Ashwani Khanna

In vivo expression of transforming growth factor-beta1 in humans: stimulation by cyclosporine.

BACKGROUND Transforming growth factor-beta1 (TGF-beta1) is an immunoregulatory and fibrogenic cytokine. In an earlier in vitro study, we demonstrated that cyclosporine (CsA) increases TGF-beta1 transcription rate in human T lymphocytes. Herein, we explored whether CsA augments the in vivo expression of TGF-beta1 in humans. METHODS The inherent difficulty in studying the in vivo effect of CsA in humans was circumvented by investigating stable end-stage renal disease patients who were preconditioned with CsA before their living donor renal transplantation. Sera and peripheral blood mononuclear cells were obtained from CsA-preconditioned patients and quantified for TGF-beta1 expression at the mRNA (by competitive polymerase chain reaction) and protein (sandwich enzyme-linked immunosorbent assay) levels. RESULTS Our studies demonstrated a significant increase in TGF-beta1 expression after CsA therapy. The stimulatory effect was unique to TGF-beta1, and CsA did not increase interleukin (IL)-10, IL-6, IL-2, or tumor necrosis factor-alpha expression. CONCLUSIONS Our first-time demonstration of a TGF-beta1-selective in vivo stimulatory effect of CsA in humans: (1) advances a TGF-beta1-centered hypothesis for the beneficial (immunosuppression) and detrimental (fibrosis, hypertension) effects of CsA use, and (2) broadens the mechanism of immunosuppressive action of CsA to include heightened expression of an endogenous immunosuppressive cytokine.Background.Transforming growth factor-β1(TGF-β1) is an immunoregulatory and fibrogenic cytokine. In an earlier in vitro study, we demonstrated that cyclosporine (CsA) increases TGF-β1transcription rate in human T lymphocytes. Herein, we explored whether CsA augments the in vivo expression of TGF-β1i

TGF-β1 DNA Polymorphisms, Protein Levels, and Blood Pressure

Transforming growth factor-beta1 (TGF-beta1), a multifunctional cytokine with fibrogenic properties, has been implicated in the pathogenesis of the vascular and target organ complications of hypertension. TGF-beta1 may also regulate blood pressure via stimulation of endothelin-1 and/or renin secretion. Herein we explored the hypothesis that circulating levels of TGF-beta1 protein (quantified using a TGF-beta1-specific sandwich ELISA) are correlates of blood pressure levels. This hypothesis was tested in 98 stable end-stage renal disease (ESRD) patients. (The use of ESRD patients as the study cohort eliminates renal function-dependent alterations in circulating levels of TGF-beta1 protein.) In addition, in view of the previously reported correlation among TGF-beta1 DNA polymorphisms and systolic blood pressure, TGF-beta1 codon 25 genotype and alleles were identified in 71 hypertensive subjects and 57 normotensives using amplification refractory mutation system polymerase chain reaction. Our studies demonstrate for the first time that TGF-beta1 levels (209+/-13 ng/mL, mean+/-SEM) are positive correlates (Pearson correlation analysis) of mean arterial pressure (P=0.008), systolic pressure (P=0.02), and diastolic pressure (P=0. 01). We also report that a higher percentage of hypertensives (92%) compared with normotensives (86%) are homozygous for the arginine allele at codon 25. Our observations support the idea that genetically determined TGF-beta1 protein concentrations may play a role in blood pressure regulation in humans.

Regulation of new DNA synthesis in mammalian cells by cyclosporine : demonstration of a transforming growth factor β-dependent mechanism of inhibition of cell growth

Immunosuppressants such as cyclosporine are considered to constrain cell growth by preventing the production of growth stimulatory cytokines (e.g., interleukin-2). The possibility exists, however, that CsA and other immunosuppressants might restrain cell growth by promoting the production of growth-inhibitory cytokines. We have explored herein the hypothesis that CsA stimulates the production of transforming growth factor-beta (TGF-beta), and restrains new DNA synthesis in mammalian cells via a TGF-beta-dependent mechanism. To investigate this new postulate independently of an IL-2-dependent mechanism, we utilized, as probes, two mammalian cell lines, distinguished by their sensitivity to growth inhibition by TGF-beta and resistance to IL-2: CCL-64 mink lung epithelial cells (CCL-64 cells) and A-549 human adenocarcinoma cells (A-549 cells). Our experimental approach revealed the following: (A) CsA and not cyclosporine H, an inactive analogue of CsA, mediates growth inhibition of TGF-beta-sensitive cells, CCL-64 cells, and A-549 cells; (B) CsA stimulates these mammalian cells to secrete TGF-beta; and (C) TGF-beta induced by CsA is biologically active in inducing cell growth inhibition (demonstrated by the reversal of CsA-associated inhibition with anti-TGF-beta monoclonal antibodies). Our observations suggest that CsA can regulate cell growth via a TGF-beta-dependent mechanism. Since the multifunctional cytokine TGF-beta can enhance extracellular matrix accumulation as well as augment endothelin production, our findings also advance a mechanism that links, via TGF-beta, the beneficial (immunosuppression) and the harmful (fibrosis, hypertension) consequences of CsA usage.

In vivo hyperexpression of transforming growth factor-beta1 in mice: stimulation by cyclosporine.

BACKGROUND We have demonstrated that cyclosporine (CsA) stimulates transforming growth factor (TGF) beta1 expression in vitro and that growth of mammalian cells can be arrested by CsA via a TGF-beta1-dependent mechanism. Herein, we have explored whether CsA stimulates TGF-beta1 hyperexpression in vivo. METHODS Four groups of B6AF1 mice were studied: group 1, control; group 2, CsA pretreatment; group 3, anti-CD3 monoclonal antibody pretreatment; and group 4, CsA plus anti-CD3 pretreatment. RESULTS CsA pretreatment augmented TGF-beta1 protein expression and increased intrarenal display of TGF-beta1 mRNA. This heightened TGF-beta1 expression was associated with an impaired T cell proliferative response. CONCLUSIONS Our observations, together, advance the hypothesis that CsA might function in vivo as an immunosuppressant not only by inhibiting the expression of proinflammatory cytokines (e.g., interleukin 2), but also by stimulating the expression of TGF-beta1, a potent immunosuppressive cytokine. Moreover, prevention of TGF-beta1 hyperexpression might prevent CsA-associated renal fibrosis, as TGF-beta1 is a fibrogenic cytokine.

Transforming growth factor (TGF)-β mimics and anti-TGF-β antibody abrogates the in vivo effects of cyclosporine : Demonstration of a direct role of TGF-β in immunosuppression and nephrotoxicity of cyclosporine

BACKGROUND Cyclosporine (CsA) has been shown to induce the expression of transforming growth factor (TGF)-beta both in vitro and in vivo. It is hypothesized that the efficacy as well as the side effects of CsA are mediated by TGF-beta. This study was planned to investigate whether anti-TGF-beta mitigated and TGF-beta reproduced the in vivo effects of CsA to directly prove this hypothesis. METHODS B6AF1 (H2b/k.d) mice were divided into groups and received the following: CsA, vehicle (olive oil), CsA + anti-TGF-beta1 antibody, TGF-beta1, or vehicle phosphate-buffered saline/bovine serum albumin. All studies were carried out at 10 and 28 days after the last day of CsA administration with the exception of the exogenous TGF-beta experiments, which were performed 5 days after exogenous TGF-beta administration. The efficacy was studied by the anti-CD3-induced ex vivo proliferation of splenocytes measured by [3H]thymidine uptake; TGF-beta protein levels were quantified by ELISA. TGF-beta, collagen, and fibronectin gene expression was studied using reverse transcriptase-polymerase chain reaction, and histopathological analysis was made on periodic acid-Schiff- and trichrome C-stained thin kidney sections. RESULTS CsA treatment resulted in decreased ex vivo proliferation of splenocytes, an increase in TGF-beta protein in the sera, and renal histopathological changes including tubular swelling, vacuolization, thrombotic microangiopathy, and increased expression of TGF-beta, collagen and fibronectin genes. All of these findings were blocked by anti-TGF-beta antibody. CONCLUSION The study demonstrates the in vivo modulation of the effects of CsA by manipulating TGF-beta levels and suggests that TGF-beta at least in part mediates CsAs beneficial and detrimental effects.

Tacrolimus induces increased expression of transforming growth factor-beta1 in mammalian lymphoid as well as nonlymphoid cells.

BACKGROUND We and others have reported that cyclosporine (CsA) induces increased expression of transforming growth factor-beta1 (TGF-beta1) in vitro as well as in vivo. In view of similarities between tacrolimus and CsA with respect to immunosuppressive mechanisms, we determined whether tacrolimus, in a fashion similar to CsA, induces TGF-beta1 hyperexpression in mammalian cells. METHODS We studied the induction of TGF-beta1 mRNA by tacrolimus using reverse transcription-polymerase chain reaction and Northern blot analysis in normal human T cells and A-549 cells (human lung adenocarcinoma cell line), a cell line used to study the biology of TGF-beta and the induction of TGF-beta1 by CsA. We also measured the induction of TGF-beta1 protein by tacrolimus in activated human T cells, peripheral blood mononuclear cells, and A-549 cells, using sandwich enzyme-linked immunosorbent assay. RESULTS A significant increase in the TGF-beta1 mRNA expression was observed after treatment of T cells or A-549 cells. Tacrolimus treatment resulted also in heightened production of TGF-beta1 protein by activated T cells, A-549 cells, or peripheral blood mononuclear cells activated with anti-CD3, phytohemagglutinin, and concanavalin A. CONCLUSIONS Our observations that tacrolimus stimulates TGF-beta1 hyperexpression in mammalian cells suggest a unifying mechanism for the immunosuppressive as well as nephrotoxic properties of tacrolimus, as the multifunctional TGF-beta1 is a potent immunosuppressive and fibrogenic cytokine.

Mechanism of the combination immunosuppressive effects of rapamycin with either cyclosporine or tacrolimus

Background. The combination of rapamycin with Cyclosporine has demonstrated beneficial effects in organ transplantation; however, the mechanism of this combination immunosuppression is not fully understood.The mechanism of action of cyclosporine and tacrolimus has been explained on the basis of their inhibition of interleukin-2 (IL-2), and induction of transforming growth factor-&bgr; (TGF-&bgr;). In this study, we explored the effects of rapamycin in combination with either tacrolimus or cyclosporine on lymphocyte proliferation, and expression of interleulin-2 (IL-2) and TGF-&bgr; Methods. Peripheral blood mononuclear cells/lymphocytes were isolated from buffy coats obtained from blood center, and activated with phytohemagglutinin (PHA) in the presence or absence of rapamycin, cyclosporine, and tacrolimus alone or in various combinations. The activation was quantified by 3H-thymidine uptake assay and using reverse transcriptase assisted polymerase chain reaction and ELISA, we studied the TGF-&bgr; and IL-2 mRNA and protein expression, respectively. Results. In this study, we report that rapamycin in combination with either tacrolimus or cyclosporine significantly inhibited the lymphocyte proliferation, IL-2 expression, and induced TGF-&bgr;, compared with these drugs alone. The levels of TGF-&bgr; and IL-2 correlated positively and negatively, respectively, with inhibition of lymphocyte proliferation. Conclusions. These novel findings provide the mechanism as well as the rationale to use these drugs in combination at subclinical dosages in organ transplantation.

Inflammation and oxidative stress induced by cigarette smoke in Lewis rat brains

Exposure to cigarette smoke has been associated with an increased risk of neurological diseases such as stroke, Alzheimers disease and multiple sclerosis. In these studies, serum and brain sections from Lewis rats or those exposed to cigarette smoke and control rats were examined for evidence of increased inflammation and oxidative stress. Immunocytochemical staining of brain sections from CS-exposed rats showed increased expression of class II MHC and, in ELISA, levels of IFN-gamma and TNF-α were higher than for non-exposed rats. In polymerase chain reaction assays there was increased interferon-gamma, TNF-α, IL-1α, IL-1β, IL-23, IL-6, IL-23, IL-17, IL-10, TGF-β, T-bet and FoxP3 gene expression with CS exposure. There was also markedly elevated MIP-1α/CCL3, less prominent MCP-1/CCL2 and no elevation of SDF-1α gene expression. Analysis of samples from CS-exposed and control rats for anti-oxidant expression showed no significant difference in serum levels of glutathione and, in brain, similar levels of superoxide dismutase and decreased thioredoxin gene expression. In contrast, there was increased brain gene expression for the pro-oxidants iNOS and the NADPH components NOX4, dual oxidase 1 and p22(phox). Nrf2 expression, which is typically triggered as a secondary response to oxidative stress, was also increased in brains from CS-exposed rats with nuclear translocation of this protein from cytoplasm demonstrated in astrocytes in association with increased expression of the aryl hydrocarbon receptor gene, an Nrf2 target. These studies, therefore, demonstrate that CS exposure in these animals can trigger multiple immune and oxidative responses that may have important roles in the pathogenesis of CNS inflammatory neurological diseases.

Cyclosporine induces the expression of the cyclin inhibitor p21.

BACKGROUND Current immunosuppression strategies involve inhibition of T cell activation and/or lymphocyte proliferation. During T cell cycle progression/activation, the expression of cyclins and cyclin dependent kinases is increased. In this study, we examined whether cyclosporine A (CsA) suppresses the cell cycle progression through the induction of the cell cycle inhibitor p21. Because CsA induces the expression of transforming growth factor (TGF)-beta, and TGF-beta induces p21 expression, we also determined whether CsAs induction of p21 is dependent on or independent of TGF-beta. METHOD Using reverse transcription assisted polymerase chain reaction and western blot analysis, we studied the induction of p21 mRNA and protein in human T cells and A-549 cells (human lung adenocarcinoma cells) by CsA. The stimulation of p21 promoter activity was studied by luciferase assay using p21-luc, chimeric plasmid DNA containing a p21 promoter segment, and luciferase reporter gene. The dependence of CsAs induction of p21 was studied using anti-TGF-beta antibody and TGF-beta altered A-549 cells. RESULTS CsA induced p21 mRNA protein expression and stimulated its promoter activity in lymphoid (T cells) and nonlymphoid (human lung adenocarcinoma, A-549 cells).CsAs induction of p21 was inhibited both by a neutralizing anti-TGF-beta antibody and in TGF-beta-altered A-549 cells, consistent with its effects on p21 requiring TGF-beta. CONCLUSION These data support the hypothesis that at least one component of CsAs antiproliferative effects may occur through the induction of p21 and that this induction is dependent on TGF-beta. Should p21 induction be a viable immunosuppressive strategy, inducing this molecule independent from the fibrogenic cytokine TGF-beta might reduce the toxicity associated with current immunosuppression.

Augmentation of the expression of proangiogenic genes in cardiomyocytes with low dose laser irradiation In vitro

BACKGROUND AND OBJECTIVE Several reports suggest that low power red laser light (LPRLL) is capable of affecting cellular processes in the absence of significant thermal effect. The objective of the present study was to determine the effect of LPRLL on proliferation of fetal cardiomyocytes in vitro and on the expression of proangiogenic genes, transforming growth factor-beta (TGF-beta), and vascular endothelial growth factor (VEGF). STUDY DESIGN/MATERIALS AND METHODS All cell cultures were irradiated with single-dose LPRLL using a He-Ne continuous wave laser (632 nm) with different doses. The effect of LPRLL on new DNA synthesis was studied by 3H thymidine-incorporation assay. VEGF and TGF-beta expression by cardiomyocytes was studied by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS We observed that a dose-dependent increase in cardiomyocytes proliferation can be obtained with LPRLL and that there is a significant increase in VEGF and TGF-beta mRNA expression by cardiomyocytes. CONCLUSIONS These data may have significant importance leading to the establishment of new methods for myocardial photoangiogenesis and photoregeneration as well as in vitro proliferation of cardiac myocytes.