Gianna Galli

Osteogenic Differentiation of Human Adipose Tissue-Derived Stem Cells Is Modulated by the miR-26a Targeting of the SMAD1 Transcription Factor†‡

The molecular mechanisms that regulate hADSC differentiation toward osteogenic precursors and subsequent bone‐forming osteoblasts is unknown. Using osteoblast precursors obtained from subcutaneous human adipose tissue, we observed that microRNA‐26a modulated late osteoblasts differentiation by targeting the SMAD1 transcription factor.

Regulation of extracellular matrix synthesis by transforming growth factor β1 in human fat-storing cells

BACKGROUND Fat storing cells (FSC) are nonparenchymal liver cells generally considered the major source of the hepatic extracellular matrix (ECM). Transforming growth factor beta 1 (TGF-beta 1) is a potent regulator of ECM synthesis in various cell types. In this study, the effect of TGF-beta 1 on procollagen types I, III, IV, laminin (Lam), and fibronectin (FN) synthesis in cultured human FSCs was analyzed. METHODS FSCs were isolated from wedge sections of normal human livers. Morphological studies were performed by immunofluorescence and electron microscopy. ECM components in human FSC cultures were measured by an enzyme-linked immunosorbent assay. The expression of messenger RNA (mRNA) was evaluated by Northern blot and in situ hybridization. RESULTS Cultured human FSCs displayed numerous fat droplets in the perinuclear zone, and immunoreactivity for vimentin and alpha-smooth muscle actin. A weak nonfibrillar staining was observed by using a polyclonal antidesmin antibody. TGF-beta 1 induced a dose-dependent increase of procollagen I, III, and FN accumulation in human FSC cultures, whereas procollagen IV and Lam production was not affected. Furthermore, TGF-beta 1 increased the expression of alpha 1 (I), alpha 1 (III) procollagen, FN and TGF-beta 1 mRNA in human FSC cultures. CONCLUSIONS These data indicate that TGF-beta 1 is able to increase the synthesis of procollagen I, III, and FN in cultured human FSCs. Moreover, TGF-beta 1 can induce its own mRNA in the same cells.

Upregulation of Mesangial Growth Factor and Extracellular Matrix Synthesis by Advanced Glycation End Products Via a Receptor-Mediated Mechanism

Enhanced advanced glycosylation end product (AGE) formation has been shown to participate in the patho-genesis of diabetes-induced glomerular injury by mediating the increased extracellular matrix (ECM) deposition and altered cell growth and turnover leading to mesangial expansion. These effects could be exerted via an AGE-receptor-mediated upregulation of growth factors, such as the IGFs and transforming growth factor-β (TGF-β). We tested this hypothesis in human and rat mesangial cells grown on nonglycated or native bovine serum albumin (BSA), glycated BSA with AGE formation (BSA-AGE), or glycated BSA in which AGE formation was prevented by the use of aminoguanidine (BSA-AM), in the presence or absence of an antibody, α-p60, directed against the p60/OST protein named AGE-receptor 1 (AGE-R1), or normal control (pre-immune) serum. The mRNA and/or protein levels of IGF-I, IGF-II, IGF receptors, IGF binding proteins (IGFBPs), TGF-β1 and the ECM components fibronectin, laminin, and collagen IV were measured, together with cell proliferation. Both human and rat mesangial cells grown on BSA-AGE showed increased IGF-I and total and bioac-tive TGF-β medium levels and enhanced IGF-I, IGF-II, and TGF-β1 gene expression, compared with cells grown on BSA, whereas total IGFBP and IGFBP-3 medium content, IGF receptor density and affinity, and IGF-I receptor transcripts were unchanged. Moreover, cells grown on BSA-AGE showed increased ECM protein and mRNA levels versus cells cultured on BSA, whereas cell proliferation was unchanged in human mesangial cells and slightly reduced in rat mesangial cells. Growing cells on BSA-AM did not affect any of the measured parameters. Co-incubation of BSA-AGE with anti-AGE-R1, but not with pre-immune serum, prevented AGE-induced increases in IGF-I, TGF-β1, and ECM production or gene expression; anti-AGE-R1 also reduced growth factor and matrix synthesis in cells grown on BSA. These results demonstrate that mesangial IGF and TGF-β1 synthesis is upregulated by AGE-modified proteins through an AGE-receptor-mediated mechanism. The parallelism with increased ECM production raises the speculation that the enhanced synthesis of these growth factors resulting from advanced nonen-zymatic glycation participates in the pathogenesis of hyperglycemia-induced mesangial expansion.

Mechanisms of glucose-enhanced extracellular matrix accumulation in rat glomerular mesangial cells

In view of the importance of mesangial extracellular matrix (ECM) accumulation in the pathogenesis of diabetic glomerulosclerosis, we investigated 1) the effects of high glucose on ECM production by rat glomerular mesangial cells in culture (study A) and 2) the mechanisms underlying these effects, particularly the role of high sugar levels irrespective of intracellular metabolism (study B1) and of excess glucose disposal via the polyol pathway and associated biochemical alterations (study B2). Cells were cultured for 4 weeks, through six to eight passages, under the experimental conditions indicated below and, at each passage, the levels of fibronectin (FN), laminin (LAM), and collagen types I (C-I), III (C-III), IV (C-IV), and VI (C-VI) in media and cell extracts were quantified by an enzyme immunoassay. In study A, medium and cell content of matrix were assessed, together with [3H]leucine and [3H]thymidine incorporation into monolayers, polyol, fructose, and myo-inositol levels and the cytosolic redox state, in cells grown in high (30 mM) D-glucose or iso-osmolar mannitol versus cells cultured in normal (5.5 mM) D-glucose. FN, LAM, C-IV, and C-VI accumulation, but not C-I and C-III accumulation, was increased by 30 mM glucose, but not by iso-osmolar mannitol, when compared with 5.5 mM glucose, starting at week 2 and, except for C-VI, persisting throughout the remaining 2 weeks, whereas no change was observed in the measured indexes of total protein synthesis and DNA synthesis/cell proliferation. At any time point, polyol levels were increased, whereas myo-inositol was reduced by high glucose; in cells grown under elevated glucose concentrations, the lactate/pyruvate (L/P) ratio, an index of the cytosolic redox state, progressively increased. In study B1, the effects of high D-glucose were compared with those of iso-osmolar concentrations of sugars that are partly or not metabolized but are capable of inducing nonenzymatic glycosylation, such as D-galactose and L-glucose, and of mannitol, which does not enter the cell. Both D-galactose and L-glucose, but not mannitol, partly mimicked D-glucose-induced ECM overproduction. Although D-galactose is metabolized via the polyol pathway and alters the cytosolic redox state, ECM changes induced by high galactose were not prevented by the use of an aldose reductase inhibitor (ARI), Alcon 1576 (14 μM). In study B2, agents interfering with intracellular metabolism of excess glucose via the polyol pathway (14 μM Alcon 1576) and associated changes in myo-inositol metabolism (1 mM myo-inositol) and cytosolic redox state (1 mM sodium pyruvate, which corrects glucose-induced polyol pathway-dependent increased NADH/NAD+) were added to cells cultured in 30 and 5.5 mM D-glucose. Alcon 1576 inhibited polyol pathway activity with decreasing efficacy during the 4-week period, whereas myo-inositol and pyruvate produced complete and persistent prevention of reduced myo-inositol levels and increased L/P ratio, respectively. High glucose-induced ECM overproduction was transiently reduced by pyruvate and, to a lesser extent, by the ARI and myo-inositol. These results suggest that 1) high glucose selectively increases accumulation of basement membrane components and 2) multiple mechanisms seem to be operating in the pathogenesis of glucose-induced ECM overproduction, including elevated sugar levels per se, possibly via nonenzymatic glycosylation, and to a lesser extent, intracellular glucose metabolism via the polyol pathway and associated changes in myo-inositol metabolism and cytosolic redox state.

The Negative Feedback-Loop between the Oncomir Mir-24-1 and Menin Modulates the Men1 Tumorigenesis by Mimicking the “Knudson’s Second Hit”

Multiple endocrine neoplasia type 1 (MEN1) syndrome is a rare hereditary cancer disorder characterized by tumors of the parathyroids, of the neuroendocrine cells, of the gastro-entero-pancreatic tract, of the anterior pituitary, and by non-endocrine neoplasms and lesions. MEN1 gene, a tumor suppressor gene, encodes menin protein. Loss of heterozygosity at 11q13 is typical of MEN1 tumors, in agreement with the Knudson’s two-hit hypothesis. In silico analysis with Target Scan, Miranda and Pictar-Vert softwares for the prediction of miRNA targets indicated miR-24-1 as capable to bind to the 3′UTR of MEN1 mRNA. We investigated this possibility by analysis of miR-24-1 expression profiles in parathyroid adenomatous tissues from MEN1 gene mutation carriers, in their sporadic non-MEN1 counterparts, and in normal parathyroid tissue. Interestingly, the MEN1 tumorigenesis seems to be under the control of a “negative feedback loop” between miR-24-1 and menin protein, that mimics the second hit of Knudson’s hypothesis and that could buffer the effect of the stochastic factors that contribute to the onset and progression of this disease. Our data show an alternative way to MEN1 tumorigenesis and, probably, to the “two-hit dogma”. The functional significance of this regulatory mechanism in MEN1 tumorigenesis is also the basis for opening future developments of RNA antagomir(s)-based strategies in the in vivo control of tumorigenesis in MEN1 carriers.

Increased activity of the insulin-like growth factor system in mesangial cells cultured in high glucose conditions. Relation to glucose-enhanced extracellular matrix production.

SummaryRecent evidence suggests that several growth factors participate in diabetic glomerular disease by mediating increased extracellular matrix accumulation and altered cell growth and turnover leading to mesangial expansion. Transforming growth factor (TGF)-β has been demonstrated to be upregulated both in vivo and in vitro, whereas studies on the activity of the renal insulin-like growth factor (IGF) system in experimental diabetes have provided conflicting results. We investigated the effects of prolonged exposure (4 weeks) of cultured human and rat mesangial cells to high (30 mmol/1) glucose vs iso-osmolar mannitol or normal (5.5 mmol/1) glucose levels on: 1) the autocrine/paracrine activity of the IGF system (as assessed by measuring IGF-I and II, IGF-I and II receptors, and IGF binding proteins); and, in parallel, on 2) TGF-βl gene expression; 3) matrix production; and 4) cell proliferation. High glucose levels progressively increased the medium content of IGF-I and the mRNA levels for IGF-I and IGF-II, increased IGF-I and IGF-II binding and IGF-I receptor gene expression, and reduced IGF binding protein production. TGF-βl transcripts and matrix accumulation and gene expression were increased in parallel, whereas cell proliferation was reduced. Iso-osmolar mannitol did not affect any of the above parameters. These experiments demonstrated that high glucose levels induce enhanced mesangial IGF activity, together with enhanced TGF-βl gene expression, increased matrix production, and reduced cell proliferation. It is possible that IGFs participate in mediating diabetes-induced changes in matrix turnover leading to mesangial expansion, by acting in a paracrine/autocrine fashion within the glomerulus. [Diabetologia (1996) 39: 775-784]

Acetaldehyde-protein adducts, but not lactate and pyruvate, stimulate gene transcription of collagen and fibronectin in hepatic fat-storing cells

Hepatic fibrosis is an important morphological feature of alcohol-induced liver injury. We previously reported that acetaldehyde, but not ethanol can stimulate type I collagen and fibronectin synthesis in cultures of rat fat-storing cells (FSC) by increasing transcription of the specific genes. The effect of lactate and pyruvate was studied on collagen I, III, fibronectin accumulation by cultured rat FSCs and it was investigated whether acetaldehyde could increase procollagen I and fibronectin gene transcription through the formation of protein adducts. Lactate and pyruvate (5, 15 and 25 mmol/l) did not significantly affect collagen I, III and fibronectin production by cultured FSCs. Pyridoxal-phosphate and p-hydroxymecuribenzoate (inhibitors of acetaldehyde-protein adduct formation) blocked the stimulatory effect of acetaldehyde on procollagen I and fibronectin gene transcription. These data suggest that ethanol may act as a liver fibrogenic factor through acetaldehyde, its immediate metabolite, whereas lactate does not seem to play a role. Acetaldehyde might stimulate gene transcription of extracellular matrix components by liver FSCs through the formation of adducts with proteins.

The Regulatory Network Menin-MicroRNA 26a As a Possible Target for RNA-Based Therapy of Bone Diseases

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression, interplaying with transcription factors in complex regulatory networks. Menin is the product of the MEN1 oncosuppressor gene, responsible for multiple endocrine neoplasia type 1 syndrome. Recent data suggest that menin functions as a general regulator of transcription. Menin expression modulates mesenchymal cell commitment to the myogenic or osteogenic lineages. The microRNA 26a (miR-26a) modulates the expression of SMAD1 protein during the osteoblastic differentiation of human adipose tissue-derived stem cells (hADSCs). We used siRNA silencing against MEN1 mRNA and pre-miR-26 mimics to study the interplay between them and to investigate the interplay between menin and miR-26a as regulators of osteogenic differentiation in the hADSCs. We found that in hADSCs the siRNA-induced silencing of MEN1 mRNA resulted in a down regulation of miR-26a, with a consequent up-regulation of SMAD1 protein. Chromatin immunoprecipitation (ChIP) showed that menin occupies the miR-26-a gene promoter, thus inducing its expression and confirming that menin is a positive regulator of miR-26a. In conclusion, results from this study evidenced, for the first time, a direct interaction between menin transcription factor and miRNA, interaction that seems to play a pivotal role during the hADSCs osteogenesis, thus suggesting a novel target for bone disease RNA-based therapy.

Methodological models for in vitro amplification and maintenance of human articular chondrocytes from elderly patients.

Articular cartilage defects, an exceedingly common problem closely correlated with advancing age, is characterized by lack of spontaneous resolution because of the limited regenerative capacity of adult articular chondrocytes. Medical and surgical therapies yield unsatisfactory short-lasting results. Recently, cultured autologous chondrocytes have been proposed as a source to promote repair of deep cartilage defects. Despite encouraging preliminary results, this approach is not yet routinely applicable in clinical practice, but for young patients. One critical points is the isolation and ex vivo expansion of large enough number of differentiated articular chondrocytes. In general, human articular chondrocytes grown in monolayer cultures tend to undergo dedifferentiation. This reversible process produces morphological changes by which cells acquire fibroblast-like features, loosing typical functional characteristics, such as the ability to synthesize type II collagen. The aim of this study was to isolate human articular chondrocytes from elderly patients and to carefully characterize their morphological, proliferative, and differentiative features. Cells were morphologically analyzed by optic and transmission electron microscopy (TEM). Production of periodic acid-schiff (PAS)-positive cellular products and of type II collagen mRNA was monitored at different cellular passages. Typical chondrocytic characteristics were also studied in a suspension culture system with cells encapsulated in alginate-polylysine-alginate (APA) membranes. Results showed that human articular chondrocytes can be expanded in monolayers for several passages, and then microencapsulated, retaining their morphological and functional characteristics. The results obtained could contribute to optimize expansion and redifferentiation sequences for applying cartilage tissue engineering in the elderly patients.

Osteodifferentiation of Human Preadipocytes Induced by Strontium Released from Hydrogels

In recent years, there has been an increasing interest in interactive application principles of biology and engineering for the development of valid biological systems for tissue regeneration, such as for the treatment of bone fractures or skeletal defects. The application of stem cells together with biomaterials releasing bioactive factors promotes the formation of bone tissue by inducing proliferation and/or cell differentiation. In this study, we used a clonal cell line from human adipose tissue-derived mesenchymal stem cells (hADSCs or preadipocytes), named PA2-E12, to evaluate the effects of strontium (Sr2+) released in the culture medium from an amidated carboxymethylcellulose (CMCA) hydrogel enriched with different Sr2+ concentrations on osteodifferentiation. The osteoinductive effect was evaluated through both the expression of alkaline phophatase (ALP) activity and the hydroxyapatite (HA) production during 42 days of induction. Present data have shown that Sr2+ released from CMCA promotes the osteodifferentiation induced by an osteogenic medium as shown by the increase of ALP activity at 7 and 14 days and of HA production at 14 days. In conclusion, the use of biomaterials able to release in situ osteoinductive agents, like Sr2+, could represent a new strategy for future applications in bone tissue engineering.