Azadeh Hosseini-Tabatabaei

Fibroblast populated collagen matrix promotes islet survival and reduces the number of islets required for diabetes reversal

Islet transplantation represents a viable treatment for type 1 diabetes. However, due to loss of substantial mass of islets early after transplantation, islets from two or more donors are required to achieve insulin independence. Islet‐extracellular matrix disengagement, which occurs during islet isolation process, leads to subsequent islet cell apoptosis and is an important contributing factor to early islet loss. In this study, we developed a fibroblast populated collagen matrix (FPCM) as a novel scaffold to improve islet cell viability and function post‐transplantation. FPCM was developed by embedding fibroblasts within type‐I collagen and used as scaffold for islet grafts. Viability and insulin secretory function of islets embedded within FPCM was evaluated in vitro and in a syngeneic murine islet transplantation model. Islets embedded within acellular matrix or naked islets were used as control. Islet cell survival and function was markedly improved particularly after embedding within FPCM. The composite scaffold significantly promoted islet isograft survival and reduced the critical islet mass required for diabetes reversal by half (from 200 to 100 islets per recipient). Fibroblast embedded within FPCM produced fibronectin and growth factors and induced islet cell proliferation. No evidence of fibroblast over‐growth within composite grafts was noticed. These results confirm that FPCM significantly promotes islet viability and functionality, enhances engraftment of islet grafts and decreases the critical islet mass needed to reverse hyperglycemia. This promising finding offers a new approach to reducing the number of islet donors per recipient and improving islet transplant outcome. J. Cell. Physiol. 226: 1813–1819, 2011.

On the benefits of silymarin in murine colitis by improving balance of destructive cytokines and reduction of toxic stress in the bowel cells

Inflammatory bowel disease (IBD) is a multifactorial disease with an unknown etiology characterized by oxidative stress, leucocyte infiltration and a rise in inflammatory cytokines. In this study, we have investigated the effects of silymarin, a mixture of several flavonolignans with established antioxidant and anti-inflammatory properties, on trinitrobenzene sulphonic acid (TNBS)-induced colitis in rats. Experimental colitis was induced in male Wistar-albino rats by delivering TNBS to the distal colon. All the medicines were administered by gavage for seven days. Thirty-six male rats were divided into six groups containing six rats in each one. Control rats received only TNBS. In the treated groups, animals were given different doses of silymarin (40, 80, and 160 mg/kg). Dexamethasone (1 mg/kg) was used as the positive treatment. Colonic status was investigated seven days post induction of colitis through macroscopic, histological, and biochemical analyses. Amelioration of the morphological signs including macroscopic damage, necrotic area, and histology were seen subsequent to treating animals with silymarin. These observations were accompanied by a significant reduction in the degree of both neutrophil infiltration, indicated by decreased myeloperoxidase activity, and lipid peroxidation, as measured by a decline in malodialdehyde content in inflamed colon as well as a decrease in levels of inflammatory cytokines (TNF-α and IL-1β). The results of the present study reveal that the beneficial effect of silymarin in bowel cells is mediated through its anti-oxidant and anti-inflammatory potentials.

Potassium Channel Openers and Improvement of Toxic Stress: Do they have Role in the Management of Inflammatory Bowel Disease?

Inflammatory bowel disease (IBD) is a progressive condition in gastrointestinal tract, which refers to two idiopathic diseases; ulcerative colitis and Crohns disease. Although certain etiology of these conditions is not known, it seems that an abnormality in reaction and regulation of the immune system plays an important role in adventure of the disease. According to the investigations, it is likely that oxidative and nitrosative stress have etiologic roles in IBD. Their destructive effects may contribute to the initiation or progression of the disease. Nowadays, the effectiveness of different medicines in the treatment of IBD has been proved, but none of them has shown a desirable result. Potassium channel openers (PCOs) are a class of drugs with various usages in the aspects of cardiovascular diseases and urinary incontinence. Their major mechanism is the opening of ATP-sensitive potassium (K-ATP) channels and contribute to the relaxation of smooth muscles. Nicorandil is a member of PCOs, with a special chemical structure. Recent investigations mention some novel effects and functions for this drug. Nicorandil reveals an anti-apoptosis property not only via a nitric oxide (NO)/cGMP-dependent mechanism, but also through activating mitochondrial K-ATP channels. Nicorandil can also elevate cGMP levels in some tissues, without direct NO generation. Gastroprotective activity via opening of the K channels, free radical scavenging, prostaglandin E2 elevation, decreasing pepsin and acid secretion, and prevention of the detrimental rise in NO has been proposed for nicorandil. According to these protective mechanisms and the role of oxidative/nitrosative stress in the expression of IBD, we herein hypothesize that nicorandil and other PCOs with similar structure can be used in the management of IBD. This approach offers new hope for the successful treatment of IBD. Further investigations on animal models are needed, to place nicorandil and similar drugs alongside IBD therapy.

Benefit of nicorandil using an immunologic murine model of experimental colitis

Inflammatory bowel disease (IBD) is a chronic inflammatory condition with an unknown etiology. Nicorandil, a potassium channel opener, has been used for many years for the treatment of angina. Recently, it has been shown that nicorandil possesses some novel traits such as anti-apoptotic, gastroprotective, free radical scavenging, and anti-inflammatory properties. Therefore, we set out to examine the possible beneficial effect of nicorandil in a rat model of IBD. Colitis was induced by rectal administration of 2,4,6-trintrobenzene sulphonic acid (TNBS) into rats. Groups of animals used in this study were sham, control, and exposure to dexamethasone, nicorandil, glibenclamid (a pure adenosine triphosphate sensitive potassium channel (KATP) blocker), or nicorandil plus glibenclamid. Drugs were administered by gavage and animals were sacrificed after 7 days. Biochemical markers, including TNF-α and IL-1β, ferric reducing/antioxidant power (FRAP), myeloperoxidase (MPO) activity and thiobarbitoric acid-reactive substance (TBARS), were measured in the homogenate of colonic tissue. Results indicate that nicorandil significantly reduces macroscopic and histological damage induced by TNBS. Nicorandil diminishes MPO activity and levels of TBARS, TNF-∢, and IL-1β in damaged colonic tissue with a concomitant increase in FRAP value (P<0.01). These effects were not reversed by coadministration of glibenclamide. In conclusion, nicorandil is able to ameliorate experimental IBD with a dose in which it does not show any anti-hypertensive effect, and the mechanism of which is partially or totally independent from KATP channels. It is hypothesized that nitric oxide donation and free-radical scavenging properties of nicorandil upregulate endothelial nitric oxide synthase may be responsible for this phenomenon. These findings suggest that nicorandil can be useful in treatment of IBD, although further investigations are needed to elucidate the mechanisms involved.

Improvement of inflammatory and toxic stress biomarkers by silymarin in a murine model of type one diabetes mellitus

Type 1 diabetes mellitus (T1DM) is characterized by an impairment of the insulin-secreting beta cells with an immunologic base. Inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and free radicals are believed to play key roles in destruction of pancreatic β cells. The present study was designed to investigate the effect of Silybum marianum seed extract (silymarin), a combination of several flavonolignans with immunomodulatory, anti-oxidant, and anti-inflammatory potential on streptozotocin (STZ)-induced T1DM in mouse. Experimental T1DM was induced in male albino mice by IV injection of multiplelow- doses of STZ for 5 days. Seventy-two male mice in separate groups received various doses of silymarin (20, 40, and 80 mg/kg) concomitant or after induction of diabetes for 21 days. Blood glucose and pancreatic biomarkers of inflammation and toxic stress (IL-1β, TNF-α, myeloperoxidase, lipid peroxidation, protein oxidation, thiol molecules, and total antioxidant capacity) were determined. Silymarin treatment reduced levels of inflammatory cytokines such as TNF-α and IL-1β and oxidative stress mediators like myeloperoxidase activity, lipid peroxidation, carbonyl and thiol content of pancreatic tissue in an almost dose dependent manner. No marked difference between the prevention of T1DM and the reversion of this disease by silymarin was found. Use of silymarin seems to be helpful in T1DM when used as pretreatment or treatment. Benefit of silymarin in human T1DM remains to be elucidated by clinical trials.

Immuno-regulatory function of indoleamine 2,3 dioxygenase through modulation of innate immune responses.

Successful long-term treatment of type-1 diabetes mainly relies on replacement of β-cells via islet transplantation. Donor shortage is one of the main obstacles preventing transplantation from becoming the treatment of choice. Although animal organs could be an alternative source for transplantation, common immunosuppressive treatments demonstrate low efficacy in preventing xenorejection. Immunoprotective effects of indoleamine 2,3-dioxygenase (IDO) on T-cell mediated allorejection has been extensively studied. Our studies revealed that IDO expression by fibroblasts, induced apoptosis in T-cells while not affecting non-immune cell survival/function. Since macrophages play a pivotal role in xenograft rejection, herein we investigated the effect of IDO-induced tryptophan deficiency/kynurenine accumulation on macrophage function/survival. Moreover, we evaluated the local immunosuppressive effect of IDO on islet-xenograft protection. Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction. Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression. To determine whether IDO-induced tryptophan starvation or kynurenine accumulation is responsible for macrophage apoptosis and inhibition of their proinflammatory activity, Raw264.7 cell viability and proinflammatory responses were evaluated in tryptophan deficient medium or in the presence of kynurenine. Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity. Next a three-dimensional islet-xenograft was engineered by embedding rat islets within either control or IDO–expressing fibroblast-populated collagen matrix. Islets morphology and immune cell infiltration were then studied in the xenografts transplanted into the C57BL/6 mouse renal sub-capsular space. Local IDO significantly decreased the number of infiltrating macrophages (11±1.47 vs. 70.5±7.57 cells/HPF), T-cells (8.75±1.03 vs. 75.75±5.72 cells/HPF) and iNOS expression in IDO-expressing xenografts versus controls. Islet morphology remained intact in IDO-expressing grafts and islets were strongly stained for insulin/glucagon compared to control. These findings support the immunosuppressive role of IDO on macrophage-mediated xeno-rejection.

Extracellular 14-3-3 from human lung epithelial cells enhances MMP-1 expression.

Airway remodelling in asthma involves various mediators modulating the production/breakdown of collagen by lung fibroblasts. Matrix metalloproteinase-1 (MMP-1) plays an important role in collagen breakdown. We recently showed that epithelial cell-derived extracellular form of 14-3-3σ is an important inducer of MMP-1 expression in skin fibroblasts. Thus, we hypothesized that 14-3-3 proteins are important regulators of MMP-1 expression in the respiratory airway. We examined the presence of extracellular 14-3-3 proteins in conditioned media obtained from primary lung epithelial cells, A549 and HS24 cells, and their effect on MMP-1 expression by lung fibroblasts (IMR-90). In addition, we evaluated IMR-90 response to 14-3-3 proteins in the presence of transforming growth factor-β1 (TGF-β1), a cytokine known to decrease MMP-1 expression by fibroblasts. Extracellular 14-3-3α/β, but not -σ, is released by the human-derived lung epithelial cell lines, A549 and HS24. Unlike dermal fibroblasts, IMR-90 cells do not produce MMP-1 in response to 14-3-3σ. Conversely, MMP-1 production was induced following treatment of IMR-90 with recombinant or lung epithelial cell-derived 14-3-3α/β. These findings were also confirmed using primary human bronchial epithelial cells and lung fibroblasts obtained from non-asthmatic patients. The MMP-1-inducing effect of 14-3-3α/β on IMR-90 was not inhibited by TGF-β1. Lung epithelial cell-derived 14-3-3α/β has a potent MMP-1-inducing effect on airway fibroblasts. Modulation of MMP-1 by 14-3-3α/β, may be important in the alteration of collagenase production associated with airway remodelling in obstructive lung diseases. Our data indicate that 14-3-3 proteins may be potential targets for future therapeutic strategies aimed at modulating tissue remodelling in asthma.

Mechanism Underlying Defective Interferon Gamma-Induced IDO Expression in Non-obese Diabetic Mouse Fibroblasts

Indoleamine 2,3-dioxygenase (IDO) can locally suppress T cell-mediated immune responses. It has been shown that defective self-tolerance in early prediabetic female non-obese diabetic (NOD) mice can be attributed to the impaired interferon-gamma (IFN-γ)- induced IDO expression in dendritic cells of these animals. As IFN-γ can induce IDO in both dendritic cells and fibroblasts, we asked the question of whether there exists a similar defect in IFN-γ-induced IDO expression in NOD mice dermal fibroblasts. To this end, we examined the effect of IFN-γ on expression of IDO and its enzymatic activity in NOD dermal fibroblasts. The results showed that fibroblasts from either prediabetic (8 wks of age) female or male, and diabetic female or male (12 and 24 wks of age respectively) NOD mice failed to express IDO in response to IFN-γ treatment. To find underlying mechanisms, we scrutinized the IFN- γ signaling pathway and investigated expression of other IFN-γ-modulated factors including major histocompatibility complex class I (MHC-I) and type I collagen (COL-I). The findings revealed a defect of signal transducer and activator of transcription 1 (STAT1) phosphorylation in NOD cells relative to that of controls. Furthermore, we found an increase in MHC-I and suppression of COL-I expression in fibroblasts from both NOD and control mice following IFN-γ treatment; indicating that the impaired response to IFN-γ in NOD fibroblasts is specific to IDO gene. Finally, we showed that an IFN-γ-independent IDO expression pathway i.e. lipopolysaccharide (LPS)-mediated-c-Jun kinase is operative in NOD mice fibroblast. In conclusion, the findings of this study for the first time indicate that IFN-γ fails to induce IDO expression in NOD dermal fibroblasts; this may partially be due to defective STAT1 phosphorylation in IFN-γ-induced-IDO signaling pathway.

Immunoprotection and Functional Improvement of Allogeneic Islets in Diabetic Mice, Using a Stable Indoleamine 2,3-Dioxygenase Producing Scaffold.

Background We have previously shown that an immunomodulatory enzyme, indoleamine 2,3-dioxygenase (IDO) in dermal fibroblasts generates a tryptophan-deficient environment that selectively inhibits proliferation and induces apoptosis of bystander CD4+ and CD8+ T cells, but not pancreatic islets. Because these immune cells are involved in islet allograft rejection, we hypothesized that transplantation of islets embedded in a novel 3-dimensional composite scaffold within which stable IDO-expressing fibroblasts serve as source of local immunosuppression would lead to normoglycemia in a streptozotocin-induced diabetic mouse model. Methods Islet grafts were prepared by embedding stable IDO-expressing fibroblasts and allogeneic islets into a protease-resistant composite scaffold. Islets function and survival were evaluated in vitro using immunohistochemistry. Allografts were transplanted under the kidney capsule of streptozotocin-induced diabetic mice; viability, function, and criteria for graft take were evaluated. Flow cytometry was performed to determine specific intragraft, draining lymph nodes and spleen T-cell population, and splenocytes alloantigen responsiveness of graft recipients. Results The results of a series of in vitro experiments revealed that IDO-expressing fibroblasts do not compromise islet function or survival. The expression of IDO suppressed the proliferation of alloantigen-stimulated splenocytes. The in vivo experiments revealed that local IDO expression delivered by lentiviral vector prolonged islet allograft survival (51.0 ± 2.9 days) by increasing the population of FOXP3+ regulatory T cells at the graft site and graft-draining lymph nodes and preventing T-cell infiltration. Conclusions This study shows that incorporation of islets within our novel matrix that is equipped with stable IDO-expressing fibroblasts prolongs allograft survival.

Topical application of aminopeptidase N-neutralizing antibody accelerates wound closure

Upon release from keratinocytes, 14-3-3 sigma (also known as stratifin) acts on the dermal fibroblast and modulates its production of extracellular matrix proteins. Subsequent to the recent identification as a receptor responsible for stratifin-mediated matrix turnover in dermal fibroblasts, aminopeptidase N has been implicated in the regulation of epidermal–dermal communication and expression of key matrix proteases and adhesion molecules. In light of the growing importance of aminopeptidase N in modulation of the fibroblast phenotype, the present study evaluates the potential of targeting the ectoenzyme in cutaneous repair, and demonstrates that neutralization of aminopeptidase N led to acceleration of wound closure. This was attributed to at least in part an increase of collagen deposition and fibroblast contractility in the granulation tissue. These findings confirmed the important role of aminopeptidase N in post-injury tissue remodeling and wound contraction.