A Novel Role for Reactive Oxygen Species in the Regulation of RhoA: Implications for Endothelial Permeability and Leukocyte Transmigration

The endothelial lining of the vasculature plays a critical role in regulating the passage of fluid, macromolecules, and cells between the blood and surrounding tissues. Vascular permeability is tightly regulated and is modulated during both physiological and pathological situations. Our laboratory is interested in the mechanisms which regulate vascular permeability and the transmigration of leukocytes during inflammation. The migration of leukocytes across the endothelial barrier is called "leukocyte transendothelial migration" (TEM). Deciphering the mechanisms which regulate TEM is important to understanding and managing inflammatory diseases. My interest in TEM is focused on pathways which involve small GTPases. My specific interest is in the role of Rho GTPases and their regulation by reactive oxygen species (ROS) during leukocyte TEM. Although ROS have been largely seen as mediators of oxidative damage, more recently, ROS have been recognized as necessary components of cell signaling pathways. Importantly, ROS have been shown to play an important role in regulating vascular permeability and TEM. In this dissertation, I show that physiological levels of ROS can directly activate RhoA in cells. In vitro studies had previously identified two critical cysteine residues in the nucleotide binding pocket of RhoA that are oxidatively modified by ROS. My work showed that this oxidative regulation of RhoA can occur in a cellular context. Before these studies, the regulation of small GTPases had almost exclusively focused on regulatory proteins. Importantly, my work identifies direct oxidative modification as a novel way to regulate RhoA activity. I extended this work by investigating the regulation of RhoA by ROS in the context of leukocyte TEM. Based on these findings and my previous work, I hypothesized that leukocyte adhesion to endothelial cells initiates ROS generation and the direct activation of RhoA to promote leukocyte TEM. In preliminary studies, I found that crosslinking of a cell adhesion molecule (ICAM- 1) stimulates RhoA and Rac1 activation. In addition the activation of RhoA appears to be dependent on ROS, as seen by studies of redox-insensitive mutants. The work presented in this dissertation lays the framework for future studies on the role of ROS during leukocyte adhesion and TEM

Direct Activation of RhoA by Reactive Oxygen Species Requires a Redox-Sensitive Motif

BACKGROUND:Rho family GTPases are critical regulators of the cytoskeleton and affect cell migration, cell-cell adhesion, and cell-matrix adhesion. As with all GTPases, their activity is determined by their guanine nucleotide-bound state. Understanding how Rho proteins are activated and inactivated has largely focused on regulatory proteins such as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). However, recent in vitro studies have indicated that GTPases may also be directly regulated by redox agents. We hypothesized that this redox-based mechanism occurs in cells and affects cytoskeletal dynamics, and in this report we conclude this is indeed a novel mechanism of regulating the GTPase RhoA. METHODOLOGY/PRINCIPAL FINDINGS:In this report, we show that RhoA can be directly activated by reactive oxygen species (ROS) in cells, and that this requires two critical cysteine residues located in a unique redox-sensitive motif within the phosphoryl binding loop. First, we show that ROS can reversibly activate RhoA and induce stress fiber formation, a well characterized readout of RhoA activity. To determine the role of cysteine residues in this mechanism of regulation, we generated cysteine to alanine RhoA mutants. Mutation of these cysteines abolishes ROS-mediated activation and stress fiber formation, indicating that these residues are critical for redox-regulation of RhoA. Importantly, these mutants maintain the ability to be activated by GEFs. CONCLUSIONS/SIGNIFICANCE:Our findings identify a novel mechanism for the regulation of RhoA in cells by ROS, which is independent of classical regulatory proteins. This mechanism of regulation may be particularly relevant in pathological conditions where ROS are generated and the cellular redox-balance altered, such as in asthma and ischemia-reperfusion injury

Redox Regulation of Ras and Rho GTPases: Mechanism and Function

Significance: Oxidation and reduction events are critical to physiological and pathological processes and are highly regulated. Herein, we present evidence for the role of Ras and Rho GTPases in controlling these events and the unique underlying mechanisms. Evidence for redox regulation of Ras GTPases that contain a redox-sensitive cysteine (X) in the conserved NKXD motif is presented, and a growing consensus supports regulation by a thiyl radical-mediated oxidation mechanism. We also discuss the debate within the literature regarding whether 2e− oxidation mechanisms also regulate Ras GTPase activity. Recent Advances: We examine the increasing in vitro and cell-based data supporting oxidant-mediated activation of Rho GTPases that contain a redox-sensitive cysteine at the end of the conserved phosphoryl-binding loop (p-loop) motif (GXXXXG[S/T]C). While this motif is distinct from Ras, these data suggest a similar 1e− oxidation-mediated activation mechanism. Critical Issues: We also review the data showing that the unique p-loop placement of the redox-sensitive cysteine in Rho GTPases supports activation by 2e− cysteine oxidation. Finally, we examine the role that Ras and Rho GTPases play in controlling key oxidant-regulating enzymes in the cell, and we speculate on a feedback mechanism. Future Directions: Given that these GTPases and redox-regulating enzymes are involved in multiple physiological and pathological processes, we discuss future experiments that may clarify the interplay between them. Antioxid. Redox Signal. 18, 250–258

Levels of Blood Biomarkers among Patients with Myocardial Infarction in Comparison to Control Group

BACKGROUND: Myocardial infarction (MI) as a term for a heart attack happens due to reduced blood flow to heart myocardium and lack of oxygen supply caused by plaques inthe interior walls of coronary arteries. With respect to the importance of MI etiology, we aimed to study the relationship of MI and blood examination variables.METHODS: This study was conducted in Mazandaran Heart Center as a hospital-based case-control Comprising 894 participants including 465 cases and 429 controls, individually matched by sex and age. Considered blood markers were analyzed using routine laboratory methods and equipment.RESULTS: Of all participants, 64.3% of the cases and 51.0% of the controls were males with a mean age of 61.2 (±13.8) in cases and 62.4 (±14.) in controls. We could not find any differences between cases and controls for total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and alkaline-phosphatase (ALP) (P>0.05). However, levels of creatine-kinase-muscle/brain (CK-MB) (P<0.0001), fasting-blood-sugar (FBS) (P<0.0001), aspartateaminotransferase (AST) (P<0.0001), alanine-transferase (ALT) (P<0.0001) and erythrocyte sedimentation rate (ESR) (P=0.001) were significantly higher in cases compared to the controls (P<0.05). Multivariable analyses revealed that the risk of MI was associated with high levels of AST (adjusted OR=24.3, 95%CI=3.5±165.6, P=0.001) and LDL (adjusted OR=7.4, 95%CI=1.0±51.8, P=0.001).CONCLUSION: Our investigation indicated that the levels of CK-MB, FBS, AST, ALT and ESR were significantly higher in patients with MI. Besides, our findings showed that the risk of MI in cases with high levels of AST and LDL was about 24 and 7 times more than the control group respectively

Porphyromonas gingivalis–dendritic cell interactions: consequences for coronary artery disease

An estimated 80 million US adults have one or more types of cardiovascular diseases. Atherosclerosis is the single most important contributor to cardiovascular diseases; however, only 50% of atherosclerosis patients have currently identified risk factors. Chronic periodontitis, a common inflammatory disease, is linked to an increased cardiovascular risk. Dendritic cells (DCs) are potent antigen presenting cells that infiltrate arterial walls and may destabilize atherosclerotic plaques in cardiovascular disease. While the source of these DCs in atherosclerotic plaques is presently unclear, we propose that dermal DCs from peripheral inflamed sites such as CP tissues are a potential source. This review will examine the role of the opportunistic oral pathogen Porphyromonas gingivalis in invading DCs and stimulating their mobilization and misdirection through the bloodstream. Based on our published observations, combined with some new data, as well as a focused review of the literature we will propose a model for how P. gingivalis may exploit DCs to gain access to systemic circulation and contribute to coronary artery disease. Our published evidence supports a significant role for P. gingivalis in subverting normal DC function, promoting a semimature, highly migratory, and immunosuppressive DC phenotype that contributes to the inflammatory development of atherosclerosis and, eventually, plaque rupture

Redox Regulation of Ras and Rho GTPases: Mechanism and Function

Significance: Oxidation and reduction events are critical to physiological and pathological processes and are highly regulated. Herein, we present evidence for the role of Ras and Rho GTPases in controlling these events and the unique underlying mechanisms. Evidence for redox regulation of Ras GTPases that contain a redox-sensitive cysteine (X) in the conserved NKXD motif is presented, and a growing consensus supports regulation by a thiyl radical-mediated oxidation mechanism. We also discuss the debate within the literature regarding whether 2e(−) oxidation mechanisms also regulate Ras GTPase activity. Recent Advances: We examine the increasing in vitro and cell-based data supporting oxidant-mediated activation of Rho GTPases that contain a redox-sensitive cysteine at the end of the conserved phosphoryl-binding loop (p-loop) motif (GXXXXG[S/T]C). While this motif is distinct from Ras, these data suggest a similar 1e(−) oxidation-mediated activation mechanism. Critical Issues: We also review the data showing that the unique p-loop placement of the redox-sensitive cysteine in Rho GTPases supports activation by 2e(−) cysteine oxidation. Finally, we examine the role that Ras and Rho GTPases play in controlling key oxidant-regulating enzymes in the cell, and we speculate on a feedback mechanism. Future Directions: Given that these GTPases and redox-regulating enzymes are involved in multiple physiological and pathological processes, we discuss future experiments that may clarify the interplay between them. Antioxid. Redox Signal. 18, 250–258

Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Using 3D-Printed PDLLA/ β-TCP Nanocomposite Scaffolds

Designing bone scaffolds containing both organic and inorganic composites simulating the architecture of the bone is the most important principle in bone tissue engineering. The objective of this study was to fabricate a composite scaffold containing poly (D, l)-lactide (PDLLA) and β-tricalcium phosphate (β-TCP) as a platform for osteogenic differentiation of adipose-derived mesenchymal stem cells. In this study, PDLLA/β-TCP scaffolds were fabricated using three-dimensional printing (3D) technology through melt excursion technique. The physicomechanical characteristics, including microstructure, mechanical properties, of the customized scaffolds were investigated. Further, the in vitro biological characteristics of manufactured scaffolds were evaluated in conjugation with buccal fat pad derived mesenchymal stem cells in terms of cell attachment, viability, proliferation, and osteogenic differentiation capacity. The 3D printed customized scaffold in this study showed proper pore size, porosity, mechanical strength, material composition, biocompatibility, and osteogenic differentiation capacity. The obtained results converge to reveal the promising features of the nanocomposite 3D printed platform for personalized bone tissue engineering

A 3D Nanostructured Calcium-Aluminum-Silicate Scaffold with Hierarchical Meso-Macroporosity for Bone Tissue Regeneration: Fabrication, Sintering Behavior, Surface Modification and in Vitro Studies

This is a comprehensive study reporting the fabrication of highly porous Gehlenite scaffold (Ca2Al2SiO7)—both with and without surface modification—for the first time. The sintering temperature of Gehlenite scaffolds was optimized. Next, the optimized Gehlenite scaffold was coated by polycaprolactone (PCL)-Forsterite (Mg2SiO4) nanocomposite to improve the scaffold’s brittleness and biological properties. 1375 °C was found to be the optimized sintering temperature by which the Gehlenite scaffold was consolidated. Different PCL and Forsterite concentrations were separately applied on the optimized scaffold to yield a complete nanocomposite coating without clogging the macroporous structure. The bioactivity, degradation rate, cell viability, attachment and proliferation of three different scaffolds—non-coated (sintered at 1375 °C), PCL-coated and PCL/Forsterite nanocomposite-coated—were scrutinized and compared to each other in vitro. Based on our results, it is concluded that the PCL-Forsterite nanocomposite-coated scaffold with desired physical, chemical and biological-related properties has a great potential for bone tissue regeneration