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ALTEX 2023Human induced pluripotent stem cells (hiPSCs) offer great opportunities within the 3R framework. In the field of toxicology, they may contribute greatly to the reduction... (Comparative Study)
Comparative Study
Human induced pluripotent stem cells (hiPSCs) offer great opportunities within the 3R framework. In the field of toxicology, they may contribute greatly to the reduction and eventually replacement of animal models. However, culturing hiPSCs as well as differentiation of hiPSCs into target cells that are used for toxicity testing depend on the presence of extracellular matrix (ECM) coating the growth surface. The most widely used ECM is MatrigelR, an animal product that is derived from mouse sarcoma. Drawbacks of Matrigel are widely recognized and include batch-to batch variations, use of animal rather than human material, and ethical concerns about its production. While alternative coatings exist, higher cost and limited characterizations may hinder their broader uptake by the scientific community. Here, we report an extensive comparison of three commercially available human ECM coatings, vitronectin, laminin-511, and laminin-521, to Matrigel in three different hiPSC lines in long-term culture (≥ 9 passages). Characterization included expression of pluripotent markers in a genome-wide transcriptomics study (TempO-Seq), capacity to differentiate into embryoid bodies, and karyotype stability assessed by analyzing copy number variations by shallow DNA sequencing. Furthermore, a low-cost, decellularized ECM produced by human neonatal dermal fibroblasts was tested. In addition, all alternative coatings were tested for hiPSC differentiation into renal podocyte-like cells in a genome-wide transcriptomics screen. Our results show that all tested coatings were highly comparable to animal-derived Matrigel for both hiPSC maintenance and differentiation into renal podocyte-like cells. Furthermore, decellularized fibroblast-ECM could be a novel, attractive low-cost coating material.
Topics: Animals; Humans; Infant, Newborn; Mice; Cell Differentiation; DNA Copy Number Variations; Extracellular Matrix; Fibroblasts; Induced Pluripotent Stem Cells; Laminin; Podocytes; Recombinant Proteins
PubMed: 35791294
DOI: 10.14573/altex.2112204 -
Journal of Thrombosis and Haemostasis :... Oct 2022Severe COVID-19 disease is associated with thrombotic complications and extensive fibrin deposition. This study investigates whether the hemostatic complications in...
BACKGROUND
Severe COVID-19 disease is associated with thrombotic complications and extensive fibrin deposition. This study investigates whether the hemostatic complications in COVID-19 disease arise due to dysregulation of the fibrinolytic system.
METHODS
This prospective study analyzed fibrinolytic profiles of 113 patients hospitalized with COVID-19 disease with 24 patients with non-COVID-19 respiratory infection and healthy controls. Antigens were quantified by Ella system or ELISA, clot lysis by turbidimetric assay, and plasminogen activator inhibitor-1 (PAI-1)/plasmin activity using chromogenic substrates. Clot structure was visualized by confocal microscopy.
RESULTS
PAI-1 and its cofactor, vitronectin, are significantly elevated in patients with COVID-19 disease compared with those with non-COVID-19 respiratory infection and healthy control groups. Thrombin activatable fibrinolysis inhibitor and tissue plasminogen activator were elevated in patients with COVID-19 disease relative to healthy controls. PAI-1 and tissue plasminogen activator (tPA) were associated with more severe COVID-19 disease severity. Clots formed from COVID-19 plasma demonstrate an altered fibrin network, with attenuated fiber length and increased branching. Functional studies reveal that plasmin generation and clot lysis were markedly attenuated in COVID-19 disease, while PAI-1 activity was elevated. Clot lysis time significantly correlated with PAI-1 levels. Stratification of COVID-19 samples according to PAI-1 levels reveals significantly faster lysis when using the PAI-1 resistant (tPA) variant, tenecteplase, over alteplase lysis.
CONCLUSION
This study shows that the suboptimal fibrinolytic response in COVID-19 disease is directly attributable to elevated levels of PAI-1, which attenuate plasmin generation. These data highlight the important prognostic potential of PAI-1 and the possibility of using pre-existing drugs, such as tenecteplase, to treat COVID-19 disease and potentially other respiratory diseases.
Topics: Carboxypeptidase B2; Chromogenic Compounds; Fibrin; Fibrinolysin; Fibrinolysis; Hemostatics; Humans; Plasminogen Activator Inhibitor 1; Prospective Studies; Tenecteplase; Thrombosis; Tissue Plasminogen Activator; Vitronectin; COVID-19 Drug Treatment
PubMed: 35780481
DOI: 10.1111/jth.15806 -
Journal of Bone Metabolism May 2022A rapid increase in bone turnover and bone loss has been observed in response to the discontinuation of denosumab. It led to an acute increase in the fracture risk,...
BACKGROUND
A rapid increase in bone turnover and bone loss has been observed in response to the discontinuation of denosumab. It led to an acute increase in the fracture risk, similar to that observed in the untreated patients. We aimed to investigate the effect of denosumab on osteoclast (OC) precursor cells compared to that of zoledronate.
METHODS
The study compared the effects of denosumab (60 mg/24-week) and zoledronate (5 mg/48-week) over 48 weeks in postmenopausal women with osteoporosis. From patients' peripheral mononuclear cells, CD14+/CD11b+/vitronectin receptor (VNR)- and CD14+/CD11b+/VNR+ cells were isolated using fluorescent-activated cell sorting, representing early and late OC precursors, respectively. The primary endpoint was the changes in OC precursors after 48 weeks of treatment.
RESULTS
Among the 23 patients, 11 were assigned to the denosumab group and 12 to the zoledronate group (mean age, 69 years). After 48 weeks, the changes in OC precursors were similar between and within the groups. Serum C-terminal telopeptide of type I collagen levels were inversely correlated with OC precursor levels after denosumab treatment (r=-0.72, P<0.001). Lumbar spine, femur neck, and total hip bone mineral density (BMD) increased in both groups. Lumbar spine BMD increased more significantly in the denosumab group than in the zoledronate group.
CONCLUSIONS
Denosumab and zoledronate treatments induced similar changes in OC precursors. During denosumab treatment, old age and suppressed bone turnover were associated with increased OC precursor cell populations. Further validation studies with prospective designs are required.
PubMed: 35718926
DOI: 10.11005/jbm.2022.29.2.93 -
Journal of Cerebral Blood Flow and... Oct 2022We found that blood vitronectin (VTN) leaks into the brain and exacerbates tissue loss after stroke by increasing pro-inflammatory IL-6 expression in female, but not...
We found that blood vitronectin (VTN) leaks into the brain and exacerbates tissue loss after stroke by increasing pro-inflammatory IL-6 expression in female, but not male, mice. VTN signals through integrins and downstream focal adhesion kinase (FAK). Here, a two day systemic treatment with a small molecule FAK inhibitor starting 6 h after middle cerebral artery occlusion reduced ipsilateral brain injury size by ∼40-45% at 7 and 14 d, as well as inflammation and motor dysfunction in wild-type female, but not male, mice. FAK inhibition also reduced IL-6 expression in the injured female striatum at 24 h by 62%. Inducible selective gene deletion of FAK in astrocytes also reduced acute IL-6 expression by 72% only in females, and mitigated infarct size by ∼80% and inflammation at 14 d after stroke. Lastly, VTN-/- females had better outcomes, but FAK inhibitor treatment had no additional protective or anti-inflammatory effects. Altogether, this suggests that VTN is detrimental in females primarily through FAK and that FAK inhibition provides neuroprotection (cerebroprotection) by reducing VTN-induced IL-6 expression in astrocytes. Thus, VTN signaling can be targeted to mitigate harmful inflammation with relevance to treatments for women with ischemic stroke, who often have worse outcomes than men.
Topics: Animals; Anti-Inflammatory Agents; Brain Ischemia; Female; Focal Adhesion Protein-Tyrosine Kinases; Humans; Inflammation; Integrins; Interleukin-6; Ischemic Stroke; Mice; Mice, Inbred C57BL; Neuroprotection; Stroke; Vitronectin
PubMed: 35702047
DOI: 10.1177/0271678X221107871 -
Cancers May 2022In epithelia, breakdown of tensional homeostasis is closely associated with E-cadherin dysfunction and disruption of tissue function and integrity. In this study, we...
In epithelia, breakdown of tensional homeostasis is closely associated with E-cadherin dysfunction and disruption of tissue function and integrity. In this study, we investigated the effect of E-cadherin mutations affecting distinct protein domains on tensional homeostasis of gastric cancer cells. We used micropattern traction microscopy to measure temporal fluctuations of cellular traction forces in AGS cells transfected with the wild-type E-cadherin or with variants affecting the extracellular, the juxtamembrane, and the intracellular domains of the protein. We focused on the dynamic aspect of tensional homeostasis, namely the ability of cells to maintain a consistent level of tension, with low temporal variability around a set point. Cells were cultured on hydrogels micropatterned with different extracellular matrix (ECM) proteins to test whether the ECM adhesion impacts cell behavior. A combination of Fibronectin and Vitronectin was used as a substrate that promotes the adhesive ability of E-cadherin dysfunctional cells, whereas Collagen VI was used to test an unfavorable ECM condition. Our results showed that mutations affecting distinct E-cadherin domains influenced differently cell tensional homeostasis, and pinpointed the juxtamembrane and intracellular regions of E-cadherin as the key players in this process. Furthermore, Fibronectin and Vitronectin might modulate cancer cell behavior towards tensional homeostasis.
PubMed: 35681670
DOI: 10.3390/cancers14112690 -
Cells May 2022The pathogenesis of age-related macular degeneration (AMD), a frequent disorder of the central retina, is incompletely understood. Genome-wide association studies (GWAS)...
The pathogenesis of age-related macular degeneration (AMD), a frequent disorder of the central retina, is incompletely understood. Genome-wide association studies (GWAS) suggest a strong contribution of genomic variation in AMD susceptibility. Nevertheless, little is known about biological mechanisms of the disease. We reported previously that the AMD-associated polymorphism rs704C > T in the vitronectin (VTN) gene influences protein expression and functional aspects of encoded vitronectin, a human blood and extracellular matrix (ECM) protein. Here, we refined the association of rs704 with AMD in 16,144 cases and 17,832 controls and noted that rs704 is carried exclusively by the neovascular AMD subtype. Interaction studies demonstrate that rs704 affects the ability of vitronectin to bind the angiogenic regulator plasminogen activator inhibitor 1 (PAI-1) but has no influence on stabilizing its active state. Western blot analysis and confocal imaging reveal a strong enrichment of PAI-1 in the ECM of cultured endothelial cells and RPE cell line ARPE-19 exposed to vitronectin. Large-scale gene expression of VTN and PAI-1 showed positive correlations and a statistically significant increase in human retinal and blood tissues aged 60 years and older. Our results suggest a mechanism by which the AMD-associated rs704 variant in combination with ageing may contribute to the vascular complications in AMD.
Topics: Aged; Angiogenesis Inhibitors; Endothelial Cells; Genome-Wide Association Study; Humans; Macular Degeneration; Middle Aged; Plasminogen Activator Inhibitor 1; Vascular Endothelial Growth Factor A; Visual Acuity; Vitronectin
PubMed: 35681461
DOI: 10.3390/cells11111766 -
Theranostics 2022In the glioblastoma (GBM) microenvironment, tumor-associated macrophages (TAMs) are prominent components and facilitate tumor growth. The exact molecular mechanisms...
In the glioblastoma (GBM) microenvironment, tumor-associated macrophages (TAMs) are prominent components and facilitate tumor growth. The exact molecular mechanisms underlying TAMs' function in promoting glioma stem cells (GSCs) maintenance and tumor growth remain largely unknown. We found a candidate molecule, transforming growth factor beta-induced (TGFBI), that was specifically expressed by TAMs and extremely low in GBM and GSC cells, and meanwhile closely related to glioma WHO grades and patient prognosis. The exact mechanism of TGFBI linking TAM functions to GSC-driven tumor growth was explored. Western blot, quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), immunohistochemistry staining (IHC) and public datasets were used to evaluate TGFBI origin and level in GBM. The response of GSCs to recombinant human TGFBI was assessed and orthotopic xenografts were established to investigate the function and mechanism . M2-like TAMs infiltration was elevated in high-grade gliomas. TGFBI was preferentially secreted by M2-like TAMs and associated with a poor prognosis for patients with GBM. TGFBI promoted the maintenance of GSCs and GBM malignant growth through integrin αvβ5-Src-Stat3 signaling and . Of clinical relevance, TGFBI was enriched in the serum and CSF of GBM patients and significantly decreased after tumor resection. TAM-derived TGFBI promotes GSC-driven tumor growth through integrin αvβ5-Src-Stat3 signaling. High serum or CSF TGFBI may serve as a potential diagnostic and prognostic bio-index for GBMs.
Topics: Brain Neoplasms; Cell Line, Tumor; Glioblastoma; Glioma; Humans; Neoplastic Stem Cells; Receptors, Vitronectin; STAT3 Transcription Factor; Transforming Growth Factor beta; Tumor Microenvironment; Tumor-Associated Macrophages
PubMed: 35673564
DOI: 10.7150/thno.69605 -
Biomedicine & Pharmacotherapy =... Aug 2022Nephrotoxicity (NT) is a renal-specific situation caused by different toxins and drugs like non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs like diclofenac (DCF)...
Nephrotoxicity (NT) is a renal-specific situation caused by different toxins and drugs like non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs like diclofenac (DCF) lead to glomerular dysfunction. Pentoxifylline (PTX) and berberine (BER) have antioxidant and anti-inflammatory properties. Thus, the objective of the present study was to investigate the ameliorative effect of PTX, BER and their combination against DCF-mediated acute NT. Induction of acute NT was done via DCF injection (150 mg/kg I.P, for 6 days) in rats. PTX 200 mg/kg, BER 200 mg/kg and their combination were administrated for 6 days prior to DCF injection and concurrently with DCF for additional 6 days. Acute NT was evaluated biochemically and histopathologically by measuring blood urea (BU), serum creatinine (SCr), kidney injury molecule-1(KIM-1), integrin (ITG), and vitronectin (VTN), interleukin (IL)-18, Neutrophil gelatinase-associated lipocalin (NGAL), glomerular filtration rate (GFR), superoxide dismutase (SOD) and glutathione (GSH) and malondialdehyde (MDA) with the scoring of histopathological alterations. PTX, BER and their combination significantly (P < 0.05) attenuated biochemical and histopathological changes in DCF-mediated acute NT by amelioration of BU, SCr, KIM-1, ITG, VTN, IL-18, NGAL, GFR, SOD, GSH, MDA and scoring of histopathological alterations. The combined effects of PTX and BER produced more significant effects (P < 0.05) than either PTX or BER when used alone against DCF-induced acute NT. In conclusion, BER and BTX were found to have potential renoprotective effects against DCF-induced NT in rats by inhibiting inflammatory reactions and oxidative stress.
Topics: Animals; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Berberine; Diclofenac; Drug-Related Side Effects and Adverse Reactions; Glutathione; Inflammation; Kidney; Lipocalin-2; Male; Oxidative Stress; Pentoxifylline; Rats; Rats, Sprague-Dawley; Renal Insufficiency; Superoxide Dismutase
PubMed: 35671584
DOI: 10.1016/j.biopha.2022.113225 -
Frontiers in Cell and Developmental... 2022The linker of nucleoskeleton and cytoskeleton (LINC) complex is composed of the inner nuclear membrane-spanning SUN proteins and the outer nuclear membrane-spanning...
The linker of nucleoskeleton and cytoskeleton (LINC) complex is composed of the inner nuclear membrane-spanning SUN proteins and the outer nuclear membrane-spanning nesprin proteins. The LINC complex physically connects the nucleus and plasma membrane the actin cytoskeleton to perform diverse functions including mechanotransduction from the extracellular environment to the nucleus. Mammalian somatic cells express two principal SUN proteins, namely SUN1 and SUN2. We have previously reported that SUN1, but not SUN2, is essential for directional cell migration; however, the underlying mechanism remains elusive. Because the balance between adhesive force and traction force is critical for cell migration, in the present study, we focused on focal adhesions (FAs) and the actin cytoskeleton. We observed that siRNA-mediated SUN1 depletion did not affect the recruitment of integrin β1, one of the ubiquitously expressed focal adhesion molecules, to the plasma membrane. Consistently, SUN1-depleted cells normally adhered to extracellular matrix proteins, including collagen, fibronectin, laminin, and vitronectin. In contrast, SUN1 depletion reduced the activation of integrin β1. Strikingly, the depletion of SUN1 interfered with the incorporation of vinculin into the focal adhesions, whereas no significant differences in the expression of vinculin were observed between wild-type and SUN1-depleted cells. In addition, SUN1 depletion suppressed the recruitment of zyxin to nascent focal adhesions. These data indicate that SUN1 is involved in the maturation of focal adhesions. Moreover, disruption of the SUN1-containing LINC complex abrogates the actin cytoskeleton and generation of intracellular traction force, despite the presence of SUN2. Thus, a physical link between the nucleus and cytoskeleton through SUN1 is required for the proper organization of actin, thereby suppressing the incorporation of vinculin and zyxin into focal adhesions and the activation of integrin β1, both of which are dependent on traction force. This study provides insights into a previously unappreciated signaling pathway from the nucleus to the cytoskeleton, which is in the opposite direction to the well-known mechanotransduction pathways from the extracellular matrix to the nucleus.
PubMed: 35663386
DOI: 10.3389/fcell.2022.885859 -
Journal of Cell Science Jun 2022The vitronectin receptor integrin αVβ5 can reside in two distinct adhesion structures - focal adhesions (FAs) and flat clathrin lattices (FCLs). Here, we investigate...
The vitronectin receptor integrin αVβ5 can reside in two distinct adhesion structures - focal adhesions (FAs) and flat clathrin lattices (FCLs). Here, we investigate the mechanism that regulates the subcellular distribution of β5 in keratinocytes and show that β5 has approximately 7- and 5-fold higher affinity for the clathrin adaptors ARH (also known as LDLRAP1) and Numb, respectively, than for the talin 1 (TLN1); all proteins that bind to the membrane-proximal NPxY motif of the β5 cytoplasmic domain. Using mass spectrometry, we identified β5 interactors, including the Rho GEFs p115Rho-GEF and GEF-H1 (also known as ARHGEF1 and ARHGEF2, respectively), and the serine protein kinase MARK2, depletion of which diminishes the clustering of β5 in FCLs. Replacement of two serine residues (S759 and S762) in the β5 cytoplasmic domain with phospho-mimetic glutamate residues causes a shift in the localization of β5 from FAs into FCLs without affecting the interactions with MARK2, p115Rho-GEF or GEF-H1. Instead, we demonstrate that changes in the actomyosin-based cellular contractility by ectopic expression of activated Rho or disruption of microtubules regulates β5 localization. Finally, we present evidence that β5 in either FAs or FCLs functions to promote adhesion to vitronectin, cell spreading, and proliferation.
Topics: Cell Adhesion; Cell Proliferation; Clathrin; Focal Adhesions; Receptors, Vitronectin; Serine
PubMed: 35532004
DOI: 10.1242/jcs.259465