Search

The presence of multipotent cells in several adult and embryo-related tissues opened new paths for their use in regenerative medicine. Extraembryonic tissues such as umbilical cord are considered a promising source of stem cells, potentially useful in therapy. The characterization of cells from the umbilical cord matrix (Wharton''s Jelly) and amniotic membrane revealed the presence of a population of mesenchymal-like cells, sharing a set of core-markers expressed by "mesenchymal stem cells". Several reports enlightened the differentiation capabilities of these cells, even if at times the lack of an extensive characterization of surface markers and immune co-stimulators expression revealed hidden pitfalls when in vivo transplantation was performed. The present work describes a novel isolation protocol for obtaining mesenchymal stem cells from the umbilical cord matrix. These cells are clonogenic, retain long telomeres, can undergo several population doublings in vitro, and can be differentiated in mature mesenchymal tissues as bone and adipose. We describe for the first time that these cells, besides expressing all of the core-markers for mesenchymal stem cells, feature also the expression, at both protein and mRNA level, of tolerogenic molecules and markers of all the three main lineages, potentially important for both their differentiative potential as well as immunological features.

Mesenchymal stem cells (MSC) are considered promising tools in regenerative medicine applications. Even if they are already applied clinically, their extended characterization is being increasingly viewed as a needed feature, in order to avoid contrasting results when translating “in vitro” experiments to “in vivo” approaches. We recently demonstrated in human MSC isolated from the umbilical cord matrix (HEMSC) the expression of novel markers indicative of their stemness, as well as differentiative and immune properties [1]. HEMSC were cultured and subject to multiple molecular and morphological analyses to determine the expression of markers of interest. Undifferentiated HEMSC expressed immunoregulatory molecules, both surface antigens and secreted cytokines. In addition to the differential expression of B7 co-stimulators (CD80+/CD86-), HEMSC expressed pregnancy-specific immunomodulatory molecules, together with non-classical type Ib MHC antigens. In fact, besides HLA-A and HLA-G, HEMSC expressed both HLA-E and HLA-F, two tolerance-promoting HLAs, involved in attenuating NK- and T-cytotoxic responses. In the continued characterization of these cells, we assessed the expression of endoderm-related molecules, suggestive of their possible transdifferentiation towards hepatocytes. In addition to HNF4α and endoderm-related GATA factors (GATA-4,-5,-6), undifferentiated cells expressed also albumin, cytokeratins 18 and 19, and further hepatocyte-enriched transcription factors (as HNF1α, and HNF1β), thus suggesting the possibility to be successfully differentiated towards hepatocytes [2]. The immunological and differentiative properties of human extraembryonic MSC are under intense investigation. Our data suggest that HEMSC bear new markers and have a hypoimmunogenic phenotype which can be a favoring factor to ensure the engraftment of “in vivo” transplanted cells.

In recent years, human mesenchymal stem cells (MSC) have been extensively studied. Their key characteristics of long-term self-renewal and a capacity to differentiate into diverse mature tissues favour their use in regenerative medicine applications. Stem cells can be found in embryonic and extra-embryonic tissues as well as in adult organs. Several reports indicate that cells of Wharton's jelly (WJ), the main component of umbilical cord extracellular matrix, are multipotent stem cells, expressing markers of bone marrow mesenchymal stem cells (BM-MSC), and giving rise to different cellular types of both connective and nervous tissues. Wharton's jelly mesenchymal stem cells (WJ-MSC) express markers previously characterized in embryonic stem cells (ESC), such as Nanog and Oct3/4A. WJ-MSC further emerge as promising hypoimmunogenic cells, due to the expression of molecules able to modulate NK cells and expand regulatory T cells populations. Moreover, it is now accepted that the differentiative capacities of such cells span all the mesoderm-derived tissues, extending to neuroectodermal as well as endodermal lineages. In this review we compare very recent data on the potential of WJ-MSC to undergo hepatocyte-like differentiation with the results obtained from other adult MSC populations. Data in the literature strongly suggest that WJ-MSC can differentiate into diverse cell types, showing a unique ability to cross lineage borders. This, together with their in vitro proliferative potential and their immunoregulatory features, renders these cells extremely promising for regenerative medicine applications in different pathological settings.

Endothelial cells are key modulators of diverse physiological processes, and their impaired function is a cause of numerous cardiovascular diseases. Under physiologic condition, the reactive oxygen and nitrogen mediators in endothelia lead to the signal propagation of the initial stimulus, by forming molecules with a longer half-life like hydrogen peroxide. Hydrogen peroxide is the focus of growing attention in endothelial biology, and consequently the enzymes involved in its generation and clearance are viewed as novel mediators of great importance. In particular, among peroxidases, myeloperoxidase is recognized as a key enzyme, capable of impairing intracellular NO reservoirs as well as producing oxidized amino acids such as 3-chlorotyrosine or 3-nitrotyrosine. This process switches the functional pathways from normal signalling to a condition characterized by oxidative and/or nitrosative stress. Understanding the molecular mechanisms involved in these stress responses in endothelium will lead to better therapeutic strategies for oxidative stress-driven cardiovascular diseases.

The presence of a reciprocal link between inflammation and oxidative/nitrosative stress has been postulated in chronic heart failure (CHF). We aimed to determine signs of nitrosative stress in serum/plasma of CHF patients. ELISA tests were used for quantification of serum/plasma levels of Nitrotyrosine (NT), H(2)O(2), total NO, nitrite (NO(2)(-)), myeloperoxidase (MPO), Tumor Necrosis Factor-alpha (TNFalpha) and pro-Brain Natriuretic Peptide (proBNP) in 66 CHF patients (9 in NYHA I, 34 NYHA II, 23 NYHA III) and in 14 age-matched healthy subjects. NT levels were higher in NYHA III CHF patients compared to NYHA II (p

Airway epithelial cells modulate bronchial remodelling in COPD and asthma. TGF-β1 up-regulation was observed in the bronchial epithelial cells of asthmatics and COPD patients. TGF-β is involved in airways remodelling mainly acting via the Smad pathway. Increased CCL5 was also observed in bronchi of patients with severe COPD. We investigated the effects of TGF-β1 on collagen type I, Smad 3-4 and 7 expression and the effects of CCL5 on TGF-β1-induced collagen production in human bronchial epithelial cells (16HBE).Cells were treated with 10ng/ml of TGF-β1, 10ng/ml of CCL5 and 10ng/ml of both TGF-β1 and CCL5 for 0,3 and 24 hours.TGF-b1 increased Smad3,Smad4 and collagen type I(p=0.0472, p=0.0433 and p

Summary There are increased numbers of activated T lymphocytes in the bronchial mucosa of stable chronic obstructive pulmonary disease (COPD) patients. T helper type 17 (Th17) cells release interleukin (IL)-17 as their effector cytokine under the control of IL-22 and IL-23. Furthermore, Th17 numbers are increased in some chronic inflammatory conditions. To investigate the expression of interleukin (IL)-17A, IL-17F, IL-21, IL-22 and IL-23 and of retinoic orphan receptor RORC2, a marker of Th17 cells, in bronchial biopsies from patients with stable COPD of different severity compared with age-matched control subjects. The expression of IL-17A, IL-17F, IL-21, IL-22, IL-23 and RORC2 was measured in the bronchial mucosa using immunohistochemistry and/or quantitative polymerase chain reaction. The number of IL-22+ and IL-23+ immunoreactive cells is increased in the bronchial epithelium of stable COPD compared with control groups. In addition, the number of IL-17A+ and IL-22+ immunoreactive cells is increased in the bronchial submucosa of stable COPD compared with control non-smokers. In all smokers, with and without disease, and in patients with COPD alone, the number of IL-22+ cells correlated significantly with the number of both CD4+ and CD8+ cells in the bronchial mucosa. RORC2 mRNA expression in the bronchial mucosa was not significantly different between smokers with normal lung function and COPD. Further, we report that endothelial cells express high levels of IL-17A and IL-22. Increased expression of the Th17-related cytokines IL-17A, IL-22 and IL-23 in COPD patients may reflect their involvement, and that of specific IL-17-producing cells, in driving the chronic inflammation seen in COPD.

Mesenchymal stem cells (MSC) are pluripotent cells which reside in several adult organs, including heart, even if heart regeneration in vivo is still a poorly comprised phenomenon. Contrasting literature reports suggest that several efforts should be made to better characterize resident or migrating MSC populations (for both markers expression and immunogenic potential) prior to their effective use for regenerative medicine applications in heart diseases. We developed a new protocol to obtain human sub-endocardial MSC (HSE-MSC) from post-infarct hearts explanted from chronic heart failure (CHF) patients undergoing heart transplantation. We characterized HSE-MSC by immunocytochemistry (ICC) and RT-PCR, to assess their adherence to the MSC phenotype. The differentiative ability towards the adicpocyte and the osteoblast lineage was assessed using standardized procedures (1). Self-renewal was evaluated by clonogenicity experiments. Karyotype analyses and telomere length assays were further performed. HSE-MSC were successfully isolated and expanded in vitro. These cells spontaneously showed the expression of several key MSC markers. ICC and RT-PCR analyses revealed the expression of c-kit (CD117), CD10, CD13, CD44, CD73, CD90, CD105, CD166, while hematopoietic/endothelial molecules (CD4, CD8, CD20, CD31, CD34, CD45) were absent. Moreover, HSE-MSC expressed key markers of the embryonic stem cell phenotype (Oct3/4 and Nanog), were further positive for the expression of transcription factors involved in heart development (GATA-4, GATA-6, Nkx2.5, Mef2C, Myocardin, ISL-1), but lacked, when kept undifferentiated, the expression of mature myocyte markers (as MYL-2). We assessed for the first time in HSE-MSC the expression of a favorable panel of markers suggesting hypoimmunogenicity of these cells, a positive feature for subsequent in vivo applications. HSE-MSC were differentiated towards the adipogenic and osteoblastic lineage, as demonstrated by histochemical stainings and RT-PCR detection of adipocyte-specific and osteoblast-specific markers. In addition, such cells were able undergoing more than 45 population doublings in culture, maintaining a normal karyotype and long telomeres. Their self-renewal was further demonstrated by clonogenicity experiments. Diseased human heart is still a source of MSC populations, even after developing chronic pathologies as CHF. HSE-MSC are capable of self-renewal, express several stem cell markers, as well as markers of the cardiac differentiation program, and have a hypoimmunogenic profile of surface markers. 1. La Rocca G et al. Histochem Cell Biol 2009 131:267-82

Increased oxidative stress has been implicated in the pathogenesis of a number of cardiovascular diseases. Recent findings suggest that myeloperoxidase (MPO) may play a key role in the initiation and maintenance of chronic heart failure (CHF) by contributing to the depletion of the intracellular reservoir of nitric oxide (NO). NO consumption through MPO activity may lead to protein chlorination or nitration, leading to tissue damage. Primary cultures of human endocardial endothelial cells (EEC) obtained at heart transplantation of patients with CHF and human umbilical vein endothelial cells (HUVEC) were subjected to oxidative stress by incubation with hydrogen peroxide at non lethal (60 microM) dose for different exposure times (3 and 6 h). Treated and control cells were tested by immunohistochemistry and RT-PCR for MPO and 3-chlorotyrosine expression. Both endothelial cell types expressed myeloperoxidase following oxidative stress, with higher levels in EEC. Moreover, 3-chlorotyrosine accumulation in treated cells alone indicated the presence of MPO-derived hypochlorous acid. Immunohistochemistry on sections from post-infarcted heart confirmed in vivo the endothelial positivity to MPO, 3-chlorotyrosine and, to a minor extent, nitrotyrosine. Immunohistochemical observations were confirmed by detection of MPO mRNA in both stimulated EEC and HUVEC cells. This study demonstrates for the first time that EEC can express MPO after oxidative stress, both in vitro and in vivo, followed by accumulation of 3-chlorotyrosine, an end product of oxidative stress. Deregulation of endothelial functions may contribute to the development of a number of cardiovascular diseases, including CHF. The results also highlight the notion that endothelium is not only a target but also a key player in oxidative-driven cardiovascular stress.

Telomeres are DNA-protein complexes playing an important role in the maintenance of genome integrity. Telomerase is the enzyme acting as a template for addition of new telomeric repeats; this addition is essential for those cellular populations that have proliferative and differentiative potential. Telomerase and associated proteins are essential in response to DNA damage. Moreover, telomere-associated proteins as TRF2 are involved in all signalling transduction pathway which drive cellular proliferation and differentiation. In somatic cells, shortening of telomeres contributes to the onset of senescence or apoptosis; tissues which require cellular renewal express telomerase activity in order to compensate telomeric shortening. Telomerase activity remains detectable in adult stem cells populations and in cells with high proliferative potential. Reactivation of telomerase activity may be a pathogenetic characteristic of neoplastic cellular population favouring tumoral proliferation and progression.

Airway epithelial cells play an important role in inflammatory, apoptotic and remodelling process associate with fibrosis and COPD. Transforming growth factor 1 (TGF-b1) is involved in airways remodelling by Smads signalling pathway. We investigated the role of TGF-b1 on type I collagen production and Smads (Smad 2-3-4-and 7) expression in bronchial epithelial cells (16HBE). Cells were treated with 1ng/ml and 10ng/ml of TGF-b1 for 0, 3 and 24 hours. With low dose of TGF-b1 we observed no significant variation on Smad2 mRNA expression for both times but a significant increased of Smad7 mRNA expression at 3h (p=0.0043) and a significant reduction of Smad3, Smad4 and Smad7 mRNA expression at 24h of treatment (p=0.0207, p=0.0381 and p=0.0087, respectively); TGF-b1 increases type I collagen mRNA expression (p=0.0190), but no significantly Smad3 activation after just 3 hours of treatment (p=0.0571). High dose of TGF-b1 increases Smad2 mRNA expression at 3h (p=0.0190), Smad3 and Smad4 mRNA (p=0.0081, p=0.0087, respectively) and type I collagen mRNA at 24h (p=0.00022); no change were observed on Smad7 mRNA level. Than we observed a time dependent phospho-Smad3 significant increase (p= 0.0286 for 3 and 24h) but a phospho-Smad2 expression reduction. These results let think that TGF-b1 is involved on type I collagen production in 16HBE in a time and dose-dependent manner and this effect is mediated more by phosphor-Smad3 than phosphor-Smad2. These data shows that low doses of TGF-b1 had not effect on inhibitor Smad7 mechanism whom balances and reduces Smad3 activation and type I collagen production, while high doses seems act on inhibitor Smad7 mRNA expression with increase on phospho-Smad3 and type I collagen expression in 16HBE.

AIMS: CD1a is a molecule belonging to the highly conserved group of CD1 proteins. Its expression in dendritic cells is related to the presentation of tumour-derived glycolipid antigens to T cells and, consequently, the development of a successful antitumour response. The aim was to investigate the presence of CD1a+ cells in both primary tumours and lymph nodes (LN) of a series of 35 invasive ductal carcinomas by both immunohistochemistry and reverse transcription-polymerase chain reaction. METHODS AND RESULTS: CD1a antigen was more expressed in N0 than N1 breast cancer (P < 0.0001) in both primary lesions and LN metastases and correlated positively and significantly with oestrogen (ER) (P = 0.0025) and progesterone (P = 0.0226) receptor (PR) status, as well as CD4+ and CD8+ T-lymphocyte infiltration. CONCLUSIONS: This is the first report to show a link between CD1a+ mononuclear cells in breast cancer and in paired LN metastases. The positive and significant correlations between the number of CD1a+ cells and positivity of the primary tumour for ER and PR suggest a possible role for CD1a as a prognostic marker for breast cancer, raising the possibility that hormone receptor-positive breast cancer patients may have a better prognosis in the presence of greater dendritic cell infiltration.

Transforming Growth Factor beta (TGFβ) cytokine plays an important role in normal pulmonary morphogenesis and function as well as in the pathogenesis of lung diseases. The principal signaling pathway downstream to activate TGFβ is the Smad pathway. Even though many studies have focused on Smads’ structural features and pathway, less is known about the possible relationship between protein and mRNA expression of Smads and lung diseases. This review will focus on Smads and sum up what is know about their role in some respiratory diseases: COPD,asthma end fibrosis

Background Exposure to cigarette smoke is considered a major risk factor for the development of lung diseases, since its causative role has been assessed in the induction and maintenance of an inflamed state in the airways. Lung fibroblasts can contribute to these processes, due to their ability to produce proinflammatory chemotactic molecules and extracellular matrix remodelling proteinases. Among proteolytic enzymes, gelatinases A and B have been studied for their role in tissue breakdown and mobilisation of matrix-derived signalling molecules. Multiple reports linked gelatinase deregulation and overexpression to the development of inflammatory chronic lung diseases such as COPD. Methods In this study we aimed to determine variations in the gelatinolytic pattern of human lung fibroblasts (HFL-1 cell line) exposed to cigarette smoke extract (CSE). Gelatinolytic activity levels were determined by using gelatin zymography for the in-gel detection of the enzymes (proenzyme and activated forms), and the subsequent semi-quantitative densitometric evaluation of lytic bands. Expression of gelatinases was evaluated also by RT-PCR, zymography of the cell lysates and by western blotting. Results CSE exposure at the doses used (1–10%) did not exert any significant cytotoxic effects on fibroblasts. Zymographic analysis showed that CSE exposure resulted in a linear decrease of the activity of gelatinase A. Control experiments allowed excluding a direct inhibitory effect of CSE on gelatinases. Zymography of cell lysates confirmed the expression of MMP-2 in all conditions. Semi-quantitative evaluation of mRNA expression allowed assessing a reduced transcription of the enzyme, as well as an increase in the expression of TIMP-2. Statistical analyses showed that the decrease of MMP-2 activity in conditioned media reached the statistical significance (p = 0.0031 for 24 h and p = 0.0012 for 48 h), while correlation analysis showed that this result was independent from CSE cytotoxicity (p = 0.7833 for both exposures). Conclusion Present work describes for the first time that, apart well characterized proinflammatory responses, human lung fibroblasts may react to CSE with a significant reduction of extracellular MMP-2 lytic activity. Therefore, fibroblasts may actively participate to the alteration of the proteolysis/antiproteolysis balance, which reflects the defective repair of the extracellular matrix. Such event should provide a further contribution to the maintenance of the inflamed state in the lungs.

BACKGROUND. The relation between smoking, chronic obstructive pulmonary disease (COPD), and lung cancer (LC) is an open field of investigation. A higher frequency of adenocarcinoma has been reported in patients with COPD. Heat shock proteins (Hsps) are implicated in tumoral cell growth and differentiation. The aim of the present study was to investigate the expression of Hsp60 and Hsp10 in bronchial biopsies from smokers with COPD and in 10 lung cancer patients and to evaluate the association between Hsps expression and carcinogenetic steps of LC. METHODS. An immunohistochemical study was performed for Hsp60 and Hsp10 in bronchial biopsies from 35 COPD (postbronchodilator forced expiratory volume in 1 second [FEV1]: 53 6 19% [mean 6 SD]) patients with a history of smoking (53 6 34 pack/years) and in 10 patients with adenocarcinoma or adenosquamous carcinoma (ASC). Immunopositivity was quantified in the bronchial epithelium and in specimens with ASC. RESULTS. In smokers with COPD, 10 out of 35 patients had a normal bronchial epithelium (NBE), 12 showed basal cell hyperplasia (BCH), 5 squamous metaplasia (SM), and 8 dysplasia (Dy). It was found that 58 6 23% and 54 6 23% of NBE and 48 6 29% and 52 6 26% of BCH expressed Hsp60 and Hsp10, respectively; in contrast, only 3 6 3% and 3.6 6 2% of SM, 1.9 6 4% and 1.1 6 2% of Dy espresse Hsp60 and Hsp10, respectively. ASC specimens were negative for Hsps proteins. Interestingly, NBE also present at the edges of ASC specimens was negative for Hsps proteins. CONCLUSIONS. The loss of Hsp60 and Hsp10 immunopositivity is related to the development and progression of bronchial cancer in smokers with COPD. BACKGROUND. The relation between smoking, chronic obstructive pulmonary disease (COPD), and lung cancer (LC) is an open field of investigation. A higher frequency of adenocarcinoma has been reported in patients with COPD. Heat shock proteins (Hsps) are implicated in tumoral cell growth and differentiation. The aim of the present study was to investigate the expression of Hsp60 and Hsp10 in bronchial biopsies from smokers with COPD and in 10 lung cancer patients and to evaluate the association between Hsps expression and carcinogenetic steps of LC. METHODS. An immunohistochemical study was performed for Hsp60 and Hsp10 in bronchial biopsies from 35 COPD (postbronchodilator forced expiratory volume in 1 second [FEV1]: 53±19% [mean ± SD]) patients with a history of smoking (53±34 pack/years) and in 10 patients with adenocarcinoma or adenosquamous carcinoma (ASC). Immunopositivity was quantified in the bronchial epithelium and in specimens with ASC. RESULTS. In smokers with COPD, 10 out of 35 patients had a normal bronchial epithelium (NBE), 12 showed basal cell hyperplasia (BCH), 5 squamous metaplasia (SM), and 8 dysplasia (Dy). It was found that 58 ± 23% and 54 ± 23% of NBE and 48 ± 29% and 52 ± 26% of BCH expressed Hsp60 and Hsp10, respectively; in contrast, only 3 ± 3% and 3.6 ± 2% of SM, 1.9 ± 4% and 1.1 ± 2% of Dy expressed Hsp60 and Hsp10, respectively. ASC specimens were negative for Hsps proteins. Interestingly, NBE also present at the edges of ASC specimens was negative for Hsps proteins. CONCLUSIONS. The loss of Hsp60 and Hsp10 immunopositivity is related to the development and progression of bronchial cancer in smokers with COPD.

Wharton’s jelly (WJ), the main constituent of umbilical cord, is a reliable source of mesenchymal stem cells (MSC). WJ-MSC show unique ability in crossing lineage borders. As other extraembryonic mesenchymal populations (placenta and amnionderived cells), WJ-MSC express several immunomodulatory molecules, essential during the initial phases of human development. Indeed, our recent work pointed out the expression of non-classical HLA molecules as HLA-G in such cells, together with a favorable combination of B7 costimulators. Very few data in literature suggest that some of the immune features of the naïve cells are maintained after performing differentiation. The aim of this work was extending the knowledge on the expression of immunomodulatory molecules by naïve and differentiated WJ-MSC. To this purpose, WJMSC underwent differentiation to osteoblasts, adipocytes and hepatocyte-like cells. Differentiated cells were characterized, by both RT-PCR, ICC and histological stains for the acquisistion of the desired phenotypical features. RT-PCR and ICC were used to investigate the differential expression of immune-related molecules in control and differentiated cells. WJ-MSC resulted expressing diverse immunomodulatory molecules which spans from non-classical type I HLAs (i.e. HLA-E, -F, -G) , to further members of the B7 family, and of the CEA superfamily, for all of which in vivo immunomodulating functions are known. In addition, we demonstrated for the first time that the expression of these molecules is maintained after performing osteogenic, adipogenic or hepatogenic differentiation. Further experiments are undergoing to better evaluating the implications of these findings in the evolving field of liver regenerative medicine.

In this review, we present recent insights on chronic heart failure (CHF) and the potential role of tumor necrosis factor (TNF)-alpha, interleukins, myeloperoxidase (MPO), and nitrosative stress in the progression of this disease process. Reactive oxygen species (ROS) are produced as a consequence of aerobic metabolism. Under physiologic conditions, their unfavourable effect in causing oxidative damage is counteracted by antioxidants. An imbalance in favour of oxidants leads to oxidative stress, and contributes to myocyte apoptosis, direct negative inotropic effects, and reduced bioavailability of nitric oxide (NO). Together, these effects lead to impaired vasodilatation of the coronary, pulmonary and peripheral vascular beds. In patients with moderate to severe forms of CHF, TNF-alpha leads to the formation of nitrotyrosine and consumption of nitric oxide by virtue of activation of myeloperoxidase. Further studies are required to better elucidate the complex interaction of oxidative stress, endothelial dysfunction and inflammatory activation in CHF. Such insights would likely lead to development of better strategies for the assessment of the disease severity by monitoring of new bio-humoral indices and better treatment approaches.