Study of endocrine disruptor effects in AVP and OT mediated behavioral and reproductive processes in female rat models.

Environmental exposures may have endocrine disruptor (ED) effects, e.g., a role for halogenated hydrocarbon chlorobenzenes in increasing vasopressin (AVP), oxytocin (OT) secretion and, in association, anxiety and aggression in male rats has been shown. Our aim is to investigate whether 1,2,4-trichlorobenzenehexachlorobenzene= 1:1 (mClB) treatment of female rats also shows ED effects and reproductive biology differences, and whether AVP may have a mediator role in this? Female Wistar rats were treated (0.1; 1.0; 10.0 µg/bwkg/day) with mClB (by gastrictube) and then 30; 60; 90 days after treatment anxiety (open field test) and aggressive (resident intruder test) behaviors AVP, OT concentrations from blood plasma samples were detected by radioimmunoassay on 30; 60; 90 days. Treated female rats were mated with untreated males. Mating success, number of newborn and maternal aggression on the neonates were monitored. Results showed that AVP, OT levels; and anxiety, aggressive behaviors; and mothers' aggression towards their offspring increased significantly in relation to the duration and the dose of mClB treatment. But mating propensity and number of offspring decreased. Patterns of AVP, OT release and anxiety, aggression behaviors, and reproductive-related behaviors were correlated. Consistent with the literature, our studies confirmed the role of AVP and OT in different behavioral effects.

Pioglitazone Protects Tubular Epithelial Cells during Kidney Fibrosis by Attenuating miRNA Dysregulation and Autophagy Dysfunction Induced by TGF-ß.

Excessive renal TGF-ß production and pro-fibrotic miRNAs are important drivers of kidney fibrosis that lack any efficient treatment. Dysfunctional autophagy might play an important role in the pathogenesis. We aimed to study the yet unknown effects of peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone (Pio) on renal autophagy and miRNA dysregulation during fibrosis. Mouse primary tubular epithelial cells (PTEC) were isolated, pre-treated with 5 µM pioglitazone, and then stimulated with 10 ng/mL TGF-ß1 for 24 h. Male 10-week-old C57Bl6 control (CTL) and TGF-ß overexpressing mice were fed with regular chow (TGF) or Pio-containing chow (20 mg/kg/day) for 5 weeks (TGF + Pio). PTEC and kidneys were evaluated for mRNA and protein expression. In PTEC, pioglitazone attenuated (p < 0.05) the TGF-ß-induced up-regulation of Col1a1 (1.4-fold), Tgfb1 (2.2-fold), Ctgf (1.5-fold), Egr2 (2.5-fold) mRNAs, miR-130a (1.6-fold), and miR-199a (1.5-fold), inhibited epithelial-to-mesenchymal transition, and rescued autophagy function. In TGF mice, pioglitazone greatly improved kidney fibrosis and related dysfunctional autophagy (increased LC3-II/I ratio and reduced SQSTM1 protein content (p < 0.05)). These were accompanied by 5-fold, 3-fold, 12-fold, and 2-fold suppression (p < 0.05) of renal Ccl2, Il6, C3, and Lgals3 mRNA expression, respectively. Our results implicate that pioglitazone counteracts multiple pro-fibrotic processes in the kidney, including autophagy dysfunction and miRNA dysregulation.

Susceptibility to kidney fibrosis in mice is associated with early growth response-2 protein and tissue inhibitor of metalloproteinase-1 expression.

Patients with chronic kidney disease and experimental animal models of kidney fibrosis manifest diverse progression rates. Genetic susceptibility may contribute to this diversity, but the causes remain largely unknown. We have previously described kidney fibrosis with a mild or severe phenotype in mice expressing transforming growth factor-beta1 (TGF-ß1) under the control of a mouse albumin promoter (Alb/TGF-ß1), on a mixed genetic background with CBAxC57Bl6 mice. Here, we aimed to examine how genetic background may influence kidney fibrosis in TGF-ß1 transgenic mice, and in the unilateral ureteral obstruction (UUO) and subtotal nephrectomy (SNX) mouse models. Congenic C57Bl6(B6)-TGFß and CBAxB6-TGFß (F1) transgenic mice were generated and survival, proteinuria, kidney histology, transcriptome and protein expressions were analyzed. We investigated the kidneys of B6 and CBA mice subjected to UUO and SNX, and the effects of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) neutralization on the fibrotic process. CBAxB6-TGFß mice developed severe kidney fibrosis and premature death, while B6-TGF-ß mice had mild fibrosis and prolonged survival. Kidney early growth response factor-2 (EGR2) and TIMP-1 expression were induced only in CBAxB6-TGFß mice. Similar strain-dependent early changes in EGR2 and TIMP-1 of mice subjected to UUO or SNX were observed. TIMP-1 neutralization in vivo hindered fibrosis both in transgenic mice and the SNX model. EGR2 over-expression in cultured HEK293 cells induced TIMP-1 while EGR2 silencing hindered TGF-ß induced TIMP-1 production in HK-2 cells and ureteral obstructed kidneys. Finally, EGR2 and TIMP1 was increased in human kidneys manifesting focal segmental glomerulosclerosis suggesting a correlation between animal studies and patient clinical settings. Thus, our observations demonstrate a strong relationship between genetic background and the progression of kidney fibrosis, which might involve early altered EGR2 and TIMP-1 response, but the relationship to patient genetics remains to be explored.

PPARγ is a gatekeeper for extracellular matrix and vascular cell homeostasis: beneficial role in pulmonary hypertension and renal/cardiac/pulmonary fibrosis.

PURPOSE OF REVIEW: Pulmonary arterial hypertension (PAH) is characterized by pulmonary arterial endothelial cell (PAEC) dysfunction and apoptosis, pulmonary arterial smooth muscle cell (PASMC) proliferation, inflammation, vasoconstriction, and metabolic disturbances that include disrupted bone morphogenetic protein receptor (BMPR2)-peroxisome proliferator-activated receptor gamma (PPARγ) axis and DNA damage. Activation of PPARγ improves many of these mechanisms, although erroneous reports on potential adverse effects of thiazolidinedione (TZD)-class PPARγ agonists reduced their clinical use in the past decade. Here, we review recent findings in heart, lung, and kidney research related to the pathobiology of vascular remodeling and tissue fibrosis, and also potential therapeutic effects of the PPARγ agonist pioglitazone. RECENT FINDINGS: Independent of its metabolic effects (improved insulin sensitivity and fatty acid handling), PPARγ activation rescues BMPR2 dysfunction, inhibits TGFß/Smad3/CTGF and TGFß/pSTAT3/pFoxO1 pathways, and induces the PPARγ/apoE axis, inhibiting vascular remodeling. PPARγ activation dampens mtDNA damage via PPARγ/UBR5/ATM pathway, improves function of endothelial progenitor cells (EPCs), and decrease renal fibrosis by repressing TGFß/pSTAT3 and TGFß/EGR1. SUMMARY: Pharmacological PPARγ activation improves many hallmarks of PAH, including dysfunction of BMPR2-PPARγ axis, PAEC, PASMC, EPC, mitochondria/metabolism, and inflammation. Recent randomized controlled trials, including IRIS (Insulin Resistance Intervention After Stroke Trial), emphasize the beneficial effects of PPARγ agonists in PAH patients, leading to recent revival for clinical use.

Cell physiological effects of glass ionomer cements on fibroblast cells.

The cytotoxicity of glass ionomer cements (GICs) was investigated using a novel, cost-effective, easy-to-perform and standardized test. GIC rings were made using in-house designed, custom-made moulds under sterile conditions; 10 with Fuji Equia and 10 with Fuji Triage capsules, placed in direct contact with primary human gingival fibroblasts (HGF) and immortalized human fibroblasts (HFF1). On day 1, 4, 14 and 21, an AlamarBlue® (resazurin) assay was completed towards determining the effects of the GICs on metabolic activities of the cells, whilst cell morphology was examined by light microscopy. The influence of the compounds released from the GIC rings on cell physiological effects (viability, proliferation and adhesion) during 24 h incubation was further investigated by impedimetry. Result trends obtained from this battery of techniques were complementary. At 100 v/v% concentration, the released compounds from Equia were strongly cytotoxic, while at lower concentration (0, 4, 20 v/v%) they were not cytotoxic. In contrast, Triage elicited only slightly transient cytotoxicity. The method proposed has been proved as being efficient, reliable and reproducible and may be useful in quick testing of the cytotoxicity of similar biomaterials by using an immortalized cell line.

The PPARγ agonist pioglitazone prevents TGF-ß induced renal fibrosis by repressing EGR-1 and STAT3.

BACKGROUND: It has been proposed that peroxisome proliferator-activated receptor-γ (PPARγ) agonists might reduce renal fibrosis, however, several studies had contradictory results. Moreover, the possible interaction of TGF-ß1, PPARγ, and transcription factors in renal fibrosis have not been investigated. We hypothesized that oral pioglitazone treatment would inhibit TGF-ß-driven renal fibrosis and its progression, by modulating profibrotic transcription factors in TGF-ß1 transgenic mice. METHODS: Male C57Bl/6 J mice (control, CTL, n = 14) and TGF-ß overexpressing transgenic mice (TGFß, n = 14, having elevated plasma TGF-ß1 level) were divided in two sets at 10 weeks of age. Mice in the first set were fed with regular rodent chow (CTL and TGFß, n = 7/group). Mice in the second set were fed with chow containing pioglitazone (at a dose of 20 mg/kg/day, CTL + Pio and TGFß+Pio, n = 7/group). After 5 weeks of treatment, blood pressure was assessed and urine samples were collected, and the kidneys were analyzed for histology, mRNA and protein expression. RESULTS: TGF-ß1 induced glomerulosclerosis and tubulointerstitial damage were significantly reduced by pioglitazone. Pioglitazone inhibited renal mRNA expression of all the profibrotic effectors: type-III collagen, TGF-ß1, CTGF and TIMP-1, and alike transcription factors cFos/cJun and protein expression of EGR-1, and STAT3 protein phosphorylation. CONCLUSIONS: Oral administration of PPARγ agonist pioglitazone significantly reduces TGF-ß1-driven renal fibrosis, via the attenuation of EGR-1, STAT3 and AP-1. This implies that PPARγ agonists might be effective in the treatment of chronic kidney disease patients.

Alterations in SCAI Expression during Cell Plasticity, Fibrosis and Cancer.

Suppressor of cancer cell invasion (SCAI) has been originally characterized as a tumor suppressor inhibiting metastasis in different human cancer cells, and it has been suggested that SCAI expression declines in tumors. The expression patterns and role of SCAI during physiological and pathophysiological processes is still poorly understood. Earlier we demonstrated that SCAI is regulating the epithelial-mesenchymal transition of proximal tubular epithelial cells, it is downregulated during renal fibrosis and it is overexpressed in Wilms' tumors. Here we bring further evidence for the involvement of SCAI during cell plasticity and we examine the prognostic value and expression patterns of SCAI in various tumors. SCAI prevented the activation of the SMA promoter induced by angiotensin II. SCAI expression decreased in a model of endothelial-mesenchymal transition and increased during iPS reprogramming of fibroblasts. During renal fibrosis SCAI expression declined, as evidenced in a rat model of renal transplant rejection and in TGF-ß1 overexpressing transgenic mice. High expression of SCAI correlated with better survival in patients with breast and lung cancers. Intriguingly, in the case of other cancers (gastric, prostate, colorectal) high SCAI expression correlated with poor survival of patients. Finally, we bring evidence for SCAI overexpression in colorectal cancer patients, irrespective of stage or metastatic status of the disease, suggesting a diverse role of SCAI in various diseases and cancer.

Sustained hyperosmolarity increses TGF-ß1 and Egr-1 expression in the rat renal medulla.

BACKGROUND: Although TGF-ß and the transcription factor Egr-1 play an important role in both kidney fibrosis and in response to acute changes of renal medullary osmolarity, their role under sustained hypo- or hyperosmolar conditions has not been elucidated. We investigated the effects of chronic hypertonicity and hypotonicity on the renal medullary TGF-ß and Egr-1 expression. METHODS: Male adult Sprague Dawley rats (n = 6/group) were treated with 15 mg/day furosemide, or the rats were water restricted to 15 ml/200 g body weight per day. Control rats had free access to water and rodent chow. Kidneys were harvested after 5 days of treament. In cultured inner medullary collecting duct (IMCD) cells, osmolarity was increased from 330 mOsm to 900 mOsm over 6 days. Analyses were performed at 330, 600 and 900 mOsm. RESULTS: Urine osmolarity has not changed due to furosemide treatment but increased 2-fold after water restriction (p < 0.05). Gene expression of TGF-ß and Egr-1 increased by 1.9-fold and 7-fold in the hypertonic medulla, respectively (p < 0.05), accompanied by 6-fold and 2-fold increased c-Fos and TIMP-1 expression, respectively (p < 0.05) and positive immunostaining for TGF-ß and Egr-1 (p < 0.05). Similarly, hyperosmolarity led to overexpression of TGF-ß and Egr-1 mRNA in IMCD cells (2.5-fold and 3.5-fold increase from 330 to 900 mOsm, respectively (p < 0.05)) accompanied by significant c-Fos and c-Jun overexpressions (p < 0.01), and increased Col3a1 and Col4a1 mRNA expression. CONCLUSION: We conclude that both TGF-ß and Egr-1 are upregulated by sustained hyperosmolarity in the rat renal medulla, and it favors the expression of extracellular matrix components.

PPARγ Links BMP2 and TGFß1 Pathways in Vascular Smooth Muscle Cells, Regulating Cell Proliferation and Glucose Metabolism.

BMP2 and TGFß1 are functional antagonists of pathological remodeling in the arteries, heart, and lung; however, the mechanisms in VSMCs, and their disturbance in pulmonary arterial hypertension (PAH), are unclear. We found a pro-proliferative TGFß1-Stat3-FoxO1 axis in VSMCs, and PPARγ as inhibitory regulator of TGFß1-Stat3-FoxO1 and TGFß1-Smad3/4, by physically interacting with Stat3 and Smad3. TGFß1 induces fibrosis-related genes and miR-130a/301b, suppressing PPARγ. Conversely, PPARγ inhibits TGFß1-induced mitochondrial activation and VSMC proliferation, and regulates two glucose metabolism-related enzymes, platelet isoform of phosphofructokinase (PFKP, a PPARγ target, via miR-331-5p) and protein phosphatase 1 regulatory subunit 3G (PPP1R3G, a Smad3 target). PPARγ knockdown/deletion in VSMCs activates TGFß1 signaling. The PPARγ agonist pioglitazone reverses PAH and inhibits the TGFß1-Stat3-FoxO1 axis in TGFß1-overexpressing mice. We identified PPARγ as a missing link between BMP2 and TGFß1 pathways in VSMCs. PPARγ activation can be beneficial in TGFß1-associated diseases, such as PAH, parenchymal lung diseases, and Marfan's syndrome.

Ductular reaction correlates with fibrogenesis but does not contribute to liver regeneration in experimental fibrosis models.

BACKGROUND AND AIMS: Ductular reaction is a standard component of fibrotic liver tissue but its function is largely unknown. It is supposed to interact with the matrix producing myofibroblasts and compensate the declining regenerative capacity of hepatocytes. The relationship between the extent of fibrosis-ductular reaction, proliferative activity of hepatocytes and ductular reaction were studied sequentially in experimental hepatic fibrosis models. METHODS: Liver fibrosis/cirrhosis was induced in wild type and TGFß overproducing transgenic mice by carbon tetrachloride and thioacetamide administration. The effect of thioacetamide was modulated by treatment with imatinib and erlotinib. The extent of ductular reaction and fibrosis was measured by morphometry following cytokeratin 19 immunofluorescent labeling and Picro Sirius staining respectively. The proliferative activity of hepatocytes and ductular reaction was evaluated by BrdU incorporation. The temporal distribution of the parameters was followed and compared within and between different experimental groups. RESULTS: There was a strong significant correlation between the extent of fibrosis and ductular reaction in each experimental group. Although imatinib and erlotinib temporarily decreased fibrosis this effect later disappeared. We could not observe negative correlation between the proliferation of hepatocytes and ductular reaction in any of the investigated models. CONCLUSIONS: The stringent connection between ductular reaction and fibrosis, which cannot be influenced by any of our treatment regimens, suggests that there is a close mutual interaction between them instead of a unidirectional causal relationship. Our results confirm a close connection between DR and fibrogenesis. However, since the two parameters changed together we could not establish a causal relationship and were unable to reveal which was the primary event. The lack of inverse correlation between the proliferation of hepatocytes and ductular reaction questions that ductular reaction can compensate for the failing regenerative activity of hepatocytes. No evidences support the persistent antifibrotic property of imatinib or erlotinib.

The Effect of Combined Treatment with the (Pro)Renin Receptor Blocker HRP and Quinapril in Type 1 Diabetic Rats.

BACKGROUND/AIMS: Diabetic nephropathy remains a major clinical problem. The effects of prorenin might be adverse, but the literature data are controversial. We compared the renal effects of the (pro)renin receptor ((P)RR) blockade and angiotensin converting enzyme (ACE) inhibition on the progression of diabetic nephropathy in rats. METHODS: Diabetes (DM) was induced by ip. streptozotocin administration in adult male Sprague-Dawley rats, followed by eight weeks of treatment with the (P)RR blocker "handle region" decoy peptide (HRP, 0,1 mg/kg/day) or with the ACE inhibitor Quinapril (Q, 50 mg/kg/day) and grouped as follows: 1. Control (n=10); 2. DM (n=8); 3. DM+HRP (n=6); 4. DM+Q (n=10); 5. DM+Q+HRP (n=10). Renal functional parameters, histology and gene expressions were evaluated. RESULTS: HRP reduced glomerulosclerosis and podocyte desmin expression, but did not affect proteinuria and tubular ERK(1/2) phosphorylation. Both Q and Q+HRP treatment reduced proteinuria, glomerular and tubular damage, tubular TGF-ß1 expression and ERK(1/2) phosphorylation to the same extent. CONCLUSION: The effects of HRP were partially beneficial on diabetic kidney lesions as HRP reduced damage but did not improve tubular damage and failed to reduce ERK(1/2) phosphorylation in rats. The combination of HRP with Quinapril had no additive effects over Quinapril monotherapy on the progression of diabetic nephropathy.

LPS-induced delayed preconditioning is mediated by Hsp90 and involves the heat shock response in mouse kidney.

INTRODUCTION: We and others demonstrated previously that preconditioning with endotoxin (LPS) protected from a subsequent lethal LPS challenge or from renal ischemia-reperfusion injury (IRI). LPS is effective in evoking the heat shock response, an ancient and essential cellular defense mechanism, which plays a role in resistance to, and recovery from diseases. Here, by using the pharmacological Hsp90 inhibitor novobiocin (NB), we investigated the role of Hsp90 and the heat shock response in LPS-induced delayed renal preconditioning. METHODS: Male C57BL/6 mice were treated with preconditioning (P: 2 mg/kg, i.p.) and subsequent lethal (L: 10 mg/kg, i.p.) doses of LPS alone or in combination with NB (100 mg/kg, i.p.). Controls received saline (C) or NB. RESULTS: Preconditioning LPS conferred protection from a subsequent lethal LPS treatment. Importantly, the protective effect of LPS preconditioning was completely abolished by a concomitant treatment with NB. LPS induced a marked heat shock protein increase as demonstrated by Western blots of Hsp70 and Hsp90. NB alone also stimulated Hsp70 and Hsp90 mRNA but not protein expression. However, Hsp70 and Hsp90 protein induction in LPS-treated mice was abolished by a concomitant NB treatment, demonstrating a NB-induced impairment of the heat shock response to LPS preconditioning. CONCLUSION: LPS-induced heat shock protein induction and tolerance to a subsequent lethal LPS treatment was prevented by the Hsp90 inhibitor, novobiocin. Our findings demonstrate a critical role of Hsp90 in LPS signaling, and a potential involvement of the heat shock response in LPS-induced preconditioning.

Characterization and role of SCAI during renal fibrosis and epithelial-to-mesenchymal transition.

During progressive tubulointerstitial fibrosis, renal tubular epithelial cells transform into α-smooth muscle actin (SMA)-expressing myofibroblasts via epithelial-to-mesenchymal transition (EMT). SMA expression is regulated by transforming growth factor (TGF)-ß1 and cell contact disruption, through signaling events targeting the serum response factor-myocardin-related transcription factor (MRTF) complex. MRTFs are important regulators of fibrosis, tumor cell invasion, and metastasis. Consistent with the role of MRTFs in tumor progression, suppressor of cancer cell invasion (SCAI) was recently identified as a negative regulator of MRTF. Herein, we studied the role of SCAI in a fibrotic EMT model established on LLC-PK1 cells. SCAI overexpression prevented SMA promoter activation induced by TGF-ß1. When co-expressed, it inhibited the stimulatory effects of MRTF-A, MRTF-B or the constitutive active forms of RhoA, Rac1, or Cdc42 on the SMA promoter. SCAI interfered with TGF-ß1-induced SMA, connective tissue growth factor, and calponin protein expression; it rescued TGF-ß1-induced E-cadherin down-regulation. IHC studies on human kidneys showed that SCAI expression is reduced during fibrosis. Kidneys of diabetic rats and mice with unilateral ureteral obstruction depicted significant loss of SCAI expression. In parallel with the decrease of SCAI protein expression, diabetic rat and mouse kidneys with unilateral ureteral obstruction showed SMA expression, as evidenced by using Western blot analysis. Finally, TGF-ß1 treatment of LLC-PK1 cells attenuated SCAI protein expression. These data suggest that SCAI is a novel transcriptional cofactor that regulates EMT and renal fibrosis.

Selective phosphodiesterase-5 (PDE-5) inhibitor vardenafil ameliorates renal damage in type 1 diabetic rats by restoring cyclic 3',5' guanosine monophosphate (cGMP) level in podocytes.

BACKGROUND: Diabetic nephropathy (DN) is characterized by podocyte damage and increased phosphodiesterase-5 (PDE-5) activity-exacerbating nitric oxide (NO)-cyclic 3',5' guanosine monophosphate (cGMP) pathway dysfunction. It has been shown that PDE-5 inhibition ameliorates DN. The role of podocytes in this mechanism remains unclear. We investigated how selective PDE-5 inhibition influences podocyte damage in streptozotocin (STZ) diabetic rats. METHODS: Male Sprague-Dawley rats (250-300 g) were injected with STZ and divided into two groups: (i) STZ control (non-treated, STZ, n=6) and (ii) STZ+vardenafil treatment (10 mg/kg/day, STZ-Vard, n=8). Non-diabetic rats served as negative controls (Control, n=7). Following 8 weeks of treatment, immunohistochemical and molecular analysis of the kidneys were performed. RESULTS: Diabetic rats had proteinuria, increased renal transforming growth factor (TGF)-ß1 expression and podocyte damage when compared with controls. Vardenafil treatment resulted in preserved podocyte cGMP levels, less proteinuria, reduced renal TGF-ß1 expression, desmin immunostaining in podocytes and restored both nephrin and podocin mRNA expression. Diabetes led to increased glomerular nitrotyrosine formation and renal neuronal nitric oxide synthase and endothelial nitric oxide synthase mRNA expression, but vardenafil did not influence these parameters. CONCLUSIONS: Our data suggest that a dysfunctional NO-cGMP pathway exacerbates podocyte damage in diabetes. In conclusion, vardenafil treatment preserves podocyte function and reduces glomerular damage, which indicates therapeutic potential in patients with DN.

Elevated systemic TGF-beta impairs aortic vasomotor function through activation of NADPH oxidase-driven superoxide production and leads to hypertension, myocardial remodeling, and increased plaque formation in apoE(-/-) mice.

The role of circulating, systemic TGF-beta levels in endothelial function is not clear. TGF-beta(1) may cause endothelial dysfunction in apolipoprotein E-deficient (apoE(-/-)) mice via stimulation of reactive oxygen species (ROS) production by the NADPH oxidase (NOX) system and aggravate aortic and heart remodeling and hypertension. Thoracic aorta (TA) were isolated from 4-mo-old control (C57Bl/6), apoE(-/-), TGF-beta(1)-overexpressing (TGFbeta(1)), and crossbred apoE(-/-) x TGFbeta(1) mice. Endothelium-dependent relaxation was measured before and after incubation with apocynin (NOX inhibitor) or superoxide dismutase (SOD; ROS scavenger). Superoxide production within the vessel wall was determined by dihydroethidine staining under confocal microscope. In 8-mo-old mice, aortic and myocardial morphometric changes, plaque formation by en face fat staining, and blood pressure were determined. Serum TGF-beta(1) levels (ELISA) were elevated in TGFbeta(1) mice without downregulation of TGF-beta-I receptor (immunohistochemistry). In the aortic wall, superoxide production was enhanced and NO-dependent relaxation diminished in apoE(-/-) x TGFbeta(1) mice but improved significantly after apocynin or SOD. Myocardial capillary density was reduced, fibrocyte density increased, aortic wall was thicker, combined lesion area was greater, and blood pressure was higher in the apoE(-/-) x TGFbeta vs. C57Bl/6 mice. Our results demonstrate that elevated circulating TGF-beta(1) causes endothelial dysfunction through NOX activation-induced oxidative stress, accelerating atherosclerosis and hypertension in apoE(-/-) mice. These findings may provide a mechanism explaining accelerated atherosclerosis in patients with elevated plasma TGFbeta(1).

Increased renoprotection with ACE inhibitor plus aldosterone antagonist as compared to monotherapies--the effect on podocytes.

BACKGROUND: Blockade of the renin-angiotensin-aldosterone system (RAAS) does not completely prevent progression of renal disease. Mineralocorticoid receptor blockade provides additional renoprotection over ACE-inhibition monotherapy. We examined the mechanisms underlying superior renoprotection in the subtotal nephrectomy (SNX) model. METHODS: Sprague-Dawley rats were randomized into six groups: (1) sham-op, (2) SNX without treatment, (3) SNX + quinapril (Q), (4) SNX + spironolactone (S), (5) SNX + combination therapy (Q+S), (6) SNX + combination hydrochlorothiazide + reserpin + hydralazine (HRH). Albuminuria and blood pressure were monitored, and kidneys were examined by morphometric and molecular methods. RESULTS: In SNX rats, albumin excretion was significantly higher than in sham-op rats. Blood pressure reduction was not significantly different between the treatment groups. All therapies (S, Q, Q+S and HRH) reduced albuminuria; the values were lowest in animals treated with Q+S. The volume density of glomerular matrix and the number of mesangial cells were significantly increased in SNX and were lowest in SNX treated with Q+S. The number of podocytes was reduced in SNX, but was normalized in SNX treated with Q+S. Glomerular volumes and podocyte volumes were significantly higher in SNX than in sham-op. Both volumes were reduced by all interventions, but almost normalized by treatment with Q+S. Expression of collagen IV, TGF-beta(1) and desmin was increased after SNX and significantly reduced by treatment with Q and Q+S. CONCLUSIONS: In subtotally nephrectomized rats, mineralocorticoid blockade provided additional renoprotection over and above ACE inhibition. Such benefit was paralleled by major changes in podocyte number and morphology and was not blood pressure dependent.

Regulation of inducible class II MHC, costimulatory molecules, and cytokine expression in TGF-beta1 knockout renal epithelial cells: effect of exogenous TGF-beta1.

As reports of mice genetically deficient for TGF-beta1 demonstrated aberrant renal class II MHC expression, we investigated inducible class II MHC expression on renal tubular epithelial cells derived from TGF-beta1 knockout (-/-) and wild-type (+/+) mice. IFN-gamma markedly upregulated class II MHC (I-A(b)) expression in both (-/-) and (+/+) tubular epithelial cells. Coincubation studies of (+/+) and (-/-) tubular epithelial cells with IFN-gamma+LPS, or pretreatment of these cells with TGF-beta1, revealed inhibition of IFN-gamma-induced I-A(b) mRNA and cell surface expression that occurred via a decrease in class II transactivator gene expression in both (+/+) and (-/-) tubular epithelial cells. In addition, ICAM-1 was constitutively expressed on both (+/+) and (-/-) tubular epithelial cells and was upregulated by IFN-gamma or IFN-gamma+LPS. ICAM-1 expression in (+/+) and (-/-) tubular epithelial cells, however, was decreased by TGF-beta1. Parallel analysis evaluating B7-1 expression detected low levels of B7-1 in unstimulated (+/+) and (-/-) tubular epithelial cells that were increased by IFN-gamma, LPS, and IFN-gamma+LPS. IFN-gamma+LPS-mediated upregulation of B7-1 was also blocked by pretreatment with TGF-beta1. Cytokine analysis detected significantly higher levels of TNF-alpha and MIP-1alpha mRNA in all treated (-/-) preparations than in (+/+) tubular epithelial cell controls. These studies demonstrate normal patterns of class II MHC, ICAM-1, and B7 expression in TGF-beta1 (-/-) tubular epithelial cells in response to IFN-gamma, LPS, and TGF-beta1. Upregulated cytokine expression at baseline and in response to proinflammatory mediators is apparent in (-/-) tubular epithelial cells, however, and suggests that dysregulation of cytokine expression in inflammatory responses may be a primary event in multifocal inflammation observed in TGF-beta1-deficient animals.

Overexpression of the type II transforming growth factor-beta receptor inhibits fibroblasts proliferation and activates extracellular signal regulated kinase and c-Jun N-terminal kinase.

Transforming growth factor-beta (TGF-beta) is a bimodal regulator of cellular growth. The cellular effects of TGF-beta depend on the intensity of signals emanating from TGF-beta receptors. Low levels of receptor activity are sufficient to stimulate cell proliferation, while higher degrees of receptor activation are associated with growth inhibition. To study the mechanisms of these effects, a tetracycline-inducible expression system was used to overexpress type II TGF-beta receptors in NIH 3T3 fibroblasts. Overexpressed type II TGF-beta receptors suppressed fibroblast proliferation elicited by TGF-beta1, fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF). Accompanying these anti-proliferative effects, increases in extracellular-signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activity were detected. Furthermore, PDGF alpha-, but not PDGF beta-receptor protein levels were reduced by type II TGF-beta receptor overexpression. In conclusion, our system is an excellent tool to study the molecular mechanisms of growth inhibition by TGF-beta in fibroblasts. Activation of JNK and ERK, or modulation of PDGF receptor expression may be involved in this process.