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Military Medical Research May 2022Cerebral ischemia-reperfusion injury (CIRI) refers to a secondary brain injury that can occur when the blood supply to the ischemic brain tissue is restored. However,...
BACKGROUND
Cerebral ischemia-reperfusion injury (CIRI) refers to a secondary brain injury that can occur when the blood supply to the ischemic brain tissue is restored. However, the mechanism underlying such injury remains elusive.
METHODS
The 150 male C57 mice underwent middle cerebral artery occlusion (MCAO) for 1 h and reperfusion for 24 h, Among them, 50 MCAO mice were further treated with Mitochondrial division inhibitor 1 (Mdivi-1) and 50 MCAO mice were further treated with N-acetylcysteine (NAC). SH-SY5Y cells were cultured in a low-glucose culture medium for 4 h under hypoxic conditions and then transferred to normal conditions for 12 h. Then, cerebral blood flow, mitochondrial structure, mitochondrial DNA (mtDNA) copy number, intracellular and mitochondrial reactive oxygen species (ROS), autophagic flux, aggresome and exosome expression profiles, cardiac tissue structure, mitochondrial length and cristae density, mtDNA and ROS content, as well as the expression of Drp1-Ser616/Drp1, RIP1/RIP3, LC3 II/LC3 I, TNF-α, IL-1β, etc., were detected under normal or Drp1 interference conditions.
RESULTS
The mtDNA content, ROS levels, and Drp1-Ser616/Drp1 were elevated by 2.2, 1.7 and 2.7 times after CIRI (P < 0.05). However, the high cytoplasmic LC3 II/I ratio and increased aggregation of p62 could be reversed by 44% and 88% by Drp1 short hairpin RNA (shRNA) (P < 0.05). The low fluorescence intensity of autophagic flux and the increased phosphorylation of RIP3 induced by CIRI could be attenuated by ROS scavenger, NAC (P < 0.05). RIP1/RIP3 inhibitor Necrostatin-1 (Nec-1) restored 75% to a low LC3 II/LC3 I ratio and enhanced 2 times to a high RFP-LC3 after Drp1 activation (P < 0.05). In addition, although CIRI-induced ROS production caused no considerable accumulation of autophagosomes (P > 0.05), it increased the packaging and extracellular secretion of exosomes containing p62 by 4 - 5 times, which could be decreased by Mdivi-1, Drp1 shRNA, and Nec-1 (P < 0.05). Furthermore, TNF-α and IL-1β increased in CIRI-derived exosomes could increase RIP3 phosphorylation in normal or oxygen-glucose deprivation/reoxygenation (OGD/R) conditions (P < 0.05).
CONCLUSIONS
CIRI activated Drp1 and accelerated the p62-mediated formation of autophagosomes while inhibiting the transition of autophagosomes to autolysosomes via the RIP1/RIP3 pathway activation. Undegraded autophagosomes were secreted extracellularly in the form of exosomes, leading to inflammatory cascades that further damaged mitochondria, resulting in excessive ROS generation and the blockage of autophagosome degradation, triggering a vicious cycle.
Topics: Animals; Brain Ischemia; Cerebral Infarction; DNA, Mitochondrial; Exosomes; Glucose; Humans; Inflammation; Male; Mice; RNA, Small Interfering; Reactive Oxygen Species; Reperfusion; Reperfusion Injury; Tumor Necrosis Factor-alpha
PubMed: 35624495
DOI: 10.1186/s40779-022-00383-2 -
STAR Protocols Sep 2020Primary hepatocytes are a vital tool in various biomedical research disciplines, serving as an model for liver physiology. Obtaining high yields of viable primary mouse...
Primary hepatocytes are a vital tool in various biomedical research disciplines, serving as an model for liver physiology. Obtaining high yields of viable primary mouse hepatocytes is technically challenging, limiting their use. Here, we present an improved protocol based on the classic two-step collagenase perfusion technique. The liver is washed by perfusion, hepatocytes are dissociated by collagenase, separated from other cells, and cultured. This protocol was optimized to significantly reduce procedure duration and improve hepatocyte yield and viability.
Topics: Animals; Cell Separation; Cells, Cultured; Collagenases; Hepatocytes; Liver; Mice; Mice, Inbred C57BL; Perfusion; Primary Cell Culture
PubMed: 33111119
DOI: 10.1016/j.xpro.2020.100086 -
American Family Physician Oct 2021Diabetes-related foot infections occur in approximately 40% of diabetes-related foot ulcers and cause significant morbidity. Clinicians should consider patient risk... (Review)
Review
Diabetes-related foot infections occur in approximately 40% of diabetes-related foot ulcers and cause significant morbidity. Clinicians should consider patient risk factors (e.g., presence of foot ulcers greater than 2 cm, uncontrolled diabetes mellitus, poor vascular perfusion, comorbid illness) when evaluating for a foot infection or osteomyelitis. Indicators of infection include erythema, induration, tenderness, warmth, and drainage. Superficial wound cultures should be avoided because of the high rate of contaminants. Deep cultures obtained through aseptic procedures (e.g., incision and drainage, debridement, bone culture) help guide treatment. Plain radiography is used for initial imaging if osteomyelitis is suspected; however, magnetic resonance imaging or computed tomography may help if radiography is inconclusive, the extent of infection is unknown, or if the infection orientation needs to be determined to help in surgical planning. Staphylococcus aureus and Streptococcus agalactiae are the most commonly isolated pathogens, although polymicrobial infections are common. Antibiotic therapy should cover commonly isolated organisms and reflect local resistance patterns, patient preference, and the severity of the foot infection. Mild and some moderate infections may be treated with oral antibiotics. Severe infections require intravenous antibiotics. Treatment duration is typically one to two weeks and is longer for slowly resolving infections or osteomyelitis. Severe or persistent infections may require surgery and specialized team-based wound care. Although widely recommended, there is little evidence on the effectiveness of primary prevention strategies. Systematic assessment, counseling, and comorbidity management are hallmarks of effective secondary prevention for diabetes-related foot infections.
Topics: Anti-Bacterial Agents; Bandages; Debridement; Diabetic Foot; Humans; Risk Factors; Severity of Illness Index; Shoes
PubMed: 34652105
DOI: No ID Found -
Nature Sep 2020Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration. This adaptability is lost in cultured adult endothelial cells,...
Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2) in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) 'resets' these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens. In three-dimensional matrices-which do not have the constraints of bioprinted scaffolds-the 'reset' vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call 'Organ-On-VascularNet'. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting.
Topics: Blood Vessels; Carcinogenesis; Cell Culture Techniques; Chromatin; Endothelial Cells; Epigenesis, Genetic; Epigenomics; Hemodynamics; Human Umbilical Vein Endothelial Cells; Humans; In Vitro Techniques; Islets of Langerhans; Models, Biological; Neoplasms; Organ Specificity; Organogenesis; Organoids; RNA-Seq; Single-Cell Analysis; Transcription Factors; Transcriptome
PubMed: 32908310
DOI: 10.1038/s41586-020-2712-z -
Theranostics 2021Cardiovascular diseases, such as myocardial infarction (MI), are the leading causes of death worldwide. Reperfusion therapy is the common standard treatment for MI....
Cardiovascular diseases, such as myocardial infarction (MI), are the leading causes of death worldwide. Reperfusion therapy is the common standard treatment for MI. However, myocardial ischemia/reperfusion (I/R) causes cardiomyocyte injury, including apoptosis and fibrosis. We aimed to investigate the effects of conditioned medium from adipose-derived stem cells (ADSC-CM) on apoptosis and fibrosis in I/R-treated hearts and hypoxia/reoxygenation (H/R)-treated cardiomyocytes and the underlying mechanisms. ADSC-CM was collected from ADSCs. The effects of intramuscular injection of ADSC-CM on cardiac function, cardiac apoptosis, and fibrosis examined by echocardiography, Evans blue/TTC staining, TUNEL assay, and Masson's trichrome staining in I/R-treated mice. We also examined the effects of ADSC-CM on apoptosis and fibrosis in H/R-treated H9c2 cells by annexin V/PI flow cytometry, TUNEL assay, and immunocytochemistry. ADSC-CM treatment significantly reduced heart damage and fibrosis of I/R-treated mice and H/R-treated cardiomyocytes. In addition, the expression of apoptosis-related proteins, such as p53 upregulated modulator of apoptosis (PUMA), p-p53 and B-cell lymphoma 2 (BCL2), as well as the fibrosis-related proteins ETS-1, fibronectin and collagen 3, were significantly reduced by ADSC-CM treatment. Moreover, we demonstrated that ADSC-CM contains a large amount of miR-221/222, which can target and regulate PUMA or ETS-1 protein levels. Furthermore, the knockdown of PUMA and ETS-1 decreased the induction of apoptosis and fibrosis, respectively. MiR-221/222 overexpression achieved similar results. We also observed that cardiac I/R markedly increased apoptosis and fibrosis in miR-221/222 knockout (KO) mice, while ADSC-CM decreased these effects. The increased phosphorylation of p38 and NF-κB not only mediated myocardial apoptosis through the PUMA/p53/BCL2 pathway but also regulated fibrosis through the ETS-1/fibronectin/collagen 3 pathway. Overall, our results show that ADSC-CM attenuates cardiac apoptosis and fibrosis by reducing PUMA and ETS-1 expression, respectively. The protective effect is mediated via the miR-221/222/p38/NF-κB pathway.
Topics: Adipose Tissue; Animals; Apoptosis; Apoptosis Regulatory Proteins; Cell Death; Culture Media, Conditioned; Fibrosis; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; MicroRNAs; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Proto-Oncogene Protein c-ets-1; Reperfusion; Reperfusion Injury; Stem Cells; Tumor Suppressor Proteins
PubMed: 33537078
DOI: 10.7150/thno.52677 -
Angiogenesis Nov 2022With recent progress in modeling liver organogenesis and regeneration, the lack of vasculature is becoming the bottleneck in progressing our ability to model human...
With recent progress in modeling liver organogenesis and regeneration, the lack of vasculature is becoming the bottleneck in progressing our ability to model human hepatic tissues in vitro. Here, we introduce a platform for routine grafting of liver and other tissues on an in vitro grown microvascular bed. The platform consists of 64 microfluidic chips patterned underneath a 384-well microtiter plate. Each chip allows the formation of a microvascular bed between two main lateral vessels by inducing angiogenesis. Chips consist of an open-top microfluidic chamber, which enables addition of a target tissue by manual or robotic pipetting. Upon grafting a liver microtissue, the microvascular bed undergoes anastomosis, resulting in a stable, perfusable vascular network. Interactions with vasculature were found in spheroids and organoids upon 7 days of co-culture with space of Disse-like architecture in between hepatocytes and endothelium. Veno-occlusive disease was induced by azathioprine exposure, leading to impeded perfusion of the vascularized spheroid. The platform holds the potential to replace animals with an in vitro alternative for routine grafting of spheroids, organoids, or (patient-derived) explants.
Topics: Animals; Azathioprine; Coculture Techniques; Humans; Liver; Microfluidics; Organoids
PubMed: 35704148
DOI: 10.1007/s10456-022-09842-9 -
Biomaterials May 2022Human cholangiocyte organoids are promising for regenerative medicine applications, such as repair of damaged bile ducts. However, organoids are typically cultured in...
Human cholangiocyte organoids are promising for regenerative medicine applications, such as repair of damaged bile ducts. However, organoids are typically cultured in mouse tumor-derived basement membrane extracts (BME), which is poorly defined, highly variable and limits the direct clinical applications of organoids in patients. Extracellular matrix (ECM)-derived hydrogels prepared from decellularized human or porcine livers are attractive alternative culture substrates. Here, the culture and expansion of human cholangiocyte organoids in liver ECM(LECM)-derived hydrogels is described. These hydrogels support proliferation of cholangiocyte organoids and maintain the cholangiocyte-like phenotype. The use of LECM hydrogels does not significantly alter the expression of selected genes or proteins, such as the cholangiocyte marker cytokeratin-7, and no species-specific effect is found between human or porcine LECM hydrogels. Proliferation rates of organoids cultured in LECM hydrogels are lower, but the differentiation capacity of the cholangiocyte organoids towards hepatocyte-like cells is not altered by the presence of tissue-specific ECM components. Moreover, human LECM extracts support the expansion of ICO in a dynamic culture set up without the need for laborious static culture of organoids in hydrogel domes. Liver ECM hydrogels can successfully replace tumor-derived BME and can potentially unlock the full clinical potential of human cholangiocyte organoids.
Topics: Animals; Extracellular Matrix; Humans; Hydrogels; Liver; Mice; Neoplasms; Organoids; Plant Extracts; Swine
PubMed: 35344800
DOI: 10.1016/j.biomaterials.2022.121473 -
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 -
ACS Omega Jan 2023Two-dimensional (2D) cell culture techniques are commonly employed to investigate biophysical and biochemical cellular responses. However, these culture methods, having... (Review)
Review
Two-dimensional (2D) cell culture techniques are commonly employed to investigate biophysical and biochemical cellular responses. However, these culture methods, having monolayer cells, lack cell-cell and cell-extracellular matrix interactions, mimicking the cell microenvironment and multicellular organization. Three-dimensional (3D) cell culture methods enable equal transportation of nutrients, gas, and growth factors among cells and their microenvironment. Therefore, 3D cultures show similar cell proliferation, apoptosis, and differentiation properties to . A spheroid is defined as self-assembled 3D cell aggregates, and it closely mimics a cell microenvironment thanks to cell-cell/matrix interactions, which enables its use in several important applications in medical and clinical research. To fabricate a spheroid, conventional methods such as liquid overlay, hanging drop, and so forth are available. However, these labor-intensive methods result in low-throughput fabrication and uncontrollable spheroid sizes. On the other hand, microfluidic methods enable inexpensive and rapid fabrication of spheroids with high precision. Furthermore, fabricated spheroids can also be cultured in microfluidic devices for controllable cell perfusion, simulation of fluid shear effects, and mimicking of the microenvironment-like conditions. This review focuses on recent microfluidic spheroid fabrication techniques and also organ-on-a-chip applications of spheroids, which are used in different disease modeling and drug development studies.
PubMed: 36743071
DOI: 10.1021/acsomega.2c06052 -
Journal of Pharmacy & Pharmaceutical... 2021We investigated the signal transduction pathway associated with growth hormone (GH)-stimulated DNA synthesis and proliferation in primary cultured hepatocytes.
BACKGROUND
We investigated the signal transduction pathway associated with growth hormone (GH)-stimulated DNA synthesis and proliferation in primary cultured hepatocytes.
METHODS
Adult rat hepatocytes were isolated from normal livers by two-step in situ collagenase perfusion to facilitate disaggregation of the adult rat liver. Then hepatocytes were cultured in serum-free Williams' medium E supplemented with GH (1-100 ng/ml) in the presence or absence of test reagents. GH-induced hepatocyte DNA synthesis and proliferation were determined, and the phosphorylation activities of Janus kinase (JAK) 2 (JAK2) (p125 kDa), p95-kDa RTK, and ERK1/2 were measured by western blotting.
RESULTS
Hepatocytes grown in serum-free defined medium proliferated within 5 h of culture in the presence of GH (100 ng/ml) in a concentration- and time-dependent manner (EC50 75 ng/ml). These proliferative effects of GH were almost completely blocked by an anti-GH receptor monoclonal antibody (85 ng/ml) and an anti-insulin-like growth factor (IGF)-I receptor monoclonal antibody. In addition, the proliferative effects of GH were significantly blocked by a JAK2 inhibitor (TG101209, 10-6 M), as well as specific signal-transducing inhibitors of phospholipase C (PLC; U-73122, 10-6 M), RTK (AG538, 10-6 M), phosphoinositide 3-kinase (PI3K; LY294002, 10-6 M), mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK; PD98059, 10-6 M), and mammalian target of rapamycin (mTOR; rapamycin, 10 ng/ml). GH significantly induced the phosphorylations of JAK2 (p125 kDa), p95-kDa IGF-I receptor tyrosine kinase (RTK), and ERK2 in this order according to western blotting analysis.
CONCLUSIONS
The proliferative action of GH is mediated by two main signaling pathways. One includes activation of the GH receptor/JAK2/PLC/Ca2+ pathway, and the other involves activation of the p95-kDa IGF-I RTK/PI3K/ERK2/mTOR pathway in primary cultures of adult rat hepatocytes.
Topics: Animals; Cell Proliferation; Cells, Cultured; DNA; Growth Hormone; Hepatocytes; Humans; Male; Rats; Rats, Wistar; Signal Transduction
PubMed: 33434118
DOI: 10.18433/jpps31586