Characterization of Dermal Stem Cells of Diabetic Patients.

Diabetic foot ulcers (DFUs) are lesions that involve loss of epithelium and dermis, sometimes involving deep structures, compartments, and bones. The aim of this work is to investigate the innate regenerative properties of dermal tissue around ulcers by the identification and analysis of resident dermal stem cells (DSCs). Dermal samples were taken at the edge of DFUs, and genes related to the wound healing process were analyzed by the real-time PCR array. The DSCs were isolated and analyzed by immunofluorescence, flow cytometry, and real-time PCR array to define their stemness properties. The gene expression profile of dermal tissue showed a dysregulation in growth factors, metalloproteinases, collagens, and integrins involved in the wound healing process. In the basal condition, diabetic DSCs adhered on the culture plate with spindle-shaped fibroblast-like morphology. They were positive to the mesenchymal stem cells markers CD44, CD73, CD90, and CD105, but negative for the hematopoietic markers CD14, CD34, CD45, and HLA-DR. In diabetic DSCs, the transcription of genes related to self-renewal and cell division were equivalent to that in normal DSCs. However, the expression of CCNA2, CCND2, CDK1, ALDH1A1, and ABCG2 was downregulated compared with that of normal DSCs. These genes are also related to cell cycle progression and stem cell maintenance. Further investigation will improve the understanding of the molecular mechanisms by which these genes together govern cell proliferation, revealing new strategies useful for future treatment of DFUs.

Bovine pericardium membrane as new tool for mesenchymal stem cells commitment.

Acellular matrices are widespread biomaterials used in surgical practice as tissue reinforcement and anatomical support to favor tissue regeneration. It is clear that a fundamental role in the regeneration of tissue is played by cell-material interaction. In this work, the interaction between a bovine pericardium membrane and human adult stem cells was investigated by microscopy analysis and gene expression analysis. Parallel cell cultures were prepared on the pericardium membrane or tissue culture plate. They were incubated in basal growth medium or in adipogenic differentiation medium to perform experiments on the seventh and the 14th day of culture. Results demonstrated that the membrane allows cell viability, adhesion, and proliferation of human stem cells. During adipogenic commitment on the membrane, the accumulation of cytoplasmatic lipid droplets and the expression of adipogenic gene PPARG, CEBPA, GLUT4, FABP4, and ADIPOQ were detected. Concurrently, a downregulation of mesenchymal stem cell gene CD29, CD90, and CD105 was detected. In basal medium, the adipogenic gene expression was upregulated, whereas the mesenchymal markers were indifferently expressed. These findings suggest that the bovine pericardium membrane is a biocompatible matrix and that their rough surface allows cell adhesion, spreading, and proliferation. The surface morphology activates mechanochemical signals that stimulate the adipogenic commitment of stem cells in basal medium and potentiate their commitment in adipogenic differentiation medium.

Metal Nanoparticles Released from Dental Implant Surfaces: Potential Contribution to Chronic Inflammation and Peri-Implant Bone Loss.

Peri-implantitis is an inflammatory disease affecting tissues surrounding dental implants. Although it represents a common complication of dental implant treatments, the underlying mechanisms have not yet been fully described. The aim of this study is to identify the role of titanium nanoparticles released form the implants on the chronic inflammation and bone lysis in the surrounding tissue. We analyzed the in vitro effect of titanium (Ti) particle exposure on mesenchymal stem cells (MSCs) and fibroblasts (FU), evaluating cell proliferation by MTT test and the generation of reactive oxygen species (ROS). Subsequently, in vivo analysis of peri-implant Ti particle distribution, histological, and molecular analyses were performed. Ti particles led to a time-dependent decrease in cell viability and increase in ROS production in both MSCs and FU. Tissue analyses revealed presence of oxidative stress, high extracellular and intracellular Ti levels and imbalanced bone turnover. High expression of ZFP467 and the presence of adipose-like tissue suggested dysregulation of the MSC population; alterations in vessel morphology were identified. The results suggest that Ti particles may induce the production of high ROS levels, recruiting abnormal quantity of neutrophils able to produce high level of metalloproteinase. This induces the degradation of collagen fibers. These events may influence MSC commitment, with an imbalance of bone regeneration.

Effects of novel antidepressant drugs on mesenchymal stem cell physiology.

It is known that users of psychotropic drugs often have weight gain, adverse effects on bone mineral density and osteoporosis, but the molecular basis for these side effects is poorly understood. The aim of this study is to evaluate the effects in vitro of duloxetine (a serotonin and norepinephrine reuptake inhibitor) and fluoxetine (a selective serotonin reuptake inhibitor) on the physiology of human adult stem cells. Adipose-derived stem cells (ADSCs) were isolated and characterized investigating phenotype morphology, expression and frequency of surface markers. Then, a non-toxic concentration of duloxetine and fluoxetine was selected to treat cells during adipogenic and osteogenic differentiation. Stemness properties and the differentiation potential of drug-treated cells were investigated by the quantification of adipogenic and osteogenic markers gene expression and histological staining. The collected data showed that the administration of a daily non-toxic dose of duloxetine and fluoxetine has not directly influenced ADSCs proliferation and their stemness properties. The treatment with duloxetine or fluoxetine did not lead to morphological alterations during adipogenic or osteogenic commitment. However, treatments with the antidepressant showed a slight difference in adipogenic gene expression timing. Furthermore, duloxetine treatment caused an advance in gene expression of early and late osteogenic markers. Fluoxetine instead caused an increase in expression of osteogenic genes compared to untreated cells. In contrast, in pre-differentiated cells, the daily treatment with duloxetine or fluoxetine did not alter the expression profile of adipogenic and osteogenic differentiation. In conclusion, a non-toxic concentration of duloxetine and fluoxetine does not alter the stemness properties of ADSCs and does not prevent the commitment of pre-differentiated ADSCs in adipocytes or osteocyte. Probably, the weight gain and osteoporotic effects associated with the use of psychotropic drugs could be closely related to the direct action of serotonin.

Collagen Fiber Array of Peritumoral Stroma Influences Epithelial-to-Mesenchymal Transition and Invasive Potential of Mammary Cancer Cells.

: Interactions of cancer cells with matrix macromolecules of the surrounding tumor stroma are critical to mediate invasion and metastasis. In this study, we reproduced the collagen mechanical barriers in vitro (i.e., basement membrane, lamina propria under basement membrane, and deeper bundled collagen fibers with different array). These were used in 3D cell cultures to define their effects on morphology and behavior of breast cancer cells with different metastatic potential (MCF-7 and MDA-MB-231) using scanning electron microscope (SEM). We demonstrated that breast cancer cells cultured in 2D and 3D cultures on different collagen substrates show different morphologies: i) a globular/spherical shape, ii) a flattened polygonal shape, and iii) elongated/fusiform and spindle-like shapes. The distribution of different cell shapes changed with the distinct collagen fiber/fibril physical array and size. Dense collagen fibers, parallel to the culture plane, do not allow the invasion of MCF-7 and MDA-MB-231 cells, which, however, show increases of microvilli and microvesicles, respectively. These novel data highlight the regulatory role of different fibrillar collagen arrays in modifying breast cancer cell shape, inducing epithelial-to-mesenchymal transition, changing matrix composition and modulating the production of extracellular vesicles. Further investigation utilizing this in vitro model will help to demonstrate the biological roles of matrix macromolecules in cancer cell invasion in vivo.

Exosome in Cardiovascular Diseases: A Complex World Full of Hope.

Exosomes are a subgroup of extracellular vesicles containing a huge number of bioactive molecules. They represent an important means of cell communication, mostly between different cell populations, with the purpose of maintaining tissue homeostasis and coordinating the adaptive response to stress. This type of intercellular communication is important in the cardiovascular field, mainly due to the fact that the heart is a complex multicellular system. Given the growing interest in the role of exosomes in cardiovascular diseases and the numerous studies published in the last few decades, we focused on the most relevant results about exosomes in the cardiovascular filed starting from their characterization, passing through the study of their function, and ending with perspectives for their use in cardiovascular therapies.

In vitro effects of interleukin (IL)-1 beta inhibition on the epithelial-to-mesenchymal transition (EMT) of renal tubular and hepatic stellate cells.

BACKGROUND: The epithelial to mesenchymal transition (EMT) is a multi-factorial biological mechanism involved in renal and hepatic fibrosis and the IL-1 beta has been assumed as a mediator of this process although data are not exhaustive. Therefore, the aim of our study was to evaluate the role of this cytokine in the EMT of renal proximal tubular epithelial cells (HK-2) and stellate cells (LX-2) and the protective/anti-fibrotic effect of its inhibition by Canakinumab (a specific human monoclonal antibody targeted against IL-1beta). METHODS: Both cell types were treated with IL-1 beta (10 ng/ml) for 6 and 24 h with and without Canakinumab (5 µg/ml). As control we used TGF-beta (10 ng/ml). Expression of EMT markers (vimentin, alpha-SMA, fibronectin) were evaluated through western blotting and immunofluorescence. Genes expression for matrix metalloproteinases (MMP)-2 was measured by Real-Time PCR and enzymatic activity by zymography. Cellular motility was assessed by scratch assay. RESULTS: IL-1 beta induced a significant up-regulation of EMT markers in both cell types and increased the MMP-2 protein expression and enzymatic activity, similarly to TGF-beta. Moreover, IL-1 beta induced a higher rate of motility in HK-2. Canakinumab prevented all these modifications in both cell types. CONCLUSIONS: Our results clearly demonstrate the role of IL-1 beta in the EMT of renal/stellate cells and it underlines, for the first time, the therapeutic potential of its specific inhibition on the prevention/minimization of organ fibrosis.

Inhibition of heparanase protects against chronic kidney dysfunction following ischemia/reperfusion injury.

Renal ischemia/reperfusion (I/R) injury occurs in patients undergoing renal transplantation and with acute kidney injury and is responsible for the development of chronic allograft dysfunction as characterized by parenchymal alteration and fibrosis. Heparanase (HPSE), an endoglycosidase that regulates EMT and macrophage polarization, is an active player in the biological response triggered by ischemia/reperfusion (I/R) injury. I/R was induced in vivo by clamping left renal artery for 30 min in wt C57BL/6J mice. Animals were daily treated and untreated with Roneparstat (an inhibitor of HPSE) and sacrificed after 8 weeks. HPSE, fibrosis, EMT-markers, inflammation and oxidative stress were evaluated by biomolecular and histological methodologies together with the evaluation of renal histology and measurement of renal function parameters. 8 weeks after I/R HPSE was upregulated both in renal parenchyma and plasma and tissue specimens showed clear evidence of renal injury and fibrosis. The inhibition of HPSE with Roneparstat-restored histology and fibrosis level comparable with that of control. I/R-injured mice showed a significant increase of EMT, inflammation and oxidative stress markers but they were significantly reduced by treatment with Roneparstat. Finally, the inhibition of HPSE in vivo almost restored renal function as measured by BUN, plasma creatinine and albuminuria. The present study points out that HPSE is actively involved in the mechanisms that regulate the development of renal fibrosis arising in the transplanted organ as a consequence of ischemia/reperfusion damage. HPSE inhibition would therefore constitute a new pharmacological strategy to reduce acute kidney injury and to prevent the chronic pro-fibrotic damage induced by I/R.

Therapeutic Potential of Autologous Adipose-Derived Stem Cells for the Treatment of Liver Disease.

Currently, the most effective therapy for liver diseases is liver transplantation, but its use is limited by organ donor shortage, economic reasons, and the requirement for lifelong immunosuppression. Mesenchymal stem cell (MSC) transplantation represents a promising alternative for treating liver pathologies in both human and veterinary medicine. Interestingly, these pathologies appear with a common clinical and pathological profile in the human and canine species; as a consequence, dogs may be a spontaneous model for clinical investigations in humans. The aim of this work was to characterize canine adipose-derived MSCs (cADSCs) and compare them to their human counterpart (hADSCs) in order to support the application of the canine model in cell-based therapy of liver diseases. Both cADSCs and hADSCs were successfully isolated from adipose tissue samples. The two cell populations shared a common fibroblast-like morphology, expression of stemness surface markers, and proliferation rate. When examining multilineage differentiation abilities, cADSCs showed lower adipogenic potential and higher osteogenic differentiation than human cells. Both cell populations retained high viability when kept in PBS at controlled temperature and up to 72 h, indicating the possibility of short-term storage and transportation. In addition, we evaluated the efficacy of autologous ADSCs transplantation in dogs with liver diseases. All animals exhibited significantly improved liver function, as evidenced by lower liver biomarkers levels measured after cells transplantation and evaluation of cytological specimens. These beneficial effects seem to be related to the immunomodulatory properties of stem cells. We therefore believe that such an approach could be a starting point for translating the results to the human clinical practice in future.

Heparanase: A Multitasking Protein Involved in Extracellular Matrix (ECM) Remodeling and Intracellular Events.

Heparanase (HPSE) has been defined as a multitasking protein that exhibits a peculiar enzymatic activity towards HS chains but which simultaneously performs other non-enzymatic functions. Through its enzymatic activity, HPSE catalyzes the cutting of the side chains of heparan sulfate (HS) proteoglycans, thus contributing to the remodeling of the extracellular matrix and of the basal membranes. Furthermore, thanks to this activity, HPSE also promotes the release and diffusion of various HS-linked molecules like growth factors, cytokines and enzymes. In addition to being an enzyme, HPSE has been shown to possess the ability to trigger different signaling pathways by interacting with transmembrane proteins. In normal tissue and in physiological conditions, HPSE exhibits only low levels of expression restricted only to keratinocytes, trophoblast, platelets and mast cells and leukocytes. On the contrary, in pathological conditions, such as in tumor progression and metastasis, inflammation and fibrosis, it is overexpressed. With this brief review, we intend to provide an update on the current knowledge about the different role of HPSE protein exerted by its enzymatic and non-enzymatic activity.

Graphene-Based Nanomaterials for Tissue Engineering in the Dental Field.

The world of dentistry is approaching graphene-based nanomaterials as substitutes for tissue engineering. Apart from its exceptional mechanical strength, electrical conductivity and thermal stability, graphene and its derivatives can be functionalized with several bioactive molecules. They can also be incorporated into different scaffolds used in regenerative dentistry, generating nanocomposites with improved characteristics. This review presents the state of the art of graphene-based nanomaterial applications in the dental field. We first discuss the interactions between cells and graphene, summarizing the available in vitro and in vivo studies concerning graphene biocompatibility and cytotoxicity. We then highlight the role of graphene-based nanomaterials in stem cell control, in terms of adhesion, proliferation and differentiation. Particular attention will be given to stem cells of dental origin, such as those isolated from dental pulp, periodontal ligament or dental follicle. The review then discusses the interactions between graphene-based nanomaterials with cells of the immune system; we also focus on the antibacterial activity of graphene nanomaterials. In the last section, we offer our perspectives on the various opportunities facing the use of graphene and its derivatives in associations with titanium dental implants, membranes for bone regeneration, resins, cements and adhesives as well as for tooth-whitening procedures.

Heparanase regulates the M1 polarization of renal macrophages and their crosstalk with renal epithelial tubular cells after ischemia/reperfusion injury.

Heparanase (HPSE) is part of the biologic network triggered by ischemia/reperfusion (I/R) injury, a complication of renal transplantation and acute kidney injury. During this period, the kidney or graft undergoes a process of macrophages recruitment and activation. HPSE may therefore control these biologic effects. We measured the ability of HPSE and its inhibitor, SST0001, to regulate macrophage polarization and the crosstalk between macrophages and HK-2 renal tubular cells during in vitro hypoxia/reoxygenation (H/R). Furthermore, we evaluated in vivo renal inflammation, macrophage polarization, and histologic changes in mice subjected to monolateral I/R and treated with SST0001 for 2 or 7 d. The in vitro experiments showed that HPSE sustained M1 macrophage polarization and modulated apoptosis, the release of damage associated molecular patterns in post-H/R tubular cells, the synthesis of proinflammatory cytokines, and the up-regulation of TLRs on both epithelial cells and macrophages. HPSE also regulated M1 polarization induced by H/R-injured tubular cells and the partial epithelial-mesenchymal transition of these epithelial cells by M1 macrophages. All these effects were prevented by inhibiting HPSE. Furthermore, the inhibition of HPSE in vivo reduced inflammation and M1 polarization in mice undergoing I/R injury, partially restored renal function and normal histology, and reduced apoptosis. These results show for the first time that HPSE regulates macrophage polarization as well as renal damage and repair after I/R. HPSE inhibitors could therefore provide a new pharmacologic approach to minimize acute kidney injury and to prevent the chronic profibrotic damages induced by I/R.-Masola, V., Zaza, G., Bellin, G., Dall'Olmo, L., Granata, S., Vischini, G., Secchi, M. F., Lupo, A., Gambaro, G., Onisto, M. Heparanase regulates the M1 polarization of renal macrophages and their crosstalk with renal epithelial tubular cells after ischemia/reperfusion injury.

Involvement of heparanase in the pathogenesis of acute kidney injury: nephroprotective effect of PG545.

Despite the high prevalence of acute kidney injury (AKI) and its association with increased morbidity and mortality, therapeutic approaches for AKI are disappointing. This is largely attributed to poor understanding of the pathogenesis of AKI. Heparanase, an endoglycosidase that cleaves heparan sulfate, is involved in extracellular matrix turnover, inflammation, kidney dysfunction, diabetes, fibrosis, angiogenesis and cancer progression. The current study examined the involvement of heparanase in the pathogenesis of ischemic reperfusion (I/R) AKI in a mouse model and the protective effect of PG545, a potent heparanase inhibitor. I/R induced tubular damage and elevation in serum creatinine and blood urea nitrogen to a higher extent in heparanase over-expressing transgenic mice vs. wild type mice. Moreover, TGF-ß, vimentin, fibronectin and α-smooth muscle actin, biomarkers of fibrosis, and TNFα, IL6 and endothelin-1, biomarkers of inflammation, were upregulated in I/R induced AKI, primarily in heparanase transgenic mice, suggesting an adverse role of heparanase in the pathogenesis of AKI. Remarkably, pretreatment of mice with PG545 abolished kidney dysfunction and the up-regulation of heparanase, pro-inflammatory (i.e., IL-6) and pro-fibrotic (i.e., TGF-ß) genes induced by I/R. The present study provides new insights into the involvement of heparanase in the pathogenesis of ischemic AKI.Our results demonstrate that heparanase plays a deleterious role in the development of renal injury and kidney dysfunction,attesting heparanase inhibition as a promising therapeutic approach for AKI.

Specific heparanase inhibition reverses glucose-induced mesothelial-to-mesenchymal transition.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells induced by high glucose (HG) levels is a major biological mechanism leading to myofibroblast accumulation in the omentum of patients on peritoneal dialysis (PD). Heparanase (HPSE), an endoglycosidase that cleaves heparan sulfate chains, is involved in the EMT of several cell lines, and may have a major role in this pro-fibrotic process potentially responsible for the failure of dialysis. Its specific inhibition may therefore plausibly minimize this pathological condition. METHODS: An in vitro study employing several biomolecular strategies was conducted to assess the role of HPSE in the HG-induced mesothelial EMT process, and to measure the effects of its specific inhibition by SST0001, a N-acetylated glycol-split heparin with a strong anti-HPSE activity. Rat mesothelial cells were grown for 6 days in HG (200 mM) culture medium with or without SST0001. Then EMT markers (VIM, α-SMA, TGF-ß) and vascular endothelial growth factor (VEGF) (a factor involved in neoangiogenesis) were measured by real-time PCR and immunofluorescence/western blotting. As a functional analysis, trans-epithelial resistance (TER) and permeability to albumin were also measured in our in vitro model using a Millicell-ERS ohmmeter and a spectrophotometer, respectively. RESULTS: Our results showed that 200 mM of glucose induced a significant gene and protein up-regulation of VEGF and all EMT markers after 6 days of culture. Intriguingly, adding SST0001 on day 3 reversed these biological and cellular effects. HPSE inhibition also restored the normal TER and permeability lost during the HG treatment. CONCLUSION: Taken together, our data confirm that HG can induce EMT of mesothelial cells, and that HPSE plays a central part in this process. Our findings also suggest that pharmacological HPSE inhibition could prove a valuable therapeutic tool for minimizing fibrosis and avoiding a rapid decline in the efficacy of dialysis in patients on PD, though clinical studies and/or trials would be needed to confirm the clinical utility of this treatment.

Treatment by Therapeutic Magnetic Resonance (TMR™) increases fibroblastic activity and keratinocyte differentiation in an in vitro model of 3D artificial skin.

This study investigated the effect of extremely low-frequency pulsed electromagnetic fields (PEMFs) on skin wound healing in an in vitro dermal-like tissue. In this study, fibroblast and endothelial cells were utilized for the in vitro reconstruction of dermal-like tissues treated for various times up to 21 days with PEMFs. The effects of PEMFs on cell proliferation (MTT test), cell ageing (ß-galactosidase test, ROS production), gene expression, the quality of the extracellular matrix and the amount of fibroblast growth factors were analysed. The high quality of the dermis products in the presence of PEMFs at the end of the study was confirmed through the high degree of organization of keratinocytes, which were subsequently seeded on the aforementioned in vitro reconstructed dermis. The cells organized themselves in well-defined multi-layers and were better organized compared with the epidermis present on the dermis that was obtained without PEMF treatment. Copyright © 2015 John Wiley & Sons, Ltd.

Transcriptomics: A Step behind the Comprehension of the Polygenic Influence on Oxidative Stress, Immune Deregulation, and Mitochondrial Dysfunction in Chronic Kidney Disease.

Chronic kidney disease (CKD) is an increasing and global health problem with a great economic burden for healthcare system. Therefore to slow down the progression of this condition is a main objective in nephrology. It has been extensively reported that microinflammation, immune system deregulation, and oxidative stress contribute to CKD progression. Additionally, dialysis worsens this clinical condition because of the contact of blood with bioincompatible dialytic devices. Numerous studies have shown the close link between immune system impairment and CKD but most have been performed using classical biomolecular strategies. These methodologies are limited in their ability to discover new elements and enable measuring the simultaneous influence of multiple factors. The "omics" techniques could overcome these gaps. For example, transcriptomics has revealed that mitochondria and inflammasome have a role in pathogenesis of CKD and are pivotal elements in the cellular alterations leading to systemic complications. We believe that a larger employment of this technique, together with other "omics" methodologies, could help clinicians to obtain new pathogenetic insights, novel diagnostic biomarkers, and therapeutic targets. Finally, transcriptomics could allow clinicians to personalize therapeutic strategies according to individual genetic background (nutrigenomic and pharmacogenomic). In this review, we analyzed the available transcriptomic studies involving CKD patients.

Heparanase: A Potential New Factor Involved in the Renal Epithelial Mesenchymal Transition (EMT) Induced by Ischemia/Reperfusion (I/R) Injury.

BACKGROUND: Ischemia/reperfusion (I/R) is an important cause of acute renal failure and delayed graft function, and it may induce chronic renal damage by activating epithelial to mesenchymal transition (EMT) of renal tubular cells. Heparanase (HPSE), an endoglycosidase that regulates FGF-2 and TGFß-induced EMT, may have an important role. Therefore, aim of this study was to evaluate its role in the I/R-induced renal pro-fibrotic machinery by employing in vitro and in vivo models. METHODS: Wild type (WT) and HPSE-silenced renal tubular cells were subjected to hypoxia and reoxygenation in the presence or absence of SST0001, an inhibitor of HPSE. In vivo, I/R injury was induced by bilateral clamping of renal arteries for 30 min in transgenic mice over-expressing HPSE (HPA-tg) and in their WT littermates. Mice were sacrificed 48 and 72 h after I/R. Gene and protein EMT markers (α-SMA, VIM and FN) were evaluated by bio-molecular and histological methodologies. RESULTS: In vitro: hypoxia/reoxygenation (H/R) significantly increased the expression of EMT-markers in WT, but not in HPSE-silenced tubular cells. Notably, EMT was prevented in WT cells by SST0001 treatment. In vivo: I/R induced a remarkable up-regulation of EMT markers in HPA-tg mice after 48-72 h. Noteworthy, these effects were absent in WT animals. CONCLUSIONS: In conclusion, our results add new insights towards understanding the renal biological mechanisms activated by I/R and they demonstrate, for the first time, that HPSE is a pivotal factor involved in the onset and development of I/R-induced EMT. It is plausible that in future the inhibition of this endoglycosidase may represent a new therapeutic approach to minimize/prevent fibrosis and slow down chronic renal disease progression in native and transplanted kidneys.

Everolimus-induced epithelial to mesenchymal transition (EMT) in bronchial/pulmonary cells: when the dosage does matter in transplantation.

BACKGROUND: Everolimus (EVE) is a mammalian target of rapamycin inhibitor (mTOR-I) widely used in transplantation that may determine some severe adverse events, including pulmonary fibrosis. The pathogenic mechanism of mTOR-I-associated pulmonary toxicity is still unclear, but epithelial to mesenchymal transition (EMT) of bronchial/pulmonary cells may play a role. METHODS: Three cell lines-human type II pneumocyte-derived A549, normal bronchial epithelial, and bronchial epithelial homozygous for the delta F508 cystic fibrosis-causing mutation-were treated with EVE or tacrolimus at different concentrations. Real-time polymerase chain reaction and immunofluorescence were used to evaluate mRNA and protein levels of EMT markers (alpha-SMA, vimentin, fibronectin). Subsequently, in 13 EVE- and 13 tacrolimus-treated patients we compared the rate of lung fibrosis, estimated by an arbitrary pulmonary fibrosis index score (PFIS). RESULTS: Biomolecular experiments demonstrated that high doses of EVE (100 nM) up-regulated EMT markers in all cell lines at both gene- and protein level. High concentrations of EVE were also able to reduce the mRNA levels of epithelial markers (E-cadherin and ZO-1) and to induce the phosphorylation of AKT. In the in vivo part of the study, PFIS was significantly higher in the EVE-group than the tacrolimus-group (p = 0.03) and correlated with trough levels (R2 = 0.35). CONCLUSIONS: Our data reveal, for the first time, a dose-dependent EVE-induced EMT in airway cells. They suggest that clinicians should employ, wherever possible, low dosages of mTOR-Is in transplant recipients, assessing periodically their pulmonary function.

Epithelial to mesenchymal transition in the liver field: the double face of Everolimus in vitro.

BACKGROUND: Everolimus (EVE), a mammalian target of rapamycin inhibitor, has been proposed as liver transplant immunosuppressive drug, gaining wide interest also for the treatment of cancer. Although an appropriate tolerance, it may induce several adverse effects, such as fibro-interstitial pneumonitis due to the acquisition of activated myofibroblasts. The exact molecular mechanism associated with epithelial to mesenchymal transition (EMT) may be crucial also in the liver context. This work examines the role and the molecular mediators of EMT in hepatic stellate cell (HSC) and human liver cancer cells (HepG2) and the potential role of EVE to maintain the epithelial phenotype rather than to act as a potential initiators of EMT. METHODS: Real time-PCR and western blot have been used to assess the capability of EVE at low-therapeutic (10 nM) and high (100 nM) dose to induce an in vitro EMT in HSC and HepG2. RESULTS: Biomolecular experiments demonstrated that low concentration of EVE (10 nM) did not modify the gene expression of alpha-smooth muscle actin (α-SMA), Vimentin (VIM), Fibronectin (FN) in both HSC and HepG2 cells, whereas EVE at 100 nM induced a significant over-expression of all the three above-mentioned genes and an increment of α-SMA and FN protein levels. Additionally, 100 nM of EVE induced a significant phosphorylation of AKT and an up-regulation of TGF-ß expression in HSC and HepG2 cells. DISCUSSION: Our data, although obtained in an in vitro model, revealed, for the first time, that high concentration of EVE may induce EMT in liver cells confirming previous published evidences obtained in renal cells. Additionally, they suggested that mTOR-I should be administered at the lowest dose able to maximize their important and specific therapeutic properties minimizing or avoiding fibrosis-related adverse effects. CONCLUSIONS: In summary, if confirmed by additional studies, our results could be useful for researchers to standardize new therapeutic immunosuppressive and anticancer drugs protocols.

Sulodexide alone or in combination with low doses of everolimus inhibits the hypoxia-mediated epithelial to mesenchymal transition in human renal proximal tubular cells.

BACKGROUND: Prolonged cold ischemia time, the period from the start of perfusion with cold preservation fluid after cessation of circulation due to arterial clamping until transplantation in the recipient, could induce epithelial-to-mesenchymal transition (EMT) in renal tubular cells, a process associated with chronic graft damage. In this context, everolimus (EVE) and sulodexide (SUL) could potentially slow down this process. METHODS: To assess whether SUL (50 µg/ml), EVE (at 5, 10, 100 nM) or their combination were able to inhibit EMT in human renal epithelial proximal tubular cells (HK-2) reoxygenated after 24 h under hypoxic conditions, we used classical biomolecular strategies. RESULTS: Hypoxia induced upregulation of alpha smooth muscle actin (α-SMA), fibronectin (FN) and vimentin at gene-expression and α-SMA and FN at protein levels. However, the addition, after reoxygenation, of SUL plus low-dose EVE (5 nM) to the cell culture reversed this condition. Moreover, SUL and EVE were able to inhibit the hypoxia-induced Akt phosphorylation in HK2 cells and their morphological changes. Similarly, SUL was able to reverse the hyper-expression of EMT markers induced by high EVE dosage (100 nM) in cells cultured under both normoxic and hypoxic conditions. CONCLUSIONS: Our data reveal, for the first time, that sulodexide, alone or combined to low doses of everolimus, may hinder EMT in renal cells following hypoxia or minimize fibrotic complications due to high dosage of mammalian target of rapamycin inhibitors.