Trans-crocetin improves amyloid-ß degradation in monocytes from Alzheimer's Disease patients.

Herbal medicines have been recently employed in research and clinical studies for the potential treatment of behavioral and psychological symptoms associated with Alzheimer's Disease (AD) and other types of dementia. The present study investigates the effect of trans-crocetin, an active constituent of Crocus sativus L., to restore in vitro the reduced ability of AD patients' monocytes to degrade amyloid-ß(1-42) (Aß42). CD14+ monocytes from 22 sporadic AD patients with moderate cognitive impairment were isolated; then, the role of trans-crocetin, purified from saffron extracts, was evaluated in terms of Aß42 degradation rate through flow cytometry, as well as expression of cathepsin B by Western blotting. We observed that low micromolar doses of trans-crocetin enhanced Aß42 degradation in AD monocytes through the upregulation of the lysosomal protease cathepsin B. CA074Me, a potent and selective cathepsin B inhibitor, counteracted such trans-crocetin-induced effect. These data suggest that the carotenoid trans-crocetin improves in vitro the clearance of Aß42 through the involvement of cathepsin B, and this could be of value in developing a new anti-amyloid strategy in AD.

Biochemical parameters of renal impairment/injury and surrogate markers of nephron number in intrauterine growth-restricted and preterm neonates at 30-40 days of postnatal corrected age.

BACKGROUND: Premature and/or intrauterine growth-restricted neonates have an increased risk of developing postnatal renal injuries in later life. Studies on renal physiology in these neonates at a corrected age of 30-40 days are scarce and mostly relate to preterm infants. The data from these studies often lack the results of correlation analyses between biochemical parameters and nephron number-data which could provide additional insight and/or improve recognition of individuals at higher risk of renal failure. METHODS: Urinary total protein and albumin levels and N-acetyl-ß-D-glucosaminidase and cathepsin B activity were evaluated in preterm and intrauterine growth-restricted infants at a corrected age of 30-40 days and compared to data from a healthy control neonate population. The data were then associated with predominant susceptibility factors of renal damage related to low nephron number, such as gestational age, birth weight, total renal volume and renal cortex volume. RESULTS: Compared to the control neonate population, we found significantly increased levels of all biochemical parameters tested in the intrauterine growth-restricted neonates, whereas in the preterm infants we observed a significant increase in cathepsin B activity, total protein level and, to a lesser extent, albumin level. Cathepsin B activity showed a significant, strong and inverse correlation with all surrogate markers of nephron number and was also strongly and positively correlated with urinary albumin level. CONCLUSIONS: At this postnatal age, we found that lower nephron number in low birth weight neonates was associated to tubular impairment/injury that could be concurrent with a dysfunction of glomerular permeability. Urinary cathepsin B activity may be a candidate marker for the early prediction of renal susceptibility to damage in low birth weight neonates.

Increased Urinary Cystatin-C Levels Correlate with Reduced Renal Volumes in Neonates with Intrauterine Growth Restriction.

BACKGROUND: Exposure to intrauterine growth retardation (IUGR) can have a negative impact on nephrogenesis resulting in limited fetal kidney development and supporting the hypothesis that IUGR represents a risk for renal function and long-term renal disease. Cystatin-C (Cys-C), a strong inhibitor of cysteine proteinases, is freely filtered by the kidney glomerulus and is reabsorbed by the tubules, where it is almost totally catabolized; what remains is subsequently eliminated in urine. In tubular diseases and in hyperfiltration conditions, it seems reasonable to postulate that Cys-C degradation would decrease, and consequently an increase in its urinary elimination would be observed. OBJECTIVES: The aim of this study was to investigate the urinary excretion of Cys-C simultaneously with the assessment of renal volumes in adequate for gestational age (AGA) and IUGR neonates in order to identify its clinical value in IUGR. METHODS: Urinary Cys-C levels were measured using the enzyme immunoassay DetectX® Human Cystatin C kit in IUGR and AGA neonates. Whole renal and renal cortex volumes were assessed with ultrasounds (Vocal II; Software, GE). RESULTS: Urinary Cys-C levels in IUGR were significantly higher than those found in AGA and were negatively correlated to reduced whole renal and renal cortex volumes. CONCLUSIONS: The increased levels of Cys-C in the urine of neonates with IUGR were significantly associated with reduced renal/renal cortex volumes, suggesting that Cys-C could be taken as a surrogate of nephron mass. It also could be used as an early biochemical marker to identify IUGR neonates at high risk of developing long-term renal disease and to select patients for monitoring during childhood.

miR128 up-regulation correlates with impaired amyloid ß(1-42) degradation in monocytes from patients with sporadic Alzheimer's disease.

Alzheimer's disease (AD), the most common form of dementia in elderly individuals, is characterized by neurofibrillary tangles, extracellular amyloid-ß (Aß) plaques and neuroinflammation. New evidence has shown that the lysosomal system might be a crossroad in which etiological factors in AD pathogenesis converge. This study shows that several lysosomal enzymes, including Cathepsin B, D, S, ß-Galactosidase, α-Mannosidase, and ß-Hexosaminidase, were less expressed in monocytes and lymphocytes from patients with a clinical diagnosis of AD dementia compared with cells from healthy controls. In vitro experiments of gain and loss of function suggest that down-regulation is a direct consequence of miR-128 up-regulation found in AD-related cells. The present study also demonstrates that miR-128 inhibition in monocytes from AD patients improves Aß(1-42) degradation. These results could contribute to clarify the molecular mechanisms that affect the imbalanced Aß production/clearance involved in the pathogenesis of AD.

Long-term miR-669a therapy alleviates chronic dilated cardiomyopathy in dystrophic mice.

BACKGROUND: Dilated cardiomyopathy (DCM) is a leading cause of chronic morbidity and mortality in muscular dystrophy (MD) patients. Current pharmacological treatments are not yet able to counteract chronic myocardial wastage, thus novel therapies are being intensely explored. MicroRNAs have been implicated as fine regulators of cardiomyopathic progression. Previously, miR-669a downregulation has been linked to the severe DCM progression displayed by Sgcb-null dystrophic mice. However, the impact of long-term overexpression of miR-669a on muscle structure and functionality of the dystrophic heart is yet unknown. METHODS AND RESULTS: Here, we demonstrate that intraventricular delivery of adeno-associated viral (AAV) vectors induces long-term (18 months) miR-669a overexpression and improves survival of Sgcb-null mice. Treated hearts display significant decrease in hypertrophic remodeling, fibrosis, and cardiomyocyte apoptosis. Moreover, miR-669a treatment increases sarcomere organization, reduces ventricular atrial natriuretic peptide (ANP) levels, and ameliorates gene/miRNA profile of DCM markers. Furthermore, long-term miR-669a overexpression significantly reduces adverse remodeling and enhances systolic fractional shortening of the left ventricle in treated dystrophic mice, without significant detrimental consequences on skeletal muscle wastage. CONCLUSIONS: Our findings provide the first evidence of long-term beneficial impact of AAV-mediated miRNA therapy in a transgenic model of severe, chronic MD-associated DCM.

Expression of cathepsins S and D signals a distinctive biochemical trait in CD34+ hematopoietic stem cells of relapsing-remitting multiple sclerosis patients.

BACKGROUND: The elucidation of mechanistic aspects of relapsing-remitting multiple sclerosis (RRMS) pathogenesis may offer valuable insights into diagnostic decisions and medical treatment. RESULTS: Two lysosomal proteases, cathepsins S and D (CatS and CatD), display an exclusive pattern of expression in CD34(+) hematopoietic stem cells (HSCs) from peripheral blood of acute MS (A-MS) patients (n = 20). While both enzymes normally exist as precursor forms in the HSCs of healthy individuals (n = 30), the same cells from A-MS patients consistently exhibit mature enzymes. Further, mature cathepsins are expressed at lower rates in stable MS subjects (S-MS, n = 15) and revert to precursor proteins after interferon-ß1a treatment (n = 5). Mature CatD and CatS were induced in HSCs of healthy donors that were either co-cultured with PBMCs of A-MS patients or exposed to their plasma, suggesting a functional involvement of soluble agents. Following HSC exposure to several cytokines known to be implicated in MS, and based on relative cytokine levels displayed in A-MS, S-MS and control individuals, we identified IL-16 as a specific cell signaling factor associated with cathepsin processing. CONCLUSIONS: These data point to an evident correlation between CatS and CatD expression and MS clinical stage, and define a biochemical trait in HSCs with functional, medical, and diagnostic relevance.

Nitric oxide depletion alters hematopoietic stem cell commitment toward immunogenic dendritic cells.

BACKGROUND: NO* is a key molecule involved in the regulation of cell survival, proliferation and differentiation in many cell types. In this study we investigated the contribution of NO* during the differentiation of human peripheral blood hemopoietic stem cells (CD34+HSCs) toward immunogenic dendritic cells (i-DCs). METHODS: We depleted autocrine NO* production, using NG-monomethyl-L-arginine monoacetate (L-NMMA) and paracrine NO', using oxy-hemoglobin (HbO2) as a NO* scavenger during in vitro differentiation of CD34+HSCs to i-DCs. We monitored the NO* level, cell proliferation, phenotype and differentiation potential. RESULTS: We found that the depletion of paracrine or autocrine NO* correlated with (I) an active proliferation state at the end of differentiation, when control cells were not proliferating; (II) a significant reduction in the expression levels of differentiative markers (CD1a and HLA-DR) with a parallel high expression of the CD34 marker (III) with a retrieved clonogenic ability compared to control cells. CONCLUSIONS: On the whole, our data indicate that the depletion of NO* during the commitment stage blocks CD34+HSC differentiation into i-DCs and maintains an undifferentiated, highly proliferating cell population, indicating/revealing a novel role for NO* in the commitment of CD34+HSCs into i-DCs. GENERAL SIGNIFICANCE: The essential finding of the present study is that NO*, produced in HSCs by NOS enzymes, may act as autocrine and paracrine effectors regulating the in vitro differentiation process of CD34+-HSCs toward i-DCs.

Biocompatible poly(L-lactide)/MWCNT nanocomposites: morphological characterization, electrical properties, and stem cell interaction.

The promising perspectives of PLLA-based nanostructured biomaterials and their relevance in tissue engineering are reported. Nanocomposites based on PLLA and MWCNTs are developed with an MWCNT content ranging from 0 to 3 wt%. The electrical properties show a percolation threshold within a range of 0.21-0.33 wt% MWCNTs, and the conductivity increases by six orders of magnitude. The surface structure shows changes with the carbon nanotube concentration. The functional role of MWCNTs incorporation in terms of interactions with adult stem cells suggests that PLLA/MWCNT nanocomposites are suitable substrates for primary stem cell culture.

Tuning multi/pluri-potent stem cell fate by electrospun poly(L-lactic acid)-calcium-deficient hydroxyapatite nanocomposite mats.

In this study, we investigated whether multipotent (human-bone-marrow-derived mesenchymal stem cells [hBM-MSCs]) and pluripotent stem cells (murine-induced pluripotent stem cells [iPSCs] and murine embryonic stem cells [ESCs]) respond to nanocomposite fibrous mats of poly(L-lactic acid) (PLLA) loaded with 1 or 8 wt % of calcium-deficient nanohydroxyapatite (d-HAp). Remarkably, the dispersion of different amounts of d-HAp to PLLA produced a set of materials (PLLA/d-HAp) with similar architectures and tunable mechanical properties. After 3 weeks of culture in the absence of soluble osteogenic factors, we observed the expression of osteogenic markers, including the deposition of bone matrix proteins, in multi/pluripotent cells only grown on PLLA/d-HAp nanocomposites, whereas the osteogenic differentiation was absent on stem-cell-neat PLLA cultures. Interestingly, this phenomenon was confined only in hBM-MSCs, murine iPSCs, and ESCs grown on direct contact with the PLLA/d-HAp mats. Altogether, these results indicate that the osteogenic differentiation effect of these electrospun PLLA/d-HAp nanocomposites was independent of the stem cell type and highlight the direct interaction of stem cell-polymeric nanocomposite and the mechanical properties acquired by the PLLA/d-HAp nanocomposites as key steps for the differentiation process.

Stem cell-biomaterial interactions for regenerative medicine.

The synergism of stem cell biology and biomaterial technology promises to have a profound impact on stem-cell-based clinical applications for tissue regeneration. Biomaterials development is rapidly advancing to display properties that, in a precise and physiological fashion, could drive stem-cell fate both in vitro and in vivo. Thus, the design of novel materials is trying to recapitulate the molecular events involved in the production, clearance and interaction of molecules within tissue in pathologic conditions and regeneration of tissue/organs. In this review we will report on the challenges behind translating stem cell biology and biomaterial innovations into novel clinical therapeutic applications for tissue and organ replacements (graphical abstract).

Knock-down of HEXA and HEXB genes correlate with the absence of the immunostimulatory function of HSC-derived dendritic cells.

In an attempt to investigate whether the genetic defect in the HEXA and HEXB genes (which causes the absence of the lysosomal ß-N-acetyl-hexosaminidase), are related to the wide inflammation in GM2 gangliosidoses (Tay-Sachs and Sandhoff disease), we have chosen the dendritic cells (DCs) as a study model. Using the RNA interference approach, we generated an in vitro model of HEXs knock-down immunogenic DCs (i-DCs) from CD34(+)-haemopoietic stem cells (CD34(+)-HSCs), thus mimicking the Tay-Sachs (HEXA-/-) and Sandhoff (HEXB-/-) cells. We showed that the absence of ß-N-acetyl-hexosaminidase activity does not alter the differentiation of i-DCs from HSCs, but it is critical for the activation of CD4(+)T cells because knock-down of HEXA or HEXB gene causes a loss of function of i-DCs. Notably, the silencing of the HEXA gene had a stronger immune inhibitory effect, thereby indicating a major involvement of ß-N-acetyl-hexosaminidase A isoenzyme within this mechanism.

Neural stem cell gene therapy ameliorates pathology and function in a mouse model of globoid cell leukodystrophy.

Murine neural stem cells (mNSCs), either naive or genetically modified to express supranormal levels of ß-galactocerebrosidase (GALC), were transplanted into the brain of Twitcher mice, a murine model of globoid cell leukodystrophy, a severe sphingolipidosis. Cells engrafted long-term into the host cytoarchitecture, producing functional GALC. Levels of enzyme activity in brain and spinal cord tissues were enhanced when GALC-overexpressing NSC were used. Enzymatic correction correlated with reduced tissue storage, decreased activation of astroglia and microglia, delayed onset of symptoms, and longer lifespan. Mechanisms underlying the therapeutic effect of mNSC included widespread enzyme distribution, cross-correction of host cells, anti-inflammatory activity, and neuroprotection. Similar cell engraftment and metabolic correction were reproduced using human NSC. Thus, NSC gene therapy rapidly reconstitutes sustained and long-lasting enzyme activity in central nervous system tissues. Combining this approach with treatments targeting the systemic disease associated with leukodystrophies may provide significant therapeutic benefit.

Development of a New Tool for 3D Modeling for Regenerative Medicine.

The effectiveness of therapeutic treatment based on regenerative medicine for degenerative diseases (i.e., neurodegenerative or cardiac diseases) requires tools allowing the visualization and analysis of the three-dimensional (3D) distribution of target drugs within the tissue. Here, we present a new computational procedure able to overcome the limitations of visual analysis emerging by the examination of a molecular signal within images of serial tissue/organ sections by using the conventional techniques. Together with the 3D anatomical reconstitution of the tissue/organ, our framework allows the detection of signals of different origins (e.g., marked generic molecules, colorimetric, or fluorimetric substrates for enzymes; microRNA; recombinant protein). Remarkably, the application does not require the employment of specific tracking reagents for the imaging analysis. We report two different representative applications: the first shows the reconstruction of a 3D model of mouse brain with the analysis of the distribution of the ß-Galactosidase, the second shows the reconstruction of a 3D mouse heart with the measurement of the cardiac volume.

Intrinsic cell memory reinforces myogenic commitment of pericyte-derived iPSCs.

Mesoangioblasts (MABs) are a subset of muscle-derived pericytes able to restore dystrophic phenotype in mice and dogs. However, their lifespan is limited and they undergo senescence after 25-30 population doublings. Recently, induced pluripotent stem cells (iPSCs) generated from reprogrammed fibroblasts have been demonstrated to have in vitro and in vivo myogenic potential when sorted for the SM/C-2.6 antigen. Furthermore, chimeric mice from mdx-iPSCs (DYS-HAC) cells showed tissue-specific expression of dystrophin. Nevertheless, myogenic differentiation protocols and the potential of iPSCs generated from different cell sources still present unanswered questions. Here we show that iPSCs generated from prospectively sorted MABs (MAB-iPSCs) are pluripotent as fibroblast-derived iPSCs (f-iPSCs). However, both teratoma formation and genetic cell manipulation assays identify a durable epigenetic memory in MAB-iPSCs, resulting in stronger myogenic commitment. Striated muscle tissue accounts for up to 70% of MAB-iPSC teratomas. Moreover, transfection with Pax3 and Pax7 induces a more robust myogenic differentiation in MAB-iPSCs than in f-iPSCs. A larger amount of CD56(+) progenitors can be sorted from the MAB-iPSCs differentiating pool and, after transplantation into αsg-KO mice, can efficiently participate to skeletal muscle regeneration and restore αsg expression. Our data strongly suggest that iPSCs are a heterogeneous population and, when generated from myogenic adult stem cells, they exhibit a stronger commitment, paving the way for creating custom-made cell protocols for muscular dystrophies.

Lysosomal ß-galactosidase and ß-hexosaminidase activities correlate with clinical stages of dementia associated with Alzheimer's disease and type 2 diabetes mellitus.

Multiple epidemiological studies have shown that individuals affected by type-2 diabetes mellitus (T2DM) carry a 2-to-5-fold higher risk of developing Alzheimer's disease (AD) when compared to non-diabetic subjects. Thus, biochemical parameters that can be easily and routinely assessed for high-confidence evaluation of diabetic conditions leading to AD (AD-T2DM) are regarded as efficient tools aimed at early diagnosis and, in turn, timely AD treatment. In this regard, the activity of lysosomal glycohydrolases may of use, in light of the implication of these enzymes in early events that underlie AD pathology and an overt correlation, in diabetes, between altered metabolic homeostasis, abnormal glycohydrolase secretion in body fluids, and occurrence of diabetic complications. Based on marked up-regulation previously shown in a peripheral, cell-based model of AD, we selected ß-Galactosidase, ß-Hexosaminidase, and α-Mannosidase to discriminate T2DM from AD-T2DM subjects. A screen of 109, 114, and 116 patients with T2DM, AD and AD-T2DM, respectively, was performed by testing enzyme activities in both blood plasma and peripheral blood mononuclear cells. Compared to age-matched, healthy controls (n = 122), ß-Galactosidase and ß-Hexosaminidase activities markedly diverged across the three groups, whereas virtually unchanged values were observed for α-Mannosidase. In particular, plasma ß-Galactosidase and ß-Hexosaminidase levels were higher in patients with AD-T2DM compared to those with T2DM, suggesting different mechanisms leading to enzyme secretion. Statistical analyses based on ROC curves showed that both ß-Galactosidase and ß-Hexosaminidase activities, either intracellular or plasma-secreted, may be used to discriminate AD patients from controls and AD-T2DM from T2DM patients.

Coordinated involvement of cathepsins S, D and cystatin C in the commitment of hematopoietic stem cells to dendritic cells.

The identity of biochemical players which underpin the commitment of CD34(+) hematopoietic stem cells to immunogenic or tolerogenic dendritic cells is largely unknown. To explore this issue, we employed a previously established cell-based system amenable to shift dendritic cell differentiation from the immunogenic into the tolerogenic pathway upon supplementation with a conventional cytokine cocktail containing thrombopoietin (TPO) and IL-16. We show that stringent regulation of cathepsins S and D, two proteases involved in antigen presentation, is crucial to engage cell commitment to either route. In response to TPO+IL-16-dependent signaling, both cathepsins undergo earlier maturation and down-regulation. Additionally, cystatin C orchestrates cathepsin S expression through a tight but reversible interaction that, based on a screen of adult stem cells from disparate origins, CD14(+) cells, primary fibroblasts and the MCF7 cell line, appears unique to CD34(+) stem cells from peripheral and cord blood. As shown by CD4(+) T cell proliferation in mixed-lymphocyte reactions, cell commitment to either pathway is disrupted upon cathepsin knockdown by RNAi. Surprisingly, similar effects were also observed upon gene overexpression, which prompts atypically accelerated maturation of cathepsins S and D in cells of the immunogenic pathway, similar to the tolerogenic route. Furthermore, RNAi studies revealed that cystatin C is a proteolytic target of cathepsin D and has a direct, causal impact on cell differentiation. Together, these findings uncover a novel biochemical cluster that is subject to time-controlled and rigorously balanced expression to mediate specific stem cell commitment at the crossroads towards tolerance or immunity.

Mechanotransduction: tuning stem cells fate.

It is a general concern that the success of regenerative medicine-based applications is based on the ability to recapitulate the molecular events that allow stem cells to repair the damaged tissue/organ. To this end biomaterials are designed to display properties that, in a precise and physiological-like fashion, could drive stem cell fate both in vitro and in vivo. The rationale is that stem cells are highly sensitive to forces and that they may convert mechanical stimuli into a chemical response. In this review, we describe novelties on stem cells and biomaterials interactions with more focus on the implication of the mechanical stimulation named mechanotransduction.

Gene expression profiling identifies ARSD as a new marker of disease progression and the sphingolipid metabolism as a potential novel metabolism in chronic lymphocytic leukemia.

BACKGROUND: Several studies demonstrated IGVH mutational status and ZAP70 expression as the most relevant prognostic markers in Chronic Lymphocytic Leukemia (CLL), suggesting the separation of two patient subgroups: with good mutated ZAP70 negative (MTZAP70(-) and poor unmutated ZAP70 positive (UMZAP70(+)) prognosis. DESIGN AND METHODS: We determined the gene expression of B cells in 112 CLL patients divided into three classes: class 1 with MTZAP70(-), class 2 with UMZAP70(+), and class 3 included both UMZAP70(-) and MTZAP70(+). RESULTS: We found LPL, AGPAT2, MBOAT1, CHPT1, AGPAT4, PLD1 genes encoding enzymes involved in lipid metabolism overexpressed in UMZAP70(+). In addition, this study identified ARSD, a gene belonging to the sphingolipid metabolism, as a new gene significantly overexpressed in UMZAP70(+) compared to MTZAP70(-). Western blots confirmed that ARSD protein levels were significantly different between the 3 classes of patients and normal controls. Statistical analysis identified a significant correlation between ARSD and IGVH; however, both ARSD protein level and IGVH were independently associated with the need for therapy of CLL patients. CONCLUSIONS: ARSD is a novel prognostic factor as the time to start therapy is shorter in patients with high levels of ARSD protein and sphingolipid metabolism could represent a new biological mechanism in CLL.

Identification of hematopoietic stem cell-specific miRNAs enables gene therapy of globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD; also known as Krabbe disease) is an invariably fatal lysosomal storage disorder caused by mutations in the galactocerebrosidase (GALC) gene. Hematopoietic stem cell (HSC)-based gene therapy is being explored for GLD; however, we found that forced GALC expression was toxic to HSCs and early progenitors, highlighting the need for improved regulation of vector expression. We used a genetic reporter strategy based on lentiviral vectors to detect microRNA activity in hematopoietic cells at single-cell resolution. We report that miR-126 and miR-130a were expressed in HSCs and early progenitors from both mice and humans, but not in differentiated progeny. Moreover, repopulating HSCs could be purified solely on the basis of miRNA expression, providing a new method relevant for human HSC isolation. By incorporating miR-126 target sequences into a GALC-expressing vector, we suppressed GALC expression in HSCs while maintaining robust expression in mature hematopoietic cells. This approach protected HSCs from GALC toxicity and allowed successful treatment of a mouse GLD model, providing a rationale to explore HSC-based gene therapy for GLD.