Acute Elution of TGFß2 Affects the Smooth Muscle Cells in a Compliance-Matched Vascular Graft.

Transforming growth factor beta 2 (TGFß2) is a pleiotropic growth factor that plays a vital role in smooth muscle cell (SMC) function. Our prior in vitro work has shown that SMC response can be modulated with TGFß2 stimulation in a dose dependent manner. In particular, we have shown that increasing concentrations of TGFß2 shift SMCs from a migratory to a synthetic behavior. In this work, electrospun compliance-matched and hypocompliant TGFß2-eluting tissue engineered vascular grafts (TEVGs) were implanted into Sprague Dawley rats for 5 days to observe SMC population and collagen production. TEVGs were fabricated using a combined computational and experimental approach that varied the ratio of gelatin:polycaprolactone to be either compliance matched or twice as stiff as rat aorta (hypocompliant). TGFß2 concentrations of 0, 10, 100 ng/mg were added to both graft types (n = 3 in each group) and imaged in vivo using ultrasound. Histological markers (SMC, macrophage, collagen, and elastin) were evaluated following explanation at 5 days. In vivo ultrasound showed that compliance-matched TEVGs became stiffer as TGFß2 increased (100 ng/mg TEVGs compared to rat aorta, p < 0.01), while all hypocompliant grafts remained stiffer than control rat aorta. In vivo velocity and diameter were also not significantly different than control vessels. The compliance-matched 10 ng/mg group had an elevated SMC signal (myosin heavy chain) compared to the 0 and 100 ng/mg grafts (p = 0.0009 and 0.0006). Compliance-matched TEVGs containing 100 ng/mg TGFß2 had an increase in collagen production (p < 0.01), general immune response (p < 0.05), and a decrease in SMC population to the 0 and 10 ng/mg groups. All hypocompliant groups were found to be similar, suggesting a lower rate of TGFß2 release in these TEVGs. Our results suggest that TGFß2 can modulate in vivo SMC phenotype over an acute implantation period, which is consistent with our prior in vitro work. To the author's knowledge, this is the first in vivo rat study that evaluates a TGFß2-eluting TEVG. Impact statement TGFß2 affects the SMCs in a vascular graft.

Modulation of the Systemic Immune Response in Suckling Rats by Breast Milk TGF-ß2, EGF and FGF21 Supplementation.

Breast milk is a rich fluid containing bioactive compounds such as specific growth factors (GF) that contribute to maturation of the immune system in early life. The aim of this study was to determine whether transforming growth factor-ß2 (TGF-ß2), epidermal growth factor (EGF) and fibroblast growth factor 21 (FGF21), compounds present in breast milk, could promote systemic immune maturation. For this purpose, newborn Wistar rats were daily supplemented with these GF by oral gavage during the suckling period (21 days of life). At day 14 and 21 of life, plasma for immunoglobulin (Ig) quantification was obtained and spleen lymphocytes were isolated, immunophenotyped and cultured to evaluate their ability to proliferate and release cytokines. The main result was obtained at day 14, when supplementation with EGF increased B cell proportion to reach levels observed at day 21. At the end of the suckling period, all GF increased the plasma levels of IgG1 and IgG2a isotypes, FGF21 balanced the Th1/Th2 cytokine response and both EGF and FGF21 modified splenic lymphocyte composition. These results suggested that the studied milk bioactive factors, mainly EGF and FGF21, may have modulatory roles in the systemic immune responses in early life, although their physiological roles remain to be established.

Effect of Oral Nutrition Supplements and TGF-ß2 on Nutrition and Inflammatory Patterns in Patients With Active Crohn's Disease.

BACKGROUND: Crohn's disease (CD) is often associated with nutrition disorders. Many nutrition therapeutic alternatives have been studied. Nevertheless, the actual role of nutrition therapy is still controversial. The objective of this study was to assess the effects of nutrition supplementation with and without transforming growth factor-beta 2 (TGF-ß2) on inflammatory, endoscopic, histopathologic, and nutrition parameters in active CD. MATERIALS AND METHODS: Thirty-eight patients were allocated into 3 groups: group 1 (patients who received only nutrition orientation), group 2 (nutrition orientation and a normoproteic, normocaloric nutrition supplement), and group 3 (nutrition orientation and the nutritional supplement with TGF-ß2). Clinical and nutrition evaluation, C-reactive protein (CRP) levels, and assessment of endoscopic and histologic parameters in the intestinal mucosa were performed before and after nutrition intervention. RESULTS: The mean follow-up period was 3 months. In the beginning of the study, groups were homogeneous regarding age, gender, CD behavior and localization, and medication in use. In the end of the study, the Clinical Disease Activity Index score was reduced in groups 2 and 3; in group 3, a reduction in CRP levels and an improvement in histologic findings were observed. Among patients who received nutritional supplement, some anthropometric patterns were improved. CONCLUSION: The results of the study indicate that nutritional supplementation improved nutrition and inflammatory patterns in patients with active CD. However, only patients receiving TGF-ß2-enriched formula showed improvement in histologic parameters and significant reduction in CRP levels.

Analysis of sphingolipids in human corneal fibroblasts from normal and keratoconus patients.

The pathophysiology of human keratoconus (KC), a bilateral progressive corneal disease leading to protrusion of the cornea, stromal thinning, and scarring, is not well-understood. In this study, we investigated a novel sphingolipid (SPL) signaling pathway through which KC may be regulated. Using human corneal fibroblasts (HCFs) and human KC cells (HKCs), we examined the SPL pathway modulation. Both cell types were stimulated by the three transforming growth factor (TGF)-ß isoforms: TGF-ß1 (T1), TGF-ß2 (T2), and TGF-ß3 (T3). All samples were analyzed using lipidomics and real-time PCR. Our data showed that HKCs have increased levels of signaling SPLs, ceramide (Cer), and sphingosine 1-phosphate (S1P). Treatment with T1 reversed the increase in Cer in HKCs and treatment with T3 reversed the increase in S1P. S1P3 receptor mRNA levels were also significantly upregulated in HKCs, but were reduced to normal levels following T3 treatment. Furthermore, stimulation with Cer and S1P led to significant upregulation of fibrotic markers in HCFs, but not in HKCs. Additionally, stimulation with a Cer synthesis inhibitor (FTY720) led to significant downregulation of specific fibrotic markers in HKCs (TGF-ß1, collagen type III, and α smooth muscle actin) without an effect on healthy HCFs, suggesting a causative role of Cer and S1P in fibrogenesis. Overall, this study suggests an association of the SPL signaling pathway in KC disease and its relation with the TGF-ß pathway.

TGF-ß2-mediated ocular hypertension is attenuated in SPARC-null mice.

PURPOSE: Transforming growth factor-ß2 (TGF-ß2) has been implicated in the pathogenesis of primary open-angle glaucoma through extracellular matrix (ECM) alteration among various mechanisms. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that regulates ECM within the trabecular meshwork (TM), and is highly upregulated by TGF-ß2. We hypothesized that, in vivo, SPARC is a critical regulatory node in TGF-ß2-mediated ocular hypertension. METHODS: Empty (Ad.empty) or TGF-ß2-containing adenovirus (Ad.TGF-ß2) was injected intravitreally into C57BL6-SV129 WT and SPARC-null mice. An initial study was performed to identify a stable period for IOP measurement under isoflurane. The IOP was measured before injection and every other day for two weeks using rebound tonometry. Additional mice were euthanized at peak IOP for immunohistochemistry. RESULTS: The IOP was stable under isoflurane during minutes 5 to 8. The IOP was significantly elevated in Ad.TGF-ß2-injected (n = 8) versus Ad.empty-injected WT (n = 8) mice and contralateral uninjected eyes during days 4 to 11 (P < 0.03). The IOPs were not significantly elevated in Ad.TGF-ß2-injected versus Ad.empty-injected SPARC-null mice. However, on day 8, the IOP of Ad.TGF-ß2-injected SPARC-null eyes was elevated compared to that of contralateral uninjected eyes (P = 0.0385). Immunohistochemistry demonstrated that TGF-ß2 stimulated increases in collagen IV, fibronectin, plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), and SPARC in WT mice, but only PAI-1 and CTGF in SPARC-null mice (P < 0.05). CONCLUSIONS: SPARC is essential to the regulation of TGF-ß2-mediated ocular hypertension. Deletion of SPARC significantly attenuates the effects of TGF-ß2 by restricting collagen IV and fibronectin expression. These data provide further evidence that SPARC may have an important role in IOP regulation and possibly glaucoma pathogenesis.

Trichostatin A, a histone deacetylase inhibitor, suppresses proliferation and epithelial-mesenchymal transition in retinal pigment epithelium cells.

The proliferation and epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells are the major pathological changes in development of proliferative vitreoretinopathy (PVR), which leads to severe visual impairment. Histone deacetylases (HDACs)-mediated epigenetic mechanisms play important roles in controlling various physiological and pathological events. However, whether HDACs are involved in the regulation of proliferation and EMT in PRE cells remains unidentified. In this study, we evaluated the expression profile of HDAC family (18 genes) and found that some of class I and class II HDACs were up-regulated in transforming growth factor-ß2 (TGF-ß2)/TGF-ß1-stimulated RPE cells. Tricostatin A (TSA), a class I and II HDAC inhibitor, suppressed the proliferation of RPE cells by G1 phase cell cycle arrest through inhibition of cyclin/CDK/p-Rb and induction of p21 and p27. In the meantime, TSA strongly prevented TGF-ß2-induced morphological changes and the up-regulation of α-SMA, collagen type I, collagen type IV, fibronectin, Snail and Slug. We also demonstrated that TSA affected not only the canonical Smad signalling pathway but also the non-canonical TGF-ß/Akt, MAPK and ERK1/2 pathways. Finally, we found that the underlying mechanism of TSA affects EMT in RPE cells also through down-regulating the Jagged/Notch signalling pathway. Therefore, this study may provide a new insight into the pathogenesis of PVR, and suggests that epigenetic treatment with HDAC inhibitors may have therapeutic value in the prevention and treatment of PVR.

Dietary transforming growth factor-beta 2 (TGF-ß2) supplementation reduces methotrexate-induced intestinal mucosal injury in a rat.

BACKGROUND/AIMS: Dietary supplementation with transforming growth factor-beta (TGF-ß) has been proven to minimize intestinal damage and facilitate regeneration after mucosal injury. In the present study, we evaluated the effects of oral TGF-ß2 supplementation on intestinal structural changes, enterocyte proliferation and apoptosis following methotrexate (MTX)-induced intestinal damage in a rat and in a cell culture model. METHODS: Caco-2 cells were treated with MTX and were incubated with increasing concentrations of TGF-ß2. Cell apoptosis was assessed using FACS analysis by annexin staining and cell viability was monitored using Trypan Blue assay. Male rats were divided into four experimental groups: Control rats, CONTR- TGF-ß rats were treated with diet enriched with TGF-ß2, MTX rats were treated with a single dose of methotrexate, and MTX- TGF-ß rats were treated with diet enriched with TGF-ß2. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined at sacrifice. Real Time PCR and Western blot were used to determine bax and bcl-2 mRNA, p-ERK, ß-catenin, IL-1B and bax protein expression. RESULTS: Treatment of MTX-pretreated Caco-2 cells with TGF-B2 resulted in increased cell viability and decreased cell apoptosis. Treatment of MTX-rats with TGF-ß2 resulted in a significant increase in bowel and mucosal weight, DNA and protein content, villus-height (ileum), crypt-depth (jejunum), decreased intestinal-injury score, decreased level of apoptosis and increased cell proliferation in jejunum and ileum compared to the untreated MTX group. MTX-TGF-ß2 rats demonstrated a lower bax mRNA and protein levels as well as increased bcl-2 mRNA levels in jejunum and ileum compared to MTX group. Treatment with TGF-ß2 also led to increased pERK, IL-1B and ß-catenin protein levels in intestinal mucosa. CONCLUSIONS: Treatment with TGF-ß2 prevents mucosal-injury, enhances p-ERK and ß-catenin induced enterocyte proliferation, inhibits enterocyte apoptosis and improves intestinal recovery following MTX-induced intestinal-mucositis in rats.

Repair of osteochondral defects with adipose stem cells and a dual growth factor-releasing scaffold in rabbits.

The purpose of this work was to evaluate the in vivo effectiveness of a TGF-beta(2) and bone morphogenetic protein (BMP)-7-immobilized porous polycaprolactone (PCL)/F127 scaffold to enhance the healing of cartilage defect. An osteochondral defect was created on the patellar groove of the right distal femur of 12 rabbits and managed by one of the following methods: filling it with the scaffold only (Group I); the scaffold seeded with adipose stem cells (ASCs) (Group II); a TGF-beta(2) and BMP-7-immobilized scaffold (Group III); and a TGF-beta(2) and BMP-7-immobilized scaffold seeded with ASCs (Group IV). Each group had three rabbits. Nine weeks after the implantation, the implanted scaffolds were filled with yellowish, dense tissue, and had distinct margins with adjacent normal cartilage. The histological findings showed infiltration of foreign-body giant cells and blood vessel, more prominently in Groups III and IV. The presence of growth factor significantly increased the ICRS Macroscopic Score (p = 0.045) while the presence of ASC did not. The ICRS Visual Histological Score was not significantly affected by the presence of either growth factors or ASCs, showing similar values in all groups. In conclusion, the use of TGF-beta(2) and BMP-7-immobilized PCL/F127 scaffolds improved gross appearances of the osteochondral defects while not actually leading to better histological results and induced a greater degree of foreign body reaction.

Dynamic alteration of low-density lipoprotein receptor after exposure to transforming growth factor-beta2 in human Tenon's capsule fibroblasts.

PURPOSE: The present study investigated dynamic alteration of low-density lipoprotein receptor and its binding and uptake of low-density lipoprotein (LDL) after exposure to transforming growth factor-beta(2) (TGF-beta(2)) in human Tenon's capsule fibroblasts. METHODS: Tenon's capsule fibroblasts obtained from elective cataract surgery patients were cultured and stimulated with different concentrations (0.1-10 ng/mL) of TGF-beta(2) for 24, 48, and 72 h. The LDLr mRNA and protein levels were analyzed by relative quantification real-time RT-PCR and Western blot analysis, respectively. The binding and uptake of DiO (3,3'-dioctadecyloxacarbocyanine)-labeled LDL was assessed by confocal microscopy. RESULTS: Real-time RT-PCR and Western blot analyses showed similar results revealing that after exposure to TGF-beta(2), the expression of protein and mRNA of LDLr occurred in a concentration-dependent and time-dependent manner with a peak at a concentration of 1.0 ng/mL at 72 h in Tenon's capsule fibroblasts. Confocal microscopy showed that DiO-LDL binding and uptake were time-dependent, reaching saturation at approximately 6 h. CONCLUSIONS: This study shows that LDLrs were overexpressed in the activated Tenon's capsule fibroblasts in a concentration-dependent and time-dependent manner after exposure to TGF-beta(2). The results suggest that LDLr in the activated Tenon's capsule fibroblasts may become a novel focus as a target receptor for controlled drug delivery, particularly in anti-scarring therapy during excessive conjunctival wound healing.

Chondrogenic differentiation of adipose tissue-derived mesenchymal stem cells: greater doses of growth factor are necessary.

There have been controversies regarding the chondrogenic potential of adipose tissue-derived mesenchymal stem cells (ATMSCs) compared with bone marrow-derived mesenchymal stem cells (BMMSCs). The purpose of this study was to confirm the hypothesis that chondrogenesis can be achieved from ATMSCs comparable to that from BMMSCs by using greater dose of currently known chondrogenic growth factors. Chondrogenesis was induced from ATMSCs by culturing them in pellets under the following conditions: #1 without growth factors (negative control); #2 5 ng/mL of TGF-beta2; #3 5 ng/mL of TGF-beta2 and 100 ng/mL of IGF-I; #4 15 ng/mL of TGF-beta2; #5 15 ng/mL of TGF-beta2 and 300 ng/mL of IGF-I; #6 25 ng/mL of TGF-beta2; #7 25 ng/mL of TGF-beta2 and 500 ng/mL of IGF-I. After 4 weeks of in vitro culture, the pellets were harvested for DNA quantification, analysis of the glycosaminoglycan content, reverse transcription, and real-time PCR for collagen type I (COL1A1), collagen type II (COL2A1), and Sox-9. Safranin-O and immunohistochemical staining for type II collagen also were carried out, and histological grading was performed based on the findings. A combination of 25 ng/mL TGF-beta2 and 500 ng/mL IGF-I produced results comparable to the positive control (BMMSCs treated with 5 ng/mL TGF-beta2) as demonstrated by DNA amount, GAG analysis, real-time PCR, and histological findings. Although ATMSCs have lower chondrogenic potentials than BMMSCs, chondrogenesis comparable to BMMSCs can be induced from ATMSCs using a greater dose combination of growth factors. These results lend a further support to the application of ATMSCs for cartilage tissue engineering.

Enteral nutrition in adult Crohn's disease: present status and perspectives.

Enteral nutrition has long been a therapeutic alternative often used in adult Crohn's disease patients to obtain remission or clinical response, especially in those not responding to conventional therapy such as corticosteroids. However, the increasing use of immunosuppressors (6-mercaptopurine and azathioprine, methotrexate, etc.), and the advent of biotherapies (especially anti-tumor necrosis factor-alpha (TNF-alpha) antibodies), decreased its use in adult Crohn's disease. Nevertheless, enteral nutrition remains of interest in patients presenting concomitant malnutrition (in particular in nonobstructed patients needing surgery), or in those intolerant or who failed to other therapeutics. In addition, recent studies provide data indicating its potential interest in maintenance therapy in selected patients groups. Finally, future research (in particular in the field of immuno- or pharmaconutrition) could lead to enteral formula's improvement, with better tolerance and acceptability, as well as increased efficacy.

Reactive oxygen species mediates the apoptosis induced by transforming growth factor beta(2) in human lens epithelial cells.

Transforming growth factor beta(2) (TGF-beta(2)), a growth regulator of human lens epithelial cells (HLECs), also regulates the death of these cells. Dose-response analysis showed that the TGF-beta(2) concentration needed to induce HLECs death (100 pg/ml) was 10 times that needed to inhibit growth in these cells (10 pg/ml). TGF-beta(2)-induced apoptosis in HLECs was preceded by an induction of reactive oxygen species (ROS) and a decrease in glutathione in the intracellular content, indicating that this factor induces oxidative stress in HLECs. Studies performed to analyze the levels of c-fos mRNA, a gene whose expression is modulated by the redox state, demonstrated that only high, apoptotic concentrations of TGF-beta(2) (100 pg/ml) produced an increase in the mRNA levels of this gene, the level of induction being similar to that found when cells were incubated in the presence of hydrogen peroxide. Finally, the cell death induced by TGF-beta(2) in HLECs was partially blocked by radical scavengers, which decreased the percentage of apoptotic cells, whereas these agents did not modify the growth-inhibitory effect elicited by TGF-beta(2) in these cells. The results presented in this paper provide evidence for the involvement of an oxidative process in the apoptosis elicited by TGF-beta(2) in HLECs.

Protection against chemotherapy induced mucositis by TGF-beta(2) in childhood cancer patients: results from a randomized cross-over study.

BACKGROUND: Mucositis is one of the most frequent and severe side-effect of chemotherapy in childhood-cancer patients for which there is no prophylaxis available. The efficacy and feasibility of a TGF-beta(2)-enriched feeding for preventing oral and gastro-intestinal-mucositis in childhood-cancer patients were studied. PROCEDURE: The study was designed as a two-period cross-over, randomized, double-blinded, placebo, controlled trial. Patients who had a high risk for developing mucositis and who would receive two comparable cycles of chemotherapy were eligible for the study. During one cycle of chemotherapy, TGF-beta(2)-enriched feeding was administered; during the other, a "placebo" (not enriched) feeding was used. WHO toxicity scales of diarrhea, oral mucositis, fever, anal lesions and nausea/vomiting were scored daily. In addition, the incidence of occurrence of blood cultures, antibiotic therapy, and interventions or diagnostics related to mucositis were measured. RESULTS: The feasibility of the study was good: 83% of the patients completed two cycles and 86% of the study-feeding was effectively consumed. Administration of TGF-beta(2) was safe as serum TGF-beta(2) did not increase, and renal and liver function were not affected during TGF-beta(2) consumption compared to normal feeding. Differences in toxicity, scored during the whole observation period and the number of days with WHO 3/4 toxicity, were not significantly different between cycles with TGF-beta(2) enriched and normal feeding. CONCLUSIONS: TGF-beta(2) administration via feeding is well tolerated and safe. Although this study might have had limitations to show potential benefit of TGF-beta(2), it does not provide evidence that TGF-beta(2) decreases the incidence or degree of mucositis induced by combination chemotherapy in childhood-cancer patients.