Profibrotic Transforming Growth Factor-beta Research Articles

Electrospun composite-coated endotracheal tubes with controlled siRNA and drug delivery to lubricate and minimize upper airway injury

Endotracheal Tubes (ETTs) maintain and secure a patent airway; however, prolonged intubation often results in unintended injury to the mucosal epithelium and inflammatory sequelae which complicate recovery. ETT design and materials used have yet to adapt to address intubation associated complications. In this study, a composite coating of electrospun polycaprolactone (PCL) fibers embedded in a four-arm polyethylene glycol acrylate matrix (4APEGA) is developed to transform the ETT from a mechanical device to a dual-purpose device capable of delivering multiple therapeutics while preserving coating integrity. Further, the composite coating system (PCL-4APEGA) is capable of sustained delivery of dexamethasone from the PCL phase and small interfering RNA (siRNA) containing polyplexes from the 4APEGA phase. The siRNA is released rapidly and targets smad3 for immediate reduction in pro-fibrotic transforming growth factor-beta 1 (TGFϐ1) signaling in the upper airway mucosa as well as suppressing long-term sequelae in inflammation from prolonged intubation. A bioreactor was used to study mucosal adhesion to the composite PCL-4APEGA coated ETTs and investigate continued mucus secretory function in ex vivo epithelial samples. The addition of the 4APEGA coating and siRNA delivery to the dexamethasone delivery was then evaluated in a swine model of intubation injury and observed to restore mechanical function of the vocal folds and maintain epithelial thickness when observed over 14 days of intubation. This study demonstrated that increase in surface lubrication paired with surface stiffness reduction significantly decreased fibrotic behavior while reducing epithelial adhesion and abrasion.

Tamoxifen Upregulates Collagenase Gene Expression in Human Dermal Fibroblasts.

Tamoxifen is a known inhibitor of fibroblast transforming growth factor beta biosynthesis and wound scar formation. Tamoxifen is also known to be an estrogen antagonist and protein kinase C (PKC) inhibitor. Cells treated with tamoxifen and other PKC/calmodulin inhibitors depolymerize their membrane focal adhesion complexes and cytoskeletal protein structures. These effects result in substrate detachment, cell shape rounding, and upregulation of collagenase synthesis and extracellular matrix degradation. The purpose of our study was to test the hypothesis that tamoxifen treatment of human foreskin fibroblasts results in alteration of cytoskeletal protein organization, cell detachment and rounding, and increased collagenase synthesis similar to known PKC/calmodulin inhibitors such as H-7. We characterized the effects of PKC/calmodulin inhibitors tamoxifen and H-7 on human dermal fibroblast morphology, cytoskeletal protein organization, and collagenase gene expression in monolayer culture and within collagen gels. We found that fibroblasts responded to tamoxifen by initiation of actin filament depolymerization followed by alteration from spindle to spheroidal shapes. This change in cell shape led to increased collagenase synthesis in cells treated with either tamoxifen or H-7 compared with controls. There was also a 23% increase of hydroxyproline release from tamoxifen-treated fibroblast-populated collagen matrices. Tamoxifen may reduce scarring by inhibiting fibroblast PKC/calmodulin activity, which down-regulates pro-fibrotic transforming growth factor beta signaling and upregulates collagenase production. These effects mimic those of the known PKC/calmodulin inhibitor H-7. Overall, these findings suggest that tamoxifen and its analogues are promising agents for clinical investigation as small molecule regulators of fibrosis and scarring disorders.

The effects of TGF-β1 and IFN-α2b on decorin, decorin isoforms and type I collagen in hypertrophic scar dermal fibroblasts.

Hypertrophic scars (HTS) develop from an excessive synthesis of structural proteins like collagen and a decreased expression of proteoglycans such as decorin. Previous research has demonstrated that decorin expression is significantly down-regulated in HTS, deep dermal tissue, and thermally injured tissue, reducing its ability to regulate pro-fibrotic transforming growth factor-beta 1 (TGF-β1) and normal fibrillogenesis. However, treatment of HTS fibroblasts with interferon-alpha 2b (IFN-α2b) has been shown to reduce excessive collagen synthesis and improve HTS by reducing serum TGF-β1 levels. The expression of decorin isoforms in HTS is currently unknown and the effects of TGF-β1 and IFN-α2b on decorin, decorin isoform expression and type 1 collagen are of great interest to our group. Dermal fibroblasts were treated with TGF-β1 and/or IFN-α2b, for 48 h. The expression and secretion of decorin, decorin isoforms and type 1 collagen were quantified with reverse transcription-quantitative polymerase chain reaction, immunofluorescence staining and enzyme-linked immunosorbent assays. The mRNA expression of decorin and each isoform was significantly reduced in HTS fibroblasts relative to normal skin. TGF-β1 decreased the mRNA expression of decorin and decorin isoforms, whereas IFN-α2b showed the opposite effect. IFN-α2b significantly inhibited TGF-β1's effect on the mRNA expression of type I collagen alpha 1 in papillary dermal fibroblasts and overall showed relative effects of inhibiting TGF-β1. These data support that a further investigation into the structural and functional roles of decorin isoforms in HTS pathogenesis is warranted and that IFN-α2b is an important agent in reducing fibrotic outcomes.

Temporal expression and spatial distribution of the proteoglycan versican during cardiac fibrosis development

Cardiac fibrosis is a central pathological feature in several cardiac diseases, but the underlying molecular players are insufficiently understood. The extracellular matrix proteoglycan versican is elevated in heart failure and suggested to be a target for treatment. However, the temporal expression and spatial distribution of versican and the versican cleavage fragment containing the neoepitope DPEAAE in cardiac fibrosis remains to be elucidated. In this study, we have examined versican during cardiac fibrosis development in a murine pressure overload model and in patients with cardiomyopathies. We found that versican, mainly the V1 isoform, was expressed immediately after induction of pressure overload, preceding collagen accumulation, and versican protein levels extended from the perivascular region into the cardiac interstitium. In addition, we found increased production of versican by collagen expressing fibroblasts, and that it was deposited extensively in the fibrotic extracellular matrix during pressure overload. In cardiac cell cultures, the expression of versican was induced by the pro-fibrotic transforming growth factor beta and mechanical stretch. Furthermore, we observed that the proteolytic cleavage of versican (DPEAAE fragment) increased in the late phase of fibrosis development during pressure overload. In patients with hypertrophic and dilated cardiomyopathies, we found elevated levels of versican and a positive correlation between versican and collagen mRNA in the heart, as well as increased cleavage of full-length protein. Taken together, the temporal expression profile and the spatial distribution of both the full-length versican and the DPEAAE fragment observed in this study indicates a role for versican in development of cardiac fibrosis.

Suppression of the fibrotic encapsulation of silicone implants by inhibiting the mechanical activation of pro-fibrotic TGF-β.

The fibrotic encapsulation of implants involves the mechanical activation of myofibroblasts and of pro-fibrotic transforming growth factor beta 1 (TGF-β1). Here, we show that both softening of the implant surfaces and inhibition of the activation of TGF-β1 reduce the fibrotic encapsulation of subcutaneous silicone implants in mice. Conventionally stiff silicones (elastic modulus, ~2 MPa) coated with a soft silicone layer (elastic modulus, ~2 kPa) reduced collagen deposition as well as myofibroblast activation without affecting the numbers of macrophages and their polarization states. Instead, fibroblasts around stiff implants exhibited enhanced intracellular stress, increased the recruitment of αv and β1 integrins, and activated TGF-β1 signalling. In vitro, the recruitment of αv integrin to focal adhesions and the activation of β1 integrin and of TGF-β were higher in myofibroblasts grown on latency-associated peptide (LAP)-coated stiff silicones than on soft silicones. Antagonizing αv integrin binding to LAP through the small-molecule inhibitor CWHM-12 suppressed active TGF-β signalling, myofibroblast activation and the fibrotic encapsulation of stiff subcutaneous implants in mice.

Basic and clinical science posters: Basic science of diabetes complications

Aims: Glucose evoked changes in profibrotic transforming growth factor-beta1 (TGFs1), increase connexin hemichannel mediated ATP release in kidney proximal tubule cells. With ATP linked to inflammation, we evaluated the efficacy of hemichannel blocker Peptide 5 in negating ATP induced inflammatory cytokine secretion in proximal tubule cells. Methods: Primary human proximal tubular epithelial cells (hPTECs), and immortalised human kidney (HK2) cells were treated with TGFs1 (10ng/ml) ± Peptide5 (25µM) for 48hrs. Carboxyfluorescein (200µM) dye uptake and ATP biosensing determined hemichannel activity and ATP release respectively. Proteome arrays determined cytokine secretion. Results: In TGFs1 treated hPTECs, carboxyfluorescein dye uptake decreased from 347.4±11.9%, (P<0.001, N=3) to 158.6±7.9% when co-incubated with Peptide 5. Parallel to this, ATP release increased from 0.16±0.1µM to 2.5±0.14µM (P<0.001, N=3) in TGF1 treated cells as compared to control. Peptide 5 significantly blunted this response, to 0.62±0.17µM (P<0.001, N=3). A proteome array determined the impact of Peptide 5 on secretion of 105 candidate chemokines and cytokines in TGFs1 treated primary cells. As an example, Peptide 5 significantly restored secretion of adiponectin from 163±8.0% (P<0.001, N=4) to 136±6.1% (P<0.05, N=4), granulocyte-macrophage colony-stimulating factor from 183±8.0% (P<0.001, N=4) to 111.3±5.9% (P<0.001, N=4) and interleukin 1-beta from 189±14.9 (P<0.001, N=4) to 109±9.6% (P<0.001, N=4) in TGF-s1 treated hPTECs, as compared to control. Conclusion: Glucose evoked changes in TGFs1 increase hemichannel mediated ATP release. Hemichannel blocker; Peptide 5 blocks ATP release and downstream cytokine and chemokine secretion, suggesting that Peptide 5 may be of future therapeutic potential in targeting inflammation of the diabetic kidney.

Anti-fibrotic Actions of Equine Interleukin-10 on Transforming Growth Factor-Beta1-Stimulated Dermal Fibroblasts Isolated From Limbs of Horses.

Fibroproliferative disorders occur in both humans and horses following skin injury. In horses, wound healing on the limb is often complicated by the formation of fibroproliferative exuberant granulation tissue, characterized by persistent expression of pro-fibrotic transforming growth factor-beta1 (TGF-β1) and deficient expression of anti-inflammatory interleukin-10 (IL-10). IL-10 has been shown to directly modulate fibrotic gene expression in human fibroblasts, so we hypothesized that equine IL-10 (eIL-10) may exert similar anti-fibrotic effects on equine dermal fibroblasts. Cell-lines were created from the limb skin of six individual horses. Recombinant eIL-10 was produced and purified, and its effects on the cells investigated in the presence and absence of equine TGF-β1 (eTGF-β1). Myofibroblast differentiation and collagen production were examined using immunofluorescent cytometry, cell contractility in a collagen gel assay, and fibrotic gene expression using quantitative PCR. In response to eTGF-β1, fibroblasts increased in contractility and expression of alpha-smooth muscle actin, collagen types 1 and 3, and matrix metalloproteinase 1, 2, and 9. Equine IL-10 limited cell contractility and production of alpha-smooth muscle actin and type 3 collagen, and decreased mRNA levels of eCol3a1 and eMMP9, while increasing that of eMMP1. Opposing effects on eTGF-βR3 and eIL-10R1 gene expression were also observed, with mRNA levels decreasing following eTGF-β1 treatment, and increasing with eIL-10 treatment. These findings indicate that eIL-10 limits the pro-fibrotic effects of eTGF-β1, potentially through the modulation of fibrotic and receptor gene expression. Further investigations are warranted to assess the therapeutic utility of eIL-10 in the treatment of exuberant granulation tissue.

Paclitaxel alleviates liver fibrosis induced by bile duct ligation in rats: Role of TGF-β1, IL-10 and c-Myc

Liver fibrosis is a global health issue that causes morbidity and mortality with no currently available treatment. It has been shown that low dose paclitaxel (PTX) can stabilize microtubules and inhibit the profibrotic transforming growth factor-beta 1 (TGF-β1) signaling pathway. In this study the effect of treatment with low dose PTX was examined using a model of cholestatic liver fibrosis. Bile-duct ligation (BDL) was induced in rats for 2 weeks then PTX (0.3 mg/kg/ip) was administered three times a week for 2 weeks. Administration of PTX ameliorated BDL-induced elevation in biomarkers of hepatocellular damage (alanine transaminase; ALT and aspartate transaminase; AST) and obstructive cholestatic injury (total bilirubin and gamma glutamyl transferase; γ-GT). PTX was able to correct the increase in liver weight to body weight ratio and the bile duct proliferation induced by BDL. Additionally, PTX treatment corrected the BDL-induced fibrosis of portal tracts, elevation of hydroxyproline content and increased alpha smooth muscle actin (α-SMA) mRNA and protein expression. This antifibrotic effect of PTX was further examined through its inhibitory effect on TGF-β1 mRNA and protein expression in addition to c-Myc mRNA expression. Furthermore, PTX rectified the BDL-induced decrease in interleukin-10 (IL-10) mRNA and protein expression. In conclusion, this study suggests that PTX at low dose has the potential to treat BDL-induced liver fibrosis in rats possibly through suppression of TGF-β1 and c-Myc and activation of IL-10 pathways.

Abstract 425: Doxorubicin Upregulation of the Fibrotic Transforming Growth Factor-beta Pathway

Childhood cancer survivors are at an increased risk of heart disease as a result of their cancer treatments. Drugs like doxorubicin (DOX) are an effective part of treatment regimens, but have been proven to cause acute and chronic cardiotoxicity (DOX tox). An under-investigated aspect of DOX tox is the interstitial fibrosis that the majority of patients develop. This project aims to better understand the pathology of DOX-induced cardiac fibrosis and the role of the pro-fibrotic transforming growth factor-beta (TGFb) signaling pathway. Research in the area of fibrosis and the effect of DOX on cardiac fibroblasts will increase our understanding of DOX tox. This understanding will allow for improved treatment of pediatric cancer patients by reducing the cardiotoxic sequelae of many standard chemotherapy regimens. Cardiac fibroblasts, isolated from 3 week old mice and treated with 5 μM DOX, showed an increase in nuclear pSMAD compared to control cells via fluorescent immunocytology (2.06 ± 0.26 vs 1.13 ± 0.15, p&lt;0.05). Mice treated with 3 mg/kg DOX injections from 2 weeks to 6 weeks of age showed increased TGFb staining in the left ventricle (1.83 ± 0.34 vs 0.87 ± 0.28, p&lt;0.05) a week after treatment ceased. A subset of mice were followed into old age and sacrificed at 80 weeks. A clear increase in TGFb was seen with age. However, 80 week mice that were exposed to DOX early in life showed a greater increase in TGFb staining compared to untreated 80 week old mice (44.50 ± 2.48 vs 30.93 ± 2.30, p&lt;0.001). Early DOX exposure causes chronic molecular changes as evidenced by acute and chronic changes in signaling molecules in cardiac tissue. Changes in collagen seen in earlier studies and increases in MMP-2 from the literature suggest a cardiac remodeling phenotype in DOX-exposed animals. This project demonstrates that DOX initiates changes to pro-fibrotic pathways, seemingly driven by the TGFb signaling pathway.

Effects of fish oil containing eicosapentaenoic acid and docosahexaenoic acid on dystrophic mdx mice hearts at later stages of dystrophy

ObjectiveIn the present study, we investigated whether omega-3 would be effective against dystrophic cardiomyopathy at later stages (13 and 17 mo) of the disease. MethodsMdx mice (8 mo old) received omega-3 oil (commercially available fish oil; FDC vitamins; omega-3) for 5 mo. Untreated-mdx mice received mineral oil. Heart and diaphragm muscle were evaluated by morphometric (fibrosis), molecular (western blot, inflammatory markers), biochemical (creatine kinase), and functional (electrocardiogram) analyses. ResultsMdx mice presented elevated plasma levels of cardiac creatine kinase (41.2 U/L in normal × 119.6 U/L in untreated-mdx mice), which were significantly decreased by omega-3 treatment. Heart fibrosis was significantly ameliorated by omega-3 treatment at 17 mo of age (untreated-mdx: 20.8% of fibrosis; omega-3-treated: 15.7% of fibrosis in right ventricle). Omega-3 improved some electrocardiogram parameters. Markers of inflammation (tumor necrosis factor alpha, matrix metalloprotease-9, and tissue inhibitor of metalloprotease 1) in mdx heart were significantly decreased by omega-3 treatment. Omega-3 increased β-dystroglycan levels in mdx heart and did not affect the levels of the profibrotic transforming growth factor beta. Omega-3 ameliorated the dystrophic diaphragm in almost all of the parameters evaluated (fibrosis, transforming growth factor beta, metalloprotease-9, and tissue inhibitor of metalloprotease 1). ConclusionsThe present study suggests that omega-3 may be useful in ameliorating dystrophic cardiomyopathy and diaphragm dystrophy in mdx mice at later stages of the disease, further supporting the use of omega-3 in DMD clinical trials.

Profibrotic transforming growth factor beta 1 and activin A are increased in nasal polyp tissue and induced in nasal polyp epithelium by cigarette smoke and Toll-like receptor 3 ligation.

The mechanism of airway remodeling in chronic rhinosinusitis with nasal polyposis (CRSwNP) remains unknown. We wished to determine whether profibrotic transforming growth factor beta 1 (TGF-β1) and activin A and their downstream signaling proteins are increased in CRSwNP and if they are regulated in epithelial cells by noxious or inflammatory stimuli. Frozen tissue from CRSwNP patients, healthy control (HC) middle turbinates, and sinus tissue from CRS without NP (CRSsNP) patients were immunostained for TGF-β1, activin A, and downstream signaling proteins. Primary nasal epithelial cells (PNECs) from HCs and CRSwNP patients were cultured in media, cigarette smoke extract (CSE), or double-stranded RNA (dsRNA) (a ligand for Toll-like receptor-3) and examined for inflammatory and profibrotic genes using real-time polymerase chain reaction (PCR). CRSwNP patients showed increased TGF-β1 and activin A in the stroma, increased TGF-β1 signaling (phosphorylated Smad2/3) in the stroma and epithelium, and increased Smad3-dependent Snail1 in the stroma. Immunostaining for TGF-β1, pSmad2/3, and Snail1 in CRSwNP patients was highly correlated. Immunostaining for pSmad2/3 and Snail1 was similar in CRSwNP and CRSsNP patients. Compared to HCs, PNECs from CRSwNP patients were more responsive to CSE and dsRNA in terms of TGF-β1 and activin A and more strongly induced by dsRNA in terms of chemokines. Increased TGF-β1 and activin A and increased downstream TGF-β1 signaling is present in CRSwNP patients, primarily in the stroma. This may contribute to features of airway remodeling previously described. PNECs from CRSwNP patients are induced to produce TGF-β1 and activin A by CSE and dsRNA, suggesting that cigarette smoke and viral infection might also contribute to airway remodeling.

Hypothyroidism and its rapid correction alter cardiac remodeling.

The cardiovascular effects of mild and overt thyroid disease include a vast array of pathological changes. As well, thyroid replacement therapy has been suggested for preserving cardiac function. However, the influence of thyroid hormones on cardiac remodeling has not been thoroughly investigated at the molecular and cellular levels. The purpose of this paper is to study the effect of hypothyroidism and thyroid replacement therapy on cardiac alterations. Thirty Wistar rats were divided into 2 groups: a control (n = 10) group and a group treated with 6-propyl-2-thiouracil (PTU) (n = 20) to induce hypothyroidism. Ten of the 20 rats in the PTU group were then treated with L-thyroxine to quickly re-establish euthyroidism. The serum levels of inflammatory markers, such as C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL6) and pro-fibrotic transforming growth factor beta 1 (TGF-β1), were significantly increased in hypothyroid rats; elevations in cardiac stress markers, brain natriuretic peptide (BNP) and cardiac troponin T (cTnT) were also noted. The expressions of cardiac remodeling genes were induced in hypothyroid rats in parallel with the development of fibrosis, and a decline in cardiac function with chamber dilation was measured by echocardiography. Rapidly reversing the hypothyroidism and restoring the euthyroid state improved cardiac function with a decrease in the levels of cardiac remodeling markers. However, this change further increased the levels of inflammatory and fibrotic markers in the plasma and heart and led to myocardial cellular infiltration. In conclusion, we showed that hypothyroidism is related to cardiac function decline, fibrosis and inflammation; most importantly, the rapid correction of hypothyroidism led to cardiac injuries. Our results might offer new insights for the management of hypothyroidism-induced heart disease.

The epithelium in idiopathic pulmonary fibrosis: breaking the barrier

Idiopathic pulmonary fibrosis is a progressive disease of unknown etiology characterized by a dysregulated wound healing response that leads to fatal accumulation of fibroblasts and extracellular matrix (ECM) in the lung, which compromises tissue architecture and lung function capacity. Injury to type II alveolar epithelial cells is thought to be the key event for the initiation of the disease, and so far both genetic factors, such as mutations in telomerase and MUC5B genes as well as environmental components, like cigarette smoking, exposure to asbestos and viral infections have been implicated as potential initiating triggers. The injured epithelium then enters a state of senescence-associated secretory phenotype whereby it produces both pro-inflammatory and pro-fibrotic factors that contribute to the wound healing process in the lung. Immune cells, like macrophages and neutrophils as well as activated myofibroblasts then perpetuate this cascade of epithelial cell apoptosis and proliferation by release of pro-fibrotic transforming growth factor beta and continuous deposition of ECM stiffens the basement membrane, altogether having a deleterious impact on epithelial cell function. In this review, we describe the role of the epithelium as both a physical and immunological barrier between environment and self in the homeostatic versus diseased lung and explore the potential mechanisms of epithelial cell injury and the impact of loss of epithelial cell permeability and function on cytokine production, inflammation, and myofibroblast activation in the fibrotic lung.

Human Amnion Epithelial Cell Transplantation Abrogates Lung Fibrosis and Augments Repair

Chronic lung disease characterized by loss of lung tissue, inflammation, and fibrosis represents a major global health burden. Cellular therapies that could restore pneumocytes and reduce inflammation and fibrosis would be a major advance in management. To determine whether human amnion epithelial cells (hAECs), isolated from term placenta and having stem cell-like and antiinflammatory properties, could adopt an alveolar epithelial phenotype and repair a murine model of bleomycin-induced lung injury. Primary hAECs were cultured in small airway growth medium to determine whether the cells could adopt an alveolar epithelial phenotype. Undifferentiated primary hAECs were also injected parenterally into SCID mice after bleomycin-induced lung injury and analyzed for production of surfactant protein (SP)-A, SP-B, SP-C, and SP-D. Mouse lungs were also analyzed for inflammation and collagen deposition. hAECs grown in small airway growth medium developed an alveolar epithelial phenotype with lamellar body formation, production of SPs A-D, and SP-D secretion. Although hAECs injected into mice lacked SPs, hAECs recovered from mouse lungs 2 weeks post-transplantation produced SPs. hAECs remained engrafted over the 4-week test period. hAEC administration reduced inflammation in association with decreased monocyte chemoattractant protein-1, tumor necrosis factor-alpha, IL-1 and -6, and profibrotic transforming growth factor-beta in mouse lungs. In addition, lung collagen content was significantly reduced by hAEC treatment as a possible consequence of increased degradation by matrix metalloproteinase-2 and down-regulation of the tissue inhibitors of matrix metalloproteinase-1 and 2. hAECs offer promise as a cellular therapy for alveolar restitution and to reduce lung inflammation and fibrosis.

Platelet-Rich Fibrin Versus Albumin in Surgical Wound Repair

To study the effects of autologous platelet-rich fibrin (PRF) versus human albumin on incisional wound breaking strength and subcutaneous collagen deposition in patients undergoing laparoscopic cholecystectomy in a randomized trial. Platelet peptidic growth factors may stimulate collagen synthesis and tissue repair. One expanded polytetrafluoroethylene (ePTFE) tube was inserted subcutaneously from the edge of each of the two 10-mm trocar incisions in 51 patients. Treatment with PRF prepared from the patient's own blood or human albumin was randomized to respective wound site by concealed allocation. On postoperative day 10, breaking strength of the incisional wounds as well as the collagen concentration, type I procollagen mRNA, type III procollagen mRNA, matrix metalloproteinase-1 mRNA, and fibroblast density in the ePTFE tubes were determined. All analyses were assessor-blinded. The trial was registered in the Current Controlled Trials Registry (ISRCTN34481461). Local PRF had no significant effect on incisional wound-breaking strength. In the ePTFE tubes, PRF treatment decreased collagen concentration by 24% (P=0.046) and type I procollagen mRNA level by 29% (P=0.003), but had no significant impact on type III procollagen mRNA, matrix metalloproteinase-1 mRNA or fibroblast infiltration. The profibrotic transforming growth factor-beta1 level increased (P<0.0001) 2-fold with PRF. Collagen concentration in albumin-treated ePTFE tubes correlated with breaking strength of the skin incisions (rs=0.48, P=0.03). PRF did not improve wound strength significantly compared with albumin but suppressed subcutaneous collagen synthesis and deposition during early repair of surgical wounds in humans. Furthermore, deposition of reparative collagen in the subcutaneous ePTFE tube model partly predicted the breaking strength of an incisional skin wound.

Mast cells decrease renal fibrosis in unilateral ureteral obstruction

Mast cells regulate both inflammatory responses and tissue repair in human diseases but there are conflicting reports on the role of these cells in the pathogenesis of various kidney diseases. Here we measured mast cell function in unilateral ureteral obstruction, a well-characterized model of renal fibrosis, using Kit(W)/Kit(W-v) mice genetically deficient in mast cells, wild-type mice, and deficient mice reconstituted by adoptive transfer with mast cells from the wild-type animals. Mast cell-deficient mice had higher levels of renal tubular damage, more stromal fibrosis, higher numbers of infiltrating ERHR3-positive macrophages and CD3-positive T cells, and higher tissue levels of profibrotic transforming growth factor-beta1 than wild-type mice or mice reconstituted by adoptive transfer of mast cells 3 weeks after ureteral obstruction. Similarly, while wild-type and adoptively transferred mice had increased alpha-smooth muscle actin and decreased E-cadherin expression, which are indicators of epithelial-mesenchymal transition, the obstructed kidneys of the mast cell-deficient mice had significant attenuation of those indicators. Thus, our study suggests that mast cells protect the kidney against fibrosis by modulation of inflammatory cell infiltration and by transforming growth factor-beta1-driven epithelial-to-mesenchymal transitions.

Increased expression of profibrotic neutral endopeptidase and bradykinin type 1 receptors in stenotic aortic valves

In aortic stenosis (AS), adverse remodelling of the valves may depend on altered local regulation of pro- and antifibrotic systems. We have recently shown that angiotensin-converting enzyme (ACE), which generates profibrotic angiotensin II and inactivates antifibrotic bradykinin (BK), is upregulated in stenotic aortic valves. Here, we analyse the expression of neutral endopeptidase (NEP), another profibrotic and BK-degrading enzyme, and of BK receptors in aortic valves in AS. Stenotic aortic valves (n = 86) were obtained at valve replacement surgery and control valves (n = 13) at cardiac transplantation. Expression levels of NEP and BK type 1 and 2 receptors (BK-1R and BK-2R) in aortic valves and in isolated valvular myofibroblasts were analysed by real-time PCR and immunohistochemistry, and NEP activity was quantified by autoradiography. NEP, BK-1R, and BK-2R mRNA levels were higher in stenotic than in non-stenotic valves (P < 0.05 for each) and the respective proteins localized to valvular endothelial cells and myofibroblasts. In stenotic valves, the proteolytic activity of NEP was significantly increased (4.5-fold, P < 0.001), and tumour necrosis factor-alpha induced the expression of NEP in cultured myofibroblasts. Finally, treatment of cultured myofibroblasts with an NEP inhibitor (phosphoramidon) downregulated the expression of profibrotic transforming growth factor-beta1, whereas addition of BK decreased the expression of collagens I and III which was reversed by a BK-2R antagonist. NEP activity is increased in stenotic aortic valves in parallel with increased expression of BK-receptors. The upregulation of NEP and BK-1R have the potential to promote valvular fibrosis and remodelling while the increase in BK-2R may represent a compensatory antifibrotic response. These findings add novel pathogenic insight and raise potential new therapeutic targets in AS.

Serum and urine interleukin-6 and transforming growth factor-beta1 in young infants with pyelonephritis

Cytokines are involved both in the acute response during pyelonephritis and in the progression to renal scarring. The aim of the present study was to assess the pro-inflammatory interleukin-6 (IL-6) and the anti-inflammatory pro-fibrotic transforming growth factor-beta1 (TGF-beta) in very young infants with pyelonephritis. Serum and urine concentrations of IL-6 and TGF-beta1 were determined by enzyme immunoassay in infants with acute pyelonephritis before antibiotic treatment and in infants with non-renal fever. IL-6 was studied in 12 infants with pyelonephritis and in eight with non-renal fever (median ages, 2 months for both groups). TGF-beta1 was studied in 11 infants with pyelonephritis and in nine with non-renal fever (median ages, 2 and 4 months, respectively). No significant differences were documented in serum concentrations of IL-6 and TGF-beta1 between patients and controls. Urine IL-6 levels were significantly higher in infants with pyelonephritis than in controls (medians, 147 and 21.4 pg/ml, respectively; P = 0.0106). The urine levels of TGF-beta1 were lower in infants with pyelonephritis than in controls, although not significantly (medians, 6.12 and 11.0 ng/ml, respectively; P = 0.0705). Our findings confirm the implication of IL-6 but not of TGF-beta1 in the pathogenesis of the early stages of pyelonephritis in young infants. Tauhe role of the pro-fibrotic TGF-beta1 in the development of renal scarring deserves further investigation.

Endostatin peptide, an inhibitor of angiogenesis, prevents the progression of peritoneal sclerosis in a mouse experimental model

Peritoneal sclerosis is a major and serious complication in patients on long-term continuous ambulatory peritoneal dialysis (PD). The involvement of angiogenesis and proangiogenic factors such as vascular endothelial growth factor (VEGF)-A in progressing peritoneal sclerosis has been reported. We previously reported the therapeutic efficacy of endostatin peptide, a potent inhibitor of angiogenesis derived from type XVIII collagen, in a mouse diabetic nephropathy model. Here, we examined the therapeutic effect of endostatin peptide in preventing progression in a mouse peritoneal sclerosis model. Male ICR mice received intraperitoneal injections of chlorhexidine gluconate (CG) every other day to induce peritoneal sclerosis. Endostatin peptide (1 or 4 mg/kg/day) was administered via subcutaneously implanted osmotic minipumps. Peritoneal sclerosis (day 24) was significantly suppressed by endostatin peptide in a dose-dependent manner. Peritoneal accumulation of type III collagen was significantly suppressed by endostatin peptide. Increase in the number of CD31(+) blood vessels, F4/80(+) monocyte/macrophage accumulation, and 5-bromodeoxyuridine(+) proliferating cells was significantly inhibited by endostatin peptide. Increase in peritoneal expression of VEGF-A, profibrotic transforming growth factor-beta1, and alpha-smooth muscle actin was suppressed by endostatin peptide. Immunoreactivity for endogenous endostatin (whole molecule) and endostatin receptor alpha5beta1-integrin was increased and colocalized to CD31(+) blood vessels in the thickened peritonea of CG-injected mice. These results demonstrate the potential use of antiangiogenic endostatin peptide as a novel therapeutic agent in preventing peritoneal sclerosis, a severe complication in patients undergoing long-term PD.

A comparison of pathomolecular markers of fibrosis and morphology in kidney from autopsies of African Americans and whites

African Americans have an increased incidence of chronic kidney disease (CKD) due to hypertension and arteriosclerosis and increased death due to coronary artery disease, compared with whites. The pathogenesis of CKD involves the increased presence and activation of myofibroblasts and macrophages, promotion of tubulointerstitial fibrosis, and effects of tubulointerstitial cell mitosis and apoptosis. We hypothesized that increased risk of hypertensive vascular disease may be identified by renal pathomolecular markers that are associated with progressive CKD. Renal sections were available from 50 autopsies of 33 African Americans (55% males) and 17 whites (76% males) undergoing forensic autopsy for unexpected death. Sclerotic glomeruli, severity of cortical fibrosis, and renal arteriolosclerosis, total glomerular number (N (glom)), average glomerular volume (V (glom)), birth weights, and blood pressure were known. Presence and locality of markers for myofibroblasts (alpha-SMA), macrophages (CD68), collagen, pro-fibrotic transforming growth factor-beta1 were scored in renal autopsies, and tubulointerstitial apoptosis was recorded. The results demonstrated a strong positive correlation between age, cortical fibrosis and alpha-SMA (p<0.05), and between CD68 and hypertension and coronary artery disease (p<0.05). The findings confirm the role of myofibroblasts and macrophages in pathogenesis of human CKD. However, the markers showed no significant relationships to V (glom), N (glom), birth weight, or race.