Hematopoietic stem cell transplantation (HSCT) is the therapeutic concept to cure the blood/immune system of patients suffering from malignancies, immunodeficiencies, red blood cell disorders, and ...inherited bone marrow failure syndromes. Yet, allogeneic HSCT bear considerable risks for the patient such as non-engraftment, or graft-versus host disease. Transplanting gene modified autologous HSCs is a promising approach not only for inherited blood/immune cell diseases, but also for the acquired immunodeficiency syndrome. However, there is emerging evidence for substantial heterogeneity of HSCs in situ as well as ex vivo that is also observed after HSCT. Thus, HSC gene modification concepts are suggested to consider that different blood disorders affect specific hematopoietic cell types. We will discuss the relevance of HSC heterogeneity for the development and manufacture of gene therapies and in exemplary diseases with a specific emphasis on the key target HSC types myeloid-biased, lymphoid-biased, and balanced HSCs.
The process of angiogenesis is involved in several pathological conditions, such as tumor growth or age-related macular degeneration. Although the available anti-angiogenic drugs have improved the ...therapy of these diseases, major drawbacks, such as unwanted side effects and resistances, still exist. Consequently, the search for new anti-angiogenic substances is still ongoing. Narciclasine, a plant alkaloid from different members of the Amaryllidaceae family, has extensively been characterized as anti-tumor compound. Beyond the field of cancer, the compound has recently been shown to possess anti-inflammatory properties. Surprisingly, potential actions of narciclasine on endothelial cells in the context of angiogenesis have been neglected so far. Thus, we aimed to analyze the effects of narciclasine on angiogenic processes in vitro and in vivo and to elucidate the underlying mechanism. Narciclasine (100–300 nM) effectively inhibited the proliferation, undirected and directed migration, network formation and angiogenic sprouting of human primary endothelial cells. Moreover, narciclasine (1 mg/kg/day) strongly reduced the VEGF-triggered angiogenesis in vivo (Matrigel plug assay in mice). Narciclasine mediated its anti-angiogenic effects in part by a RhoA-independent activation of the Rho kinase ROCK. Most importantly, however, the compound reduced the de novo protein synthesis in endothelial cells by approx. 50% without exhibiting considerable cytotoxic effects. As a consequence, narciclasine diminished the presence of proteins with a short half-life, such as the VEGF receptor 2, which is the basis for its anti-angiogenic effects. Taken together, our study highlights narciclasine as an interesting anti-angiogenic compound that is worth to be further evaluated in preclinical studies.
•Narciclasine decreases key features of angiogenesis in cultured endothelial cells.•Narciclasine strongly reduces the VEGF-triggered angiogenesis in vivo.•The anti-angiogenic action is linked to a RhoA-independent activation of the ROCK.•The major mode of action of narciclasine is the reduction of VEGFR2 protein levels.•This reduction is based on the inhibition of de novo protein synthesis.
Background Bone marrow (BM) transplantation is a life-saving therapy for hematological diseases, and the BM harbors also highly useful (progenitor) cell types for novel cell therapies manufacture. ...Yet, the BM collection technique is not standardized. Methods Benchmarking our collection efficiency to BM collections worldwide (N = 1248), we noted a great variability of total nucleated cell (TNC) yields in BM products (HPC-M) with superior performance of our center, where we have implemented a small volume aspirate policy. Thus, we next prospectively aimed to assess the impact of BM collection technique on HPC-M quality. For each BM collection (N = 20 donors), small volume (3 mL) and large volume (10 mL) BM aspirates were sampled at 3 time points and analyzed for cell composition. Results Compared to large volume aspirates, small volume aspirates concentrated more TNCs, immune cells, platelets, hematopoietic stem/progenitor cells, mesenchymal stromal cells (MSCs), and endothelial progenitors. Inversely, the hemoglobin concentration was higher in large volume aspirates indicating more hemoglobin loss. Manufacturing and dosing scenarios showed that small volume aspirates save up to 42% BM volume and 44% hemoglobin for HPC-M donors. Moreover, MSC production efficiency can be increased by more than 150%. Conclusions We propose to consider small volume BM aspiration as standard technique for BM collection. Keywords: Bone marrow, Collection, Transplantation, Stem cells, Anemia, MSCs
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Classic histology is the gold standard for vascular network imaging and analysis. The method however is laborious and prone to artefacts. Here, the suitability of ultramicroscopy (UM) and ...micro-computed tomography (CT) was studied to establish potential alternatives to histology.
The vasculature of murine organs (kidney, heart and atherosclerotic carotid arteries) was visualized using conventional 2D microscopy, 3D light sheet ultramicroscopy (UM) and micro-CT. Moreover, spheroid-based human endothelial cell vessel formation in mice was quantified. Fluorescently labeled Isolectin GS-IB4 A647 was used for
labeling of vasculature for UM analysis, and analyses were performed ex vivo after sample preparation. For CT imaging, animals were perfused postmortem with radiopaque contrast agent.
Using UM imaging, 3D vascular network information could be obtained in samples of animals receiving
injection of the fluorescently labeled Isolectin GS-IB4. Resolution was sufficient to measure single endothelial cell integration into capillaries in the spheroid-based matrigel plug assay. Because of the selective staining of the endothelium, imaging of larger vessels yielded less favorable results. Using micro-CT or even nano-CT, imaging of capillaries was impossible due to insufficient X-ray absorption and thus insufficient signal-to-noise ratio. Identification of lumen in murine arteries using micro-CT was in contrast superior to UM.
UM and micro-CT are two complementary techniques. Whereas UM is ideal for imaging and especially quantifying capillary networks and arterioles, larger vascular structures are easier and faster to quantify and visualize using micro-CT. 3D information of both techniques is superior to 2D histology. UM and micro-CT together may open a new field of clinical pathology diagnosis.
Transfusion of packed red blood cells (pRBCs) saves lives, but iron overload limits survival of chronically transfused patients. Quality control methods, which involve entering pRBC units and ...removing them from the blood supply, reveal that hemoglobin (38.5–79.9 g) and heme iron (133.42–276.89 mg) vary substantially between pRBCs. Yet, neither hemoglobin nor iron content can be quantified for individual clinically used pRBCs leading to rules of thumb for pRBC transfusions. Keeping their integrity, the authors seek to predict hemoglobin/iron content of any given pRBC unit applying eight machine learning models on 6,058 pRBCs. Based on thirteen features routinely collected during blood donation, production and quality control testing, the model with best trade‐off between performance and complexity in hemoglobin/iron content prediction is identified. Validation of this model in an independent cohort of 2637 pRBCs confirms an adjusted R2 > 0.9 corresponding to a mean absolute prediction error of ≤1.43 g hemoglobin/4.96 mg iron (associated standard deviation: ≤1.13 g hemoglobin/3.92 mg iron). Such unprecedented precise prediction enables reliable pRBC dosing per pharmaceutically active agent, and monitoring iron uptake in patients and individual iron loss in donors. The model is implemented in a free open source web application to facilitate clinical application.
Hemoglobin and iron content vary substantially between packed red blood cell (pRBC) units, leading to rules of thumb for blood transfusions. Machine learning now allows to precisely predict hemoglobin and iron content of individual pRBC units, enabling for the first time reliable pRBC dosing per pharmaceutically active agent, and monitoring iron uptake in patients and individual iron loss in donors.
BACKGROUND:The angiogenic function of endothelial cells is regulated by numerous mechanisms, but the impact of long noncoding RNAs (lncRNAs) has hardly been studied. We set out to identify novel and ...functionally important endothelial lncRNAs.
METHODS:Epigenetically controlled lncRNAs in human umbilical vein endothelial cells were searched by exon-array analysis after knockdown of the histone demethylase JARID1B. Molecular mechanisms were investigated by RNA pulldown and immunoprecipitation, mass spectrometry, microarray, several knockdown approaches, CRISPR-Cas9, assay for transposase-accessible chromatin sequencing, and chromatin immunoprecipitation in human umbilical vein endothelial cells. Patient samples from lung and tumors were studied for MANTIS expression.
RESULTS:A search for epigenetically controlled endothelial lncRNAs yielded lncRNA n342419, here termed MANTIS, as the most strongly regulated lncRNA. Controlled by the histone demethylase JARID1B, MANTIS was downregulated in patients with idiopathic pulmonary arterial hypertension and in rats treated with monocrotaline, whereas it was upregulated in carotid arteries of Macaca fascicularis subjected to atherosclerosis regression diet, and in endothelial cells isolated from human glioblastoma patients. CRISPR/Cas9-mediated deletion or silencing of MANTIS with small interfering RNAs or GapmeRs inhibited angiogenic sprouting and alignment of endothelial cells in response to shear stress. Mechanistically, the nuclear-localized MANTIS lncRNA interacted with BRG1, the catalytic subunit of the switch/sucrose nonfermentable chromatin-remodeling complex. This interaction was required for nucleosome remodeling by keeping the ATPase function of BRG1 active. Thereby, the transcription of key endothelial genes such as SOX18, SMAD6, and COUP-TFII was regulated by ensuring efficient RNA polymerase II machinery binding.
CONCLUSION:MANTIS is a differentially regulated novel lncRNA facilitating endothelial angiogenic function.
OBJECTIVE—Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases contribute to angiogenesis and vascular repair. NADPH oxidase organizer 1 (NoxO1) is a ...cytosolic protein facilitating assembly of constitutively active NADPH oxidases. We speculate that NoxO1 also contributes to basal reactive oxygen species formation in the vascular system and thus modulates angiogenesis.
APPROACH AND RESULTS—A NoxO1 knockout mouse was generated, and angiogenesis was studied in cultured cells and in vivo. Angiogenesis of the developing retina and after femoral artery ligation was increased in NoxO1 when compared with wild-type animals. Spheroid outgrowth assays revealed greater angiogenic capacity of NoxO1 lung endothelial cells (LECs) and a more tip-cell–like phenotype than wild-type LECs. Usually signaling by the Notch pathway switches endothelial cells from a tip into a stalk cell phenotype. NoxO1 LECs exhibited attenuated Notch signaling as a consequence of an attenuated release of the Notch intracellular domain on ligand stimulation. This release is mediated by proteolytic cleavage involving the α-secretase ADAM17. For maximal activity, ADAM17 has to be oxidized, and overexpression of NoxO1 promoted this mode of activation. Moreover, the activity of ADAM17 was reduced in NoxO1 LECs when compared with wild-type LECs.
CONCLUSIONS—NoxO1 stimulates α-secretase activity probably through reactive oxygen species–mediated oxidation. Deletion of NoxO1 attenuates Notch signaling and thereby promotes a tip-cell phenotype that results in increased angiogenesis.
PDF
