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Human Reproduction (Oxford, England) Jun 2024What is the pathological mechanism involved in a thin endometrium, particularly under ischaemic conditions?
Cytoskeletal and inter-cellular junction remodelling in endometrial organoids under oxygen-glucose deprivation: a new potential pathological mechanism for thin endometria.
STUDY QUESTION
What is the pathological mechanism involved in a thin endometrium, particularly under ischaemic conditions?
SUMMARY ANSWER
Endometrial dysfunction in patients with thin endometrium primarily results from remodelling in cytoskeletons and cellular junctions of endometrial epithelial cells under ischemic conditions.
WHAT IS KNOWN ALREADY
A healthy endometrium is essential for successful embryo implantation and subsequent pregnancy; ischemic conditions in a thin endometrium compromise fertility outcomes.
STUDY DESIGN, SIZE, DURATION
We recruited 10 patients with thin endometrium and 15 patients with healthy endometrium. Doppler ultrasound and immunohistochemical results confirmed the presence of insufficient endometrial blood perfusion in patients with thin endometrium. Organoids were constructed using healthy endometrial tissue and cultured under oxygen-glucose deprivation (OGD) conditions for 24 h. The morphological, transcriptomic, protein expression, and signaling pathway changes in the OGD organoids were observed. These findings were validated in both thin endometrial tissue and healthy endometrial tissue samples.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Endometrial thickness and blood flow were measured during the late follicular phase using transvaginal Doppler ultrasound. Endometrial tissue was obtained via hysteroscopy. Fresh endometrial tissues were used for the generation and culture of human endometrial organoids. Organoids were cultured in an appropriate medium and subjected to OGD to simulate ischemic conditions. Apoptosis and cell death were assessed using Annexin-V/propidium iodide staining. Immunofluorescence analysis, RNA sequencing, western blotting, simple westerns, immunohistochemistry, and electron microscopy were conducted to evaluate cellular and molecular changes.
MAIN RESULTS AND THE ROLE OF CHANCE
Patients with thin endometrium showed significantly reduced endometrial thickness and altered blood flow patterns compared to those with healthy endometrium. Immunohistochemical staining revealed fewer CD34-positive blood vessels and glands in the thin endometrium group. Organoids cultured under OGD conditions exhibited significant morphological changes, increased apoptosis, and cell death. RNA-seq identified differentially expressed genes related to cytoskeletal remodeling and stress responses. OGD induced a strong cytoskeletal reorganization, mediated by the RhoA/ROCK signaling pathway. Additionally, electron microscopy indicated compromised epithelial integrity and abnormal cell junctions in thin endometrial tissues. Upregulation of hypoxia markers (HIF-1α and HIF-2α) and activation of the RhoA/ROCK pathway were also observed in thin endometrial tissues, suggesting ischemia and hypoxia as underlying mechanisms.
LARGE SCALE DATA
none.
LIMITATIONS AND REASONS FOR CAUTION
The study was conducted in an in vitro model, which may not fully replicate the complexity of in vivo conditions.
WIDER IMPLICATIONS OF THE FINDINGS
This research provides a new three-dimensional in vitro model of thin endometrium, as well as novel insights into the pathophysiological mechanisms of endometrial ischaemia in thin endometrium, offering potential avenues for identifying therapeutic targets for treating fertility issues related to thin endometrium.
STUDY FUNDING/COMPETING INTEREST(S)
This study was supported by the National Natural Science Foundation of China (81925013); National Key Research and Development Project of China (2022YFC2702500, 2021YFC2700303, 2021YFC2700601); the Capital Health Research and Development Project (SF2022-1-4092); the National Natural Science Foundation of China (82288102, 81925013, 82225019, 82192873); Special Project on Capital Clinical Diagnosis and Treatment Technology Research and Transformation Application (Z211100002921054); the Frontiers Medical Center, Tianfu Jincheng Laboratory Foundation(TFJC2023010001). The authors declare that no competing interests exist.
PubMed: 38915267
DOI: 10.1093/humrep/deae137 -
Molecular Biology Reports Jun 2024Mesenchymal Stem Cells, mesodermal origin and multipotent stem cells, have ability to differentiate into vascular endothelial cells. The cells are squamous in... (Review)
Review
Mesenchymal Stem Cells, mesodermal origin and multipotent stem cells, have ability to differentiate into vascular endothelial cells. The cells are squamous in morphology, inlining, and protecting blood vessel tissue, as well as maintaining homeostatic conditions. ECs are essential in vascularization and blood vessels formation. The differentiation process, generally carried out in 2D culture systems, were relied on growth factors induction. Therefore, an artificial extracellular matrix with relevant mechanical properties is essential to build 3D culture models. Various 3D fabrication techniques, such as hydrogel-based and fibrous scaffolds, scaffold-free, and co-culture to endothelial cells were reviewed and summarized to gain insights. The obtained MSCs-derived ECs are shown by the expression of endothelial gene markers and tubule-like structure. In order to mimicking relevant vascular tissue, 3D-bioprinting facilitates to form more complex microstructures. In addition, a microfluidic chip with adequate flow rate allows medium perfusion, providing mechanical cues like shear stress to the artificial vascular vessels.
Topics: Humans; Mesenchymal Stem Cells; Cell Differentiation; Endothelial Cells; Cell Culture Techniques, Three Dimensional; Tissue Scaffolds; Tissue Engineering; Animals; Hydrogels; Cell Culture Techniques; Coculture Techniques; Extracellular Matrix
PubMed: 38913199
DOI: 10.1007/s11033-024-09743-8 -
Resuscitation Plus Sep 2024Minimizing cardiac arrest times is critical in extracorporeal cardiopulmonary resuscitation (ECPR). Pre-primed extra corporeal membrane oxygenator (ECMO) is used for...
AIM
Minimizing cardiac arrest times is critical in extracorporeal cardiopulmonary resuscitation (ECPR). Pre-primed extra corporeal membrane oxygenator (ECMO) is used for this, but knowledge is limited to experimental studies. We prospectively investigated oxygenator function and sterility in dry plus wet pre-priming in a clinical setting.
METHODS
This prospective clinical study included 107 ECMO circuits used at Sahlgrenska University Hospital between October 2019 and December 2021. Circuits underwent dry set-up, followed by wet priming when the previous wet-primed circuit was used. Sterility was assessed by culturing the priming solution. Oxygenator function parameters, including sweep gas flow, fraction of oxygen (FiO), and oxygenator resistance, were analyzed at ECMO initiation and during treatment using linear mixed models.
RESULTS
Median total set-up time was 14 days (range 0-97), with a median wet prime time of 6 days (range 0-57). 103 of 105 circuits with culture results were negative, two showed bacterial growth (coagulase-negative staphylococci and ). Wet prime time did not significantly affect initial oxygenator function. Oxygenator resistance and FiO increased during ECMO treatment (0.035 mmHg/L min (95 % confidence interval (CI) 0.015-0.055) < 0.001, and 2.19 % (0.92-3.46) = 0.009), but these changes were unrelated to wet prime time.
CONCLUSION
Wet pre-priming of ECMO circuits for up to 57 days did not affect oxygenator function. The low incidence of bacterial growth (1.9 %) suggests that pre-primed ECMO generally maintain sterility and can facilitate rapid ECPR initiation. However, bacterial growth highlights the need for caution in non-urgent cases. Culturing the circuit at initiation can mitigate this risk.
PubMed: 38912528
DOI: 10.1016/j.resplu.2024.100680 -
Frontiers in Physiology 2024This study was undertaken to explore the potential therapeutic effects of Tongyang Huoxue Granules (TYHX) on sinoatrial node (SAN) dysfunction, a cardiac disorder...
INTRODUCTION
This study was undertaken to explore the potential therapeutic effects of Tongyang Huoxue Granules (TYHX) on sinoatrial node (SAN) dysfunction, a cardiac disorder characterized by impaired impulse generation or conduction. The research question addressed whether TYHX could positively influence SAN ion channel function, specifically targeting the sodium-calcium exchanger ( ) and L-type calcium channel ( ) of the SAN.
METHODS
Sinoatrial node cells (SANCs) were isolated and cultured from neonatal Japanese big-eared white rabbits within 24 h of birth. The study encompassed five groups: Control, H/R (hypoxia/reoxygenation), H/R+100 μg/mL TYHX, H/R+200 μg/mL TYHX, and H/R+400 μg/mL TYHX. The H/R model, simulating hypoxia/reoxygenation stress, was induced within 5 days of culture. Whole-cell patch clamp technique was employed to record currents following a 3-min perfusion and stabilization period with TYHX.
RESULTS
TYHX administration demonstrated improvements in the ignition phase of impaired SANCs. The half-maximal effective dose of TYHX, as determined by SANC beating frequency, was found to be 323.63 μg/mL. Inward current density of increased in response to TYHX (200 and 400 μg/mL), while TYHX enhanced current density in H/R SANCs, with 400 μg/mL exhibiting greater efficacy. Additionally, TYHX regulated the gating mechanisms of by right-shifting the steady-state inactivation curve and accelerating recovery from inactivation. Notably, TYHX increased the activation time constant under 200 and 400 μg/mL, prolonged the fast inactivation time constant τ1 with 400 μg/mL, and extended the slow inactivation time constant τ2 with 100 and 400 μg/mL.
DISCUSSION AND CONCLUSION
The findings suggest that TYHX may hold promise as a therapeutic intervention for sinus node dysfunction, offering potential avenues for drug development aimed at safeguarding SAN function.
PubMed: 38911325
DOI: 10.3389/fphys.2024.1402478 -
Cell Stem Cell Jun 2024The intricate anatomical structure and high cellular density of the myocardium complicate the bioengineering of perfusable vascular networks within cardiac tissues....
The intricate anatomical structure and high cellular density of the myocardium complicate the bioengineering of perfusable vascular networks within cardiac tissues. In vivo neonatal studies highlight the key role of resident cardiac macrophages in post-injury regeneration and angiogenesis. Here, we integrate human pluripotent stem-cell-derived primitive yolk-sac-like macrophages within vascularized heart-on-chip platforms. Macrophage incorporation profoundly impacted the functionality and perfusability of microvascularized cardiac tissues up to 2 weeks of culture. Macrophages mitigated tissue cytotoxicity and the release of cell-free mitochondrial DNA (mtDNA), while upregulating the secretion of pro-angiogenic, matrix remodeling, and cardioprotective cytokines. Bulk RNA sequencing (RNA-seq) revealed an upregulation of cardiac maturation and angiogenesis genes. Further, single-nuclei RNA sequencing (snRNA-seq) and secretome data suggest that macrophages may prime stromal cells for vascular development by inducing insulin like growth factor binding protein 7 (IGFBP7) and hepatocyte growth factor (HGF) expression. Our results underscore the vital role of primitive macrophages in the long-term vascularization of cardiac tissues, offering insights for therapy and advancing heart-on-a-chip technologies.
PubMed: 38908380
DOI: 10.1016/j.stem.2024.05.011 -
Biomedicine & Pharmacotherapy =... Jun 2024Peptide-functionalized hydrogel is one of commonly used biomaterials to introduce hydrogel-induced vessel regeneration. Despite many reports about the discoveries of...
Peptide-functionalized hydrogel is one of commonly used biomaterials to introduce hydrogel-induced vessel regeneration. Despite many reports about the discoveries of high-active peptides (or ligands) for regeneration, the study on the conjugating methods for the hydrogel functionalization with peptides is limited. Here, we compared the vasculogenic efficacy of the peptide-functionalized hydrogels prepared by two commonly used conjugating methods, 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC) and Click methods, through cell models, organ-on-chips models, animal models, and RNA sequencing analysis. Two vascular-related cell types, the human umbilical vein endothelial cells (HUVECs) and the adipose-derived stem cells (ADSCs), have been cultured on the hydrogel surfaces prepared by EDC/Click methods. It showed that the hydrogels prepared by Click method supported the higher vasculogenic activities while the ones made by EDC method compromised the peptide activities on hydrogels. The vasculogenesis assays further revealed that hydrogels prepared by Click method promoted a better vascular network formation. In a critical ischemic hindlimb model, only the peptide-functionalized hydrogels prepared by Click method successfully salvaged the ischemic limb, significantly improved blood perfusion, and enhanced the functional recoveries (through gait analysis and animal behavior studies). RNA sequencing studies revealed that the hydrogels prepared by Click method significantly promoted the PI3K-AKT pathway activation compared to the hydrogels prepared by EDC method. All the results suggested that EDC method compromised the functions of the peptides, while Click method preserved the vascular regenerating capacities of the peptides on the hydrogels, illustrating the importance of the conjugating method during the preparation of the peptide-functionalized hydrogels.
PubMed: 38906023
DOI: 10.1016/j.biopha.2024.116959 -
Biotechnology Progress Jun 2024The first downstream processing step in the purification of a biopharmaceutical protein secreted into mammalian cell culture fluid is the primary clarification of the...
The first downstream processing step in the purification of a biopharmaceutical protein secreted into mammalian cell culture fluid is the primary clarification of the culture fluid. As cell densities in the fed-batch and increasingly more common perfusion bioreactors have increased over last two decades through intensified upstream bioreactor production processes, the traditional primary clarification unit operations of centrifugation and/or microfiltration become more challenging with issues like frequent desludging, cell disruption due to shear damage and quick fouling of membranes. We have developed a novel compact cell settler device exploiting the enhanced sedimentation on inclined surfaces and demonstrated that this settler device can be adapted easily to remove and contain cells or cell clumps from the clarified supernatant collected via the top effluent of the settler. In this work, we present high product recovery results during primary clarification of mammalian cell culture supernatant using our novel single-use disposable BioSettler150 while processing about 10 L of cell culture broth within short processing times of about 4 h.
PubMed: 38898736
DOI: 10.1002/btpr.3489 -
Nature Communications Jun 2024Hepatocellular carcinoma frequently recurs after surgery, necessitating personalized clinical approaches based on tumor avatar models. However, location-dependent oxygen...
Hepatocellular carcinoma frequently recurs after surgery, necessitating personalized clinical approaches based on tumor avatar models. However, location-dependent oxygen concentrations resulting from the dual hepatic vascular supply drive the inherent heterogeneity of the tumor microenvironment, which presents challenges in developing an avatar model. In this study, tissue samples from 12 patients with hepatocellular carcinoma are cultured directly on a chip and separated based on preference of oxygen concentration. Establishing a dual gradient system with drug perfusion perpendicular to the oxygen gradient enables the simultaneous separation of cells and evaluation of drug responsiveness. The results are further cross-validated by implanting the chips into mice at various oxygen levels using a patient-derived xenograft model. Hepatocellular carcinoma cells exposed to hypoxia exhibit invasive and recurrent characteristics that mirror clinical outcomes. This chip provides valuable insights into treatment prognosis by identifying the dominant hepatocellular carcinoma type in each patient, potentially guiding personalized therapeutic interventions.
Topics: Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Animals; Mice; Oxygen; Tumor Microenvironment; Cell Line, Tumor; Male; Female; Xenograft Model Antitumor Assays; Middle Aged; Lab-On-A-Chip Devices
PubMed: 38879551
DOI: 10.1038/s41467-024-49386-8 -
Applied Biochemistry and Biotechnology Jun 2024Although it is crucial to promptly restore blood perfusion to revive the ischemic myocardium, reperfusion itself can paradoxically contribute to the electrical...
Although it is crucial to promptly restore blood perfusion to revive the ischemic myocardium, reperfusion itself can paradoxically contribute to the electrical instability and arrhythmias of the myocardium. Several studies have revealed that cardiac fibroblasts can impact cardiac electrophysiology through various mechanisms including the deposition of extracellular matrix, release of chemical mediators, and direct electrical coupling with myocytes. Previously, we have shown that hypoxia/reoxygenation (H/R)-treated rat fibroblasts conditional medium (H/R-FCM) could decrease the spontaneous beating frequency of rat neonatal cardiomyocytes and downregulate the expression of gap junction proteins. However, the specific mechanism by which H/R-FCM affects the gap junctions requires further investigation. H/R-FCM was obtained by culturing confluent rat cardiac fibroblasts (RCF) for 4 h under hypoxic conditions. Gap junction function, hemichannel activity, and expression of Cx43 were examined upon treatment with H/R-FCM. Gelatin zymography was performed to detect matrix metalloproteinase (MMP) activity in the conditioned medium. The effect of H/R-FCM and MMP2 inhibitors on cardiac electrophysiology and arrhythmias was investigated with an isolated rat ischemia/reperfusion (I/R) model. H/R-FCM treatment impaired gap junction function, downregulated Cx43 expression, and increased hemichannel activity in rat cardiomyocytes (H9c2). The adverse effect of H/R-FCM on gap junction, which was confirmed by the cardiomyocyte H/R model, was involved in the activation of MMP2. MMP2 inhibition could partially attenuate the detrimental effects of I/R on myocardial electrophysiological indices and arrhythmia susceptibility. Our study indicates that inhibition of MMP2 may be a promising therapeutic target for the treatment of reperfusion arrhythmia.
PubMed: 38878160
DOI: 10.1007/s12010-024-04986-4 -
Scientific Reports Jun 2024Here we report the effects of low-intensity pulsed ultrasound (LIPUS) on symptoms in peripheral arterial disease patients with Buerger disease. A double-blinded and... (Randomized Controlled Trial)
Randomized Controlled Trial
Here we report the effects of low-intensity pulsed ultrasound (LIPUS) on symptoms in peripheral arterial disease patients with Buerger disease. A double-blinded and randomized study with active and inactive LIPUS was conducted. We assessed symptoms in leg circulation during a 24-week period of LIPUS irradiation in 12 patients with Buerger disease. Twelve patients without LIPUS irradiation served as controls. The pain intensity on visual analog score was significantly decreased after 24-week LIPUS treatment. Skin perfusion pressure was significantly increased in patients who received LIPUS treatment. There was no significant difference in symptoms and perfusion parameters in the control group. No severe adverse effects were observed in any of the patients who underwent LIPUS treatment. LIPUS is noninvasive, safe and effective option for improving symptoms in patients with Buerger disease.
Topics: Humans; Male; Female; Double-Blind Method; Middle Aged; Thromboangiitis Obliterans; Ultrasonic Therapy; Adult; Ultrasonic Waves; Treatment Outcome; Skin; Aged
PubMed: 38871832
DOI: 10.1038/s41598-024-64118-0