Computational Model of MicroRNA Control of HIF-VEGF Pathway: Insights into the Pathophysiology of Ischemic Vascular Disease and Cancer

<div><p>HRMs (hypoxia-responsive miRNAs) are a specific group of microRNAs that are regulated by hypoxia. Recent studies revealed that several HRMs including let-7 family miRNAs were highly induced in response to HIF (hypoxia-inducible factor) stabilization in hypoxia, and they potently participated in angiogenesis by targeting AGO1 (argonaute 1) and upregulating VEGF (vascular endothelial growth factor). Here we constructed a novel computational model of microRNA control of HIF-VEGF pathway in endothelial cells to quantitatively investigate the role of HRMs in modulating the cellular adaptation to hypoxia. The model parameters were optimized and the simulations based on these parameters were validated against several published <i>in vitro</i> experimental data. To advance the mechanistic understanding of oxygen sensing in hypoxia, we demonstrated that the rate of HIF-1α nuclear import substantially influences its stabilization and the formation of HIF-1 transcription factor complex. We described the biological feedback loops involving let-7 and AGO1 in which the impact of external perturbations were minimized; as a pair of master regulators when low oxygen tension was sensed, they coordinated the critical process of VEGF desuppression in a controlled manner. Prompted by the model-motivated discoveries, we proposed and assessed novel pathway-specific therapeutics that modulate angiogenesis by adjusting VEGF synthesis in tumor and ischemic cardiovascular disease. Through simulations that capture the complex interactions between miRNAs and miRNA-processing molecules, this model explores an innovative perspective about the distinctive yet integrated roles of different miRNAs in angiogenesis, and it will help future research to elucidate the dysregulated miRNA profiles found in cancer and various cardiovascular diseases.</p></div

Development of a Bimetallic Pd-Ni/HZSM‑5 Catalyst for the Tandem Limonene Dehydrogenation and Fatty Acid Deoxygenation to Alkanes and Arenes for Use as Biojet Fuel

A tandem process involving the dehydroaromatization of the terpene limonene and the hydrodeoxygenation of stearic acid has been found to be efficiently catalyzed by Pd-Ni/HZSM-5. The process involves the generation of <i>p</i>-cymene from terpene with concomitant formation of H₂, which leads to the one-pot hydrodeoxygenation of stearic acid to C₁₇ and C₁₈ alkanes; these products can be used as kerosene additives for aviation fuel. Screening a wide range of catalysts, the bimetallic Pd-Ni/HZSM-5 catalyst is the most efficient, leading to quantitative conversion of stearic acid to alkanes in limonene at 280 °C at a H₂ pressure of 2 bar after 120 min. It has been found that single Ni or Pd catalysts lead to a poor conversion of stearic acid in limonene at a H₂ pressure of 2 bar. The combination of physically mixed Pd- and Ni-sites onto different supports (Pd/HZSM-5 or Pd/C, and Ni/HZSM-5, Ni/HY, or Ni/HBEA) leads to catalysts which show satisfactory conversion to <i>p</i>-cymene but generally have very low stearic acid conversion rates. Directly incorporating Pd and Ni onto the HZSM-5 scaffold forms the Pd-Ni/HZSM-5 bimetallic catalyst, which demonstrates a remarkable improvement in stearic acid conversion to C₁₇ and C₁₈ alkane products. In this catalyst system, Pd is shown to be the active site for limonene dehydroaromatization, while Ni catalyzes the separate stearic acid hydrodeoxygenation. The acidity of HZSM-5 (modified by the Si/Al ratios) influences the performance of the Pd-Ni bimetallic catalyst, and the proper pore size of HZSM-5 prevents side-reactions from limonene condensation. In addition, the alloyed Pd-Ni nanoparticles (optimized with higher Pd/Ni ratios) on the external surface of HZSM-5 enhance internal H^• transfer between the two metals, thereby increasing the rate of stearic acid hydrodeoxygenation. The catalytic compatibility of the Pd and Ni sites, coupled with the proper pore sizes and optimized level of Brönsted acid sites in HZSM-5, result in the design of a multifunctional catalyst that is efficient for both steps of the cascade reaction. Hence, a bimetallic 5%Pd-10%Ni/HZSM-5 catalyst has been developed that allows for a simple approach for producing aromatics and hydrocarbon components present in biojet fuel derived from two biomass resources

Let-7 represses Dicer and AGO1 that both limit miR-15a expression in hypoxia.

<p>(A) Overexpression of AGO1 mRNA by 0.01, 0.04 and 0.08 μM in hypoxia leads to short-term rises in the expression levels of total miR-15a (B) by promoting the association of free form miR-15a with AGO1 to make more miR-15a RISCs. (C) Dicer processing is a limiting step in the production of miR-15a in hypoxia. Introducing both Dicer and AGO1 mRNAs at the beginning of simulation results in elevated miR-15a abundance compared to adding AGO1 mRNA alone. (D) When let-7 no longer inhibits Dicer translation, an overexpression in Dicer mRNA generates a remarkable change in the expression profiles of Dicer with respect to the control situation. With let-7 mediated Dicer silencing, the response of Dicer mRNA overexpression is significantly attenuated. (E) Relative expression of non-translatable VEGF mRNA associated with miR-15a RISC and (F) translatable VEGF mRNA in response to different treatment strategies. Hypoxia causes an initial increase in the binding between VEGF mRNA and miR-15a RISC because of the rapid HIF-1-activated VEGF transcription, but the impact of AGO1 silencing becomes dominated later that, in the long run, miR-15a-bound VEGF is reduced compared to the normoxic level. In hypoxia, enforced let-7 overexpression or AGO1 silencing modestly increases the amount of translatable VEGF, while let-7 antagonists or AGO1 overexpression can remarkably blunt VEGF production.</p

HIF-1α synthesis in hypoxia.

<p>HIF-1α level at time zero is the steady state normoxic (21% O₂) level. (A) Total HIF-1α expression profiles are highly sensitive to oxygen availability. (B) When PHD2 initial concentration is in excess, an oxygen-dependent, switch-like behavior in the amount of hydroxylated HIF-1α is observed. As simulation span increases, a steep reduction in HIF-1α hydroxylation occurs between 2% to 4% O₂. (C) TTP is responsible for the delayed drop (initial overshoot) in the induction of HIF-1α in hypoxia. By increasing the dose of a simulated siRNA that silences TTP expression (assuming siRNA binds TTP mRNA potently with a K_d of 1 nM), the duration of the initial overshoot is lengthened. (D) Varying the rate of HIF-1α import from cytoplasm into nucleus (k_forward) affects the overall HIF-1α profile in hypoxia. (E) Larger k_forward values of HIF-1α nuclear import result in higher levels of HIF-1 transcription factor complex formed. (F) Smaller k_forward values lead to lower total HIF-1α levels in normoxia and in hypoxia, while the majority of induced HIF-1α is located only in the cytoplasm and unable to form transcription complex with HIF-1β. (D-F) Magnitude of k_forward is set to 10%, 50%, 200% and 500% of its original value respectively in the comparisons. For each k_forward value, steady state levels of all species, after the model is simulated in normoxia for a long time span, are collected and considered a new set of initial conditions.</p

Proposed model scheme of the miR control of HIF-VEGF pathway.

<p>HIF-1α is stabilized in hypoxia and the HIF-1 dimer complex transcriptionally induces let-7 production. Mature let-7 represses AGO1 and leads to a global desuppression of VEGF. Model components in the colored backgrounds correspond to the four modules: blue/O₂ sensing, pink/VEGF repression by miR-15a, orange/HIF-dependent transcription, green/let-7 biogenesis and targeting. Species whose names end with an N subscript are located inside the nucleus; reactions that point to red signs indicate degradation. The symbols v# refer to the 57 chemical reactions listed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004612#pcbi.1004612.s001" target="_blank">S1 Table</a>.</p

Translational repression of VEGF in normoxia and let-7 mediated VEGF desuppression in hypoxia in ECs.

<p>(A) VEGF mRNA were targeted by miRISC and inaccessible for translation in normoxia. (B) HIF-1 proteins that were stabilized in hypoxia induced let-7 biogenesis, which led to the downregulation of AGO1 mRNA, protein and miRISC formation. VEGF mRNA were desuppressed and ready for translation because of reduced miRISC activities.</p

Sensitivity analysis of key species in the pathway.

<p>Sensitivity of (A) cytoplasmic HIF1-α, (B) free form AGO1, (C) free form let-7, and (D) VEGF to variations in different sets of kinetic parameters (direct production and degradation rates excluded). (A-D) Kf(X/Y) stands for the forward reaction constant of species X binding species Y; Vm(X) stands for the speed of reaction X; Kf(X) stands for the forward rate constant of species X dissociation. Detailed description of each parameter is available in the supplemental information. (E) At different O₂ levels, TTP mRNA overexpression is tested as an anti-angiogenic therapy <i>in silico</i> compared to miR-based therapeutic strategies. (F) Affinity of O₂ binding with PHD2 or FIH and HIF-1α translocation rate contribute to the trend of HIF-1α stabilization in hypoxia. (G) Relative downregulation of AGO1 in hypoxia is influenced by its binding with let-7. (F-G) Parameters are set to 500% of their original values in the comparisons. For each new value, steady state levels of all species, after the model is simulated in normoxia for a long time span, are collected and considered a new set of initial conditions. HIF-1α and AGO1 levels in hypoxia are normalized with respect to their concentrations at time zero (normoxic steady states).</p

Let-7 and AGO1 mutually control each other in hypoxia.

<p>(A) Varying the cellular AGO1 abundance by antagonizing or overexpressing its mRNA changes the let-7 availability. (B) Let-7s that are in association with AGO1 are less prone to degradation, so a decrease in the binding strength of let-7 toward AGO1 causes more let-7 to be degraded. Consequently, (C) let-7-mediated activity including AGO1 repression is downregulated, allowing additional AGO1 protein synthesis. (B-C) Association rate of AGO1 and let-7 (k_forward) is adjusted to 10%, 20%, and 50% of its original value respectively in the comparisons. (D) AGO1 overexpression leads to an early upstroke in its time course profile but its steady state level changes insignificantly. (E) After 4 hours, almost all the additional AGO1 mRNAs (e.g. 0.01 μM and 0.04 μM) being introduced in the beginning are fully shuttled into the p-body to become inaccessible for translation. (F) In hypoxia, VEGF mRNA released from miRISC, in combination with HIF induction, boosts the pool of free form VEGF mRNA. A simulated AGO1 overexpression rescues the drop in miRISC level and drives free form VEGF mRNA back into miR-mediated repression. (A, D-F) The model assumes that in AGO1 silencing, siRNA binds AGO1 mRNA potently with a K_d of 1 nM; AGO1 overexpression is simplified as an increase of certain amount in the initial concentration of AGO1 mRNA.</p

Nontraditional Approaches To Enable High-Energy and Long-Life Lithium–Sulfur Batteries

ConspectusLithium–sulfur (Li–S) batteries are promising for automotive applications due to their high theoretical energy density (2600 Wh/kg). In addition, the natural abundance of sulfur could mitigate the global raw material supply chain challenge of commercial lithium-ion batteries that use critical elements, such as nickel and cobalt. However, due to persistent polysulfide shuttling and uncontrolled lithium dendrite growth, Li–S batteries using nonencapsulated sulfur cathodes and conventional ether-based electrolytes suffer from rapid cell degradation upon cycling. Despite significant improvements in recent decades, there is still a big gap between lab research and commercialization of the technology. To date, the reported cell energy densities and cycling life of practical Li–S pouch cells remain largely unsatisfactory.Traditional approaches to improving Li–S performance are primarily focused on confining polysulfides using electronically conductive hosts. However, these micro- and mesoporous hosts suffer from limited pore volume to accommodate high sulfur loading and the associated volume change during cycling. Moreover, they fail to balance adsorption–conversion of polysulfides during charge–discharge, leading to the formation of massive dead sulfur. Such hosts are themselves electrochemically inactive, which decreases the practical energy density. In contrast, a series of nontraditional approaches, paired with advances in multiscale mechanistic understanding, have recently demonstrated exciting performance outcomes not only in conventional coin cells but also in practical pouch cells.In this Account, we first introduce our novel cathode design strategies to overcome polysulfide shuttling and sluggish redox kinetics in thick S cathodes via selenium–sulfur chemistry and cathode host engineering. Next, we gain a mechanistic understanding of Li–S batteries in various types of electrolytes via a series of spectroscopic, nuclear magnetic resonance, and electrochemical methods. Meanwhile, a novel cathode solid electrolyte interphase encapsulation strategy via nonviscous highly fluorinated ether-based electrolyte is introduced. The established selection rule by investigating how solvating power retards the shuttle effect and induces robust cathode/solid-electrolyte interphase formation is also included. We then discuss how the synergistic interactions between rational cathode structures and electrolytes can be exploited to tailor the reaction pathways and kinetics of S cathodes under high mass loading and lean electrolyte conditions. In addition, a novel interlayer design to simultaneously overcome degradation processes (polysulfide shuttling and lithium dendrite formation) and accelerate redox reaction kinetics is presented. Finally, this Account concludes with an overview of the challenges and strategies to develop Li–S pouch cells with high practical energy density, long cycle life, and fast-charging capability

Ni-Catalyzed Cleavage of Aryl Ethers in the Aqueous Phase

A novel Ni/SiO₂-catalyzed route for selective cleavage of ether bonds of (lignin-derived) aromatic ethers and hydrogenation of the oxygen-containing intermediates at 120 °C in presence of 6 bar H₂ in the aqueous phase is reported. The C–O bonds of α-O-4 and β-O-4 linkages are cleaved by hydrogenolysis on Ni, while the C–O bond of the 4-O-5 linkage is cleaved via parallel hydrogenolysis and hydrolysis. The difference is attributed to the fact that the C_aliphatic–OH fragments generated from hydrolysis of α-O-4 and β-O-4 linkages can undergo further hydrogenolysis, while phenol (produced by hydrolysis of the 4-O-5 linkage) is hydrogenated to produce cyclohexanol under conditions investigated. The apparent activation energies, <i>E</i>_a(α-O-4) < <i>E</i>_a(β-O-4) < <i>E</i>_a(4-O-5), vary proportionally with the bond dissociation energies. In the conversion of β-O-4 and 4-O-5 ether bonds, C–O bond cleavage is the rate-determining step, with the reactants competing with hydrogen for active sites, leading to a maximum reaction rate as a function of the H₂ pressure. For the very fast C–O bond cleavage of the α-O-4 linkage, increasing the H₂ pressure increases the rate-determining product desorption under the conditions tested