Bone marrow mononuclear cells attenuate fibrosis development after severe acute kidney injury

One of the early phases that lead to fibrosis progression is inflammation. Once this stage is resolved, fibrosis might be prevented. Bone marrow mononuclear cells (BMMCs) are emerging as a new therapy for several pathologies, including autoimmune diseases, because they enact immunosuppression. In this study we aimed to evaluate the role of BMMC administration in a model of kidney fibrosis induced by an acute injury. C57Bl6 mice were subjected to unilateral severe ischemia by clamping the left renal pedicle for 1 h. BMMCs were isolated from femurs and tibia, and after 6 h of reperfusion, 1 x 10(6) cells were administrated intraperitoneally. At 24 h after surgery, treated animals showed a significant decrease in creatinine and urea levels when compared with untreated animals. Different administration routes were tested. Moreover, interferon (IFN) receptor knockout BMMCs were used, as this receptor is necessary for BMMC activation. Labeled BMMCs were found in ischemic kidney on FACS analysis. This improved outcome was associated with modulation of inflammation in the kidney and systemic modulation, as determined by cytokine expression profiling. Despite non-amelioration of functional parameters, kidney mRNA expression of interleukin (IL)-6 at 6 weeks was lower in BMMC-treated animals, as were levels of collagen 1, connective tissue growth factor (CTGF), transforming growth factor-beta (TGF-beta) and vimentin. Protective molecules, such as IL-10, heme oxygenase 1 (HO-1) and bone morphogenetic 7 (BMP-7), were increased in treated animals after 6 weeks. Moreover, Masson and Picrosirius red staining analyses showed less fibrotic areas in the kidneys of treated animals. Thus, early modulation of inflammation by BMMCs after an ischemic injury leads to reduced fibrosis through modulation of early inflammation.

Mesenchymal Stem Cells Attenuate Renal Fibrosis Through Immune Modulation and Remodeling Properties in a Rat Remnant Kidney Model

Mesenchymal stem cells (MSCs) have regenerative properties in acute kidney injury, but their role in chronic kidney diseases is still unknown. More specifically, it is not known whether MSCs halt fibrosis. The purpose of this work was to investigate the role of MSCs in fibrogenesis using a model of chronic renal failure. MSCs were obtained from the tibias and femurs of male Wistar-EPM rats. Female Wistar rats were subjected to the remnant model, and 2 vertical bar x vertical bar 10(5) MSCs were intravenously administrated to each rat every other week for 8 weeks or only once and followed for 12 weeks. SRY gene expression was observed in female rats treated with male MSCs, and immune localization of CD73(+)CD90(+) cells at 8 weeks was also assessed. Serum and urine analyses showed an amelioration of functional parameters in MSC-treated animals at 8 weeks, but not at 12 weeks. Masson`s trichrome and Sirius red staining demonstrated reduced levels of fibrosis in MSC-treated animals. These results were corroborated by reduced vimentin, type I collagen, transforming growth factor beta, fibroblast specific protein 1 (FSP-1), monocyte chemoattractant protein 1, and Smad3 mRNA expression and alpha smooth muscle actin and FSP-1 protein expression. Renal interleukin (IL)-6 and tumor necrosis factor alpha mRNA expression levels were significantly decreased after MSC treatment, whereas IL-4 and IL-10 expression levels were increased. All serum cytokine expression levels were decreased in MSC-treated animals. Taken together, these results suggested that MSC therapy can indeed modulate the inflammatory response that follows the initial phase of a chronic renal injury. The immunosuppressive and remodeling properties of MSCs may be involved in the decreased fibrosis in the kidney. STEM CELLS 2009;27:3063-3073

Allogeneic apoptotic thymocyte-stimulated dendritic cells expand functional regulatory T cells

P>Dendritic cells (DCs) play an important role in the clearance of apoptotic cells. The removal of apoptotic cells leads to peripheral tolerance, although their role is still not clear. We show that the uptake of apoptotic thymocytes by DCs converts these cells into tolerogenic DCs resistant to maturation by lipopolysaccharide, modulating the production of interleukin-12 and up-regulating the expression of transforming growth factor-beta(1) latency associated peptide. We also observed that DCs pulsed with apoptotic cells in the allogeneic context were more efficient in the expansion of regulatory T cells (Tregs), and that this expansion requires contact between DCs and the T cell. The Tregs sorted from in vitro culture suppressed the proliferation of splenocytes in vitro in a specific and non-specific manner. In the in vivo model, the transfer of CD4+ CD25- cells to Nude mice induced autoimmunity, with cell infiltrate found in the stomach, colon, liver and kidneys. The co-transfer of CD4+ CD25- and CD4+ CD25+ prevented the presence of cell infiltrates in several organs and increased the total cell count in lymph nodes. Our data indicate that apoptotic cells have an important role in peripheral tolerance via induction of tolerogenic DCs and CD4+ CD25+ Foxp3+ cells that present regulatory functions.

Vascular Endothelial Growth Factor Expression in Invasive Papillary Thyroid Carcinoma

Background: The vascular endothelial growth factor (VEGF) is a major promoter of endothelial growth and migration. Some studies have shown a correlation between expression of this growth factor and prognosis in several cancers, including well-differentiated thyroid cancer. Aim: We studied VEGF expression, local invasiveness, and other prognostic factors in papillary thyroid carcinoma (PTC) to test the hypothesis that the expression of VEGF is correlated with the degree of invasion of PTC. Patients and Methods: Clinical and pathological data of 76 patients with PTC were retrospectively reviewed. Group 1 consisted of patients with gross locally invasive tumors, group 2 consisted of patients with only invasion of the thyroid capsule, and group 3 consisted of patients with noninvasive PTC. Results: VEGF expression was noted within the tumor in all groups of PTC patients but was absent in the surrounding normal tissue. Older patients had higher expression of VEGF than younger patients. The age of patients with strong reaction to VEGF was 46 +/- 14 (mean +/- standard deviation), and that in patients with a weaker reaction was 39 +/- 16 (p<0.05). Only 20% of patients with a follicular variant of PTC had a strong reaction to VEGF compared with 68% of patients with classical PTC (p<0.01). Conclusions: VEGF expression appears to be an early event in the development of PTC. Whether VEGF expression promotes the progression of PTC is not known, but the answer to this question may be important in view of its greater expression in older patients, a group whose prognosis in PTC is worse.

Vascular Endothelial Growth Factor Haplotypes Associated with Childhood Obesity

Expansion of adipose tissue in obesity is associated with angiogenesis and adipose tissue mass depends on neovascularization. Vascular endothelial growth factor (VEGF) is the main angiogenic factor in the adipose tissue, and VEGF expression is tightly regulated at both transcriptional and translational levels. However, no previous study has tested the hypothesis that genetic polymorphisms in the VEGF gene could affect susceptibility to obesity. To test this hypothesis, we compared the distribution of genotypes and haplotypes including three VEGF genetic polymorphisms in obese children and adolescents with those found in healthy controls. We studied 172 healthy children and adolescents and 113 obese children and adolescents. Genotypes of three clinically relevant VEGF polymorphisms in the promoter region (C-2578A, G-1154A, and G-634C) of the VEGF gene were determined by TaqMan allele discrimination assay and real-time polymerase chain reaction. VEGF haplotypes were inferred using Haplo. stats and PHASE 2.1 programs. We found no differences in the distributions of VEGF genotypes and alleles (p > 0.05). However, the CAG haplotype was more frequent in the obese group than in the control group (4% versus 0%, respectively, in white subjects; p = 0.008; odds ratio 10.148 (95% confidence interval: 1.098-93.788). Our findings suggest that VEGF haplotypes affect susceptibility to obesity in children and adolescents.

Interethnic Differences in the Distribution of Clinically Relevant Vascular Endothelial Growth Factor Genetic Polymorphisms

Vascular endothelial growth factor (VEGF) is a homodimeric glycoprotein produced mostly in endothelial cells and its transcription is regulated by a variety of growth factors and cytokines. VEGF plays many relevant roles, and three functional polymorphisms in the promoter region of the VEGF gene (C-2578A, G-1154A, and G-634C) have been associated with disease conditions. Although some studies suggest that interethnic differences exist in the distribution of these variants, no previous study has examined this hypothesis in admixed populations. We examined the distribution of these three clinically relevant VEGF single-nucleotide polymorphisms in 175 white and 185 black subjects. We have also estimated the haplotype distribution and assessed associations between these variants. Although the A-2578 and A-1154 variants were more common in whites (39% and 29%, respectively) than in blacks (29% and 16%, respectively; both p < 0.05), no significant interethnic differences were found with regards to the G-634C polymorphism. While the haplotype including the C-2578, G-1154, and G-634 variants was the most common in both ethnic groups, it was more common in blacks than in whites (p < 0.05). The haplotype including the C-2578, A-1154, and G-634 alleles and the haplotype including the C-2578, A-1154, and C-634 alleles were more common in whites than in blacks (both p < 0.05). These results show marked interethnic differences in the distribution of genetic variants of VEGF that may explain, at least in part, interethnic disparities in the susceptibility to cardiovascular diseases.

Vascular Endothelial Growth Factor Genetic Polymorphisms and Haplotypes in Women with Migraine

Vascular endothelial growth factor (VEGF) production is regulated by growth factors and inflammatory cytokines, and VEGF plays a role in migraine. We examined for the first time whether three functional polymorphisms in the promoter region of VEGF gene (C(-2578)A, G(-1154A), and G(-634C)) and VEGF haplotypes are associated with migraine. We studied 114 healthy women without migraine and 175 women with migraine (129 without aura, and 46 with aura). We found no differences in the distributions of VEGF genotypes and alleles (p > 0.05). However, the CAC haplotype was more frequent in controls than in migraine patients, and the AGC haplotype was more frequent in patients with migraine with aura than in controls (both p < 0.05). These findings suggest that VEGF haplotypes affect susceptibility to migraine.

Research Smad2 and Smad6 as predictors of overall survival in oral squamous cell carcinoma patients

Background: To test if the expression of Smad1-8 mRNAs were predictive of survival in patients with oral squamous cell carcinoma (SCC). Patients and Methods: We analyzed, prospectively, the expression of Smad1-8, by means of Ribonuclease Protection Assay in 48 primary, operable, oral SCC. In addition, 21 larynx, 10 oropharynx and 4 hypopharynx SCC and 65 matched adjacent mucosa, available for study, were also included. For survival analysis, patients were categorized as positive or negative for each Smad, according to median mRNA expression. We also performed real-time quantitative PCR (QRTPCR) to asses the pattern of TGF beta 1, TGF beta 2, TGF beta 3 in oral SCC. Results: Our results showed that Smad2 and Smad6 mRNA expression were both associated with survival in Oral SCC patients. Cox Multivariate analysis revealed that Smad6 positivity and Smad2 negativity were both predictive of good prognosis for oral SCC patients, independent of lymph nodal status (P = 0.003 and P = 0.029, respectively). In addition, simultaneously Smad2(-) and Smad6(+) oral SCC group of patients did not reach median overall survival (mOS) whereas the mOS of Smad2(+)/Smad6(-) subgroup was 11.6 months (P = 0.004, univariate analysis). Regarding to TGF beta isoforms, we found that Smad2 mRNA and TGF beta 1 mRNA were inversely correlated (p = 0.05, R = -0.33), and that seven of the eight TGF beta 1(+) patients were Smad2(-). In larynx SCC, Smad7(-) patients did not reach mOS whereas mOS of Smad7(+) patients were only 7.0 months (P = 0.04). No other correlations were found among Smad expression, clinico-pathological characteristics and survival in oral, larynx, hypopharynx, oropharynx or the entire head and neck SCC population. Conclusion: Smad6 together with Smad2 may be prognostic factors, independent of nodal status in oral SCC after curative resection. The underlying mechanism which involves aberrant TGF beta signaling should be better clarified in the future.

In Vivo Effects on the Expression of Vascular Endothelial Growth Factor-A(165) Messenger Ribonucleic Acid of an Infrared Diode Laser Associated or Not with a Visible Red Diode Laser

Objective: This study investigated and correlated the kinetic expression of vascular endothelial growth factor (VEGF)-A(165) messenger ribonucleic acid (mRNA) with the associated use or not of an infrared laser and a visible red laser during the wound healing in rats. Background Data: There is a lack of scientific evidence demonstrating the influence of low-level laser therapy (LLLT) on the expression of VEGF mRNA in vivo. Materials and Methods: Forty-five Wistar rats were randomly allocated to one of three groups: I (n = 5, nonoperated animals), II (n = 25, operated animals), and III (n = 25, animals operated and subjected to laser irradiation). A surgical wound was performed using a scalpel in the right side of the tongue of operated animals. In group III, two sessions of laser irradiation were performed, one right after the surgical procedure (infrared laser, 780 nm, 70mW, 35 J/cm(2)) and the other 48 h later (visible red laser, 660 nm, 40mW, 5J/cm(2)). Five animals each were sacrificed 1, 3, 5, and 7 days postoperatively in groups II and III, and samples of tongue tissue were obtained. The animals of group I were sacrificed on day 7. Total RNA was extracted using guanidine-isothiocyanate-phenol-chloroform method. The results of horizontal electrophoresis after reverse transcription polymerase chain reaction permitted the ratio of VEGF-A(165) mRNA and glyceraldehyde 3-phosphate dehydrogenase mRNA expression for groups I, II, and III to be assessed (two-way analysis of variance and Tukey test, p<0.05). Results: The expression of VEGF-A(165) mRNA in group II (0.770 +/- 0.098) was statistically greater than that observed in groups I (0.523 +/- 0.164) and III (0.504 +/- 0.069) in the first day after surgery (p<0.05). Significant differences between the groups were not observed in other time periods. Conclusion: LLLT influenced the expression of VEGF-A(165) mRNA during wound healing after a surgical procedure on the tongue of Wistar rats.

Modulation of small leucine-rich proteoglycans (SLRPs) expression in the mouse uterus by estradiol and progesterone

Background: We have previously demonstrated that four members of the family of small leucine-rich-proteoglycans (SLRPs) of the extracellular matrix (ECM), named decorin, biglycan, lumican and fibromodulin, are deeply remodeled in mouse uterine tissues along the estrous cycle and early pregnancy. It is known that the combined action of estrogen (E2) and progesterone (P4) orchestrates the estrous cycle and prepares the endometrium for pregnancy, modulating synthesis, deposition and degradation of various molecules. Indeed, we showed that versican, another proteoglycan of the ECM, is under hormonal control in the uterine tissues. Methods: E2 and/or medroxiprogesterone acetate (MPA) were used to demonstrate, by real time PCR and immunoperoxidase staining, respectively, their effects on mRNA expression and protein deposition of these SLRPs, in the uterine tissues. Results: Decorin and lumican were constitutively expressed and deposited in the ECM in the absence of the ovarian hormones, whereas deposition of biglycan and fibromodulin were abolished from the uterine ECM in the non-treated group. Interestingly, ovariectomy promoted an increase in decorin, lumican and fibromodulin mRNA levels, while biglycan mRNA conspicuously decreased. Hormone replacement with E2 and/or MPA differentially modulates their expression and deposition. Conclusions: The patterns of expression of these SLRPs in the uterine tissues were found to be hormone-dependent and uterine compartment-related. These results reinforce the existence of subpopulations of endometrial fibroblasts, localized into distinct functional uterine compartments, resembling the organization into basal and functional layers of the human endometrium.

Sustained release carriers used to delivery bone morphogenetic proteins in the bone healing process

Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor beta superfamily, especially BMP-2, induce bone formation in vivo, and clinical application in repair of bone fractures and defects is expected. However, appropriate systems to delivery BMPs for practical use need to be developed with the objective to heal cartilage and bone-related diseases in medical, dental and veterinary practice. Thus, the aim of this article was to present an overview of the principals carriers used to delivery BMPs and alternative delivery systems for these proteins.

Suppressive action of melatonin on the TH-2 immune response in rats infected with Trypanosoma cruzi

Control of the acute phase of Trypanosoma cruzi infection is critically dependent on cytokine-mediated macrophage activation to intracellular killing, natural killer (NK) cells, CD4(+) T cells, CD8(+) T cells and B cells. Cell-mediated immunity in T. cruzi infection is also modulated by cytokines, but in addition to parasite-specific responses, autoimmunity can be also triggered. Importantly, cytokines may also play a role in the cell-mediated immunity of infected subjects. Here we studied the role of cytokines in the regulation of innate and adaptive immunity during the acute phase of T. cruzi infection in Wistar rats. Melatonin is an effective regulator of the immune system. Macrophages and T lymphocytes, which have melatonin receptors, are target cells for the immunomodulatory function of melatonin. In this paper melatonin was orally given via two protocols: prior to and concomitant with infection. Both treatments were highly effective against T. cruzi with enhanced action for the concomitant treatment. The data suggest an up-regulation of the TH-1 immune response as all analyzed parameters, interleukin (IL)-4, IL-10, transforming growth factor-beta 1 and splenocyte proliferation, displayed reduced levels as compared with the untreated counterparts. However, the direct effects of melatonin on immune cells have not been fully investigated during T. cruzi infection. We conclude that in light of the current results, melatonin exerted important therapeutic benefits through its immune regulatory effects.

Imatinib mesylate ameliorates the dystrophic phenotype in exercised mdx mice

Myofiber degeneration, inflammation, and fibrosis are remarkable features of Duchenne muscular dystrophy. We hypothesized that the administration of imatinib mesylate, an inhibitor of tyrosine kinase and TGF-beta pro-fibrogenic activity, could improve the muscular conditions in mdx mice. Four-week old mdx mice were treated and exercised for 6 weeks. Gastrocnemius and diaphragm histopathology, strength, creatine kinase, and cytokine levels were evaluated. The treated group presented increased muscular strength and decreased CK levels, injured myofibers, and inflammatory infiltrates. Pro-inflammatory cytokines and TGF-beta were also reduced, while IL-10 was increased, suggesting an immunomodulatory effect of imatinib, which can ameliorate the dystrophic phenotype in mdx mice. (C) 2009 Elsevier B.V. All rights reserved.

Fibroblast Growth Factor 23 in Hemodialysis Patients: Effects of Phosphate Binder, Calcitriol and Calcium Concentration in the Dialysate

Background: Fibroblast growth factor 23 (FGF23) concentrations increase early in chronic kidney disease (CKD), and the influence of current CKD-mineral and bone disorder (MBD) therapies on serum FGF23 levels is still under investigation. Methods: In this post-hoc analysis of a randomized clinical trial, phosphate binders and calcitriol were washed out of 72 hemodialysis patients who were then submitted to bone biopsy, coronary tomography and biochemical measures, including FGF23. They were randomized to receive sevelamer or calcium acetate for 1 year and the prescription of calcitriol and the calcium concentration in the dialysate were adjusted according to serum calcium, phosphate and PTH and bone biopsy diagnosis. Results: At baseline, bone biopsy showed that 58.3% had low-turnover bone disease, whereas 38.9% had high-turnover bone disease, with no significant differences between them with regard to FGF23. Median baseline FGF23 serum levels were elevated and correlated positively with serum phosphate. After 1 year, serum FGF23 decreased significantly. Repeated measures ANOVA analysis showed that the use of a 3.5-mEq/l calcium concentration in the dialysate, as well as the administration of calcitriol and a calcium-based phosphate binder were associated with higher final serum FGF23 levels. Conclusions: Taken together, our results confirm that the current CKD-MBD therapies have an effect on serum levels of FGF23. Since FGF23 is emerging as a potential treatment target, our findings should be taken into account in the decision on how to manage CKD-MBD therapy. Copyright (C) 2010 S. Karger AG, Basel

Pentoxifylline reduces pro-inflammatory and increases anti-inflammatory activity in patients with coronary artery disease - A randomized placebo-controlled study

The balance between different immunological stimuli is essential in the progression and stabilization of atherosclerotic plaques. Immune regulation has been suggested as potential target for the treatment of atherosclerotic disease. We sought to determine whether treatment with pentoxifylline, a phosphodiesterase inhibitor with immunomodulating properties, could reduce the pro-inflammatory response observed in patients with acute coronary syndromes (ACS) and increase anti-inflammatory activity. In a double-blind, prospective, placebo-controlled study, 64 patients with ACS were randomized to receive pentoxifylline 400 mg TID or placebo for 6 months. Analysis of the pro-inflammatory markers, Greactive protein (CRP), interleukin (IL)-6, IL-12, interferon-gamma and tumor necrosis factor (TNF)-alpha and the anti-inflammatory cytokines, transforming growth factor (TGF)-beta 1 and IL-10 were done at baseline, 1 and 6 months. Pentoxifylline treatment significantly reduced the adjusted levels of CRP and TNF-alpha compared to placebo after 6 months (P=0.04 and P < 0.01, respectively). IL-12 increase was significantly less pronounced with pentoxifylline (P=0.04). The levels of the anti-inflammatory cytokine, IL-10, also declined significantly less in the pentoxifylline group compared to placebo (P < 0.01) with a trend towards a higher increase of TGF-beta 1 in the former group (P=0.16). Pentoxifylline reduces pro-inflammatory and increases anti-inflammatory response in patients with ACS and may have beneficial clinical effects on cardiovascular events. (c) 2006 Elsevier Ireland Ltd. All rights reserved.