Proinflammatory interleukins' production by adipose tissue-derived mesenchymal stromal cells: the impact of cell culture conditions and cell-to-cell interaction.

The impact of culture conditions and interaction with activated peripheral blood mononuclear cells on the interleukin (IL) gene expression profile and proinflammatory IL-6 and IL-8 production by adipose-derived stromal cells (ASCs) was investigated. A microarray analysis revealed a wide range of IL genes either under standard (20%) or hypoxic (5%) O2 concentrations, some highly up-regulated at hypoxia. IL-6 and IL-8 production was inversely dependent on cell culture density. In early (first-third) passages, IL-6 and IL-8 concentration was higher at 20% O2 and in late (8th-12th) passages under 5% O2. Interaction between ASCs and mononuclear cells in indirect setting was accompanied with a significant decrease of IL-6 and did not result in the elevation of IL-8 concentration. Thereby, the production of proinflammatory interleukins (IL-6 and IL-8) may be affected by the ASC intrinsic features (density in culture, and duration of expansion), as well as by microenvironmental factors, such as hypoxia and the presence of blood-borne cells. These data are important for elucidating ASC paracrine activity regulation in vitro. They would also be on demand for optimisation of the cell therapy protocols, based on the application of ASC biologically active substances. SIGNIFICANCE PARAGRAPH: Ex vivo expansion is widely used for increasing the number of adipose-derived stromal cells (ASCs) and improving of their quality. The present study was designed to elucidate the particular factors influencing the interleukin production in ASCs. The presented data specified the parameters (i.e. cell density, duration of cultivation, hypoxia, etc.) that should be taken in mind when ASCs are intended to be used in protocols implying their paracrine activity. These data would be of considerable interest for researchers and clinicians working in the biomedical science.

Adipose tissue-derived stromal cells retain immunosuppressive and angiogenic activity after coculture with cord blood hematopoietic precursors.

Adipose-tissue derived stromal cells (ASCs) are currently considered as a full value alternative source of bone marrow MSCs for prevention of graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation due to their immunosuppressive potential. Besides, ASCs are known to support ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). Ex vivo expansion enables to amplify significantly the number of HSPCs of different commitment. Mononuclear cells (MNCs) from cord blood (cb) contain HSPCs and are easily assessed. The rarity of those HSPCs is a serious limitation of its application in cell therapy. Here we expanded cbMNCs in stroma-dependent setting to generate heterocellular associates consisting of ASCs and undifferentiated and low committed hematopoietic cbHSPCs. A part of cbHSPCs in associates demonstrated a primitive phenotype confirmed by formation of "cobblestone areas". ASCs associated with cbHSPCs demonstrated up-regulation of immunosuppressive indoleamine 2,3-dioxygenase (IDO), leukemia inhibitory factor (LIF), cyclooxygenase-2 (PTGS2) genes. ASC-cbHSPCs as well as ASCs provoked the suppression of HLA-DR activation and apoptosis of mitogen-stimulated T cells. VEGF transcription and secretion were elevated providing stimulation of blood vessel formation in ovo. Thus, ASCs retain immunosuppressive and proangiogenic capacities evidencing "third party" potential along with the effective support of ex vivo expansion of cbHSPCs. Above functions expand the relevance of ASCs for needs of regenerative medicine.

Hematopoiesis-supportive function of growth-arrested human adipose-tissue stromal cells under physiological hypoxia.

Ex vivo expansion of hematopoietic progenitors is considered as an attractive tool to increase the number of stem and progenitor cells (HSPCs) for cell therapy. The efficacy of ex vivo expansion is strongly depends on the feeder cell activity to mimic hematopoietic microenvironment. Here we demonstrated, that combination of mitomycin C-induced growth arrest and tissue-related O2 (physiological hypoxia) modulated stromal capacity of adipose tissue derived stromal cells (ASCs). Growth arrest did not affect viability, stromal phenotype and multilineage potential of ASCs permanently expanded at tissue-related O2. Meanwhile, the PCR analysis revealed an up-regulation of genes, encoded molecules of cell-cell (ICAM1, HCAM/CD44) and cell-matrix adhesion (ITGs), extracellular matrix production (COLs) and remodeling (MMPs, HAS1) in growth-arrested ASCs at physiological hypoxia in comparison with ambient O2 (20%). The number of ICAM-1 positive ASCs was increased under low O2 as well. These alterations contributed into the ex vivo expansion of cord blood HSPCs providing the preferential production of primitive HSPCs. The number of cobblestone area forming cell (CAFC) colonies was 1.5-fold higher at physiological hypoxia (p < 0.05). CAFCs considered as long-term culture-initiating cells (LTC-IC) known to support long-term hematopoiesis restoration in vivo. The presented data may be applicable in the development of upscale protocols of HSPC expansion.

Stromal and Hematopoietic Progenitors from C57/BI/6N Murine Bone Marrow After 30-Day "BION-M1" Spaceflight.

Elucidation of the spaceflight (SF) effects on the adult stem and progenitor cells is an important goal in space biology and medicine. A unique opportunity for this was provided by project "BION-M1". The purpose of this study was to evaluate the effects of 30-day SF on biosatellite, 7-day recovery (SFR), and subsequent ground control (GC) experiment on the mononuclear cells (MNCs) from C57/BI/6N murine tibia bone marrow. Also, hematopoietic and stromal precursor functions were characterized ex vivo. There was no significant difference in the total MNC number between experimental groups. After SF, immunophenotyping revealed an increase of large-sized CD45+MNCs corresponded to committed hematopoietic progenitors. The total hematopoietic colony-forming unit (CFU) number decreased after SF and did not restore after 7 day of recovery due to predominant reduction of bi- and multipotent CFUs and primitive burst-forming units in favor of unipotent CFUs. Functional activity of stromal precursors in vitro was only slightly altered. SF cells displayed the enhanced expression of alkaline phosphatase. The data of the GC experiment demonstrated the preservation of the functional activity of progenitor cells from mice bone marrow. The activation of erythropoiesis in expense of burst-forming units of erythrocytes elevation was detected. After 7 days of recovery, the number of colony-forming units of fibroblast (CFUs-f) was similar to the vivarium control, while the proliferative activity of bone marrow stromal precursors decreased. The present study demonstrated that certain hematopoietic progenitors are susceptible to SF factors, while the stromal precursors displayed a certain degree of resistance. These data indicate mild and reversible alterations of bone marrow progenitors after SF.

Anti-atherosclerotic effects of garlic preparation in freeze injury model of atherosclerosis in cholesterol-fed rabbits.

BACKGROUND: Garlic (Allium sativum L.) is one of the most popular substances used to reduce various risks associated with cardiovascular disease. However, little is known on the direct effects of garlic on atherosclerosis. PURPOSE: In the present study we have examined the effect of per oral administration of the time-released garlic herbal preparation on serum atherogenicity and formation of intimal thickening after freeze injury in cholesterol-fed rabbits. METHODS: Group 1 rabbits maintained on the standard cholesterol-rich diet served as the control. Group 2 rabbits were fed the cholesterol-rich diet and treated with garlic preparation containing 300 mg garlic powder. RESULTS: Local thickening of the aortic media (i.e., the neointima formation) in the freeze injury zone was observed in all the rabbits. Regular garlic preparation therapy prevented the neointima formation and the accumulation of free and esterified cholesterol, triglycerides, phospholipids and collagen in the neointima, the effects being statistically significant. Garlic preparation also decreased serum lipid content by 1.5-fold and lowered atherogenic activity of blood serum (ability to induce lipid accumulation in cultured cells) induced by cholesterol-rich diet. CONCLUSION: The results obtained indicate that garlic preparation prevents the development of cholesterol-induced experimental atherosclerosis and possesses the direct anti-atherogenic activity.

Human adipose-tissue derived stromal cells in combination with hypoxia effectively support ex vivo expansion of cord blood haematopoietic progenitors.

The optimisation of haematopoietic stem and progenitor cell expansion is on demand in modern cell therapy. In this work, haematopoietic stem/progenitor cells (HSPCs) have been selected from unmanipulated cord blood mononuclear cells (cbMNCs) due to adhesion to human adipose-tissue derived stromal cells (ASCs) under standard (20%) and tissue-related (5%) oxygen. ASCs efficiently maintained viability and supported further HSPC expansion at 20% and 5% O2. During co-culture with ASCs, a new floating population of differently committed HSPCs (HSPCs-1) grew. This suspension was enriched with СD34+ cells up to 6 (20% O2) and 8 (5% O2) times. Functional analysis of HSPCs-1 revealed cobble-stone area forming cells (CAFCs) and lineage-restricted colony-forming cells (CFCs). The number of CFCs was 1.6 times higher at tissue-related O2, than in standard cultivation (20% O2). This increase was related to a rise in the number of multipotent precursors - BFU-E, CFU-GEMM and CFU-GM. These changes were at least partly ensured by the increased concentration of MCP-1 and IL-8 at 5% O2. In summary, our data demonstrated that human ASCs enables the selection of functionally active HSPCs from unfractionated cbMNCs, the further expansion of which without exogenous cytokines provides enrichment with CD34+ cells. ASCs efficiently support the viability and proliferation of cord blood haematopoietic progenitors of different commitment at standard and tissue-related O2 levels at the expense of direct and paracrine cell-to-cell interactions.

Lipid regulators during atherogenesis: expression of LXR, PPAR, and SREBP mRNA in the human aorta.

Transcription factors LXRs, PPARs, and SREBPs have been implicated in a multitude of physiological and pathological processes including atherogenesis. However, little is known about the regulation of these transcription factors at different stages of atherosclerosis progression. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to compare the contents of mRNAs in pairs intact-injured aorta fragments taken from the same donors. Only minor changes in LXRα, LXRß, PPARα, PPARγ, SREBP1, and SREBP2 mRNA levels were found in initial lesions as compared with intact non-diseased tissue. The contents of all mRNAs but SREBP2 mRNA were found to be progressively up-regulated in fatty streaks and fibrous lipoid plaques. These changes were only partially reproduced in cultured macrophages upon lipid loading. Wave-shaped changes in abundance of correlations between given group of mRNAs and 28 atherosclerosis-related mRNA species in the course of atherogenesis were observed. The impact of specific mRNA correlations on the total correlations also significantly varied between different lesion types. The study suggests that the extent and forms of LXR/PPAR/SREBP participation in intima functions vary nonlinear in individual fashion in atherogenesis. We speculate that the observed changes in mRNAs expression and coupling reflect shifts in lipid ligands availability and cellular composition in the course of atherosclerosis progression.

Correlation between lipid deposition, immune-inflammatory cell content and MHC class II expression in diffuse intimal thickening of the human aorta.

Inflammatory reactions driven by an accumulation in the intima of immune-inflammatory cells and focal lipid depositions are the hallmarks of atherogenesis. It is commonly accepted that immune-inflammatory cell accumulation and lipid deposition are associated with the very earlier stage of atherosclerosis but no study has yet focused on the determination of quantitative values of this association. The present study examined correlations between lipid deposition, immune-inflammatory cell content and major histocompatibility complex (MHC) class II molecule HLA-DR expression in diffuse intimal thickening (DIT), which is thought to represent the earliest macroscopic manifestation of atherosclerosis. In parallel consecutive tissue sections of DIT, lipids were examined by chromatographic analysis (including triglycerides, cholesteryl esters, free cholesterol and phospholipids), histochemically, using Oil Red O staining, and by electron microscopy. Immune-inflammatory cells and HLA-DR expression were examined immunohistochemically in consecutive sections of the same tissue specimens. The study revealed that lipids exhibited a non-uniform distribution throughout the intima. In the juxtaluminal sublayer, lipids were localized both intracellularly and extracellularly, whereas in the juxtamedial musculoelastic sublayer, lipids were present predominantly along elastic fibers. Lipid deposits were found to positively correlate with HLA-DR expression (r=0.79; p<0.001). The study also identified a positive correlation between lipid deposition and immune-inflammatory cell content but the correlation values varied between different sublayers of the tunica intima. The correlation between lipid deposition and immune-inflammatory cell content in the juxtaluminal sublayer of the intima was notably stronger (r=0.69; p<0.001) than in the juxtamedial musculoelastic layer (r=0.28; p<0.001). The findings of the present study support a view that lipid accumulation in the intima plays a role in the initiation of inflammatory reaction and that at the pre-lesional stage in the development of atherosclerosis, lipid-associated immune cell activation might occur primarily in the juxtaluminal portion of the intima.

Time-released garlic powder tablets lower systolic and diastolic blood pressure in men with mild and moderate arterial hypertension.

Numerous clinical investigations that have focused on the hypotensive effects of garlic-based preparations have led to controversial results that may be partially because of differences in the composition of the preparations and in the biological responses they induce. It is possible that garlic powder tablets with a prolonged mode of action could induce more potent biological effects. In this double-blind, placebo-controlled trial with an active control arm, the hypotensive action of time-released garlic powder tablets (Allicor) was compared with that of regular garlic pills (Kwai) in 84 men with mild or moderate arterial hypertension. After an 8-week placebo treatment run-in phase, patients were randomized either to 600 mg Allicor (n=30) or to placebo (n=20) daily for 8 weeks. In addition, in the open-label branch, patients received either 2400 mg Allicor daily (n=18) or 900 mg Kwai daily (n=16). Allicor treatment (600 mg daily) resulted in a reduction of both systolic and diastolic blood pressures by 7.0 mm Hg (95% confidence interval (95% CI): 5.3-8.7) and 3.8 mm Hg (95% CI: 2.7-4.8), respectively. Increasing the Allicor dosage to 2400 mg daily did not provide any additional benefit. Treatment with Kwai resulted in the same decrease in systolic blood pressure (5.4 mm Hg, 95% CI: 1.9-8.8) as that seen with Allicor, but no decrease in diastolic blood pressure was observed with Kwai. Different effects of Allicor and Kwai on diastolic blood pressure may be because of the prolonged action of Allicor, which allows better bioavailability of the vasoactive constituents of garlic powder. The results of this study show that time-released garlic powder tablets are more effective for the treatment of mild and moderate arterial hypertension than are regular garlic supplements.

Lipid-lowering effects of time-released garlic powder tablets in double-blinded placebo-controlled randomized study.

AIM: Clinical investigations of the effects of garlic preparations in hypercholesterolemia have demonstrated somewhat controversial results. These discrepancies may be due to the differences of the composition of garlic preparations and the biological response they may induce. The study was undertaken to test the hypothesis that garlic powder tablets with a prolonged mode of action promise potent biological effects. METHODS: The lipid-lowering effects of time-released garlic powder tablets, Allicor (600 mg daily), were investigated in a double-blinded placebo-controlled randomized study in 42 men aged 35-70 with mild hypercholesterolemia. RESULTS: Allicor treatment resulted in a moderate but statistically significant decrease in total cholesterol level that was observed after 8 and 12 weeks of active treatment. By the end of the study, total cholesterol in Allicor-treated patients had fallen by 7.6% (p=0.004) as compared to the level at randomization, and was 11.5% lower than the placebo group (p=0.005). LDL cholesterol in Allicor-treated patients fell by 11.8% (p=0.002) and 13.8% (p=0.009), respectively. HDL cholesterol also increased significantly after 8 and 12 weeks of treatment. By the end of the study, HDL cholesterol in Allicor-treated patients had increased by 11.5% (p=0.013). CONCLUSION: The obtained results are in good agreement with trials that have demonstrated the cardioprotective action of garlic preparations and may be due to the use of a time-released form of garlic powder tablets that provides a prolonged biological effect.