-
Annals of Neurology Mar 2016CADASIL is a genetic paradigm of cerebral small vessel disease caused by NOTCH3 mutations that stereotypically lead to the extracellular deposition of NOTCH3 ectodomain...
OBJECTIVE
CADASIL is a genetic paradigm of cerebral small vessel disease caused by NOTCH3 mutations that stereotypically lead to the extracellular deposition of NOTCH3 ectodomain (Notch3(ECD) ) on the vessels. TIMP3 and vitronectin are 2 extracellular matrix proteins that abnormally accumulate in Notch3(ECD) -containing deposits on brain vessels of mice and patients with CADASIL. Herein, we investigated whether increased levels of TIMP3 and vitronectin are responsible for aspects of CADASIL disease phenotypes.
METHODS
Timp3 and vitronectin expression were genetically reduced in TgNotch3(R169C) mice, a well-established preclinical model of CADASIL. A mouse overexpressing human TIMP3 (TgBAC-TIMP3) was developed. Disease-related phenotypes, including cerebral blood flow (CBF) deficits, white matter lesions, and Notch3(ECD) deposition, were evaluated between 6 and 20 months of age.
RESULTS
CBF responses to neural activity (functional hyperemia), topical application of vasodilators, and decreases in blood pressure (CBF autoregulation) were similarly reduced in TgNotch3(R169C) and TgBAC-TIMP3 mice, and myogenic responses of brain arteries were likewise attenuated. These defects were rescued in TgNotch3(R169C) mice by haploinsufficiency of Timp3, although the number of white matter lesions was unaffected. In contrast, haploinsufficiency or loss of vitronectin in TgNotch3(R169C) mice ameliorated white matter lesions, although CBF responses were unchanged. Amelioration of cerebrovascular reactivity or white matter lesions in these mice was not associated with reduced Notch3(ECD) deposition in brain vessels.
INTERPRETATION
Elevated levels of TIMP3 and vitronectin, acting downstream of Notch3(ECD) deposition, play a role in CADASIL, producing divergent influences on early CBF deficits and later white matter lesions.
Topics: Animals; Brain; CADASIL; Cerebrovascular Circulation; Humans; Infant; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Tissue Inhibitor of Metalloproteinases; Vitronectin; Tissue Inhibitor of Metalloproteinase-4
PubMed: 26648042
DOI: 10.1002/ana.24573 -
Biomolecules Jul 2023Adsorbing toxins from the blood to augment membrane-based hemodialysis is an active area of research. Films composed of β-cyclodextrin-co-(methacryloyloxy)ethyl...
Adsorbing toxins from the blood to augment membrane-based hemodialysis is an active area of research. Films composed of β-cyclodextrin-co-(methacryloyloxy)ethyl phosphorylcholine (p(PMβCD-co-MPC)) with various monomer ratios were formed on magnetic nanoparticles and characterized. Surface chemistry effects on protein denaturation were evaluated and indicated that unmodified magnetic nanoparticles greatly perturbed the structure of proteins compared to coated particles. Plasma clotting assays were conducted to investigate the stability of plasma in the presence of particles, where a 2:2 monomer ratio yielded the best results for a given total surface area of particles. Total protein adsorption results revealed that modified surfaces exhibited reduced protein adsorption compared to bare particles, and pure MPC showed the lowest adsorption. Immunoblot results showed that fibrinogen, α1-antitrypsin, vitronectin, prekallikrein, antithrombin, albumin, and C3 correlated with film composition. Hemocompatibility testing with whole blood illustrated that the 1:3 ratio of CD to MPC had a negative impact on platelets, as evidenced by the increased activation, reduced response to an agonist, and reduced platelet count. Other formulations had statistically significant effects on platelet activation, but no formulation yielded apparent adverse effects on hemostasis. For the first time, p(PMβCD-co-MPC)-coated MNP were synthesized and their general hemocompatibility assessed.
Topics: Phosphorylcholine; Magnetite Nanoparticles; Adsorption; Antithrombin III; Blood Coagulation
PubMed: 37627230
DOI: 10.3390/biom13081165 -
Mediators of Inflammation 2023Vitronectin (VTN) has been reported to trigger cell pyroptosis to aggravate inflammation in our previous study. However, the function of VTN in inflammatory bowel...
OBJECTIVE
Vitronectin (VTN) has been reported to trigger cell pyroptosis to aggravate inflammation in our previous study. However, the function of VTN in inflammatory bowel disease (IBD) remains to be addressed.
METHODS
Real-time PCR and western blotting were performed to analyze VTN-regulated intestinal epithelial cell (IEC) differentiation through ferroptosis, and immunofluorescence (IF), luciferase, and chromatin immunoprecipitation were used to identify whether VTN-modulated ferroptosis is dependent on phosphodiesterase 4 (PDE4)/protein kinase A (PKA)/cyclic adenosine monophosphate-response element-binding protein (CREB) cascade pathway. experiment in mice and a pilot study in patients with IBD were used to confirm inhibition of PDE4-alleviated IECs ferroptosis, leading to cell differentiation during mucosal healing.
RESULTS
Herein, we found that caudal-related homeobox transcription factor 2-mediated IECs differentiation was impaired in response to VTN, which was attributed to enhanced ferroptosis characterized by decreased glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 expression. Inhibition of ferroptosis in IECs rescued the inhibitory effect of VTN on cell differentiation. Further analysis showed that VTN triggered phosphorylation of PDE4, leading to inhibit PKA/CREB activation and CREB nuclear translocation, which further reduced GPX4 transactivation. Endogenous PKA interacted with CREB, and this interaction was destroyed in response to VTN stimulation. What is more, overexpression of CREB in CaCO cells overcame the promotion of VTN on ferroptosis. Most importantly, inhibition of PDE4 by roflumilast or dipyridamole could alleviate dextran sulfate sodium-induced colitis in mice and in a pilot clinical study confirmed by IF.
CONCLUSIONS
These findings demonstrated that highly expressed VTN disrupted IECs differentiation through PDE4-mediated ferroptosis in IBD, suggesting targeting PDE4 could be a promising therapeutic strategy for patients with IBD.
Topics: Mice; Animals; Vitronectin; Cyclic Nucleotide Phosphodiesterases, Type 4; Ferroptosis; Pilot Projects; Inflammatory Bowel Diseases; Cell Differentiation
PubMed: 37501933
DOI: 10.1155/2023/6623329 -
BMC Molecular and Cell Biology Nov 2019Brucella is a facultative intracellular pathogen responsible for zoonotic disease brucellosis. Little is known about the molecular basis of Brucella adherence to host...
BACKGROUND
Brucella is a facultative intracellular pathogen responsible for zoonotic disease brucellosis. Little is known about the molecular basis of Brucella adherence to host cells. In the present study, the possible role of Bp26 protein as an adhesin was explored. The ability of Brucella protein Bp26 to bind to extracellular matrix (ECM) proteins was determined by enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI).
RESULTS
ELISA experiments showed that Bp26 bound in a dose-dependent manner to both immobilized type I collagen and vitronectin. Bp26 bound weakly to soluble fibronectin but did not bind to immobilized fibronectin. No binding to laminin was detected. Biolayer interferometry showed high binding affinity of Bp26 to immobilized type I collagen and no binding to fibronectin or laminin. Mapping of Bp26 antigenic epitopes by biotinylated overlapping peptides spanning the entire sequence of Bp26 using anti Bp26 mouse serum led to the identification of five linear epitopes. Collagen and vitronectin bound to peptides from several regions of Bp26, with many of the binding sites for the ligands overlapping. The strongest binding for anti-Bp26 mouse serum, collagen and vitronectin was to the peptides at the C-terminus of Bp26. Fibronectin did not bind to any of the peptides, although it bound to the whole Bp26 protein.
CONCLUSIONS
Our results highlight the possible role of Bp26 protein in the adhesion process of Brucella to host cells through ECM components. This study revealed that Bp26 binds to both immobilized and soluble type I collagen and vitronectin. It also binds to soluble but not immobilized fibronectin. However, Bp26 does not bind to laminin. These are novel findings that offer insight into understanding the interplay between Brucella and host target cells, which may aid in future identification of a new target for diagnosis and/or vaccine development and prevention of brucellosis.
Topics: Adhesins, Bacterial; Collagen; Enzyme-Linked Immunosorbent Assay; Epitope Mapping; Extracellular Matrix Proteins; Fibronectins; Host Microbial Interactions; Laminin; Membrane Proteins; Protein Binding
PubMed: 31783731
DOI: 10.1186/s12860-019-0239-7 -
Stroke May 2020Background and Purpose- Women have worse stroke outcomes than men, especially after menopause. Few studies have focused on female-specific mechanisms, other than...
Background and Purpose- Women have worse stroke outcomes than men, especially after menopause. Few studies have focused on female-specific mechanisms, other than hormones. We investigated the role of the blood protein VTN (vitronectin) after ischemic stroke in mice. Methods- Adult male and female VTN knockout and wild-type littermates and C57BL/6 mice received a middle cerebral artery occlusion and the injured brain tissue analyzed 24 hours to 3 weeks later for cell loss and inflammation, as well as neurological function. Blood VTN levels were measured before and after stroke. Results- Intravenously injected VTN leaked extensively from bloodstream into brain infarct and penumbra by 24 hours after stroke. Strikingly, VTN was detrimental in female, but not male, mice, as shown by reduced brain injury (26.2±2.6% versus 13.4±3.8%; =0.018; n=6 and 5) and forelimb dysfunction in female VTN knockout mice. Stroke increased plasma VTN 2- to 8-fold at 24 hours in females (36±4 versus 145±24 μg/mL; <0.0001; n=10 and 7), but not males (62±8 versus 68±6; >0.99; n=10 and 7), and returned to control levels by 7 days. Individually variable VTN levels at 24 hours correlated with stroke-induced brain injury at 7 days only in females. VTN promoted stroke-induced microglia/macrophage activation and leukocyte infiltration in females. Proinflammatory IL (interleukin)-6 greatly increased in the striatum at 24 hours in wild-type mice but was increased ≈60% less in female (739±159 versus 268±111; =0.02; n=7 and 6), but not male (889±178 versus 1179±295; =0.73; n=10 and 11), knockout mice. In individual wild-type females, plasma VTN levels correlated with striatal IL-6 expression at 24 hours. The female-specific effect of VTN-induced IL-6 expression following stroke was not due to gonadal hormones, as shown by ovariectomy and castration. Lastly, intrastriatal injection of IL-6 in female mice immediately before stroke reversed the VTN knockout phenotypes of reduced brain injury and microglia/macrophage activation. Conclusions- VTN plays a novel sexually dimorphic detrimental pathophysiological role in females and might ultimately be a therapeutic target to improve stroke outcomes in women.
Topics: Animals; Blood-Brain Barrier; Ciliary Neurotrophic Factor; Female; Infarction, Middle Cerebral Artery; Inflammation; Interleukin-6; Leukemia Inhibitory Factor; Macrophages; Male; Mice; Mice, Knockout; Microglia; RNA, Messenger; Sex Factors; Stroke; Vitronectin
PubMed: 32312218
DOI: 10.1161/STROKEAHA.120.029036 -
International Journal of Clinical and... 2015Vitronectin (Vn), a multifunctional adhesive protein, is found in association with tumor progression, angiogenesis and metastasis in a variety of (human) tumors. But no...
Vitronectin (Vn), a multifunctional adhesive protein, is found in association with tumor progression, angiogenesis and metastasis in a variety of (human) tumors. But no studies concerning its correlation to osteosarcoma prognosis were found. Hence, we aimed to investigate the prognostic value of Vitronectin (Vn) in osteosarcoma. Here, we studied the expression of VN in the tumor tissues from 67 patients with osteosarcoma and 20 patients with osteochondroma using immunohistochemistry and estimated the effects of VN expression in osteosarcoma on progression-free survival (PFS) and overall survival (OS) using the Kaplan-Meier curve and COX proportional hazards regression model. Increased expression of VN in osteosarcoma tissue compared to no VN expression in osteochondroma tissue was shown in immunohistochemical assay. No associations were observed between VN expression and osteosarcoma patients' gender (P = 0.675), age (P = 0.813), tumor size (P = 0.436), histologic subtype (P = 0.0.543) or tumor location (P = 0.456). Univariate survival analysis demonstrated significant correlations of high VN expression with shorter PFS (P = 0.002) and OS (P = 0.001); multivariate survival analysis revealed high VN expression as a significant independent prognostic indicator for shorter PFS (HR 2.788, P = 0.003) and OS (HR2.817, P = 0.003). In conclusion, the high expression of VN in tumor cells independently indicated poor clinical prognosis in patients with osteosarcoma, other than large tumor size and non-neoadjuvant chemoradiotherapy, suggesting that VN may serve as a potential therapeutic target in osteosarcoma.
Topics: Adolescent; Adult; Aged; Biomarkers, Tumor; Bone Neoplasms; Chemoradiotherapy, Adjuvant; Chi-Square Distribution; Child; Disease Progression; Disease-Free Survival; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Middle Aged; Multivariate Analysis; Neoadjuvant Therapy; Osteosarcoma; Predictive Value of Tests; Proportional Hazards Models; Risk Factors; Time Factors; Treatment Outcome; Tumor Burden; Up-Regulation; Vitronectin; Young Adult
PubMed: 26617861
DOI: No ID Found -
Experimental and Therapeutic Medicine Jan 2022Neurogenic bladder (NGB) is an important complication of urinary tract dysfunction after spinal cord injury (SCI). However, using urodynamics and urography to guide...
Neurogenic bladder (NGB) is an important complication of urinary tract dysfunction after spinal cord injury (SCI). However, using urodynamics and urography to guide therapy remains invasive and complicated. Therefore, the present study aimed to identify potential noninvasive biomarkers from urinary exosomes that can facilitate diagnosis and guide prognosis of patients with NGB subsequent to SCI. Urinary exosomes were isolated, and their proteome profile was analyzed by mass spectrometry. Transmission electron microscopy and Nanoparticle Tracking Analysis confirmed the size and morphological characteristics of urinary exosomes. In addition, bioinformatics analysis and parallel reaction monitoring (PRM) were used to screen candidate biomarkers. The selected biomarkers were validated using western blotting and ELISA. Mass spectrometry identified 134 upregulated proteins and 99 downregulated proteins between the vesicoureteral reflux (VUR) and non-VUR groups. A total of 18 candidate proteins were selected for PRM validation, but only vitronectin (VTN) and α-1 type I collagen (COL1A1) demonstrated significant differences. In the validation experiments using western blotting and ELISA, VTN was exclusively highly expressed in VUR patients compared with non-VUR patients. However, the ELISA results of COL1A1 revealed no significant difference when a larger sample size was used. Furthermore, a receiver operating characteristic curve of ELISA-based VTN demonstrated an area under the curve of 0.795 and 80% sensitivity at a threshold set to give 82.9% specificity. Collectively, these results suggested that VTN in urinary exosomes may be used as a biomarker to predict the progression and guide the prognosis of NGB.
PubMed: 34934436
DOI: 10.3892/etm.2021.10988 -
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 -
Molecular & Cellular Proteomics : MCP Sep 2023The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating...
The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N-Acetylgalactosamine are recognized by ASGPR, while terminal mannose, fucose, and N-Acetylglucosamine are recognized by MRC1. The effects of ASGPR and MRC1 deficiency on the N-glycosylation of individual circulating proteins have been studied. However, the impact on the homeostasis of the major plasma glycoproteins is debated and their glycosylation has not been mapped with high molecular resolution in this context. Therefore, we evaluated the total plasma N-glycome and plasma proteome of ASGR1 and MRC1 deficient mice. ASGPR deficiency resulted in an increase in O-acetylation of sialic acids accompanied by higher levels of apolipoprotein D, haptoglobin, and vitronectin. MRC1 deficiency decreased fucosylation without affecting the abundance of the major circulating glycoproteins. Our findings confirm that concentrations and N-glycosylation of the major plasma proteins are tightly controlled and further suggest that glycan-binding receptors have redundancy, allowing compensation for the loss of one major clearance receptor.
Topics: Mice; Animals; Asialoglycoprotein Receptor; Mannose Receptor; Glycoproteins; Glycosylation; Protein Processing, Post-Translational; Carrier Proteins; Mannose
PubMed: 37414249
DOI: 10.1016/j.mcpro.2023.100615 -
Acta Biomaterialia Jan 2023Targeted drug delivery requires -among others- specific interaction of nanocarriers with cell surface receptors enabling efficient internalization into the targeted...
Targeted drug delivery requires -among others- specific interaction of nanocarriers with cell surface receptors enabling efficient internalization into the targeted cells. Thus, identification of receptors allowing efficient nanocarrier uptake is essential to improve the design of targeted nanomedicines. Here we used methods based on cell surface biotinylation to identify cell surface receptors mediating nanoparticle uptake by cells. We used human brain and liver endothelial cells as representative examples of cells typically showing very low and very high nanoparticle uptake, respectively. Amino-modified and carboxylated silica were used as model nanoparticles usually associated with high and low uptake into cells, respectively, and carrying different coronas after exposure in full human plasma. Using cell surface biotinylation of live cells and receptor pull-down assays, we compared the receptors internalized in control untreated cells and those internalized upon exposure to nanoparticles. In this way, we identified receptors associated with (high) nanoparticle uptake. The candidate receptors were further validated by decorating the nanoparticles with an artificial corona consisting of the respective receptor ligands. We found that a vitronectin corona can be used to target integrin receptors and strongly enhances nanoparticle uptake in brain and liver endothelial cells. The increased uptake was maintained in the presence of serum, suggesting that the vitronectin-corona could resist interaction and competition with serum. Furthermore, plasminogen-coated nanoparticles promoted uptake in endothelial cells of the liver, but not of the brain. The presented approach using reversible biotinylation of cell surface receptors in live cells allows for receptor-based targeting of nanocarriers that are instrumental in nanoparticle uptake, which can be exploited for targeted drug delivery. STATEMENT OF SIGNIFICANCE: In order to deliver drugs to their site of action, drug-loaded nanocarriers can be targeted to cell receptors enabling efficient uptake into target cells. Thus, methods to identify nanocarrier receptors are invaluable. Here we used reversible biotinylation of live cells and receptor pull-down approaches for receptor identification. By comparative analysis of the individual receptors internalized in untreated cells and cells exposed to nanoparticles, we identified receptors enabling high nanoparticle uptake into liver and brain endothelial cells. Their role was confirmed by decorating nanoparticles with an artificial corona composed of the receptor ligands. In conclusion, live cell reversible biotinylation of cell surface proteins is a powerful tool for the identification of potential receptors for receptor-based targeting of nanocarriers.
Topics: Humans; Endothelial Cells; Biotinylation; Vitronectin; Receptors, Cell Surface; Nanoparticles
PubMed: 36371002
DOI: 10.1016/j.actbio.2022.11.010