Search results for: interstitial fibrosis

Authors: M. Markova, E. Filova, O. Kaplan, R. Matejka, L. Bacakova

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The porcine pericardium is used as a material for cardiac and aortic valves substitutes. Current biological aortic heart valve prosthesis have a limited lifetime period because they undergo degeneration. In order to make them more biocompatible and prolong their lifetime it is necessary to reseed the decellularized prostheses with endothelial cells and with valve interstitial cells. The endothelialization of the prosthesis-surface may be supported by suitable chemical surface modification of the prosthesis. The aim of this study is to prepare bioactive fibrin layers which would both support endothelialization of porcine pericardium and enhance differentiation and maturation of the endothelial cells seeded. As a material for surface adjustments we used layers of fibrin with/without heparin and some of them with adsorbed or chemically bound FGF2, VEGF or their combination. Fibrin assemblies were prepared in 24-well cell culture plate and were seeded with HSVEC (Human Saphenous Vein Endothelial Cells) at a density of 20,000 cells per well in EGM-2 medium with 0.5% FS and without heparin, without FGF2 and without VEGF; medium was supplemented with aprotinin (200 U/mL). As a control, surface polystyrene (PS) was used. Fibrin was also used as homogeneous impregnation of the decellularized porcine pericardium throughout the scaffolds. Morphology, density, and viability of the seeded endothelial cells were observed from micrographs after staining the samples by LIVE/DEAD cytotoxicity/viability assay kit on the days 1, 3, and 7. Endothelial cells were immunocytochemically stained for proteins involved in cell adhesion, i.e. alphaV integrin, vinculin, and VE-cadherin, markers of endothelial cells differentiation and maturation, i.e. von Willebrand factor and CD31, and for extracellular matrix proteins typically produced by endothelial cells, i.e. type IV collagen and laminin. The staining intensities were subsequently quantified using a software. HSVEC cells grew on each of the prepared surfaces better than on control surface. They reached confluency. The highest cell densities were obtained on the surface of fibrin with heparin and both grow factors used together. Intensity of alphaV integrins staining was highest on samples with remained fibrin layer, i.e. on layers with lower cell densities, i.e. on fibrin without heparin. Vinculin staining was apparent, but was rather diffuse, on fibrin with both FGF2 and VEGF and on control PS. Endothelial cells on all samples were positively stained for von Willebrand factor and CD31. VE-cadherin receptors clusters were best developed on fibrin with heparin and growth factors. Significantly stronger staining of type IV collagen was observed on fibrin with heparin and both growth factors. Endothelial cells on all samples produced laminin-1. Decellularized pericardium was homogeneously filled with fibrin structures. These fibrin-modified pericardium samples will be further seeded with cells and cultured in a bioreactor. Fibrin layers with/without heparin and with adsorbed or chemically bound FGF2, VEGF or their combination are good surfaces for endothelialization of cardiovascular prostheses or porcine pericardium based heart valves. Supported by the Ministry of Health, grants No15-29153A and 15-32497A, and the Grant Agency of the Czech Republic, project No. P108/12/G108.

Keywords: aortic valves prosthesis, FGF2, heparin, HSVEC cells, VEGF

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Authors: Kristina Horvat Blazinovic

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In the context of a work of art, one can talk about the idea-concept-term-intention expressed by the artist by using various forms of repetition (external, material, visible repetition). Such repetitions of elements (images in space or moving visual and sound images in time) suggest a "covert", "latent" ("dressed") repetition – i.e., "hidden", "latent" term-intention-idea. Repeating in this way reveals a "deeper truth" that the viewer needs to decode and which is hidden "under" the technical manifestation of the multiplied images. It is not only images, sounds, and screens that are repeated - something else is repeated through them as well, even if, in some cases, the very idea of repetition is repeated. This paper examines serial images and single-channel or multi-channel artwork in the field of video/film art and video installations, which in a way implies the concept of repetition and multiplication. Moving or static images and screens (as multi-screens) are repeated in time and space. The categories of the real and the symbolic partly refer to the Lacan registers of reality, i.e., the Imaginary - Symbolic – Real trinity that represents the orders within which human subjectivity is established. Authors such as Bruce Nauman, VALIE EXPORT, Ragnar Kjartansson, Wolf Vostell, Shirin Neshat, Paul Sharits, Harun Farocki, Dalibor Martinis, Andy Warhol, Douglas Gordon, Bill Viola, Frank Gillette, and Ira Schneider, and Marina Abramovic problematize, in different ways, the concept and procedures of multiplication - repetition, but not in the sense of "copying" and "repetition" of reality or the original, but of repeated repetitions of the simulacrum. Referential works of art are often connected by the theme of the traumatic. Repetitions of images and situations are a response to the traumatic (experience) - repetition itself is a symptom of trauma. On the other hand, repeating and multiplying traumatic images results in a new traumatic effect or cancels it. Reflections on repetition as a temporal and spatial phenomenon are in line with the chapters that link philosophical considerations of space and time and experience temporality with their manifestation in works of art. The observations about time and the relation of perception and memory are according to Henry Bergson and his conception of duration (durée) as "quality of quantity." The video works intended to be displayed as a video loop, express the idea of infinite duration ("pure time," according to Bergson). The Loop wants to be always present - to fixate in time. Wholeness is unrecognizable because the intention is to make the effect infinitely cyclic. Reflections on time and space end with considerations about the occurrence and effects of time and space intervals as places and moments "between" – the points of connection and separation, of continuity and stopping - by reference to the "interval theory" of Soviet filmmaker DzigaVertov. The scale of opportunities that can be explored in interval mode is wide. Intervals represent the perception of time and space in the form of pauses, interruptions, breaks (e.g., emotional, dramatic, or rhythmic) denote emptiness or silence, distance, proximity, interstitial space, or a gap between various states.

Keywords: video installation, performance, repetition, multi-screen, real and symbolic, loop, video art, interval, video time

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Authors: Hanan M. Hassan, Laila Abouzeid, Lamya H. Al-Wahaibi, George S. G. Shehatou, Ali A. El-Emam

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Cancer is a major public health problem and the second leading cause of death worldwide. In 2020, cancer diagnosis and treatment have been negatively affected by the coronavirus 2019 (COVID-19) pandemic. During the quarantine, because of the limited access to healthcare and avoiding exposure to COVID-19 as a contagious disease; patients of cancer suffered deferments in follow-up and treatment regimens leading to substantial worsening of disease, death, and increased healthcare costs. Thus, this study is designed to investigate the molecular mechanisms by which adamantne derivatives attenuate hepatocllular carcinoma experimentally and theoretically. There is a close association between increased resistance to anticancer drugs and defective apoptosis that considered a causative factor for oncogenesis. Cancer cells use different molecular pathways to inhibit apoptosis, BAX and Bcl-2 proteins have essential roles in the progression or inhibition of intrinsic apoptotic pathways triggered by mitochondrial dysfunction. Therefore, their balance ratio can promote the cellular apoptotic fate. In this study, the in vitro cytotoxic effects of seven synthetic adamantyl isothiorea derivatives were evaluated against five human tumor cell lines by MTT assay. Compounds 5 and 6 showed the best results, mostly against hepatocellular carcinoma (HCC). Hence, in vivo studies were performed in male Sprague-Dawley (SD) rats in which experimental hepatocellular carcinoma was induced with thioacetamide (TAA) (200 mg/kg, i.p., twice weekly) for 16 weeks. The most promising compounds, 5 and 6, were administered to treat liver cancer rats at a dose of 10 mg/kg/day for an additional two weeks, and the effects were compared with doxorubicin (DR), the anticancer drug. Hepatocellular carcinoma was evidenced by a dramatic increase in liver indices, oxidative stress markers, and immunohistochemical studies that were accompanied by a plethora of inflammatory mediators and alterations in the apoptotic cascade. Our results showed that treatment with adamantane derivatives 5 and 6 significantly suppressed fibrosis, inflammation, and other histopathological insults resulting in the diminished formation of hepatocyte tumorigenesis. Moreover, administration of the tested compounds resulted in amelioration of EGFR protein expression, upregulation of BAX, and lessening down of Bcl-2 levels that prove their role as apoptosis inducers. Also, the docking simulations performed for adamantane showed good fit and binding to the EGFR protein through hydrogen bond formation with conservative amino acids, which gives a shred of strong evidence for its hepatoprotective effect. In most analyses, the effects of compound 6 were more comparable to DR than compound 5. Our findings suggest that adamantane derivatives 5 and 6 are shown to have cytotoxic activity against HCC in vitro and in vivo, by more than one mechanism, possibly by inhibiting the TLR4-MyD88-NF-κB pathway and targeting EGFR signaling.

Keywords: adamantane, EGFR, HCC, apoptosis

Procedia PDF Downloads 125

Authors: Mei Chen, Yingping Hou, Michelle Hao, Soheil Aghamohammadzadeh, Esteban Terzo, Roger Clark, Vijay Modur

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Background: Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a genetic skin condition characterized by skin tearing and unremitting blistering upon minimal trauma. Repeated blistering, fibrosis, and scarring lead to aggressive squamous cell carcinoma later in life. RDEB is caused by mutations in the COL7A1 gene encoding collagen type VII (C7), the major component of anchoring fibrils mediating epidermis-dermis adherence. Nonsense mutations in the COL7A1 gene of a subset of RDEB patients leads to premature termination codons (PTC). Similarly, most Junctional Epidermolysis Bullosa (JEB) cases are caused by nonsense mutations in the LAMB3 gene encoding the β3 subunit of laminin 332. Currently, there is an unmet need for the treatment of RDEB and JEB. Zikani Therapeutics has discovered an array of macrocyclic compounds with ring structures similar to macrolide antibiotics that can facilitate readthrough activity of nonsense mutations in the COL7A1 and LAMB3 genes by acting as Ribosome Modulating Agents (RMAs). The medicinal chemistry synthetic advancements of these macrocyclic compounds have allowed targeting the human ribosome while preserving the structural elements responsible for the safety and pharmacokinetic profile of clinically used macrolide antibiotics. Methods: C7 expression was used as a measure of readthrough activity by immunoblot assays in two primary human fibroblasts from RDEB patients (R578X/R578X and R163X/R1683X-COL7A1). Similarly, immunoblot assays in C325X/c.629-12T > A-LAMB3 keratinocytes were used to measure readthrough activity for JEB. The relative readthrough activity of each compound was measured relative to Gentamicin. An imaging-based fibroblast migration assay was used as an assessment of C7 functionality in RDEB-fibroblasts over 16-20 hrs. The incubation period for the above experiments was 48 hrs for RDEB fibroblasts and 72 hours for JEB keratinocytes. Results: 9 RMAs demonstrated increased protein expression in both patient RDEB fibroblasts. The highest readthrough activity at tested concentrations without cytotoxicities increased protein expression up to 179% of Gentamicin (400 µg/ml), with favored readthrough activity in R163X/R1683X-COL7A1 fibroblasts. Concurrent with protein expression, fibroblast hypermotility phenotype observed in RDEB was rescued by reducing motility by ~35% to WT levels (the same level as 690 µM Gentamicin treated cells). Laminin β3 expression was also shown to be increased by 6 RMAs in keratinocytes to 33-83% of (400 µg/ml) Gentamicin. Conclusions: To date, 9 RMAs have been identified that enhance the expression of functional C7 in a mutation-dependent manner in two different RDEB patient fibroblast backgrounds (R578X/R578X and R163X/R1683X-COL7A1). A further 6 RMAs have been identified that enhance the readthrough of C325X-LAMB3 in JEB patient keratinocytes. Based on the clinical trial conducted by us with topical gentamycin in 2017, Zikani’s RMAs achieve clinically significant levels of read-through for the treatment of recessive dystrophic and Junctional Epidermolysis Bullosa.

Keywords: epidermolysis bullosa, nonsense mutation, readthrough, ribosome modulation

Procedia PDF Downloads 76

Authors: Imdad Ullah Khan, Yongseok Yoon, Kyeung Uk Choi, Kwang Rae Jo, Namyul Kim, Eunbee Lee, Wan Hee Kim, Oh-Kyeong Kweon

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The primary spinal cord injury (SCI) causes mechanical damage to the neurons and blood vessels. It leads to secondary SCI, which activates multiple pathological pathways, which expand neuronal damage at the injury site. It is characterized by vascular disruption, ischemia, excitotoxicity, oxidation, inflammation, and apoptotic cell death. It causes nerve demyelination and disruption of axons, which perpetuate a loss of impulse conduction through the injured spinal cord. It also leads to the production of myelin inhibitory molecules, which with a concomitant formation of an astroglial scar, impede axonal regeneration. The pivotal role regarding the neuronal necrosis is played by oxidation and inflammation. During an early stage of spinal cord injury, there occurs an abundant expression of reactive oxygen species (ROS) due to defective mitochondrial metabolism and abundant migration of phagocytes (macrophages, neutrophils). ROS cause lipid peroxidation of the cell membrane, and cell death. Abundant migration of neutrophils, macrophages, and lymphocytes collectively produce pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-1beta (IL-1β), matrix metalloproteinase, superoxide dismutase, and myeloperoxidases which synergize neuronal apoptosis. Therefore, it is crucial to control inflammation and oxidation injury to minimize the nerve cell death during secondary spinal cord injury. Therefore, in response to oxidation and inflammation, heme oxygenase-1 (HO-1) is induced by the resident cells to ameliorate the milieu. In the meanwhile, neurotrophic factors are induced to promote neuroregeneration. However, it seems that anti-stress enzyme (HO-1) and neurotrophic factor (BDNF) do not significantly combat the pathological events during secondary spinal cord injury. Therefore, optimum healing can be induced if anti-inflammatory and neurotrophic factors are administered in a higher amount through an exogenous source. During the first experiment, the inflammation and neuroregeneration were selectively targeted. HO-1 expressing MSCs (HO-1 MSCs) and BDNF expressing MSCs (BDNF MSC) were co-transplanted in one group (combination group) of dogs with subacute spinal cord injury to selectively control the expression of inflammatory cytokines by HO-1 and induce neuroregeneration by BDNF. We compared the combination group with the HO-1 MSCs group, BDNF MSCs group, and GFP MSCs group. We found that the combination group showed significant improvement in functional recovery. It showed increased expression of neural markers and growth-associated proteins (GAP-43) than in other groups, which depicts enhanced neuroregeneration/neural sparing due to reduced expression of pro-inflammatory cytokines such as TNF-alpha, IL-6 and COX-2; and increased expression of anti-inflammatory markers such as IL-10 and HO-1. Histopathological study revealed reduced intra-parenchymal fibrosis in the injured spinal cord segment in the combination group than in other groups. Thus it was concluded that selectively targeting the inflammation and neuronal growth with the combined use of HO-1 MSCs and BDNF MSCs more favorably promote healing of the SCI. HO-1 MSCs play a role in controlling the inflammation, which favors the BDNF induced neuroregeneration at the injured spinal cord segment of dogs.

Keywords: HO-1 MSCs, BDNF MSCs, neuroregeneration, inflammation, anti-inflammation, spinal cord injury, dogs

Procedia PDF Downloads 98

Authors: A. C. Facin, M. C. Maronezi , M. P. Menezes, G. L. Montanhim, L. Pavan, M. A. R. Feliciano, R. P. Nociti, R. A. R. Uscategui, P. C. Moraes

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The incidence of brachycephalic syndrome (BS) in the clinical routine of small animals has increased significantly giving the higher proportion of brachycephalic pets in the last years and has been considered as an animal welfare problem. The treatment of BS is surgical and the clinical signs related can be considerably attenuated. Nevertheless, the systemic effects of the BS are still poorly reported and little is known about these when the surgical correction is not performed early. Affected dogs are more likely to develop cardiopulmonary, gastrointestinal and sleep disorders in which the chronic hypoxemia plays a major role. This syndrome is compared with the obstructive sleep apnea (OSA) in humans, both considered as causes of systemic and metabolic dysfunction. Among the several consequences of the BS little is known if the syndrome also affects the hepatic tissue of brachycephalic patients. Elastography is a promising ultrasound technique that evaluates tissue elasticity and has been recently used with the purpose of diagnosis of liver fibrosis. In medicine, it is a growing concern regarding the hepatic injury of patients affected by OSA. This prospective study hypothesizes if there is any consequence of BS in the hepatic parenchyma of brachycephalic dogs that don’t receive any surgical treatment. This study was conducted following the approval of the Animal Ethics and Welfare Committee of the Faculdade de Ciências Agrárias e Veterinárias, UNESP, Campus Jaboticabal, Brazil (protocol no 17944/2017) and funded by Sao Paulo Research Foundation (FAPESP, process no 2017/24809-4). The methodology was based in ARFI elastography using the ACUSON S2000/SIEMENS device, with convex multifrequential transducer and specific software as well as clinical evaluation of the syndrome, in order to determine if they can be used as a prognostic non-invasive tool. On quantitative elastography, it was collected three measures of shear wave velocity (meters per second) and depth in centimeters in the left lateral, left medial, right lateral, right medial and caudate lobe of the liver. The brachycephalic patients, 16 pugs and 30 french bulldogs, were classified using a previously established 4-point functional grading system based on clinical evaluation before and after a 3-minute exercise tolerance test already established and validated. The control group was based on the same features collected in 22 beagles. The software R version 3.3.0 was used for the analysis and the significance level was set at 0.05. The data were analysed for normality of residuals and homogeneity of variances by Shapiro-Wilks test. Comparisons of parametric continuous variables between breeds were performed by using ANOVA with a post hoc test for pair wise comparison. The preliminary results show significant statistic differences between the brachycephalic groups and the control group in all lobes analysed (p ≤ 0,05), with higher values of shear wave velocities in the hepatic tissue of brachycephalic dogs. In this context, the results obtained in this study contributes to the understanding of BS as well as its consequences in our patients, reflecting in evidence that one more systemic consequence of the syndrome may occur in brachycephalic patients, which was not related in the veterinary literature yet.

Keywords: airway obstruction, brachycephalic airway obstructive syndrome, hepatic injury, obstructive sleep apnea

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