High Molecular Complex Research Articles

The regulation of necroptosis by post-translational modifications.

Necroptosis is a caspase-independent, lytic form of programmed cell death whose errant activation has been widely implicated in many pathologies. The pathway relies on the assembly of the apical protein kinases, RIPK1 and RIPK3, into a high molecular weight cytoplasmic complex, termed the necrosome, downstream of death receptor or pathogen detector ligation. The necrosome serves as a platform for RIPK3-mediated phosphorylation of the terminal effector, the MLKL pseudokinase, which induces its oligomerization, translocation to, and perturbation of, the plasma membrane to cause cell death. Over the past 10 years, knowledge of the post-translational modifications that govern RIPK1, RIPK3 and MLKL conformation, activity, interactions, stability and localization has rapidly expanded. Here, we review current knowledge of the functions of phosphorylation, ubiquitylation, GlcNAcylation, proteolytic cleavage, and disulfide bonding in regulating necroptotic signaling. Post-translational modifications serve a broad array of functions in modulating RIPK1 engagement in, or exclusion from, cell death signaling, whereas the bulk of identified RIPK3 and MLKL modifications promote their necroptotic functions. An enhanced understanding of the modifying enzymes that tune RIPK1, RIPK3, and MLKL necroptotic functions will prove valuable in efforts to therapeutically modulate necroptosis.

Program extracorporeal photophoresis in complex treatment of psoriatic arthritis

The results of complex treatment with the programmed procedures of extracorporeal photopheresis and methotrexate in patients with psoriatic arthritis for 12 months are presented. It has been established that program extracorporeal photopheresis in combination with basic therapy with methotrexate in patients with psoriatic arthritis have a significant effect on the relief of articular syndrome (reduction of morning stiffness, pain intensity, joint swelling). It was revealed that the inclusion of programmed extracorporeal photopheresis and methotrexate in the complex treatment of psoriatic arthritis reduces the level of circulating low molecular weight, medium molecular weight and high molecular weight immune complexes and in most cases leads to clinical and immunological remission. This is confirmed by a decrease in complaints of the severity of articular syndrome and a decrease in the clinical and laboratory activity of the disease (a significant decrease in the level of C-reactive protein, erythrocyte sedimentation rate). A scheme has been developed for the programmed use of extracorporeal photopheresis procedures in the complex treatment of psoriatic arthritis, which consists of 2 procedures every other day with an interval of 2,5-3 months for 12 months. Combined therapy with the programmed application of extracorporeal photopheresis procedures and methotrexate prolongs the period of disease remission and can be recommended for patients with psoriatic arthritis with high activity, insufficient effect of basic therapy and intolerance to higher doses of immunosuppressive drugs.

HIV-2 Vif and foamy virus Bet antagonize APOBEC3B by different mechanisms

The family of human APOBEC3 (A3) restriction factors is formed by seven different proteins, A3A–D and A3F–H. Among these A3s, A3B harbors strong restriction activity against several retroviruses, such as SIV, and MLV. How lentiviruses and other retroviruses, prevalent in many primate species, counteract A3B is poorly understood.In this study, we found that A3B strongly inhibited SIVmac and HIV-2 infectivity, which was antagonized by their Vif proteins. Both SIVmac and HIV-2 Vifs diminished the protein level of A3B in viral producer cells, and hindered A3B incorporation into viral particles. We observed that HIV-2 Vif binds A3B and induces its degradation by assembly of an A3-Vif-CUL5-ElonginB/C E3-ligase complex. A3B and HIV-2 Vif localize and interact in the nucleus.In addition, we also found that the accessory protein Bet of prototype foamy virus (PFV) significantly antagonized the anti-SIVmac activity of A3B. Like Vif, Bet prevented the incorporation of A3B into viral particles. However, in contrast to Vif Bet did not induce the degradation of A3B. Rather, Bet binds A3B to block formation of high molecular weight A3B complexes and induces A3B cytoplasmic trapping.In summary, these findings indicate that A3B is recognized by diverse retroviruses and counteracted by virus-specific pathways that could be targeted to inhibit A3B mutating activity in cancers.

Characterization of membrane-bound metalloproteins in the anaerobic ammonium-oxidizing bacterium “Candidatus Kuenenia stuttgartiensis” strain CSTR1

Membrane-bound metalloproteins are the basis of biological energy conservation via respiratory processes, however, their biochemical characterization is difficult. Here, we followed a gel-based proteomics and metallomics approach to identify membrane-associated metalloproteins in the anaerobic ammonium-oxidizing “Candidatus Kuenenia stuttgartiensis” strain CSTR1. Membrane-associated protein complexes were separated by two dimensional Blue Native/SDS gel electrophoresis and subunits were identified by mass spectrometry; protein-bound metal ions were quantified from the gel by connecting either a desolvating nebulizer system or laser ablation to inductively coupled plasma triple quadrupole mass spectrometry (ICP-QqQ-MS). We identified most protein complexes predicted to be involved in anaerobic ammonium oxidation and carbon fixation. The ICP-QqQ-MS data showed the presence of Fe and Zn in a wide range of high molecular weight protein complexes (230–800 kDa). Mo was prominently found in gel slices with proteins of a size of 500–650 kDa, whereas Ni was only found using the desolvating nebulizer system in the protein range of 350–500 kDa. The detected protein complexes and their metal content were consistent with genome annotations. Gel-based metalloproteomics is a sensitive and reliable approach for the characterization of metalloproteins and could be used to characterize many multimeric metalloprotein complexes in biological systems.

Metabolic regulation of Thioredoxin interacting protein (Txnip)-containing high molecular protein complexes

Metabolic regulation of Thioredoxin interacting protein (Txnip)-containing high molecular protein complexes

Human CLPB forms ATP-dependent complexes in the mitochondrial intermembrane space.

Human caseinolytic peptidase B protein homolog (CLPB), also known as suppressor of potassium transport defect 3 (SKD3), is a broadly-expressed member of the family of ATPases associated with diverse cellular activities (AAA+). Mutations in the human CLPB gene cause 3-methylglutaconic aciduria type VII. CLPB is upregulated in acute myeloid leukemia (AML), where it contributes to anti-cancer drug resistance. The biological function of CLPB in human cells and mechanistic links to the clinical phenotypes are currently unknown. Herein, subcellular fractionation of human HEK-293 and BT-549 cells showed that a single 57-kDa form of CLPB was present in the mitochondria and not in the cytosolic fraction. Immunofluorescence staining of HEK-293 and BT-549 cells with anti-CLPB antibody co-localized with the mitochondrial staining using a MitoTracker dye. In purified intact mitochondria, CLPB was protected against externally added proteinase K, but it was susceptible to degradation after disruption of the outer membrane, indicating that CLPB resides in the mitochondrial intermembrane space. Overexpressed CLPB, while properly trafficked to the mitochondria, appeared to form large clusters/aggregates that were resistant to extraction with non-ionic detergents and were readily visualized by immunofluorescence microscopy. Importantly, endogenous CLPB formed high molecular weight protein complexes in an ATP-dependent manner that were detected by blue native polyacrylamide gel electrophoresis. These results demonstrate that ATP induces a structural change in CLPB and controls its ability to self-associate or form complexes with other proteins in the intermembrane space of mitochondria.

Changes of plasma fibronectin and fibronectin-fibrin complexes in dams of stillborn dairy calves

BackgroundFibronectin (FN) is a large (450–500 kDa), multidomain and multifunctional glycoprotein existing in mammalian tissues. Some fibronectin (FN) molecular forms might be involved in biological processes occurring within the perinatal period, such as tissue remodeling, coagulation, and repair.ResultsIn this study fibronectin (FN) and fibrinogen (Fb) concentrations and FN-fibrin complexes occurrence and its relative amounts with increasing high molecular masses were respectively determined by ELISA, heat precipitation, and SDS-agarose-immunoblotting methods. Plasma samples from three groups of dams with: 1) singleton stillborn calf without or with negligible autolytic changes in internal organs (DSBn), 2) singleton stillborn calf with advanced autolytic changes in internal organs (DSBa), 3) singleton live-born control calf (DC), and 4) a group of cows during mid to late lactation (LC) were analyzed. Maternal plasma FN concentration in the DSBn and DSBa groups was significantly lower than in the LC group. The plasma samples of DSBa showed a significantly lower FN concentration than in the DC group. Plasma Fb concentration was significantly higher in the DSBa and DSBn, than in the LC group. FN immunoblotting of the cow plasma samples revealed, besides an FN-dimer band, the presence of supramolecular FN-fibrin bands corresponding to FN-fibrin complexes with increasing molecular masses: up to 5 bands from 750 kDa to 1900 kDa in the DSBn and DSBa plasma samples, two bands of 750 and 1000 kDa in the DC group, and only the smallest one of 750 kDa in the LC group.ConclusionsThe observed low FN concentration and occurrence of supramolecular FN-fibrin complexes (1000 kDa and more) in the maternal plasma comparing to cows in lactation might have been associated with periparturient changes in tissues. The presence in maternal plasma of high-molecular FN-fibrin complexes (1300–1900 kDa) arouse the question if this is the consequence of calf perinatal mortality.

Structural, physicochemical and emulsifying properties of sweet potato pectin treated by high hydrostatic pressure and/or pectinase: a comparative study.

Sweet potato (Ipomoea batatas L.) is the sixth most important food crop in the world, and China is the largest producer. Large amounts of sweet potato residues are generated during starch extraction, leading to environmental pollution and resource waste. However, these residues can be used as a viable source for pectin extraction. As a natural biopolymer with high molecular weight and complex structure, the usefulness of pectin has been limited, and it needs to be modified in order to improve its physicochemical properties, thus expanding its applications in the food industry. Therefore, the reported study was conducted to modify sweet potato pectin (SPP) using high hydrostatic pressure (HHP) and/or pectinase treatment, and to determine the effects of such treatment on structural, physicochemical and emulsifying properties. The results demonstrated that the molecular weight of SPP decreased following HHP and pectinase treatment, which was evidenced using scanning electron microscopy and atomic force microscopy. The degree of esterification was also decreased, confirmed by decreased intensity of the peak at 1739 cm-1 in the Fourier transform infrared spectrum and decreased peaks at 3.6 and 3.8 ppm in the 1 H NMR spectrum. Moreover, the content of monosaccharides and uronic acids increased and emulsifying properties improved after HHP and pectinase treatment. HHP-assisted pectinase treatment could be used as novel technique for the modification of pectin to give better emulsifying properties with great potential for application in the food industry. © 2020 Society of Chemical Industry.

Mucilage Extracted from Dragon Fruit Peel (Hylocereus undatus) as Flocculant for Treatment of Dye Wastewater by Coagulation and Flocculation Process

Dye wastewater from textile industries shows very low biodegradability due to high molecular weight and complex structures of dyes. So far, the most simple method for treatment of this type of wastewater has been coagulation and flocculation. This study determined the removal of turbidity and other pollutants from dye wastewater by mucilage extracted from the peel of dragon fruit (Hylocereus undatus) and its effect in reducing synthetic chemical polyaluminum chloride (PACl) used in coagulation and flocculation (CF) process. The removal of turbidity in a sequent CF process using PACl and dragon fruit mucilage was investigated based on Jar tests. Maximum coagulation efficiencies of PACl were typically observed at pH 4.0-6.0 and PACl concentrations of about 100-150 mg/L depending on types of wastewater, whereas optimal settling times were 30-60 minutes, respectively. The addition of dragon fruit mucilage (0.5-50 mg/L) after PACl (75-245 mg/L) resulted in turbidity removal efficiencies up to 95%. The addition of mucilage extracted from dragon fruit peels was proven to increase turbidity removal efficiency and decrease PACl use. The increase of turbidity removal was often estimated at 10-32%, whereas PACl used was about 3-10% less compared to total PACl needed for obtaining comparable efficiency when used alone. The flocculation activity of mucilage was also compared to polyacrylamide (PAM)—a synthetically organic flocculant. Since the peel of a dragon fruit is an abundant agriculture waste in Vietnam, using its extracted mucilage as a flocculant is an environmentally friendly method.

Influence of microwave on chromium complex composition in tanning liquor

Since microwave irradiation could promote hydrolysis and olation of chromium tanning liquor, but the influence of microwave on chromium complex component in the liquor was still unknown. Chromium sulphate solution (0% basicity) and 33% basicity chromium tanning liquor were subjected microwave (MW) and water bath (WB) heating, and the samples without any warming were regarded as control. Ion exchange chromatography (IEC) and gel filtration chromatography (GFC) were used to measure the charge composition and molecular size of chromium complexes in each sample. FT-IR was used to characterize the structure of chromium complexes in each composition separated by IEC. Moreover, the chromium tanning liquor after warming was used in hide powder tanning trials to illustrate whether microwave would affect its tanning ability. The results show there are more high positive charge and large molecular size complexes in chromium tanning liquor after warming but the phenomena are more significant in MW samples compared with WB due to non-thermal effect of microwave. In addition, microwave has more powerful effect on 33% basicity chromium tanning liquor hydrolysis and olation to generate larger molecular size complexes. In FT-IR results, the combination pattern between chromium and ligands are changed after warming but there is no difference between WB and MW. The chromium exhaustion and thermal stability of hide powder tanned with chromium tanning liquor after microwave irradiation are both higher. It could conclude that both thermal and non-thermal effects of microwave promote the process together, and the non-thermal effect leads to more high positive charge and large molecular size complexes and has stronger influence on high polarity system. In short, this work would provide theoretical basis for applying microwave in tanning agent modification and chrome tanning process further.Graphical abstract

Insecticidal Activity of Photorhabdus luminescens 0805-P2R Against Plutella xylostella.

Photorhabdus luminescens is an entomopathogenic rod-shaped bacterium infected with insect nematodes of the Heterorhabditidae family. It kills insects through the secretion of high molecular weight toxin complexes. In this study, Plutella xylostella larvae were orally administered P. luminescens for bioassay. After incubation in Luria-Bertani (LB) medium for a sufficiently long period, the mortality rates of P. xylostella observed after diluting the fermentation broth 50 times and diluting the supernatant 5 times were 18.89% and 91.11%, respectively. Retentates measuring more than 70kDa showed 88% mortality after ultrafiltration (UF) membrane treatment. Thus, the supernatant of P. luminescens had insecticidal activity, and the main insecticidal toxin complexes had a molecular weight exceeding 70kDa. The L9 (34) Taguchi orthogonal experimental optimized medium mode-predicted insecticidal activity levels were 84% and 119% in the 50-fold diluted fermentation broth and 5-fold diluted supernatant, respectively. Moreover, the insecticidal activity was improved to 92.2% in the 100-fold diluted fermentation broth and to 97.8% in the 10-fold diluted supernatant in the experiments. All combinations tested showed clear indications of lethality, including swelling, vesicle formation, cytoplasm vacuolization, and brush border membrane lysis. Thus, these results promote the use of P. luminescens 0805-P2R as a potent biopesticide to effectively control P. xylostella.

Dominative role of MMP-14 over MMP-15 in human urinary bladder carcinoma on the basis of its enhanced specific activity.

Background:Human urinary bladder cancer is one of the most common cancers worldwide with the mortality rate of approximately 165,000 people annually. The modulation of extracellular matrix is a crucial event in the metastatic spread, among others in angiogenesis. It is initiated and prolonged by the cascade of matrix metalloproteinases. MMP-14 and MMP-15 are associated with a high degree of malignancy, aggressiveness, and survival prognosis by the activation of other matrix metalloproteinases (MMPs). This study was aimed at evaluating the expression and the activity of selected transmembrane metalloproteinases at different stages of human urinary bladder cancer.Methods:Western blot and enzyme linked immunosorbent assay (ELISA) method were used to evaluate the expression and content of MMPs and TIMP-1. The activity of studied enzymes was determined with fluorometric method.Results:Both transmembrane metalloproteinases are found in healthy or cancerous tissue in high molecular complexes of human urinary bladder. MMP-14 dominates over MMP-15, particularly in high-grade urinary bladder cancer. Their contents significantly change with the grade of bladder tumor. The amount of MMP-14 increases with increasing grade of tumor. MMP-15 content decreases in high-grade bladder cancer. With increasing grade of urinary bladder cancer their actual activity (per kg of total protein content) is varying in different ways. In all examined tissues, the specific activity of MMP-15 (per kg of the enzyme content) is much higher in comparison to MMP-14. Human urinary bladder cancer contains higher TIMP-1 amounts than control tissue but with the decrease with an increase in tumor grade.Conclusion:Comparison of investigated enzymes’ activity and the inhibitor content suggests it opposite effects, higher suppression of MMP-14 than MMP-15 activity in low-grade bladder cancer and reverse TIMP-1 action in high-grade cancer. The MMP-14 activity determination in urinary bladder cancer tissue may be used as a predictor of a risk of metastasis.

Enzymatic Oxidation of Lignin: Challenges and Barriers Toward Practical Applications

AbstractLignocellulosic biomass represents perhaps the most abundant renewable resource with a potential to replace fossil‐based feedstock for sustainable energy, chemical and materials production. Among the three major lignocellulosic biomass components (i. e. cellulose, hemicellulose and lignin), lignin is a macromolecule with an aromatic skeleton with a variety of functional groups (e. g. hydroxyl, methoxy, carbonyl, double bond) and carries a higher energy density. The unique structure makes lignin an intriguing substrate for energy, chemicals and materials productions. However, the high molecular weight and complex macromolecular structure have made lignin a challenging substrate to be transformed by many conversion methods. Microbial enzyme degradation and modification of lignin have been subjected to a significant amount research in the last a few decades. Yet so far little success has been demonstrated to merit the use of enzymatic technology for lignin transformation at a commercial scale. This paper provides an updated review of the development of lignin degrading/modifying enzymes with an emphasis on identifying the key barriers and challenges toward practical applications of microbial enzymes for lignin valorization with a hope to generate new insights and direction that can overcome these challenges.

GLIPR1L1 is an IZUMO-binding protein required for optimal fertilization in the mouse

BackgroundThe sperm protein IZUMO1 (Izumo sperm-egg fusion 1) and its recently identified binding partner on the oolemma, IZUMO1R, are among the first ligand-receptor pairs shown to be essential for gamete recognition and adhesion. However, the IZUMO1-IZUMO1R interaction does not appear to be directly responsible for promoting the fusion of the gamete membranes, suggesting that this critical phase of the fertilization cascade requires the concerted action of alternative fusogenic machinery. It has therefore been proposed that IZUMO1 may play a secondary role in the organization and/or stabilization of higher-order heteromeric complexes in spermatozoa that are required for membrane fusion.ResultsHere, we show that fertilization-competent (acrosome reacted) mouse spermatozoa harbor several high molecular weight protein complexes, a subset of which are readily able to adhere to solubilized oolemmal proteins. At least two of these complexes contain IZUMO1 in partnership with GLI pathogenesis-related 1 like 1 (GLIPR1L1). This interaction is associated with lipid rafts and is dynamically remodeled upon the induction of acrosomal exocytosis in preparation for sperm adhesion to the oolemma. Accordingly, the selective ablation of GLIPR1L1 leads to compromised sperm function characterized by a reduced ability to undergo the acrosome reaction and a failure of IZUMO1 redistribution.ConclusionsCollectively, this study characterizes multimeric protein complexes on the sperm surface and identifies GLIPRL1L1 as a physiologically relevant regulator of IZUMO1 function and the fertilization process.

Thioredoxin interacting protein (Txnip) forms redox sensitive high molecular weight nucleoprotein complexes

Thioredoxin interacting protein (Txnip) is an α-arrestin protein that regulates pleiotropic biological responses. Txnip acts as a cancer suppressor and is a critical regulator of energy metabolism. To investigate molecular mechanisms involving Txnip, we searched for its protein binding partners using tandem affinity purification and proteomics analyses and identified several viable candidates, including HSP90, HSP70, and Prp31. We showed, by native PAGE, that Txnip is involved in the formation of high molecular weight complexes (1000–1300 kDa) in the nuclear fraction of cells treated with glucose and bortezomib. DTT treatment partly dissolved these high molecular weight complexes, suggesting that Txnip forms redox sensitive high-order nucleoprotein complexes. RNAse treatment slightly decreased the complex and RNA-seq showed differential expression of RNAs in the complex between Txnip protein overexpressing and control cells, indicating the involvement of RNAs in the complex. These results collectively provide a model whereby Txnip exerts its functions through multiple binding partners, forming transient higher-order complexes to regulate other signaling molecules.

AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading.

RNA interference (RNAi) is mediated by small, 20-24-nt-long, non-coding regulatory (s)RNAs such as micro (mi) and small interfering (si) RNAs via the action of ARGONAUTE (AGO) proteins. High-throughput sequencing of size-separated sRNA pools of plant crude extracts revealed that the majority of the canonical miRNAs were associated with high molecular weight RNA-induced silencing complexes co-migrating with AGO1 (HMW RISC). In contrast, the majority of 24-nt-long siRNAs were found in association with low molecular weight complexes co-migrating with AGO4 (LMW RISC). Intriguingly, we identified a large set of cytoplasmic sRNAs, including mature miRNA sequences, in the low molecular size range corresponding to protein-unbound sRNAs. By comparing the RISC-loaded and protein-unbound pools of miRNAs, we identified miRNAs with highly different loading efficiencies. Expression of selected miRNAs in transient and transgenic systems validated their altered loading abilities implying that this process is controlled by information associated with the diverse miRNA precursors. We also showed that the availability of AGO proteins is a limiting factor determining the loading efficiency of miRNAs. Our data reveal the existence of a regulatory checkpoint determining the RISC-loading efficiencies of various miRNAs by sorting only a subset of the produced miRNAs into the biologically active RISCs.

PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity.

Regulated proinsulin biosynthesis, disulfide bond formation and ER redox homeostasis are essential to prevent Type two diabetes. In ß cells, protein disulfide isomerase A1 (PDIA1/P4HB), the most abundant ER oxidoreductase of over 17 members, can interact with proinsulin to influence disulfide maturation. Here we find Pdia1 is required for optimal insulin production under metabolic stress in vivo. ß cell-specific Pdia1 deletion in young high-fat diet fed mice or aged mice exacerbated glucose intolerance with inadequate insulinemia and increased the proinsulin/insulin ratio in both serum and islets compared to wildtype mice. Ultrastructural abnormalities in Pdia1-null ß cells include diminished insulin granule content, ER vesiculation and distention, mitochondrial swelling and nuclear condensation. Furthermore, Pdia1 deletion increased accumulation of disulfide-linked high molecular weight proinsulin complexes and islet vulnerability to oxidative stress. These findings demonstrate that PDIA1 contributes to oxidative maturation of proinsulin in the ER to support insulin production and ß cell health.

Defective endoplasmic reticulum export causes proinsulin misfolding in pancreatic β cells

Endoplasmic reticulum (ER) homeostasis is essential for cell function. Increasing evidence indicates that, efficient protein ER export is important for ER homeostasis. However, the consequence of impaired ER export remains largely unknown. Herein, we found that defective ER protein transport caused by either Sar1 mutants or brefeldin A impaired proinsulin oxidative folding in the ER of β-cells. Misfolded proinsulin formed aberrant disulfide-linked dimers and high molecular weight proinsulin complexes, and induced ER stress. Limiting proinsulin load to the ER alleviated ER stress, indicating that misfolded proinsulin is a direct cause of ER stress. This study revealed significance of efficient ER export in maintaining ER protein homeostasis and native folding of proinsulin. Given the fact that proinsulin misfolding plays an important role in diabetes, this study suggests that enhancing ER export may be a potential therapeutic target to prevent/delay β-cell failure caused by proinsulin misfolding and ER stress.

Suppressed Expression but Not Activity of Collagenases MMP-1 and MMP-13 in Human Renal Carcinoma

Background: Collagenases are enzymes starting collagen degradation. The role of collagenases in renal carcinoma development is not well understood. Objective: Evaluation of collagen content and collagenase expression and activity in human kidney cancers. Methods: Collagen content was measured by the hydroxyproline assay. The expression and the content of collagenases were evaluated by Western blotting and ELISA. Fluorogenic substrate was used to measure enzyme activity. Results: Collagen content significantly decreases with the progression of kidney cancer. Both collagenases are first present in high molecular complexes in both control and cancer tissue. The healthy part of the kidney contains similar amounts of both collagenases. Collagenase content decreased significantly in tumor tissue with increasing cancer stage. MMP-13 activity is much higher than that of MMP-1 in all tissues investigated. We observed increasing collagenase activity (MMP-1 and MMP-13) with increasing renal cancer grade. Conclusions: The lower content and higher activity of the collagenases investigated in cancer tissue indicate that most of these enzymes are in active form in renal carcinoma. The lower collagen content in cancer tissue can be explained at least in part by increased collagenase activity.

Oscillatory shear potentiates latent TGF-\u03b21 activation more than steady shear as demonstrated by a novel force generator

Cardiovascular mechanical stresses trigger physiological and pathological cellular reactions including secretion of Transforming Growth Factor β1 ubiquitously in a latent form (LTGF-β1). While complex shear stresses can activate LTGF-β1, the mechanisms underlying LTGF-β1 activation remain unclear. We hypothesized that different types of shear stress differentially activate LTGF-β1. We designed a custom-built cone-and-plate device to generate steady shear (SS) forces, which are physiologic, or oscillatory shear (OSS) forces characteristic of pathologic states, by abruptly changing rotation directions. We then measured LTGF-β1 activation in platelet releasates. We modeled and measured flow profile changes between SS and OSS by computational fluid dynamics (CFD) simulations. We found a spike in shear rate during abrupt changes in rotation direction. OSS activated TGF-β1 levels significantly more than SS at all shear rates. OSS altered oxidation of free thiols to form more high molecular weight protein complex(es) than SS, a potential mechanism of shear-dependent LTGF-β1 activation. Increasing viscosity in platelet releasates produced higher shear stress and higher LTGF-β1 activation. OSS-generated active TGF-β1 stimulated higher pSmad2 signaling and endothelial to mesenchymal transition (EndoMT)-related genes PAI-1, collagen, and periostin expression in endothelial cells. Overall, our data suggest variable TGF-β1 activation and signaling occurs with competing blood flow patterns in the vasculature to generate complex shear stress, which activates higher levels of TGF-β1 to drive vascular remodeling.