Virgil Paunescu

Tumour-associated fibroblasts and mesenchymal stem cells: more similarities than differences

Tumour‐associated fibroblasts (TAFs) are part of the tumour stroma, providing functional and structural support for tumour progression and development. The origin and biology of TAFs are poorly understood, but within the tumour environment, TAFs become activated and secrete different paracrine and autocrine factors involved in tumorigenesis. It has been shown that bone marrow mesenchymal stem cells (MSCs) can be recruited into the tumours, where they proliferate and acquire a TAF‐like phenotype. We attempted to determine to what extent TAFs characteristics in vitro juxtapose to MSCs’ definition, and we showed that TAFs and MSCs share immunophenotypic similarities, including the presence of certain cell surface molecules [human leukocyte antigen‐DR subregion (HLA‐DR), CD29, CD44, CD73, CD90, CD106 and CD117]; the expression of cytoskeleton and extracellular matrix proteins, such as vimentin, α‐smooth muscle actin, nestin and trilineage differentiation potential (to adipocytes, chondrocytes and osteoblasts). When compared to MSCs, production of cytokines, chemokines and growth factors showed a significant increase in TAFs for vascular endothelial growth factor, transforming growth factor‐β1, interleukins (IL‐4, IL‐10) and tumour necrosis factor α. Proliferation rate was highly increased in TAFs and fibroblast cell lines used in our study, compared to MSCs, whereas ultrastructural details differentiated the two cell types by the presence of cytoplasmic elongations, lamellar content lysosomes and intermediate filaments. Our results provide supportive evidence to the fact that TAFs derive from MSCs and could be a subset of ‘specialized’ MSCs.

Telocytes within human skeletal muscle stem cell niche.

Human skeletal muscle tissue displays specific cellular architecture easily damaged during individual existence, requiring multiple resources for regeneration. Congruent with local prerequisites, heterogeneous muscle stem cells (MuSCs) are present in the muscle interstitium. In this study, we aimed to characterize the properties of human muscle interstitial cells that had the characteristic morphology of telocytes (TCs). Immunocytochemistry and immunofluorescence showed that cells with TC morphology stained positive for c‐kit/CD117 and VEGF. C‐kit positive TCs were separated with magnetic‐activated cell sorting, cultured in vitro and expanded for study. These cells exhibited high proliferation capacity (60% expressed endoglin/CD105 and 80% expressed nuclear Ki67). They also exhibited pluripotent capacity limited to Oct4 nuclear staining. In addition, 90% of c‐kit positive TCs expressed VEGF. C‐kit negative cells in the MuSCs population exhibited fibroblast‐like morphology, low trilineage differential potential and negative VEGF staining. These results suggested that c‐kit/CD117 positive TCs represented a unique cell type within the MuSC niche.

Health Effects of Toxic Organic Substances from Coal: Toward “Panendemic” Nephropathy

Coal contains myriad organic compounds, some known to be toxic and others that are potentially toxic. Toxic organic compounds found in coal of particular interest include: i) condensed aromatic structures (e.g., polycyclic aromatic hydrocarbons), which can act as mutagens, cancer promoters, and endocrine disrupters; ii) aromatic amines, which have probable nephrotoxic activity; and iii) heterocyclic compounds, which may be carcinogenic and nephrotoxic. Toxic organic compounds can be leached from coal into water supplies, and longterm human exposure to these compounds may lead to disease occurrence, including cancer and renal disease. Despite these potential hazards, little is known about the impact and toxicity of organic substances derived from coal in water supplies. One example of a disease hypothesized to be linked to coal-derived toxic organic compounds in water supplies is Balkan endemic nephropathy (BEN). In this paper, we summarize results from our studies linking BEN to the leaching of toxic organic compounds from low rank (lignite) Pliocene coal deposits into water supplies (well and spring water) of the rural villages where the disease occurs. We also introduce the idea of panendemic nephropathy (PEN) for BEN-like diseases that are linked to coal-derived toxic organic compounds in water supplies, but that occur outside the Balkans. Preliminary results supporting the PEN hypothesis are presented, with results from proposed PEN areas in Wyoming (WY) and Louisiana (LA). Results of toxicological studies of the effects of organic compounds isolated from water supplies in BEN and PEN areas on human cell cultures are also discussed. China, India, Turkey, and Portugal represent other areas where BEN-like diseases may occur, as a result of the presence of extensive low rank coal deposits and rural populations using untreated water in contact with coal in these nations.

Bone marrow derived cells in the tumour microenvironment contain cells with primitive haematopoietic phenotype.

Infiltration of bone marrow derived cells is part of the angiogenic switch required for uncontrolled tumour growth. However, the nature of the tumour‐infiltrating cells from bone marrow has not been fully elucidated. To investigate the phenotype of bone marrow derived cells within a tumour, we employed the Lewis lung carcinoma (LLC) murine tumour model. We followed bone marrow derivation of tumour‐infiltrating cells through transplantation of CD45.2 bone marrow cells into pre‐irradiated CD45.1 mice. We found robust CD45.2 donor type chimerism in bone marrow and blood of CD45.1 recipient tumour‐bearing mice. Flow cytometric analysis of LLC tumours showed, in addition to previously described pro‐angiogenic CD45+VEGFR2+‘endothelial progenitor cells’ (EPC), or CD45+Tie2+‘Tie2‐expressing monocytes’ (TEM), incorporation of donor type lineage marker negative (Lin−) and Lin−Sca1+ undifferentiated haematopoietic cell types. Immunohistochemical analysis confirmed the extravasal location of the primitive haematopoietic cells. Flow‐cytometric sorting of bone marrow cells and subsequent analysis in haematopoietic colony‐forming assays revealed that cells with a Lin−Sca1+ phenotype, which were initially negative for VEGFR2 and Tie2, gave rise to VEGFR2+ and/or Tie2+ cells. Moreover, Lin− bone marrow cells pre‐labelled with the membrane dye PKH26 (a red fluorochrome) and transplanted i.v. into tumour‐bearing mice were found to extravasate and incorporate into LLC tumours within 24 hrs. Thus, primitive haematopoietic precursors which are thought to be precursors of EPC and TEMs, constitute a part of the tumour microenvironment. This makes them an attractive target cell population for tumour‐directed cellular therapies.

A simple device for intubation of rats.

Endotracheal intubation of rats is often necessary for lengthy survival surgeries, but the animals small size and the lack of suitable equipment may complicate the procedure. The authors describe the construction and use of a simple device for the easy intubation of rats, requiring no expensive, specialized equipment.

Endothelial cells from hematopoietic stem cells are functionally different from those of human umbilical vein

Hematopoietic stem cells have a remarkable plastic capacity, which allows them to differentiate into various cells, such as immune cells, nervous cells, muscle cells, bone and cartilaginous cells. The aim of this study was to show the capacity of stem cells to differentiate into endothelial cells, in culture, after addition of endothelial cells growth suplement (ECGS). We also compared the behavior of these cells with that of endothelial cells obtained from human umbilical vein (HUVEC). CD34+ cells obtained by immunomagnetic separation from human umbilical cord and placental blood were used. After 12‐15 days of culture in a medium containing ECGS, the cells showed morphological changes characteristic to endothelial cells and immunocytochemical analysis revealed the presence of CD31 surface antigen and von Willebrand factor. The flow‐cytometric analysis of endothelial cells adhesion molecules (ECAM) showed that endothelial cells derived from CD34+ cells expressed CD54/ICAM‐1 9.65 ± 0.2% and CD106/VCAM 7.73±0.3%, values similar to those expressed by HUVECs. After TNF incubation, ECAM expression increased only in HUVECs. These data demonstrate that a fraction of circulating CD34+ cells may develop some endothelial cell characteristics when cultured with ECGS, but they are functionally different from HUVECs.

Possible health impacts of naturally occurring uptake of aristolochic acids by maize and cucumber roots: links to the etiology of endemic (Balkan) nephropathy

The original publication of the article includes errors in the affiliation of Vuk Maksimović, Calin A. Tatu and Jelena D. Maksimović and in the Acknowledgments. The correct affiliation and Acknowledgments appear in this erratum. Acknowledgments This work was supported by the Serbian Ministry of Science (grant number 173040). All the experiments were performed on equipment provided by the Institute for Multidisciplinary Research and Serbian Ministry of Science. Part of this work was supported by grants from the US Geological Survey (Reston, VA, USA), University of Medicine and Pharmacy, Timisoara, Romania, and NATO (CLG grant # 980104).

Adipocytes differentiated in vitro from rat mesenchymal stem cells lack essential free fatty acids compared to adult adipocytes.

Adult bone marrow mesenchymal stem cells (BMSCs) can be differentiated in vitro to become adipocyte-like cells with lipid vacuoles, similar to adipocytes derived from adult adipose tissue. Little is known regarding the composition of free fatty acids (FFAs) of the in vitro-differentiated adipocytes, or whether it resembles that of native adult adipocytes. We used gas chromatography-mass spectrometry to identify FFA species in BMSC-derived adipocytes and compared them with FFAs found in adipocytes derived from adult adipose tissue. We found that adult adipocytes contained significant percentages of saturated and monounsaturated FFAs, including palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1); some polyunsaturated FFAs, such as linoleic acid (C18:2), a small percentage of arachidonic acid (C20:4), and very little linolenic acid (C18:3). In comparison, 80%-90% confluent BMSCs contained comparable percentages of palmitic and oleic acids, significantly more arachidonic and stearic acids, very little linoleic acid, and no linolenic acid. After differentiation, compared with adult adipocytes, BMSC-derived adipocytes contained a comparable percentage of palmitic acid, more stearic and arachidonic acids, less oleic acid, almost no linoleic acid, and no detectable linolenic acid. This composition was quite similar to that of undifferentiated BMSCs. The differentiation medium contained only palmitic and stearic acids, with traces of oleic acid; it did not contain the essential polyunsaturated fatty acids. Thus, the composition of FFAs in BMSC-derived adipocytes was altered compared with adult adipocytes. BMSC-derived adipocytes had an altered composition of saturated and monounsaturated FFAs and lacked essential FFAs that may directly affect signaling related to their lipolysis/lipogenesis functions.

Arachidonic acid accumulates in the stromal macrophages during thymus involution in diabetes

Diabetes is a debilitating disease with chronic evolution that affects many tissues and organs over its course. Thymus is an organ that is affected early after the onset of diabetes, gradually involuting until it loses most of its thymocyte populations. We show evidence of accumulating free fatty acids with generation of eicosanoids in the diabetic thymus and we present a possible mechanism for the involution of the organ during the disease. Young rats were injected with streptozotocin and their thymuses examined for cell death by flow cytometry and TUNEL reaction. Accumulation of lipids in the diabetic thymus was investigated by histology and electron microscopy. The identity and quantitation of accumulating lipids was done with gas chromatography–mass spectrometry and liquid chromatography. The expression and dynamics of the enzymes were monitored via immunohistochemistry. Diabetes causes thymus involution by elevating the thymocyte apoptosis. Exposure of thymocytes to elevated concentration of glucose causes apoptosis. After the onset of diabetes, there is a gradual accumulation of free fatty acids in the stromal macrophages including arachidonic acid, the substrate for eicosanoids. The eicosanoids do not cause thymocyte apoptosis but administration of a cyclooxygenase inhibitor reduces the staining for ED1, a macrophage marker whose intensity correlates with phagocytic activity. Diabetes causes thymus involution that is accompanied by accumulation of free fatty acids in the thymic macrophages. Excess glucose is able to induce thymocyte apoptosis but eicosanoids are involved in the chemoattraction of macrophage to remove the dead thymocytes.

Improvement of ursolic and oleanolic acids' antitumor activity by complexation with hydrophilic cyclodextrins.

Ursolic and oleanolic acids have been brought into the spotlight of research due to their chemopreventive, anti-inflammatory and immunomodulatory properties. The most important disadvantage of ursolic and oleanolic acids is their weak water solubility which limits their bioavailability. Pentacyclic triterpenes can form inclusion complexes with different types of cyclodextrins which provide the hydrophilic matrix requested for the molecular dispersion of drugs in order to become more water soluble. The aim of the current study is the complexation of ursolic and oleanolic acids with hydrophilic cyclodextrins in order to achieve an improvement of their pharmacological effect. After the virtual screening of the binding affinities between ursolic and oleanolic acids and various cyclodextrins, 2-hydroxypropyl-β-cyclodextrin and 2-hydroxypropil-γ-cyclodextrin were selected as host-molecules for the inclusion complexation. Using the scanning electron microscopy, differential scanning calorimetry and X-ray diffraction the formation of real inclusion complexes between ursolic and oleanolic acids and the two cyclodextrins was confirmed. The anti-proliferative potential of the complexes was tested in vitro on several melanoma cell lines, using the pure compounds as reference. The complexes exhibited higher in vitro anti-proliferative activity as compared to the pure compounds; this improvement was significant for ursolic acid complexes, the highest activity being reported for the 2-hydroxypropil-γ-cyclodextrin complex. Weaker results were recorded for the oleanolic acid complexes where 2-hydroxypropyl-β-cyclodextrin proved to be the most fitted inclusion partner. The entrapment of the two active compounds inside ramified hydrophilic cyclodextrins proved to be a suitable option to increase their anti-proliferative activity.