Search results for: endothelial cells

Authors: Adel Dalilottojari, Bahman Delalat, Frances J. Harding, Michaelia P. Cockshell, Claudine S. Bonder, Nicolas H. Voelcker

Abstract:

Endothelial Progenitor Cell (EPC) based therapies continue to be of interest to treat ischemic events based on their proven role to promote blood vessel formation and thus tissue re-vascularisation. Current strategies for the production of clinical-grade EPCs requires the in vitro isolation of EPCs from peripheral blood followed by cell expansion to provide sufficient quantities EPCs for cell therapy. This study aims to examine the use of different biomolecules to significantly improve the current strategy of EPC capture and expansion on collagen type I (Col I). In this study, four different biomolecules were immobilised on a surface and then investigated for their capacity to support EPC capture and proliferation. First, a cell microarray platform was fabricated by coating a glass surface with epoxy functional allyl glycidyl ether plasma polymer (AGEpp) to mediate biomolecule binding. The four candidate biomolecules tested were Col I, collagen type II (Col II), collagen type IV (Col IV) and vascular endothelial growth factor A (VEGF-A), which were arrayed on the epoxy-functionalised surface using a non-contact printer. The surrounding area between the printed biomolecules was passivated with polyethylene glycol-bisamine (A-PEG) to prevent non-specific cell attachment. EPCs were seeded onto the microarray platform and cell numbers quantified after 1 h (to determine capture) and 72 h (to determine proliferation). All of the extracellular matrix (ECM) biomolecules printed demonstrated an ability to capture EPCs within 1 h of cell seeding with Col II exhibiting the highest level of attachment when compared to the other biomolecules. Interestingly, Col IV exhibited the highest increase in EPC expansion after 72 h when compared to Col I, Col II and VEGF-A. These results provide information for significant improvement in the capture and expansion of human EPC for further application.

Keywords: biomolecules, cell microarray platform, cell therapy, endothelial progenitor cells, high throughput screening

Procedia PDF Downloads 288

Authors: Valeria Arcucci, Musarat Ishaq, Steven A. Stacker, Greg J. Goodall, Marc G. Achen

Abstract:

Metastasis is the lethal aspect of cancer for most patients. Remodelling of lymphatic vessels associated with a tumour is a key initial step in metastasis because it facilitates the entry of cancer cells into the lymphatic vasculature and their spread to lymph nodes and distant organs. Although it is clear that vascular endothelial growth factors (VEGFs), such as VEGF-C and VEGF-D, are key drivers of lymphatic remodelling, the means by which many signaling pathways in endothelial cells are coordinately regulated to drive growth and remodelling of lymphatics in cancer is not understood. We seek to understand the broader molecular mechanisms that control cancer metastasis, and are focusing on microRNAs, which coordinately regulate signaling pathways involved in complex biological responses in health and disease. Here, using small RNA sequencing, we found that a specific microRNA, miR-132, is upregulated in expression in lymphatic endothelial cells (LECs) in response to the lymphangiogenic growth factors. Interestingly, ectopic expression of miR-132 in LECs in vitro stimulated proliferation and tube formation of these cells. Moreover, miR-132 is expressed in lymphatic vessels of a subset of human breast tumours which were previously found to express high levels of VEGF-D by immunohistochemical analysis on tumour tissue microarrays. In order to dissect the complexity of regulation by miR-132 in lymphatic biology, we performed Argonaute HITS-CLIP, which led us to identify the miR-132-mRNA interactome in LECs. We found that this microRNA in LECs is involved in the control of many different pathways mainly involved in cell proliferation and regulation of the extracellular matrix and cell-cell junctions. We are now exploring the functional significance of miR-132 targets in the biology of LECs using biochemical techniques, functional in vitro cell assays and in vivo lymphangiogenesis assays. This project will ultimately define the molecular regulation of lymphatic remodelling by miR-132, and thereby identify potential therapeutic targets for drugs designed to restrict the growth and remodelling of tumour lymphatics resulting in metastatic spread.

Keywords: argonaute HITS-CLIP, cancer, lymphatic remodelling, miR-132, VEGF

Procedia PDF Downloads 127

Authors: Narine Ghazaryan, Joana Catarina Macedo, Sara Vaz, Naira Ayvazyan, Elsa Logarinho

Abstract:

Macrovipera lebetina obtusa (MLO) is a poisonous snake in Armenia. Obtustatin represents the shortest known monomeric disintegrin, isolated from the snake venom of MLO, and is known to specifically inhibit α1β1 integrin. Its oncostatic effect is due to the inhibition of angiogenesis, which likely arises from α1β1 integrin inhibition in the endothelial cells. To explore the therapeutic potential of the MLO snake venom and obtustatin, we studied activity of obtustatin and MLO venom in vitro, by testing their efficacy in human dermal microvascular endothelial cells (HMVEC-D) and in vivo, using chick embryo chorioallantoic membrane assay (CAM assay). Our in vitro results showed that obtustatin in comparison with MLO venom did not exhibit cytotoxic activity in HMVEC-D cells in comparison to MLO venom. But in vivo results have shown that 4µg /embryo (90 µM) of obtustatin inhibited angiogenesis induced by FGF2 by 17% while MLO snake venom induced 22% reduction of the angiogenic index. The concentration of obtustatin in the crude MLO venom was 0.3 nM, which is 300.000 times less than the concentration of the obtustatin itself. Given this enormous difference in concentration, it is likely that some components of the crude venom contribute to the observed anti-angiogenic effect. Hypotheses will be ascertained to justify this action: components in the MLO venom may increase obtustatin efficacy or have independent but synergic anti-angiogenic activities.

Keywords: angiogenesis, alpa1 beta 1 integrin, Macrovipera lebetina obtusa, obtustatin

Procedia PDF Downloads 196

Authors: Jolene M. Helena, Annie M. Joubert, Peace Mabeta, Magdalena Coetzee, Roy Lakier, Anne E. Mercier

Abstract:

Targeting the distant tumour and its microenvironment whilst preserving bone density is important in improving the outcomes of patients with bone metastases. 2-Ethyl-3-O-sulphamoyl-estra1,3,5(10)16-tetraene (ESE-16) is an in-silico-designed 2- methoxyestradiol analogue which aimed at enhancing the parent compound’s cytotoxicity and providing a more favourable pharmacokinetic profile. In this study, the potential radiosensitization effects of ESE-16 were investigated in an in vitro bone metastasis model consisting of murine pre-osteoblastic (MC3T3-E1) and pre-osteoclastic (RAW 264.7) bone cells, metastatic prostate (DU 145) and breast (MDA-MB-231) cancer cells, as well as human umbilical vein endothelial cells (HUVECs). Cytotoxicity studies were conducted on all cell lines via spectrophotometric quantification of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide. The experimental set-up consisted of flow cytometric analysis of cell cycle progression and apoptosis detection (Annexin V-fluorescein isothiocyanate) to determine the lowest ESE-16 and radiation doses to induce apoptosis and significantly reduce cell viability. Subsequent experiments entailed a 24-hour low-dose ESE-16-exposure followed by a single dose of radiation. Termination proceeded 2, 24 or 48 hours thereafter. The effect of the combination treatment was investigated on osteoclasts via tartrate-resistant acid phosphatase (TRAP) activity- and actin ring formation assays. Tumour cell experiments included investigation of mitotic indices via haematoxylin and eosin staining; pro-apoptotic signalling via spectrophotometric quantification of caspase 3; deoxyribonucleic acid (DNA) damage via micronuclei analysis and histone H2A.X phosphorylation (γ-H2A.X); and Western blot analyses of bone morphogenetic protein-7 and matrix metalloproteinase-9. HUVEC experiments included flow cytometric quantification of cell cycle progression and free radical production; fluorescent examination of cytoskeletal morphology; invasion and migration studies on an xCELLigence platform; and Western blot analyses of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor receptor 1 and 2. Tumour cells yielded half-maximal growth inhibitory concentration (GI50) values in the nanomolar range. ESE-16 concentrations of 235 nM (DU 145) and 176 nM (MDA-MB-231) and a radiation dose of 4 Gy were found to be significant in cell cycle and apoptosis experiments. Bone and endothelial cells were exposed to the same doses as DU 145 cells. Cytotoxicity studies on bone cells reported that RAW 264.7 cells were more sensitive to the combination treatment than MC3T3-E1 cells. Mature osteoclasts were more sensitive than pre-osteoclasts with respect to TRAP activity. However, actin ring morphology was retained. The mitotic arrest was evident in tumour and endothelial cells in the mitotic index and cell cycle experiments. Increased caspase 3 activity and superoxide production indicated pro-apoptotic signalling in tumour and endothelial cells. Increased micronuclei numbers and γ-H2A.X foci indicated increased DNA damage in tumour cells. Compromised actin and tubulin morphologies and decreased invasion and migration were observed in endothelial cells. Western blot analyses revealed reduced metastatic and angiogenic signalling. ESE-16-induced radiosensitization inhibits metastatic signalling and tumour cell survival whilst preferentially preserving bone cells. This low-dose combination treatment strategy may promote the quality of life of patients with metastatic bone disease. Future studies will include 3-dimensional in-vitro and murine in-vivo models.

Keywords: angiogenesis, apoptosis, bone metastasis, cancer, cell migration, cytoskeleton, DNA damage, ESE-16, radiosensitization.

Procedia PDF Downloads 161

Authors: Daphne Meza, Louie Abejar, David A. Rubenstein, Wei Yin

Abstract:

Endothelial cell (ECs) morphology and function is highly impacted by the mechanical stresses these cells experience in vivo. Any change in the mechanical environment can trigger pathological EC responses. A detailed understanding of EC morphological response and function upon subjection to individual and simultaneous mechanical stimuli is needed for advancement in mechanobiology and preventive medicine. To investigate this, a programmable device capable of simultaneously applying physiological fluid shear stress (FSS) and cyclic strain (CS) has been developed, characterized and validated. Its validation was performed both experimentally, through tracer tracking, and theoretically, through the use of a computational fluid dynamics model. The effectiveness of the device was evaluated through EC morphology changes under mechanical loading conditions. Changes in cell morphology were evaluated through: cell and nucleus elongation, cell alignment and junctional actin production. The results demonstrated that the combined FSS-CS stimulation induced visible changes in EC morphology. Upon simultaneous fluid shear stress and biaxial tensile strain stimulation, cells were elongated and generally aligned with the flow direction, with stress fibers highlighted along the cell junctions. The concurrent stimulation from shear stress and biaxial cyclic stretch led to a significant increase in cell elongation compared to untreated cells. This, however, was significantly lower than that induced by shear stress alone, indicating that the biaxial tensile strain may counteract the elongating effect of shear stress to maintain the shape of ECs. A similar trend was seen in alignment, where the alignment induced by the concurrent application of shear stress and cyclic stretch fell in between that induced by shear stress and tensile stretch alone, indicating the opposite role shear stress and tensile strain may play in cell alignment. Junctional actin accumulation was increased upon shear stress alone or simultaneously with tensile stretch. Tensile stretch alone did not change junctional actin accumulation, indicating the dominant role of shear stress in damaging EC junctions. These results demonstrate that the shearing-stretching device is capable of applying well characterized dynamic shear stress and tensile strain to cultured ECs. Using this device, EC response to altered mechanical environment in vivo can be characterized in vitro.

Keywords: cyclic stretch, endothelial cells, fluid shear stress, vascular biology

Procedia PDF Downloads 377

Authors: Anjali Roy, Mirza S. Baig

Abstract:

Atherosclerosis is a chronic inflammatory disease characterized by the formation of lipid rich plaque enriched with necrotic core, modified lipid accumulation, smooth muscle cells, endothelial cells, leucocytes and macrophages. Macrophage foam cells play a critical role in the occurrence and development of inflammatory atherosclerotic plaque. Foam cells are the fat-laden macrophages in the initial stage atherosclerotic lesion formation. Foam cells are an indication of plaque build-up, or atherosclerosis, which is commonly associated with increased risk of heart attack and stroke as a result of arterial narrowing and hardening. The mechanisms that drive atherosclerotic plaque progression remain largely unknown. Dissecting the molecular mechanism involved in process of macrophage foam cell formation will help to develop therapeutic interventions for atherosclerosis. To investigate the mechanism, we studied the role of nitric oxide synthase 1(NOS1)-mediated nitric oxide (NO) on low-density lipoprotein (LDL) uptake by bone marrow derived macrophages (BMDM). Using confocal microscopy, we found that incubation of macrophages with NOS1 inhibitor, TRIM (1-(2-Trifluoromethylphenyl) imidazole) or L-NAME (N omega-nitro-L-arginine methyl ester) prior to LDL treatment significantly reduces the LDL uptake by BMDM. Further, addition of NO donor (DEA NONOate) in NOS1 inhibitor treated macrophages recovers the LDL uptake. Our data strongly suggest that NOS1 derived NO regulates LDL uptake by macrophages and foam cell formation. Moreover, we also checked proinflammatory cytokine mRNA expression through real time PCR in BMDM treated with LDL and copper oxidized LDL (OxLDL) in presences and absences of inhibitor. Normal LDL does not evoke cytokine expression whereas OxLDL induced proinflammatory cytokine expression which significantly reduced in presences of NOS1 inhibitor. Rapid NOS-1-derived NO and its stable derivative formation act as signaling agents for inducible NOS-2 expression in endothelial cells, leading to endothelial vascular wall lining disruption and dysfunctioning. This study highlights the role of NOS1 as critical players of foam cell formation and would reveal much about the key molecular proteins involved in atherosclerosis. Thus, targeting NOS1 would be a useful strategy in reducing LDL uptake by macrophages at early stage of disease and hence dampening the atherosclerosis progression.

Keywords: atherosclerosis, NOS1, inflammation, oxidized LDL

Procedia PDF Downloads 127

Authors: Shaimaa Eltanani, Thangal Yumnamcha, Ahmed S. Ibrahim

Abstract:

Purpose: Mitochondria dysfunction is central to breaking the barrier integrity of retinal endothelial cells (RECs) in various blinding eye diseases such as diabetic retinopathy and retinopathy of prematurity. Therefore, we aimed to dissect the role of different mitochondrial components, specifically, those of oxidative phosphorylation (OxPhos), in maintaining the barrier function of RECs. Methods: Electric cell-substrate impedance sensing (ECIS) technology was used to assess in real-time the role of different mitochondrial components in the total impedance (Z) of human RECs (HRECs) and its components; the capacitance (C) and the total resistance (R). HRECs were treated with specific mitochondrial inhibitors that target different steps in OxPhos: Rotenone for complex I; Oligomycin for ATP synthase; and FCCP for uncoupling OxPhos. Furthermore, data were modeled to investigate the effects of these inhibitors on the three parameters that govern the total resistance of cells: cell-cell interactions (Rb), cell-matrix interactions (α), and cell membrane permeability (Cm). Results: Rotenone (1 µM) produced the greatest reduction in the Z, followed by FCCP (1 µM), whereas no reduction in the Z was observed after the treatment with Oligomycin (1 µM). Following this further, we deconvoluted the effect of these inhibitors on Rb, α, and Cm. Firstly, rotenone (1 µM) completely abolished the resistance contribution of Rb, as the Rb became zero immediately after the treatment. Secondly, FCCP (1 µM) eliminated the resistance contribution of Rb only after 2.5 hours and increased Cm without considerable effect on α. Lastly, Oligomycin had the lowest impact among these inhibitors on Rb, which became similar to the control group at the end of the experiment without noticeable effects on Cm or α. Conclusion: These results demonstrate differential roles for complex I, complex V, and coupling of OxPhos in maintaining the barrier functionality of HRECs, in which complex I being the most important component in regulating the barrier functionality and the spreading behavior of HRECs. Such differences can be used in investigating gene expression as well as for screening selective agents that improve the functionality of complex I to be used in the therapeutic approach for treating REC-related retinal diseases.

Keywords: human retinal endothelial cells (hrecs), rotenone, oligomycin, fccp, oxidative phosphorylation, oxphos, capacitance, impedance, ecis modeling, rb resistance, α resistance, and barrier integrity

Procedia PDF Downloads 99

Authors: Alisan Kayabolen, Dilek Keskin, Ferit Avcu, Andac Aykan, Fatih Zor, Aysen Tezcaner

Abstract:

Adipose tissue engineering is a promising field for regeneration of soft tissue defects. However, only very thin implants can be used in vivo since vascularization is still a problem for thick implants. Another problem is finding a biocompatible scaffold with good mechanical properties. In this study, the aim is to develop a thick vascularized adipose tissue that will integrate with the host, and perform its in vitro and in vivo characterizations. For this purpose, a hydrogel of decellularized adipose tissue (DAT) and fibroin was produced, and both endothelial cells and adipocytes that were differentiated from adipose derived stem cells were encapsulated in this hydrogel. Mixing DAT with fibroin allowed rapid gel formation by vortexing. It also provided to adjust mechanical strength by changing fibroin to DAT ratio. Based on compression tests, gels of DAT/fibroin ratio with similar mechanical properties to adipose tissue was selected for cell culture experiments. In vitro characterizations showed that DAT is not cytotoxic; on the contrary, it has many natural ECM components which provide biocompatibility and bioactivity. Subcutaneous implantation of hydrogels resulted with no immunogenic reaction or infection. Moreover, localized empty hydrogels gelled successfully around host vessel with required shape. Implantations of cell encapsulated hydrogels and histological analyses are under study. It is expected that endothelial cells inside the hydrogel will form a capillary network and they will bind to the host vessel passing through hydrogel.

Keywords: adipose tissue engineering, decellularization, encapsulation, hydrogel, vascularization

Procedia PDF Downloads 527

Authors: Ashit Syngle, Nidhi Garg, Pawan Krishan

Abstract:

Objective: Bone marrow derived stem cells, endothelial progenitor cells (EPCs), protect against atherosclerotic vascular damage. However, EPCs are depleted in AS and contribute to the enhanced cardiovascular risk. Statins have a protective effect in CAD and diabetes by enhancing the proliferation, migration and survival of EPCs. Therapeutic potential of augmenting EPCs to treat the heightened cardiovascular risk of AS has not yet been exploited. We aimed to investigate the effect of rosuvastatin on EPCs population and inflammation in AS. Methods: 30 AS patients were randomized to receive 6 months of treatment with rosuvastatin (10 mg/day, n=15) and placebo (n=15) as an adjunct to existing stable anti-rheumatic drugs. EPCs (CD34+/CD133+) were quantified by Flow Cytometry. Inflammatory measures (BASDAI, BASFI, CRP and ESR), pro-inflammatory cytokines (TNF-α, IL-6 and IL-1) and lipids were measured at baseline and after treatment. Results: At baseline, inflammatory measures and pro-inflammatory cytokines were elevated and EPCs depleted among both groups. EPCs increased significantly (p < 0.01) after treatment with rosuvastatin. At 6 months, BASDAI, BASFI, ESR, CRP, TNF-α, and IL-6 improved significantly in rosuvastatin group. Significant negative correlation was observed between EPCs and BASDAI, CRP and IL-6 after rosuvastatin treatment. Conclusion: First study to show that rosuvastatin augments EPCs population in AS. This defines a novel mechanism of rosuvastatin treatment in AS: the augmentation of EPCs with improvement in proinflammatory cytokines and inflammatory disease activity. The augmentation of EPCs by rosuvastatin may provide a novel strategy to prevent cardiovascular events in AS.

Keywords: ankylosing spondylitis, Endothelial Progenitor Cells, inflammation, pro-inflammatory cytokines, rosuvastatin

Procedia PDF Downloads 353

Authors: Yousuf Al Suleimani, Ahmed Al Mahruqi

Abstract:

A typical cannabinoid O-1602 induces vasorelaxation and activates the orphan G protein-coupled receptor GPR55 in human endothelial cells. The aim of this study is to characterize the mechanisms of endothelium-independent relaxation of O-1602 in the rat small mesenteric artery using wire myograph. In endothelium-denuded vessels, O-1602 partially produced concentration-dependent vasorelaxation. In vessels depleted of intracellular Ca2+ (by EGTA and methoxamine), CaCl2 produced concentration-dependent contraction. Preincubation with O-1602 (at 10 µM and 30 µM) abolished the contractile responses (P<0.01). The putative antagonist at novel “endothelial anandamide receptor” O-1918 (10 µM) significantly reversed the inhibitory effect of O-1602 on CaCl2-induced vasoconstriction. It is likely that the mechanism of endothelium-independent vasorelaxation to O-1602 is mediated by interfering with Ca2+ entry via an O-1918-sensitive pathway.

Keywords: O-1602, endothelium, vasorelaxation, calcium

Procedia PDF Downloads 358

Authors: Wedad Azhar

Abstract:

Vitamin D is an essential fat-soluble vitamin that is required for the maintenance of good health. It is obtained either through exposure to sunlight (ultraviolet B radiation) or through dietary sources. The role of vitamin D is beyond bone health. Indeed, it plays a critical role in the immune system and a broad range of organ functions such as the cardiovascular system. Moreover, vitamin D plays a critical role in the endothelial function, which is one of the main indicators of atherosclerosis. This study is investigating the correlation between vitamin D status and endothelial function in preventing and treating atherosclerosis especially in country that has ample of sunshine but yet, Saudis from suffering from this issue vitamin D deficiency and insufficiency. Ninety participants from both genders and aged 40 to 60will be involved. The participants will be categorised into three groups: the control group will be healthy persons, patients at risk of developing atherosclerosis, patients formally diagnosed atherosclerosis. Half of the participants in each group should already have been taking vitamin D supplementations. Fasting blood samples will be taken from the participants for biochemical assays. Endothelial function will be assist by flow-mediated dilation of the brachial artery. Participants will be asked to complete a questionnaire on their social and economic status, education level, daily exposure to sunlight, smoking status, consumption of supplements and medication, and a food frequency of vitamin D intake. The data will be analysed using SPSS.

Keywords: atherosclerosis, endothelial function, nutrition, vitamin D

Procedia PDF Downloads 294

Authors: Thomas Louis, Irina Primac, Florent Morfoisse, Tania Durre, Silvia Blacher, Agnes Noel

Abstract:

In vitro and in vivo lymphangiogenesis assays are essential for the identification of potential lymphangiogenic agents and the screening of pharmacological inhibitors. In the present study, we analyse three biological models: in vitro lymphatic endothelial cell spheroids, in vivo ear sponge assay, and in vivo lymph node colonisation by tumour cells. These assays provide suitable 3D models to test pro- and anti-lymphangiogenic factors or drugs. 3D images were acquired by confocal laser scanning and light sheet fluorescence microscopy. Virtual scan microscopy followed by 3D reconstruction by image aligning methods was also used to obtain 3D images of whole large sponge and ganglion samples. 3D reconstruction, image segmentation, skeletonisation, and other image processing algorithms are described. Fixed and time-lapse imaging techniques are used to analyse lymphatic endothelial cell spheroids behaviour. The study of cell spatial distribution in spheroid models enables to detect interactions between cells and to identify invasion hierarchy and guidance patterns. Global measurements such as volume, length, and density of lymphatic vessels are measured in both in vivo models. Branching density and tortuosity evaluation are also proposed to determine structure complexity. Those properties combined with vessel spatial distribution are evaluated in order to determine lymphangiogenesis extent. Lymphatic endothelial cell invasion and lymphangiogenesis were evaluated under various experimental conditions. The comparison of these conditions enables to identify lymphangiogenic agents and to better comprehend their roles in the lymphangiogenesis process. The proposed methodology is validated by its application on the three presented models.

Keywords: 3D image segmentation, 3D image skeletonisation, cell invasion, confocal microscopy, ear sponges, light sheet microscopy, lymph nodes, lymphangiogenesis, spheroids

Procedia PDF Downloads 375

Authors: Anit S. Malhotra, Ajay Duseja, Neelam Chadha

Abstract:

Endothelial dysfunction is a precursor to atherosclerosis, and flow-mediated dilatation (FMD) in the brachial artery is the commonest method to evaluate endothelial function in humans. Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver disorders encountered in clinical practice. An earlier meta-analysis had quantitatively assessed the degree of endothelial dysfunction using FMD. However, the largest study investigating the relation of FMD with NAFLD was published after that meta-analysis. In addition, that meta-analysis did not include some studies, including one from our centre. Therefore, an updating the previous meta-analysis was considered important. We searched PubMed, Cochrane Library, Embase, Scopus, SCI, Google Scholar, conference proceedings, and references of included studies till June 2017 to identify observational studies evaluating endothelial function using FMD in patients with non-alcoholic fatty liver disease. Data was analyzed using MedCalc. Fourteen studies were found eligible for inclusion in the meta-analysis. Patients with NAFLD had lower brachial artery FMD as compared to controls, standardized mean difference (random effects model) being –1.279%; 95% confidence interval (CI), –1.478 to –0.914. The effect size became smaller after addition of the recent study with the largest sample size was included compared with the earlier meta-analysis. In conclusion, patients with NAFLD had low FMD values indicating that they are at a higher risk of cardiovascular disease although our results suggest the effect size is not as large as reported previously.

Keywords: endothelial dysfunction, flow-mediated dilatation, meta-analysis, non-alcoholic fatty liver disease

Procedia PDF Downloads 190

Authors: Leila Rashki Ghaleno, Mohamad Amin Hajari, Leila Montazeri, Abdolhossein Shahverdi, Mojtaba Rezazadeh Valojerdi

Abstract:

Background: Spermatogonia stem cells (SSCs) exhibit pluripotency, enabling them to undergo differentiation into many cell lineages, including neurons, glia, endothelial cells, and hepatocytes when cultured in vitro. Although the specific mechanisms are not yet fully understood, it has been observed that biopolymer agents, such as hyaluronic acid (HA) and alginate (Alg), have the potential to induce transdifferentiation of SSCs. The current work aimed to examine the process of in vitro spermatogenesis and the conversion of mouse testicular cells into hepatocytes and erythrocyte-like cells utilizing the HA-Alg hydrogel. Method: After being extracted from the testes of a 5-day postpartum mouse (5 DPP), the testicular cells were separated into two enzymatic stages and then put into a composite hydrogel containing 0.5% HA and 1% alginate. On days 14 and 28 of culture, the colonies' growth, the cells' viability, and their histology were assessed. Result: Despite observing significant cell proliferation on day 14 and the development of circular-shaped organoids on day 28, it was noted that the organoids generated in the HA-Alg medium tended to maintain their circular morphology on day 28. Notably, the testicular cells underwent transdifferentiation into cell types resembling erythrocytes and hepatocytes. The hepatocyte-like cells exhibited the presence of glycogen and lipid deposits, indicating their hepatocyte-like characteristics. Interestingly, immunostaining analysis revealed the secretion of albumin and the presence of VEGFR on day 14. However, on day 28, albumin expression was not detected, while the expression of Sox9 (a marker for hepatocytes), Vegf, CD34, and C-kit (markers for erythrocytes) showed increased levels in the gene expression evaluation. Conclusion: The present findings indicated that HA-Alg could be a potent and effective agent for the transdifferentiation of testis cells into erythrocyte and hepatocyte-like cells, as recent studies have confirmed the transformation of SSCs into hepatocyte cells during in vitro culture.

Keywords: 3D culture, mouse testicular cell, hyaluronic acid, liver organoids

Procedia PDF Downloads 70

Authors: Sahar M. El-Gowilly, Mai M. Helmy, Hanan M. El-Gowelli

Abstract:

Methotrexate (MTX) is widely used in treatment of cancers and autoimmune diseases. However, nephrotoxicity is one of its most important side effects. The peroxisome proliferator-activated receptor gamma agonist, pioglitazone, is known to exert antiinflammatory and reno-protective effects in various kidney injuries. The purpose of this study was to investigate the potential involvement of endothelial damage in MTX-induced renal injury and to elaborate the possible protective effect of pioglitazone against MTX-induced endothelial impairment. Compared with saline-treated rats, treatment with MTX (7 mg/kg for 3 day) caused significant elevations in serum levels of urea and creatinine, increased renal nitrate/nitrite level and impaired renovascular responsiveness of isolated perfused kidney to endothelium-dependent vasodilations induced by acetylcholine (0.01-2.43 nmol) and isoprenaline (1µmol). These effects were abolished by concurrent treatment with pioglitazone (2.5 mg/kg, for 5 days starting two days before MTX). Alternatively, MTX treatment did not affect endothelium-independent renovascular relaxation induced by sodium nitroprusside (0.001-10 μmole). The possibility that alterations in renal antioxidants, circulating cytokine and apoptotic factor (Fas) levels contributed to MTX-pioglitazone interaction was assessed. Pioglitazone treatment abrogated renal oxidative stress (decreased reduced glutathione and catalase activity and increased malondialdehyde), elevated serum cytokine (interleukin-6, interleukin-10, tumor necrosis factor-alpha and transforming growth factor-beta1) and Fas induced by MTX. Histologically, MTX caused defused tubular cells swelling and vacuolization associated with endothelial damage in renal arterioles. These effects disappeared upon co-treated with pioglitazone. Collectively, pioglitazone abolished MTX-induced endothelium dysfunction and nephrotoxicity via ameliorating oxidative stress and rectifying cytokines and Fas abnormalities caused by MTX.

Keywords: methotrexate, pioglitazone, endothelium, kidney

Procedia PDF Downloads 497

Authors: Chro Kawyan

Abstract:

Endothelin-1 (ET-1), the principal individual from the newfound mammalian endothelin group of organically dynamic peptides, was initially distinguished as a 21 buildup powerful vasoconstrictor peptide in vascular endothelial cells. However, it has since been demonstrated to have a wide range of pharmacological activities in tissues both inside and outside the cardiovascular system. Additionally, peptides that have a striking resemblance to ET-1 have been identified as the primary toxic component of snake venom. In addition, late examinations have proposed that warm blooded creatures, including people, produce three unmistakable individuals from this peptide family, ET-1, ET-2 and ET-J, which might have various profiles of organic action and may follow up on particular subtypes of endothelin receptor. Masashi Yanagisawa and Tomoh Masaki survey the ongoing status of the organic chemistry and sub-atomic science of endothelin.

Keywords: thelin, microbiology, molecular biology, cell

Procedia PDF Downloads 70

Authors: Edite Kulmane, Mara Pilmane, Romans Lacis

Abstract:

Introduction: Acquired heart diseases remain one of the leading health care problems in the world. Changes in myocardium of the diseased hearts are complex and pathogenesis is still not fully clear. The aim of this study was to identify appearance and distribution of apoptosis, homeostasis regulating factors, and innervation and ischemia markers in right atrial tissue in different acquired heart diseases. Methods: During elective open heart surgery were taken right atrial tissue fragments from 12 patients. All patients were operated because of acquired heart diseases- aortic valve stenosis (5 patients), coronary heart disease (5 patients), coronary heart disease and secondary mitral insufficiency (1 patient) and mitral disease (1 patient). The mean age was (mean±SD) 70,2±7,0 years (range 58-83 years). The tissues were stained with haematoxylin and eosin methods for routine light-microscopical examination and for immunohistochemical detection of protein gene peptide 9.5 (PGP 9.5), human atrial natriuretic peptide (hANUP), vascular endothelial growth factor (VEGF), chromogranin A and endothelin. Apoptosis was detected by TUNEL method. Results: All specimens showed degeneration of cardiomyocytes with lysis of myofibrils, diffuse vacuolization especially in perinuclear region, different size of cells and their nuclei. The severe invasion of connective tissue was observed in main part of all fragments. The apoptotic index ranged from 24 to 91%. One specimen showed region of newly performed microvessels with cube shaped endotheliocytes that were positive for PGP 9.5, endothelin, chromogranin A and VEGF. From all fragments, taken from patients with coronary heart disease, there were observed numerous PGP 9.5-containing nerve fibres, except in patient with secondary mitral insufficiency, who showed just few PGP 9.5 positive nerves. In majority of specimens there were regions observed with cube shaped mixed -VEGF immunoreactive endocardial and epicardial cells. Only VEGF positive endothelial cells were observed just in few specimens. There was no significant difference of hANUP secreting cells among all specimens. All patients operated due to the coronary heart disease moderate to numerous number of chromogranin A positive cells were seen while in patients with aortic valve stenosis tissue demonstrated just few factor positive cells. Conclusions: Complex detection of different factors may indicate selectively disordered morphopathogenetical event of heart disease: decrease of PGP 9.5 nerves suggests the decreased innervation of organ; increased apoptosis indicates the cell death without ingrowth of connective tissue; persistent presence of hANUP proves the unchanged homeostasis of cardiomyocytes probably supported by expression of chromogranins. Finally, decrease of VEGF detects the regions of affected blood vessels in heart affected by acquired heart disease.

Keywords: heart, apoptosis, protein-gene peptide 9.5, atrial natriuretic peptide, vascular endothelial growth factor, chromogranin A, endothelin

Procedia PDF Downloads 294

Authors: Michelle Maurer, Antonia Last, Mark S. Gresnigt, Bernhard Hube, Alexander S. Mosig

Abstract:

The intestinal epithelium forms an essential barrier to prevent translocation of microorganisms, toxins or other potentially harmful molecules into the bloodstream. In particular, dendritic cells of the intestinal epithelium orchestrate an adapted response of immune tolerance to commensals and immune defense against invading pathogens. Systemic inflammation is typically associated with a dysregulation of this adapted immune response and is accompanied by a disruption of the epithelial and endothelial gut barrier which enables dissemination of pathogens within the human body. To understand the pathophysiological mechanisms underlying the inflammation-associated gut barrier breakdown, it is crucial to elucidate the complex interplay of the host and the intestinal microbiome. A microfluidically perfused three-dimensional intestine-on-chip model was established to emulate these processes in the presence of immune cells, commensal bacteria, and facultative pathogens. Multi-organ tissue flow (MOTiF) biochips made from polystyrene were used for microfluidic perfusion of the intestinal tissue model. The biochips are composed of two chambers separated by a microporous membrane. Each chamber is connected to inlet and outlet channels allowing independent perfusion of the individual channels and application of microfluidic shear stress. Human umbilical vein endothelial cells (HUVECs), monocyte-derived macrophages and intestinal epithelial cells (Caco-2) were assembled on the biochip membrane. Following 7 – 14 days of growth in the presence of physiological flow conditions, the epithelium was colonized with the commensal bacterium Lactobacillus rhamnosus, while the endothelium was perfused with peripheral blood mononuclear cells (PBMCs). Additionally, L. rhamnosus was co-cultivated with the opportunistic fungal pathogen Candida albicans. Within one week of perfusion, the epithelial cells formed self-organized and well-polarized villus- and crypt-like structures that resemble essential morphological characteristics of the human intestine. Dendritic cells were differentiated in the epithelial tissue that specifically responds to bacterial lipopolysaccharide (LPS) challenge. LPS is well-tolerated at the luminal epithelial side of the intestinal model without signs of tissue damage or induction of an inflammatory response, even in the presence of circulating PBMC at the endothelial lining. In contrast, LPS stimulation at the endothelial side of the intestinal model triggered the release of pro-inflammatory cytokines such as TNF, IL-1β, IL-6, and IL-8 via activation of macrophages residing in the endothelium. Perfusion of the endothelium with PBMCs led to an enhanced cytokine release. L. rhamnosus colonization of the model was tolerated in the immune competent tissue model and was demonstrated to reduce damage induced by C. albicans infection. A microfluidic intestine-on-chip model was developed to mimic a systemic infection with a dysregulated immune response under physiological conditions. The model facilitates the colonization of commensal bacteria and co-cultivation with facultative pathogenic microorganisms. Both, commensal bacteria alone and facultative pathogens controlled by commensals, are tolerated by the host and contribute to cell signaling. The human intestine-on-chip model represents a promising tool to mimic microphysiological conditions of the human intestine and paves the way for more detailed in vitro studies of host-microbiota interactions under physiologically relevant conditions.

Keywords: host-microbiota interaction, immune tolerance, microfluidics, organ-on-chip

Procedia PDF Downloads 130

Authors: Fakhrosadat Sajjadian, Ramin Ghasemi Shayan

Abstract:

The tumor microenvironment plays an fundamental part in tumor start, movement, metastasis, and treatment resistance. It varies from ordinary tissue in terms of its extracellular network, vascular and lymphatic arrange, as well as physiological conditions. The clinical application of atomic cancer imaging is regularly prevented by the tall commercialization costs of focused on imaging operators as well as the constrained clinical applications and little showcase measure of a few operators. . Since numerous cancer types share comparable characteristics of the tumor microenvironment, the capacity to target these biomarkers has the potential to supply clinically translatable atomic imaging advances for numerous types encompassing cancer and broad clinical applications. Noteworthy advance has been made in focusing on the tumor microenvironment for atomic cancer imaging. In this survey, we summarize the standards and methodologies of later progresses in atomic imaging of the tumor microenvironment, utilizing distinctive imaging modalities for early discovery and conclusion of cancer. To conclude, The tumor microenvironment (TME) encompassing tumor cells could be a profoundly energetic and heterogeneous composition of safe cells, fibroblasts, forerunner cells, endothelial cells, flagging atoms and extracellular network (ECM) components.

Keywords: molecular, imaging, TME, medicine

Procedia PDF Downloads 45

Authors: Adil Elrayah, Jie Weng, Esra Suliman

Abstract:

The mineral in human bone is not pure stoichiometric calcium phosphate (Ca/P) as it is partially substituted by in organic elements. In this study, the copper ions (Cu²⁺) substituted hydroxyapatite (CuHA) powder has been synthesized by the co-precipitation method. The CuHA powder has been used to fabricate CuHA fiber scaffolds by sol-gel process and the following sinter process. The resulted CuHA fibers have slightly different microstructure (i.e. porosity) compared to HA fiber scaffold, which is denser. The mechanical properties test was used to evaluate CuHA, and the results showed decreases in both compression strength and hardness tests. Moreover, the in vitro used endothelial cells to evaluate the angiogenesis of CuHA. The result illustrated that the viability of endothelial cell on CuHA fiber scaffold surfaces tends to antigenic behavior. The results obtained with CuHA scaffold give this material benefit in biological applications such as antimicrobial, antitumor, antigens, compacts, filling cavities of the tooth and for the deposition of metal implants anti-tumor, anti-cancer, bone filler, and scaffold.

Keywords: fiber scaffold, copper ions, hydroxyapatite, in vitro, mechanical property

Procedia PDF Downloads 155

Authors: Manali Jain, Neeta Singh, Raunaq Fatima, Soniya Nityanand, Mandakini Pradhan, Chandra Prakash Chaturvedi

Abstract:

Isolated Congenital Heart Defect (ICHD) is the major cause of neonatal death worldwide among all forms of CHDs. A significant proportion of fetuses with ICHD die in the neonatal period if no treatment is provided. Recently, stem cell therapies have emerged as a potential approach to ameliorate ICHD in children. ICHD is characterized by cardiac structural abnormalities during embryogenesis due to alterations in the cardiomyogenic properties of a pool of cardiac progenitors/ stem cells associated with fetal heart development. The stem cells present in the amniotic fluid (AF) are of fetal origin and may reflect the physiological and pathological changes in the fetus during embryogenesis. Therefore, in the present study, the cardiomyogenic potential of AF-MSCs derived from fetuses with ICHD (ICHD AF-MSCs) has been evaluated and compared with that of AF-MSCs of structurally normal fetuses (normal AF-MSCs). Normal and ICHD AF-MSC were analyzed for the expression of cardiac progenitor markers viz., stage-specific embryonic antigen-1 (SSEA-1), vascular endothelial growth factor 2 (VEGFR-2) and platelet-derived growth factor receptor-alpha (PDGFR-α) by flow cytometry. The immunophenotypic characterization revealed that ICHD AF-MSCs have significantly lower expression of cardiac progenitor markers VEGFR-2 (0.14% ± 0.6 vs.48.80% ± 0.9; p <0.01), SSEA-1 (70.86% ± 2.4 vs. 88.36% ±2.7; p <0.01), and PDGFR-α (3.92% ± 1.8 vs. 47.59% ± 3.09; p <0.01) in comparison to normal AF-MSCs. Upon induction with 5’-azacytidine for 21 days, ICHD AF-MSCs showed a significantly down-regulated expression of cardiac transcription factors such as GATA-4 (0.4 ± 0.1 vs. 6.8 ± 1.2; p<0.01), ISL-1 (2.3± 0.6 vs. 14.3 ± 1.12; p<0.01), NK-x 2-5 (1.1 ± 0.3 vs. 14.1 ±2.8; p<0.01), TBX-5 (0.4 ± 0.07 vs. 4.4 ± 0.3; p<0.001), and TBX-18 (1.3 ± 0.2 vs. 4.19 ± 0.3; p<0.01) when compared with the normal AF-MSCs. Furthermore, immunocytochemical staining revealed that both types of AF-MSCs could differentiate into cardiovascular lineages and express cardiomyogenic, endothelial, and smooth muscle actin markers, viz., cardiac troponin (cTNT), CD31, and alpha-smooth muscle actin (α-SMA). However, normal AF-MSCs showed an enhanced expression of cTNT (p<0.001), CD31 (p<0.01), and α-SMA (p<0.05), compared to ICHD AF-MSCs. Overall, these results suggest that the ICHD-AF-MSCs have a defective cardiomyogenic differentiation potential and that the defects in these stem cells may have a role in the pathogenesis of ICHD.

Keywords: amniotic fluid, cardiomyogenic potential, isolated congenital heart defect, mesenchymal stem cells

Procedia PDF Downloads 102

Authors: Liang Ning, Chung Hau Yin

Abstract:

Angiogenesis is the process of new blood vessel development. It has been recognized as a therapeutic target for blocking cancer growth four decades ago. Vascular sprouting is initiated by pro-angiogenic factors. Vascular endothelial cell growth factor (VEGF) plays a central role in angiogenic initiation, many patients with cancer or ocular neovascularization have been benefited from anti-VEGF therapy. Emerging approaches impacting in the later stages of vessel remodeling and maturation are expected to improve clinical efficacy. TIE receptor as well as the corresponding angiopoietin ligands, were identified as another endothelial cell specific receptor tyrosine kinase signaling system. Much efforts were made to reduce the activity of angiopoietin-TIE receptor axis. Two eudesmane-type sesquiterpenes from laggera alata, namely, 15-dihydrocostic acid and ilicic acid were found with strong anti-angiogenic properties in zebrafish model. Meanwhile, the mRNA expression levels of VEGFR2 and TIE2 pathway related genes were down-regulated in the sesquiterpenes treated zebrafish embryos. Besides, in human umbilical vein endothelial cells (HUVECs), the sesquiterpenes have the ability to inhibit VEGF-induced HUVECs proliferation and migration at non-toxic concentration. Moreover, angiopoietin-2 induced TIE2 phosphorylation was inhibited by the sesquiterpenes, the inhibitory effect was detected in angiopoietin-1 induced HUVECs proliferation as well. Thus, we hypothesized the anti-angiogenic activity of the compounds may via the inhibition of VEGF and TIE2 related pathways. How the compounds come into play as the pathways inhibitors need to be evaluated in the future.

Keywords: Laggera alata, eudesmane-type sesquiterpene, anti-angiogenesis, VEGF, angiopoietin, TIE2

Procedia PDF Downloads 209

Authors: Roman Major, Klaudia Trembecka- Wojciga, Juergen Markus Lackner, Boguslaw Major

Abstract:

The future and the development of science is therefore seen in interdisciplinary areas such as bio medical engineering. Self-assembled structures, similar to stem cell niches would inhibit fast division process and subsequently capture the stem cells from the blood flow. By means of surface topography and the stiffness as well as micro structure progenitor cells should be differentiated towards the formation of endothelial cells monolayer which effectively will inhibit activation of the coagulation cascade. The idea of the material surface development met the interest of the clinical institutions, which support the development of science in this area and are waiting for scientific solutions that could contribute to the development of heart assist systems. This would improve the efficiency of the treatment of patients with myocardial failure, supported with artificial heart assist systems. Innovative materials would enable the redesign, in the post project activity, construction of ventricular heart assist.

Keywords: bio-inspired materials, electron microscopy, haemocompatibility, niche-like structures, thin coatings

Procedia PDF Downloads 476

Authors: Jung Park, Anca Mazare, Klaus Von Der Mark, Patrik Schmuki

Abstract:

In clinical application of TiO2 implants on tooth and hip replacement, migration, adhesion and differentiation of neighboring mesenchymal stem cells onto implant surfaces are critical steps for successful bone regeneration. In a recent decade, accumulated attention has been paid on nanoscale electrochemical surface modifications on TiO2 layer for improving bone-TiO2 surface integration. We generated, on titanium surfaces, self-assembled layers of vertically oriented TiO2 nanotubes with defined diameters between 15 and 100 nm and here we show that mesenchymal stem cells finely sense TiO2 nanotubular geometry and quickly decide their cell fate either to differentiation into osteoblasts or to programmed cell death (apoptosis) on TiO2 nanotube layers. These cell fate decisions are critically dependent on nanotube size differences (15-100nm in diameters) of TiO2 nanotubes sensing by integrin clustering. We further demonstrate that nanoscale topography-sensing is feasible not only in mesenchymal stem cells but rather seems as generalized nanoscale microenvironment-cell interaction mechanism in several cell types composing bone tissue network including osteoblasts, osteoclast, endothelial cells and hematopoietic stem cells. Additionally we discuss the synergistic effect of simultaneous stimulation by nanotube-bound growth factor and nanoscale topographic cues on enhanced bone regeneration.

Keywords: TiO2 nanotube, stem cell fate decision, nano-scale microenvironment, bone regeneration

Procedia PDF Downloads 429

Authors: Hamid Reza Khodaei, Behnaz Mahdavi, Alireza Banitaba

Abstract:

Nitric oxide (NO) plays an important role in all sexual activities of animals. It is made in body from NO syntheses enzyme and L-arginin molecule. NO can make band with sulfur-iron complexes and due to production of steroid sexual hormones related to enzymes which have this complex, NO can change the activity of these enzymes. NO affects many cells including endothelial cells of veins, macrophages and mast cells. These cells are found in testis leydig cells and therefore are important source of NO in testis tissue. Minimizing damages to sperm at the time of sperm freezing and thawing is really important. The goal of this study was to determine the function of NO before freezing and its effects on quality and viability of sperms after thawing and incubation. 4 Holstein bulls were selected from the age of 4, and artificial insemination was done for 3 weeks (2 times a week). Treatments were 0, 10, 50 and 100 nm of sodium nitroprusside (SNP). Data analysis was performed by SAS98 program. Also, mean comparison was done using Duncan's multiple ranges test (P<0.05). Concentrations used were found to increase motility and viability of spermatozoa at 1, 2 and 3 hours after thawing significantly (P<0.05) but there was no significant difference at zero time. SNP levels reduced the amount of lipid peroxidation in sperm membrane, increased acrosome health and improved samples membranes especially in 50 and 100 nm treatments. According to results, adding SNP to semen diluents increases motility and viability of spermatozoa. Also, it reduces lipid peroxidation in sperm membrane and improves sperm function.

Keywords: sperm motility, nitric oxide, lipid peroxidation, spermatozoa

Procedia PDF Downloads 656

Authors: Samir Dekali, David Crouzier

Abstract:

Due to their new physico-chemical properties engineered nanoparticles (ENPs) are increasingly employed in numerous industrial sectors (such as electronics, textile, aerospace, cosmetics, pharmaceuticals, food industry, etc). These new physico-chemical properties can also represent a threat for the human health. Consumers can notably be exposed involuntarily by different routes such as inhalation, ingestion or through the skin. Several studies recently reported a possible biodistribution of these ENPs on the blood-brain barrier (BBB). Consequently, there is a great need for developing BBB in vitro models representative of the in vivo situation and capable of rapidly and accurately assessing ENPs toxic effects and their potential translocation through this barrier. In this study, several in vitro models established with micro-endothelial brain cell lines of different origins (bEnd.3 mouse cell line or a new human cell line) co-cultivated or not with astrocytic cells (C6 rat or C8-B4 mouse cell lines) on Transwells® were compared using different endpoints: trans-endothelial resistance, permeability of the Lucifer yellow and protein junction labeling. Impact of NIST diesel exhaust particles on BBB cell viability is also discussed.

Keywords: nanoparticles, blood-brain barrier, diesel exhaust particles, toxicology

Procedia PDF Downloads 439

Authors: Hawraa Zbeeb, Hala Khalifeh, Mohamad Khalil, Francesca Storace, Francesca Baldini, Giulio Lupidi, Laura Vergani

Abstract:

Sarcopoterium spinosum, a widely distributed spiny shrub belonging to the Rosaceae family, is rich in essential and beneficial constituents. In fact, S. spinosum fruits and roots are traditionally used as herbal medicine in the eastern Mediterranean landscape, and this shrub is mentioned as a medicinal plant in a large number of ethnobotanical surveys. Aqueous root extracts from S. spinosum are used by traditional medicinal practitioners for weight loss treatment of diabetes and pain. Moreover, the anti-diabetic activity of S. spinosum root extract has been reported in different studies, but the beneficial effects of aerial parts, especially fruits, have not been elucidated yet. The aim of the present study was to investigate the in vitro antioxidant and lipid-lowering properties of an ethanolic extract from S. spinosum fruits using both hepatic (FaO) and endothelial (HECV) cells in an attempt to evaluate its possible employment as a nutraceutical supplement. First of all, in vitro spectrophotometric assays were employed to characterize the extract. The total phenol content (TPC) was evaluated by Folin–Ciocalteu spectrophotometric method and the radical scavenging activity was tested by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays. After that, the beneficial effects of the extract were tested on cells. FaO cells treated for 3 hours with 0.75 mM oleate/palmitate mix (1:2 molar ratio) mimic in vitro a moderate hepato-steatosis. HECV cells exposed for 1 hour to 100 µM H₂O₂ mimic an oxidative insult leading to oxidative stress conditions. After the metabolic and oxidative insult, both cell lines were treated with increasing concentrations of the S. spinosum extract (1, 10, 25 µg/mL) for 24 hours. The results showed the S. spinosum ethanolic extract is rather rich in phenols (TPC of 18.6 mgGAE/g dry extracts). Moreover, the extract showed a good scavenging ability in vitro (IC₅₀ 15.9 µg/ml and 10.9 µg/ml measured by DPPH and ABTS assays, respectively). When the extract was tested on cells, the results showed that it could ameliorate some markers of cell dysfunction. The three concentrations of the extract led to a significant decrease in the intracellular triglyceride (TG) content in steatotic FaO cells measured by spectrophotometric assay. On the other hand, HECV cells treated with increasing concentrations of the extract did not result in a significant decrease in both lipid peroxidation measured by the Thiobarbituric Acid Reactive Substances (TBARS) assay, and in reactive oxygen species (ROS) production measured by fluorometric analysis after DCF staining. Interestingly, the ethanolic extract was able to accelerate the wound repair of confluent HECV cells with respect to H₂O₂-insulted cells as measured by T-scratch assay. Taken together, these results seem to indicate that the ethanol extract from S. spinosum fruits is rich in phenol compounds and plays considerable lipid-lowering activity in vitro on steatotic hepatocytes and accelerates wound healing repair on endothelial cells. In light of that, the ethanolic extract from S. spinosum fruits could be a potential candidate for nutraceutical applications.

Keywords: antioxidant activity, ethanolic extract, lipid-lowering activity, phenolic compounds, Sarcopoterium spinosum fruits

Procedia PDF Downloads 173

Authors: Sang Chul Lee, Gi-Young Park, Dong Rak Kwon

Abstract:

Objective: The aim of this study was to investigate regenerative effects of ultrasound (US)-guided injection with human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and/or polydeoxyribonucleotide (PDRN) injection in a chronic traumatic full-thickness rotator cuff tendon tear (FTRCTT) in a rabbit model. Material and Methods: Rabbits (n = 32) were allocated into 4 groups. After a 5-mm sized FTRCTT just proximal to the insertion site on the subscapularis tendon was created by excision, the wound was immediately covered by silicone tube to prevent natural healing. After 6 weeks, 4 injections (0.2 mL normal saline, G1; 0.2 mL PDRN, G2; 0.2 mL UCB-MSCs, G3; and 0.2 mL UCB-MSCs with 0.2ml PDRN, G4) were injected into FTRCTT under US guidance. We evaluated gross morphologic changes on all rabbits after sacrifice. Masson’s trichrome, anti-type 1 collagen antibody, bromodeoxyuridine, proliferating cell nuclear antigen, vascular endothelial growth factor and platelet endothelial cell adhesion molecule stain were performed to evaluate histological changes. Motion analysis was also performed. Results: The gross morphologic mean tendon tear size in G3 and 4 was significantly smaller than that of G1 and 2 (p < .05). However, there were no significant differences in tendon tear size between G3 and 4. In G4, newly regenerated collagen type 1 fibers, proliferating cells activity, angiogenesis, walking distance, fast walking time, and mean walking speed were greater than in the other three groups on histological examination and motion analysis. Conclusion: Co-injection of UCB-MSCs and PDRN was more effective than UCB-MSCs injection alone in histological and motion analysis in a rabbit model of chronic traumatic FTRCTT. However, there was no significant difference in gross morphologic change of tendon tear between UCB-MSCs with/without PDRN injection. The results of this study regarding the combination of UCB-MSCs and PDRN are worth additional investigations.

Keywords: mesenchymal stem cell, umbilical cord, polydeoxyribonucleotides, shoulder, rotator cuff, ultrasonography, injections

Procedia PDF Downloads 184

Authors: W. Baumgartner, M. Welti, N. Hild, S. C. Hess, W. J. Stark, G. Meier Bürgisser, P. Giovanoli, J. Buschmann

Abstract:

Perfusion bioreactors are used to solve problems in tissue engineering in terms of sufficient nutrient and oxygen supply. Such problems especially occur in critical size grafts because vascularization is often too slow after implantation ending up in necrotic cores. Biominerizable and biocompatible nanocomposite materials are attractive and suitable scaffold materials for bone tissue engineering because they offer mineral components in organic carriers – mimicking natural bone tissue. In addition, human adipose derived stem cells (ASCs) can potentially be used to increase bone healing as they are capable of differentiating towards osteoblasts or endothelial cells among others. In the present study, electrospun nanocomposite disks of poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/a-CaP) were seeded with human ASCs and eight disks were stacked in a bioreactor running with normal culture medium (no differentiation supplements). Under continuous perfusion and uniaxial cyclic compression, load-displacement curves as a function of time were assessed. Stiffness and energy dissipation were recorded. Moreover, stem cell densities in the layers of the piled scaffold were determined as well as their morphologies and differentiation status (endothelial cell differentiation, chondrogenesis and osteogenesis). While the stiffness of the cell free constructs increased over time caused by the transformation of the a-CaP nanoparticles into flake-like apatite, ASC-seeded constructs showed a constant stiffness. Stem cell density gradients were histologically determined with a linear increase in the flow direction from the bottom to the top of the 3.5 mm high pile (r2 > 0.95). Cell morphology was influenced by the flow rate, with stem cells getting more roundish at higher flow rates. Less than 1 % osteogenesis was found upon osteopontin immunostaining at the end of the experiment (9 days), while no endothelial cell differentiation and no chondrogenesis was triggered under these conditions. All ASCs had mainly remained in their original pluripotent status within this time frame. In summary, we have fabricated a critical size bone graft based on a biominerizable bone-biomimetic nanocomposite with preserved stiffness when seeded with human ASCs. The special feature of this bone graft was that ASC densities inside the piled construct varied with a linear gradient, which is a good starting point for tissue engineering interfaces such as bone-cartilage where the bone tissue is cell rich while the cartilage exhibits low cell densities. As such, this tissue-engineered graft may act as a bone-cartilage interface after the corresponding differentiation of the ASCs.

Keywords: bioreactor, bone, cartilage, nanocomposite, stem cell gradient

Procedia PDF Downloads 306

Authors: Dalia Atallah, Lamiaa Ahmed, Hala Zaki, Mahmoud Khattab

Abstract:

Background: Isoprenaline (ISO) administration induces myocardial damage via oxidative stress and endothelial dysfunction. Atorvastatin (ATV) treatment improves both oxidative stress and endothelial dysfunction yet recent studies have reported a pro-oxidant effect upon ATV administration on both clinical and experimental studies. The present study was directed to investigate the effect of ATV pre-treatment and treatment on ISO-induced myocardial damage. Methods: Male rats were divided into five groups (n = 10). Rats were given ISO (5mg/kg/day, i.p.) for one week with or without ATV (10mg/kg/day, p.o.). ATV was given either as pre-treatment for one week before its co-administration with ISO for another week or as a treatment for two weeks at the end of the ISO administration. At the end of the experiment, the electrocardiographic examination was done and blood was isolated for the estimation of plasma creatine kinase MB (CK-MB) activity. Rats were then sacrificed and the whole ventricles were isolated for histological examination and the estimation of lipid peroxides as malondialdehyde (MDA) level, reduced glutathione (GSH) level, catalase activity, total nitrate-nitrite (NOx), as well as the estimation of both endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) protein expression. Results: ISO-induced myocardial damage showed a significant elevation in ST segment, an increase in CK-MB activity, as well as increased oxidative stress biomarkers. Also, ISO-treated rats showed a significant decrease in myocardial NOx level and eNOS as well as degeneration in the myocardium. ATV pre-treatment didn’t show any protection to ISO-treated rats. On the other hand, ATV treatment showed a significant decrease in both the elevated ST wave and CK-MB activity. Moreover, ATV Treatment succeeded to improve oxidative stress biomarkers, tissue NOx, and eNOS protein expression, as well as amelioration of the histological alterations. Conclusion: Pre-treatment with ATV failed to protect against ISO-induced damage. This might suggest a synergistic pro-oxidant effect upon administration of the pro-oxidant ISO along with ATV as demonstrated by the increased oxidative stress and endothelial dysfunction. On the other side, ATV treatment succeeded to significantly improve oxidative stress biomarkers, endothelial dysfunction and myocardial degeneration.

Keywords: atorvastatin, endothelial dysfunction, isoprenaline, oxidative stress

Procedia PDF Downloads 446