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Biomedicine & Pharmacotherapy =... Mar 2024Pulmonary hypertension (PH) is a progressive and fatal cardiopulmonary disease characterized by vascular remodeling and is associated with endothelial-to-mesenchymal...
BACKGROUND
Pulmonary hypertension (PH) is a progressive and fatal cardiopulmonary disease characterized by vascular remodeling and is associated with endothelial-to-mesenchymal transition (EndoMT). The pigment epithelium-derived factor (PEDF), a secretory protein widely distributed in multiple organs, has been shown to demonstrate anti-EndoMT activity in cardiovascular diseases. In the present study, the role of PEDF in PH was investigated.
METHODS
For PEDF overexpression, Sprague Dawley rats were infected with an adeno-associated virus through injection via the internal jugular vein. To establish PH models, the animals were subjected to monocrotaline or Sugen/hypoxia. Four weeks later, pulmonary artery angiography was performed, and hemodynamic parameters, right ventricular function, and vascular remodeling were evaluated. EndoMT and cell proliferation in the pulmonary arteries were assessed via immunofluorescence staining. Moreover, pulmonary artery endothelial cells (PAECs) isolated from experimental PH rats were cultured to investigate the underlying molecular mechanisms involved.
RESULTS
PEDF expression was significantly downregulated in PAECs from PH patients and PH model rats. Overexpressed PEDF alleviated the development of PH by improving pulmonary artery morphology and perfusion, reducing pulmonary artery pressure, improving right ventricular function, and alleviating vascular remodeling. PEDF inhibits EndoMT and reduces excessive PAEC proliferation. Moreover, PEDF overexpression reduced EndoMT in cultured PAECs by competitively inhibiting the binding of wnt to LRP6 and downregulating phosphorylation at the 1490 site of LRP6.
CONCLUSIONS
Our findings suggest that PEDF may be a potential therapeutic target for PH. We also found that PEDF can inhibit EndoMT in PAECs and may exert these effects by inhibiting the Wnt/LRP6/β-catenin pathway.
Topics: Humans; Rats; Animals; Hypertension, Pulmonary; Monocrotaline; Rats, Sprague-Dawley; Endothelial Cells; Vascular Remodeling; Serpins; Indoles; Eye Proteins; Nerve Growth Factors; Pyrroles
PubMed: 38377738
DOI: 10.1016/j.biopha.2024.116303 -
BioRxiv : the Preprint Server For... Feb 2024Organoids are powerful models of tissue physiology, yet their applications remain limited due to a lack of complex tissue morphology and high organoid-to-organoid...
Organoids are powerful models of tissue physiology, yet their applications remain limited due to a lack of complex tissue morphology and high organoid-to-organoid structural variability. To address these limitations we developed a soft, composite yield-stress extracellular matrix that supports freeform 3D bioprinting of cell slurries at tissue-like densities. Combined with a custom piezoelectric printhead, this platform allows more reproducible and complex morphogenesis from uniform and spatially organized organoid "seeds." At 4 °C the material exhibits reversible yield-stress behavior to support long printing times without compromising cell viability. When transferred to cell culture at 37 °C, the material cross-links and exhibits similar viscoelasticity and plasticity to basement membrane extracts such as Matrigel. We use this setup for high-throughput generation of intestinal and salivary gland organoid arrays that are morphologically indistinguishable from those grown in pure Matrigel, but exhibit dramatically improved homogeneity in organoid size, shape, maturation time, and budding efficiency. The reproducibility of organoid structure afforded by this approach increases the sensitivity of assays by orders of magnitude, requiring less input material and reducing analysis times. The flexibility of this approach additionally enabled the fabrication of perfusable intestinal organoid tubes. Combined, these advances lay the foundation for the efficient design of complex tissue morphologies in both space and time.
PubMed: 38370663
DOI: 10.1101/2024.02.01.578324 -
Nature Communications Feb 2024The development of vascular networks in microfluidic chips is crucial for the long-term culture of three-dimensional cell aggregates such as spheroids, organoids,...
The development of vascular networks in microfluidic chips is crucial for the long-term culture of three-dimensional cell aggregates such as spheroids, organoids, tumoroids, or tissue explants. Despite rapid advancement in microvascular network systems and organoid technologies, vascularizing organoids-on-chips remains a challenge in tissue engineering. Most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical set-ups. Considering these constraints, we develop a platform to establish and monitor the formation of endothelial networks around mesenchymal and pancreatic islet spheroids, as well as blood vessel organoids generated from pluripotent stem cells, cultured for up to 30 days on-chip. We show that these networks establish functional connections with the endothelium-rich spheroids and vascular organoids, as they successfully provide intravascular perfusion to these structures. We find that organoid growth, maturation, and function are enhanced when cultured on-chip using our vascularization method. This microphysiological system represents a viable organ-on-chip model to vascularize diverse biological 3D tissues and sets the stage to establish organoid perfusions using advanced microfluidics.
Topics: Microfluidics; Organoids; Tissue Engineering; Endothelium; Islets of Langerhans
PubMed: 38365780
DOI: 10.1038/s41467-024-45710-4 -
Lab on a Chip Mar 2024Human microphysiological systems, such as organs on chips, are an emerging technology for modeling human physiology in a preclinical setting to understand the mechanism...
Gravity-perfused airway-on-a-chip optimized for quantitative BSL-3 studies of SARS-CoV-2 infection: barrier permeability, cytokine production, immunohistochemistry, and viral load assays.
Human microphysiological systems, such as organs on chips, are an emerging technology for modeling human physiology in a preclinical setting to understand the mechanism of action of drugs, to evaluate the efficacy of treatment options for human disease and impairment, and to assess drug toxicity. By using human cells co-cultured in three-dimensional constructs, organ chips can provide greater fidelity to the human cellular condition than their two-dimensional predecessors. However, with the rise of SARS-CoV-2 and the global COVID-19 pandemic, it became clear that many microphysiological systems were not compatible with or optimized for studies of infectious disease and operation in a Biosafety Level 3 (BSL-3) environment. Given that one of the early sites of SARS-CoV-2 infection is the airway, we created a human airway organ chip that could operate in a BSL-3 space with high throughput and minimal manipulation, while retaining the necessary physical and physiological components to recapitulate tissue response to infectious agents and the immune response to infection.
Topics: Humans; COVID-19; SARS-CoV-2; Viral Load; Pandemics; Immunohistochemistry; Cytokines; Lab-On-A-Chip Devices
PubMed: 38362777
DOI: 10.1039/d3lc00894k -
Hypertension (Dallas, Tex. : 1979) Apr 2024The relationship between placental pathology and the maternal syndrome of preeclampsia is incompletely characterized. Mismatch between placental nutrient supply and...
BACKGROUND
The relationship between placental pathology and the maternal syndrome of preeclampsia is incompletely characterized. Mismatch between placental nutrient supply and fetal demands induces stress in the syncytiotrophoblast, the layer of placenta in direct contact with maternal blood. Such stress alters the content and increases the release of syncytiotrophoblast extracellular vesicles (STB-EVs) into the maternal circulation. We have previously shown 5'-tRNA fragments (5'-tRFs) constitute the majority of small RNA in STB-EVs in healthy pregnancy. 5'-tRFs are produced in response to stress. We hypothesized STB-EV 5'-tRF release might change in preeclampsia.
METHODS
We perfused placentas from 8 women with early-onset preeclampsia and 6 controls, comparing small RNA expression in STB-EVs. We used membrane-affinity columns to isolate maternal plasma vesicles and investigate placental 5'-tRFs in vivo. We quantified 5'-tRFs from circulating STB-EVs using a placental alkaline phosphatase immunoassay. 5'-tRFs and scrambled RNA controls were added to monocyte, macrophage and endothelial cells in culture to investigate transcriptional responses.
RESULTS
5'-tRFs constitute the majority of small RNA in STB-EVs from both preeclampsia and normal pregnancies. More than 900 small RNA fragments are differentially expressed in preeclampsia STB-EVs. Preeclampsia-dysregulated 5'-tRFs are detectable in maternal plasma, where we identified a placentally derived load. 5'-tRF-Glu-CTC, the most abundant preeclampsia-upregulated 5'-tRF in perfusion STB-EVs, is also increased in preeclampsia STB-EVs from maternal plasma. 5'-tRF-Glu-CTC induced inflammation in macrophages but not monocytes. The conditioned media from 5'-tRF-Glu-CTC-activated macrophages reduced eNOS (endothelial NO synthase) expression in endothelial cells.
CONCLUSIONS
Increased release of syncytiotrophoblast-derived vesicle-bound 5'-tRF-Glu-CTC contributes to preeclampsia pathophysiology.
Topics: Pregnancy; Female; Humans; Placenta; Pre-Eclampsia; Endothelial Cells; Trophoblasts; Extracellular Vesicles; RNA, Transfer; Macrophages; Inflammation
PubMed: 38362745
DOI: 10.1161/HYPERTENSIONAHA.123.22292 -
Frontiers in Immunology 2024Xenotransplantation offers a promising alternative to circumvent the lack of donated human organs available for transplantation. Different attempts to improve the...
Xenotransplantation offers a promising alternative to circumvent the lack of donated human organs available for transplantation. Different attempts to improve the survival of xenografts led to the generation of transgenic pigs expressing various combinations of human protective genes or knocked out for specific antigens. Currently, testing the efficiency of porcine organs carrying different genetic modifications in preventing xenogeneic immune responses completely relies on assays, humanized mouse models, or non-human primate transplantation models. However, these tests are often associated with major concerns due to reproducibility and generation of insufficient data as well as they raise ethical, logistical, and economic issues. In this study, we investigated the feasibility of specifically assessing the strength of human T-cell responses towards the kidneys of wild-type (WT) or transgenic pigs overexpressing human programmed death-1 ligand 1 (hPD-L1) during kidney perfusion (EVKP). Human T cells were shown to adhere to the endothelium and transmigrate into WT and hPD-L1 kidneys. However, transcript levels of TNF-a and IFN-y as well as cytotoxic molecules such as granzyme B and perforin secreted by human T cells were significantly decreased in the tissue of hPD-L1 kidneys in comparison to WT kidneys. These results were confirmed via assays using renal endothelial cells (ECs) isolated from WT and hPD-L1 transgenic pigs. Both CD4 and CD8 T cells showed significantly lower proliferation rates after exposure to hPD-L1 porcine renal ECs in comparison to WT ECs. In addition, the secretion of pro-inflammatory cytokines was significantly reduced in cultures using hPD-L1 ECs in comparison to WT ECs. Remarkably, hPD-L1 EC survival was significantly increased in cytotoxic assays. This study demonstrates the feasibility of evaluating the human response of specific immune subsets such as human T cells towards the whole xenograft during EVKP. This may represent a robust strategy to assess the potency of different genetic modifications to prevent xenogeneic immune responses and thereby predict the risk of immune rejection of new genetically engineered xenografts.
Topics: Mice; Animals; Swine; Humans; B7-H1 Antigen; CD8-Positive T-Lymphocytes; Endothelial Cells; Reproducibility of Results; Animals, Genetically Modified; Lymphocyte Activation; Kidney
PubMed: 38352884
DOI: 10.3389/fimmu.2024.1279050 -
PloS One 2024Heart failure (HF) is the last stage in the progression of various cardiovascular diseases. Although it is documented that CD151 contributes to regulate the myocardial...
BACKGROUND
Heart failure (HF) is the last stage in the progression of various cardiovascular diseases. Although it is documented that CD151 contributes to regulate the myocardial infarction, the function of CD151 on HF and involved mechanisms are still unclear.
METHOD AND RESULTS
In the present study, we found that the recombinant adeno-associated virus (rAAV)-mediated endothelial cell-specific knockdown of CD151-transfected mice improved transverse aortic constriction (TAC)-induced cardiac function, attenuated myocardial hypertrophy and fibrosis, and increased coronary perfusion, whereas overexpression of the CD151 protein aggravated cardiac dysfunction and showed the opposite effects. In vitro, the cardiomyocytes hypertrophy induced by PE were significantly improved, while the proliferation and migration of cardiac fibroblasts (CFs) were significantly reduced, when co-cultured with the CD151-silenced endothelial cells (ECs). To further explore the mechanisms, the exosomes from the CD151-silenced ECs were taken by cardiomyocyte (CMs) and CFs, verified the intercellular communication. And the protective effects of CD151-silenced ECs were inhibited when exosome inhibitor (GW4869) was added. Additionally, a quantitative proteomics method was used to identify potential proteins in CD151-silenced EC exosomes. We found that the suppression of CD151 could regulate the PPAR signaling pathway via exosomes.
CONCLUSION
Our observations suggest that the downregulation of CD151 is an important positive regulator of cardiac function of heart failure, which can regulate exosome-stored proteins to play a role in the cellular interaction on the CMs and CFs. Modulating the exosome levels of ECs by reducing CD151 expression may offer novel therapeutic strategies and targets for HF treatment.
Topics: Mice; Animals; Myocytes, Cardiac; Endothelial Cells; Down-Regulation; Exosomes; Cardiomegaly; Heart Failure
PubMed: 38349935
DOI: 10.1371/journal.pone.0297121 -
Journal of Biotechnology Mar 2024Controlling high-mannose (HM) content of therapeutic proteins during process intensification, reformulation for subcutaneous delivery, antibody-drug conjugate or...
Controlling high-mannose (HM) content of therapeutic proteins during process intensification, reformulation for subcutaneous delivery, antibody-drug conjugate or biosimilar manufacturing represents an ongoing challenge. Even though a range of glycosylation levers to increase HM content exist, modulators specially increasing M5 glycans are still scarce. Several compounds of the polyether ionophore family were screened for their ability to selectively increase M5 glycans of mAb products and compared to the well-known α-mannosidase I inhibitor kifunensine known to increase mainly M8-M9 glycans. Maduramycin, amongst other promising polyether ionophores, showed the desired effect on different cell lines. For fed-batch processes, a double bolus addition modulator feed strategy was developed maximizing the effect on glycosylation by minimizing impact on culture performance. Further, a continuous feeding strategy for steady-state perfusion processes was successfully developed, enabling consistent product quality at elevated HM glycan levels. With kifunensine and maduramycin showing inverse effects on the relative HM distribution, a combined usage of these modulators was further evaluated to fine-tune a desired HM glycan pattern. The discovered HM modulators expand the current HM modulating toolbox for biotherapeutics. Their application not only for fed-batch processes, but also steady-state perfusion processes, make them a universal tool with regards to fully continuous manufacturing processes.
Topics: Animals; Glycosylation; Perfusion; Lactones; Mammals; Mannose; Polyether Polyketides; Polysaccharides
PubMed: 38340899
DOI: 10.1016/j.jbiotec.2024.02.003 -
Scientific Reports Feb 2024Microfabricated organ-on-a-chips are rapidly becoming the gold standard for the testing of safety and efficacy of therapeutics. A broad range of designs has emerged, but...
Microfabricated organ-on-a-chips are rapidly becoming the gold standard for the testing of safety and efficacy of therapeutics. A broad range of designs has emerged, but recreating microvascularised tissue models remains difficult in many cases. This is particularly relevant to mimic the systemic delivery of therapeutics, to capture the complex multi-step processes associated with trans-endothelial transport or diffusion, uptake by targeted tissues and associated metabolic response. In this report, we describe the formation of microvascularised cardiac spheroids embedded in microfluidic chips. Different protocols used for embedding spheroids within vascularised multi-compartment microfluidic chips were investigated first to identify the importance of the spheroid processing, and co-culture with pericytes on the integration of the spheroid within the microvascular networks formed. The architecture of the resulting models, the expression of cardiac and endothelial markers and the perfusion of the system was then investigated. This confirmed the excellent stability of the vascular networks formed, as well as the persistent expression of cardiomyocyte markers such as cTNT and the assembly of striated F-actin, myosin and α-actinin cytoskeletal networks typically associated with contractility and beating. The ability to retain beating over prolonged periods of time was quantified, over 25 days, demonstrating not only perfusability but also functional performance of the tissue model. Finally, as a proof-of-concept of therapeutic testing, the toxicity of one therapeutic associated with cardiac disfunction was evaluated, identifying differences between direct in vitro testing on suspended spheroids and vascularised models.
Topics: Spheroids, Cellular; Cell Culture Techniques; Microfluidics; Coculture Techniques; Lab-On-A-Chip Devices
PubMed: 38336810
DOI: 10.1038/s41598-024-53678-w -
Cells Jan 2024Human pluripotent stem cells have been employed in generating organoids, yet their immaturity compared to fetal organs and the limited induction of all constituent cell...
Human pluripotent stem cells have been employed in generating organoids, yet their immaturity compared to fetal organs and the limited induction of all constituent cell types remain challenges. Porcine fetal progenitor cells have emerged as promising candidates for co-culturing with human progenitor cells in regeneration and xenotransplantation research. This study focused on identifying proper preservation methods for porcine fetal kidneys, hearts, and livers, aiming to optimize their potential as cell sources. Extracted from fetal microminiature pigs, these organs were dissociated before and after cryopreservation-thawing, with subsequent cell quality evaluations. Kidney cells, dissociated and aggregated after vitrification in a whole-organ form, were successfully differentiated into glomeruli and tubules in vivo. In contrast, freezing hearts and livers before dissociation yielded suboptimal results. Heart cells, frozen after dissociation, exhibited pulsating heart muscle cells similar to non-frozen hearts. As for liver cells, we developed a direct tissue perfusion technique and successfully obtained highly viable liver parenchymal cells. Freezing dissociated liver cells, although inferior to their non-frozen counterparts, maintained the ability for colony formation. The findings of this study provide valuable insights into suitable preservation methods for porcine fetal cells from kidneys, hearts, and livers, contributing to the advancement of regeneration and xenotransplantation research.
Topics: Animals; Humans; Swine; Regenerative Medicine; Cryopreservation; Freezing; Vitrification; Pluripotent Stem Cells
PubMed: 38334620
DOI: 10.3390/cells13030228