease in infants and children. Renal insufficiency is due to impaired growth and maturation in the developing kidney with obstruction. Congenital obstructive nephropathy leads to cytokine mediated inflammation and the development of interstitial fibrosis. The Janus kinase-2 (JAK-2) and Signal Transducer and Activator of Transcription'-3 (STAT3) are involved in cytokine production, inflammation, and interstitial fibrosis. METHODS: We studied the role of JAK2/STAT3 in a model of congenital obstructive nephropathy using unilateral ureteral obstruction (UUO) in neonatal mice at the second day of life. Cytokine production, inflammation, and interstitial fibrosis were analyzed in obstructed and sham operated kidneys of neonatal mice treated with or without JAK2/STAT3 inhibitor Tyrphostin AG490. To mimic obstruction and distension, proximal tubular cells were stretched in vitro. RESULTS: We show that STAT3 is highly activated in the developing kidney with obstruction and in proximal tubular cells following stretch. JAK2/STAT3 activation mediates cytokine release and leukocyte recruitment into neonatal kidneys after UUO. Pharmacological blockade of JAK2/STAT3 by Tyrphostin AG490 reduced inflammation, tubular apoptosis, and interstitial fibrosis. JAK2/STAT3 blockade decreased pro-inflammatory and profibrotic mediators in tubular cells. CONCLUSION: Our findings provide evidence that JAK2/STAT3 mediates inflammation and fibrosis in the developing kidney with obstruction. Blocking JAK2/STAT3 may prove beneficial in congenital obstructive nephropathy in children.

Photographic estimation of wild boar damage to alpine grazing pastures in the Carpathian Mountains of central Romania.

Observations of wild boar damage to alpine grazing pastures in Romania's Carpathian Mountains were collected using photographs of the slopes from vantage points. We mapped the rooted areas and then used GIS software to estimate the relative proportions of the total grazing areas visible in the photographs that were damaged by wild boar. The amounts of damage from our two demonstration pastures were 11.2 and 13.5 %. Pastures are rented for summer grazing with grazing density monitored. Wild boar damage essentially decreases the economic benefit received for the cost of the grazing rights. This paper appears to be the first documentation of the very direct costs to livestock owners from significant wild boar rooting within rented pastures. The photographic method we present provides a quick and efficient means to quantify damage to alpine grazing pastures and may have broad application for mountainous areas where swine damage (or other disturbance) occurs and there is sufficient visibility of the damaged habitat.

Arterial wall stress controls NFAT5 activity in vascular smooth muscle cells.

BACKGROUND: Nuclear factor of activated T-cells 5 (NFAT5) has recently been described to control the phenotype of vascular smooth muscle cells (VSMCs). Although an increase in wall stress or stretch (eg, elicited by hypertension) is a prototypic determinant of VSMC activation, the impact of this biomechanical force on the activity of NFAT5 is unknown. This study intended to reveal the function of NFAT5 and to explore potential signal transduction pathways leading to its activation in stretch-stimulated VSMCs. METHODS AND RESULTS: Human arterial VSMCs were exposed to biomechanical stretch and subjected to immunofluorescence and protein-biochemical analyses. Stretch promoted the translocation of NFAT5 to the nucleus within 24 hours. While the protein abundance of NFAT5 was regulated through activation of c-Jun N-terminal kinase under these conditions, its translocation required prior activation of palmitoyltransferases. DNA microarray and ChiP analyses identified the matrix molecule tenascin-C as a prominent transcriptional target of NFAT5 under these conditions that stimulates migration of VSMCs. Analyses of isolated mouse femoral arteries exposed to hypertensive perfusion conditions verified that NFAT5 translocation to the nucleus is followed by an increase in tenascin-C abundance in the vessel wall. CONCLUSIONS: Collectively, our data suggest that biomechanical stretch is sufficient to activate NFAT5 both in native and cultured VSMCs where it regulates the expression of tenascin-C. This may contribute to an improved migratory activity of VSMCs and thus promote maladaptive vascular remodeling processes such as hypertension-induced arterial stiffening.

Mechanism of stretch-induced activation of the mechanotransducer zyxin in vascular cells.

Vascular cells respond to supraphysiological amounts of stretch with a characteristic phenotypic change that results in dysfunctional remodeling of the affected arteries. Although the pathophysiological consequences of stretch-induced signaling are well characterized, the mechanism of mechanotransduction is unclear. We focused on the mechanotransducer zyxin, which translocates to the nucleus to drive gene expression in response to stretch. In cultured human endothelial cells and perfused femoral arteries isolated from wild-type and several knockout mouse strains, we characterized a multistep signaling pathway whereby stretch led to a transient receptor potential channel 3-mediated release of the endothelial vasoconstrictor peptide endothelin-1 (ET-1). ET-1, through autocrine activation of its B-type receptor, elicited the release of pro-atrial natriuretic peptide (ANP), which caused the autocrine activation of the ANP receptor guanylyl cyclase A (GC-A). Activation of GC-A, in turn, led to protein kinase G-mediated phosphorylation of zyxin at serine 142, thereby triggering the translocation of zyxin to the nucleus, where it was required for stretch-induced gene expression. Thus, we have identified a stretch-induced signaling pathway in vascular cells that leads to the activation of zyxin, a cytoskeletal protein specifically involved in transducing mechanical stimuli.

Zinc finger motif-1 antagonizes PDGF-BB-induced growth and dedifferentiation of vascular smooth muscle cells.

Zinc finger motif-1 (ZFM1) represses proinflammatory gene expression in vascular smooth muscle cells (SMCs) at a global level and thus may also be involved in the attenuation of growth factor-induced phenotype changes in these cells. Using human primary cultured thymus vein SMCs, we have investigated the molecular mechanism by which a potent SMC mitogen, platelet-derived growth factor-BB (PDGF-BB), causes a rapid decrease in ZFM1 expression in a concentration-dependent manner and consequences thereof. Reporter gene analyses and chromatin immunoprecipitation showed that PDGF-BB-induced ZFM1 repression occurs at the level of transcription through replacement of the activating transcription factor Sp1 by Egr-1. The subsequent drop in ZFM1 abundance disinhibits SMC proliferation, migration, and synthetic gene expression in a concerted manner. Stabilizing ZFM1 levels in a PDGF-BB-independent way with a GFP-ZFM1 expression construct or by using Egr-1-specific decoy oligonucleotides abrogates all PDGF-BB effects. Conversely, siRNA-mediated knockdown of ZFM1 alone not only increases the sensitivity of SMCs for PDGF-BB, but even mimics PDGF-BB-induced proliferation and gene expression. Our findings suggest that ZFM1 is an important factor for the stabilization of a contractile SMC phenotype under basal or mildly activating conditions and that, as a prerequisite for efficient action, PDGF-BB must repress ZFM1 expression to alter the SMC phenotype.

Zyxin mediation of stretch-induced gene expression in human endothelial cells.

RATIONALE: Prolonged exposure to enhanced stretch, such as in hypertension, triggers endothelial dysfunction, a hallmark of pathological vascular remodeling processes. Despite its clinical relevance, little is known about stretch-induced gene expression in endothelial cells. OBJECTIVE: Here, we have characterized a new stretch-inducible signaling pathway and the subsequent changes in endothelial gene expression in response to stretch. METHODS AND RESULTS: Using human primary endothelial cells, we observed that the protein zyxin translocates from focal adhesions to the nucleus solely in response to stretch. There, it orchestrates complex changes in gene expression by interacting with a novel cis-acting element found in all zyxin-regulated genes analyzed so far. By way of DNA microarray pathway analyses, stretch-induced changes in endothelial cell gene expression were systematically explored, revealing that zyxin mainly regulates proinflammatory pathways. CONCLUSIONS: Stretch appears to be an important factor in the development of endothelial dysfunction with zyxin as a potential therapeutic target to interfere with these early changes in endothelial cell phenotype.

Disinhibition of SOD-2 expression to compensate for a genetically determined NO deficit in endothelial cells--brief report.

OBJECTIVE: Homozygosity for the -786C-variant of the human nos-3 gene is a risk factor for coronary artery disease (CAD). Interestingly, affected individuals develop CAD more frequently but not earlier than the general population. METHODS AND RESULTS: Genotyped primary human umbilical vein endothelial cells (ECs) were exposed to fluid shear stress (FSS) and analyzed for nitric oxide (NO) and superoxide anion (O(2)(-)) formation as well as mRNA and protein expression of different antioxidant enzymes. Dysfunctional CC-genotype ECs failed to upregulate NO synthase expression in response to FSS and exhibited a reduced NO synthesis capacity when compared to functionally intact TT-genotype ECs. However, only CC-genotype ECs responded to FSS with an Egr-1-mediated increase in manganese-containing superoxide dismutase (SOD-2) expression, shielding them from endothelin-1-induced oxidative stress in a NO-independent manner. CONCLUSIONS: This FSS-induced rise in SOD-2 expression in CC-genotype ECs effectively stabilizes their antiatherosclerotic phenotype and may explain not only the comparatively slow onset of CAD in homozygous carriers of the C-allele of the nos-3 gene but also define a general strategy for preventing endothelial dysfunction at the outset of atherosclerosis.

T-786C polymorphism of the NOS-3 gene and the endothelial cell response to fluid shear stress-a proteome analysis.

Endothelial dysfunction is a common denominator of cardiovascular disease. Central to endothelial dysfunction is a decrease in the bioavailability of nitric oxide (NO) synthesized by endothelial NO synthase (NOS-3). In vivo, the level of fluid shear stress (FSS) exerted by the flowing blood determines NOS-3 expression. However, in contrast to the -786T variant of the nos-3 gene, the -786C variant is not sensitive to shear stress. Consequently, cells homozygous for this variant have an inadequate capacity to synthesize NO. Therefore, we have compared shear stress-induced protein expression in human primary cultured endothelial cells with TT or CC genotype. Cells with the CC genotype exhibited a greatly reduced FSS-induced NOS-3 expression as well as a diminished NO synthesis capacity when compared to TT genotype cells. Proteome changes in response to FSS (30 dyn/cm(2) for 24 h) were monitored by 2D-gel electrophoresis/densitometry/mass spectrometry. Of a total of 14 FSS-sensitive proteins, 8 were identically expressed in all cells. Four proteins, all of them part of the NO-dependent endoplasmic reticulum-stress response, were up-regulated by FSS only in cells with TT genotype. In contrast, CC genotype cells responded to FSS with a unique increase in manganese-containing superoxide dismutase expression. These differences in protein expression may (i) reflect the low bioavailability of NO in cells homozygous for the -786C variant of the nos-3 gene and (ii) point to a mechanism by which this deficit is counterbalanced by protecting the less abundant NO from rapid degradation.

The -786C/T single-nucleotide polymorphism in the promoter of the gene for endothelial nitric oxide synthase: insensitivity to physiologic stimuli as a risk factor for rheumatoid arthritis.

OBJECTIVE: Shear stress is the main physiologic stimulus for the expression of NOS3, the gene for human endothelial nitric oxide synthase. Interestingly, a promoter variant of the NOS3 gene, the -786C variant, is insensitive to shear stress, and individuals homozygous for this single-nucleotide polymorphism (SNP) have an increased risk of developing coronary artery disease. The cytokine interleukin-10 (IL-10) is also capable of up-regulating endothelial NOS3 expression through binding of the transcription factor STAT-3 to a nearby promoter sequence. The aim of this study was to explore the possibility that the -786C variant of the NOS3 gene is also insensitive to IL-10 and that individuals with the -786C/C genotype are more prone to developing rheumatoid arthritis (RA). METHODS: Endothelial cells were isolated from human umbilical cord veins, clonally expanded, and analyzed for NOS3 and IL-12 expression by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Umbilical cord arteries and blood samples from RA patients were genotyped for the -786C/T SNP of the NOS3gene. RESULTS: In contrast to cells of other genotypes, endothelial cells of the -786C/C genotype did not reveal an increase in NOS3 expression upon exposure to IL-10, and the cytokine failed to suppress IL-12 expression upon stimulation of CD40. Preincubation of these cells with a 16-mer C-type decoy oligonucleotide fully reconstituted the defective IL-10-induced suppression of IL-12 synthesis. The frequency of the -786C/C genotype was significantly higher in the 596 RA patients than in the general population (19.1% versus 12.1%; P < 0.0001). CONCLUSION: Individuals with the -786C/C genotype have an increased risk of developing RA. This may be explained by the IL-10 insensitivity of the C-type NOS3 gene promoter and the resulting failure to subdue CD40-mediated proinflammatory gene expression.

Shear stress insensitivity of endothelial nitric oxide synthase expression as a genetic risk factor for coronary heart disease.

Coronary heart disease (CHD) is based on the development of atherosclerosis in coronary arteries. Shear stress-induced endothelial nitric oxide (NO) release not only contributes to local blood pressure control but also effectively helps to retard atherosclerosis. Therefore, functionally relevant polymorphisms in the endothelial NO synthase (NOS-3) gene may contribute to the development of CHD. NOS-3 expression was analyzed in endothelial cells isolated from umbilical cords genotyped for the -786C/T single nucleotide polymorphism (SNP) of the human nos-3 gene. Moreover, NO-dependent relaxation was examined in segments of saphenous vein isolated from genotyped patients undergoing aortocoronary bypass surgery, and patients subjected to quantitative coronary angiography were genotyped to verify an association between this SNP and CHD. Shear stress-induced NOS-3 mRNA and protein expression was present in TT and CT genotype cells but absent in cells with CC genotype. Pretreatment of these cells with a decoy oligonucleotide comprising position -800 to -779 of the C-type nos-3 promoter reconstituted shear stress-induced NOS-3 expression. These results were confirmed by reporter gene analysis with the corresponding nos-3 promoter luciferase constructs. In addition, the NO-mediated relaxant response of vein grafts from CC genotype patients was significantly attenuated as compared with the CT or TT genotype, and in CHD-positive patients, the CC genotype was significantly more frequent (19.0%) than in CHD-negative patients (4.4%). The -786C/T SNP of the nos-3 gene thus constitutes a genetic risk factor for CHD, presumably due to binding of an inhibitory transcription factor to the C-type promoter blocking shear stress-dependent maintenance of NOS-3 expression.

Focal adhesion protein zyxin is a mechanosensitive modulator of gene expression in vascular smooth muscle cells.

Excessive deformation of vascular smooth muscle cells (SMCs) caused by a prolonged increase in blood pressure (eg, in hypertension) results in an adaptive remodeling of the vessel wall that is characterized by SMC hypertrophy or hyperplasia and contributes to fixation of the increase in blood pressure. The onset of this process is characterized by a unique change in gene expression in the SMC. However, thus far, no transcription factor has been identified that specifically mediates mechanosensitive gene expression in these cells. Therefore, the role of a putative mechanotransducer, the cytoskeletal protein zyxin, was investigated in rat aortic cultured SMCs. Immunofluorescence and Western blot analysis revealed that on exposure to cyclic stretch, but not to osmotic stress or treatment with proinflammatory cytokines, zyxin dissociates from focal adhesions and accumulates in the nucleus. Unlike zyxin, vinculin, another focal adhesion-associated protein, did not translocate. Moreover, antisense oligonucleotide downregulation of zyxin protein abundance suggested that zyxin accumulation in the nucleus is a prerequisite for mechanosensitive gene expression in these cells. Thus, stretch-induced endothelin B receptor expression, for example, was attenuated, whereas that of tenascin-C was augmented after zyxin suppression. The data are consistent with a role for zyxin in transducing mechanical stimuli from the cell membrane to the nucleus in vascular SMCs and in controlling the expression of mechanosensitive genes that have been implicated in hypertension-induced arterial remodeling.

Interleukin-10 induction of nitric-oxide synthase expression attenuates CD40-mediated interleukin-12 synthesis in human endothelial cells.

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine in Th1 cell-mediated chronic inflammatory diseases such as, e.g. Crohn's disease. Moreover, IL-10 has been shown to limit the progression of atherosclerosis, presumably by influencing endothelial cell function. Here we demonstrate that under pro-inflammatory conditions expression of the human IL-10 receptor gene is enhanced in endothelial cells in vitro and in vivo. Subsequent exposure to IL-10 results in an up-regulation of both endothelial nitric-oxide synthase (NOS-3) expression and activity. Gel mobility shift analyses and decoy oligonucleotide experiments suggest that this effect of IL-10 is mediated through activation of the transcription factor STAT-3 (signal transducer and activator of transcription-3). One functional consequence of IL-10 up-regulation of NOS-3 abundance in cultured endothelial cells is the attenuation of CD154-induced IL-12 p40 expression. Moreover, CD154-induced IL-12 p40 expression is enhanced after blockade of NOS-3 activity but attenuated in the presence of exogenous nitric oxide. Increased NOS-3 expression may, thus, be one mechanism by which IL-10 exerts its anti-inflammatory effects in Th1 cell-mediated chronic inflammatory diseases.

Cytokine-induced down-regulation of zfm1/splicing factor-1 promotes smooth muscle cell proliferation.

One hallmark of inflammation is the proliferation of bystander cells such as vascular smooth muscle cells (SMC), a process governed by growth factors and cytokines. Whereas cytokine induction of gene products promoting inflammation and proliferation is well characterized, little is known about the concomitant down-regulation of potentially counter-regulatory gene products in these cells. By employing the suppression subtractive hybridization-PCR technique, RNA isolated from rat aortic SMC treated with the cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) was subtracted from RNA of control cells. Eleven genes were identified, the expression of which fell by 44-77%. One, the transcriptional repressor splicing factor-1 or zfm1, was characterized further. Antisense oligonucleotide suppression of zfm1 protein synthesis mimicked the stimulatory effects of IL-1 beta and TNF alpha on SMC proliferation and expression of the chemokine MCP-1 and the vascular cell adhesion molecule-1. Moreover, in an in vivo mouse model of atherosclerosis, zfm1 abundance was decreased in proliferating arterial SMC. These findings suggest a role for zfm1 in controlling both proliferation and expression of pro-inflammatory gene products in SMC. Therefore, cytokine-induced down-regulation of zfm1 expression may contribute to the pathogenesis of hyperproliferative inflammatory diseases.