Melanoma is the cancer with the highest increase in incidence, and transformation of radial growth to vertical growth (i.e., noninvasive to invasive) melanoma is required for invasive disease and metastasis. We have previously shown that p42/p44 MAP kinase is activated in radial growth melanoma, suggesting that further signaling events are required for vertical growth melanoma. The molecular events that accompany this transformation are not well understood. Akt, a signaling molecule downstream of PI3K, was introduced into the radial growth WM35 melanoma in order to test whether Akt overexpression is sufficient to accomplish this transformation. Overexpression of Akt led to upregulation of VEGF, increased production of superoxide ROS, and the switch to a more pronounced glycolytic metabolism. Subcutaneous implantation of WM35 cells overexpressing Akt led to rapidly growing tumors in vivo, while vector control cells did not form tumors. We demonstrated that Akt was associated with malignant transformation of melanoma through at least 2 mechanisms. First, Akt may stabilize cells with extensive mitochondrial DNA mutation, which can generate ROS. Second, Akt can induce expression of the ROS-generating enzyme NOX4. Akt thus serves as a molecular switch that increases angiogenesis and the generation of superoxide, fostering more aggressive tumor behavior. Targeting Akt and ROS may be of therapeutic importance in treatment of advanced melanoma.

The COVID-19 pandemic has highlighted and amplified family caregiving obligations for many clinical investigators and other biomedical researchers. Unpredictable access to daycare, schools, assisted living facilities, informal networks, and other sources of care of children, older adults, or those with special needs has been harrowing. The National Academies of Science, Engineering, and Medicine emphasized such challenges will impair the vitality of the scientific workforce, calling for research and action to bolster resources for those facing family caregiving responsibilities as they pursue careers in fields that include academic medicine (1).

Reactive oxygen species (ROS) produced in the neuronal, renal and vascular systems not only influence cardiovascular physiology, but are also strongly implicated in pathological signaling leading to hypertension. Different sources of ROS have been identified, ranging from xanthine-xanthine oxidase and mitochondria to NADPH oxidase (Nox) enzymes. Out of seven Nox family members, Nox1, Nox2, Nox4 (and Nox5 in humans) influence the cardiovascular system. Their activation processes, cell and tissue distribution vary widely, adding complexity to understanding their functional roles. Whether these systems act collectively or independently in disease conditions is unclear, but recently, feed forward mechanisms have been established between ROS sources. Studies published in Hypertension over the last few years are the focus of this review and they provide a framework with which to consider the roles of Nox enzymes in neuronal, renal and vascular hypertensive mechanisms, as well as cardiac remodeling, and their relationships with other ROS-generating systems.

Rationale The type I subclass of Coronins, a family of actin binding proteins, regulates various actin dependent cellular processes including migration. However, the existence and role of coronins in vascular smooth muscle cell (VSMC) migration has yet to be determined. Objective The goal of the present study was to define the mechanism by which coronins regulate platelet-derived growth factor (PDGF)-induced VSMC migration. Methods and Results Coronin 1B (Coro1B) and 1C (Coro1C) were both found to be expressed in VSMCs at the mRNA and protein levels. Down regulation of Coro1B by siRNA increases PDGF-induced migration, while down regulation of Coro1C has no effect. We confirmed through kymograph analysis that the Coro1B-downregulation-mediated increase in migration is directly linked to increased lamellipodial protraction rate and protrusion distance in VSMC. In other cell types, coronins exert their effects on lamellipodia dynamics by an inhibitory interaction with the ARP2/3 complex, which is disrupted by the phosphorylation of Coro1B. We found that PDGF induces phosphorylation of Coro1B on serine-2 via PKCε, leading to a decrease in the interaction of Coro1B with the ARP2/3 complex. VSMCs transfected with a phospho-deficient S2A Coro1B mutant showed decreased migration in response to PDGF, suggesting that the phosphorylation of Coro1B is required for the promotion of migration by PDGF. In both the rat and mouse Coro1B phosphorylation was increased in response to vessel injury in vivo. Conclusions Our data support that phosphorylation of Coro1B and the subsequent reduced interaction with ARP2/3 complex participate in PDGF-induced VSMC migration, an important step in vascular lesion formation.

The NADPH oxidase (Nox) enzymes are critical mediators of cardiovascular physiology and pathophysiology. These proteins are expressed in virtually all cardiovascular cells, and regulate such diverse functions as differentiation, proliferation, apoptosis, senescence, inflammatory responses and oxygen sensing. They target a number of important signaling molecules, including kinases, phosphatases, transcription factors, ion channels and proteins that regulate the cytoskeleton. Nox enzymes have been implicated in many different cardiovascular pathologies: atherosclerosis, hypertension, cardiac hypertrophy and remodeling, angiogenesis and collateral formation, stroke and heart failure. In this review, we discuss in detail the biochemistry of Nox enzymes expressed in the cardiovascular system (Nox1, 2, 4 and 5), their roles in cardiovascular cell biology, and their contributions to disease development.

Background: NADPH oxidases (Nox) regulate vascular physiology and contribute to the pathogenesis of vascular disease. In vascular smooth muscle cells (VSMCs), the interactions of individual Nox homologues with regulatory proteins are poorly defined. Objective: The objective of this study was to identify novel NADPH oxidase regulatory proteins. Methods and Results: Using a yeast 2 hybrid screen, we identified a novel binding partner, Poldip2, and demonstrate that it associates with p22phox, Nox1 and Nox4 and co-localizes with p22phox at sites of Nox4 localization. Poldip2 increases Nox4 enzymatic activity by 3-fold and positively regulates basal reactive oxygen species (ROS) production in VSMCs (O2•−: 86.3±15.6% increase; H2O2: 40.7±4.5% increase). Overexpression of Poldip2 activates Rho (180.2±24.8% increase), strengthens focal adhesions and increases stress fiber formation. These phenotypic changes are blocked by dominant negative Rho. In contrast, depletion of either Poldip2 or Nox4 results in a loss of these structures, which is rescued by adding back active Rho. Cell migration, which requires dynamic cytoskeletal remodeling, is impaired by either excess (70.1±14.7% decrease) or insufficient Poldip2 (63.5±5.9% decrease). Conclusion: These results suggest that Poldip2 associates with p22phox to activate Nox4, leading to regulation of focal adhesion turnover and VSMC migration, thus linking ROS production and cytoskeletal remodeling. Poldip2 may be a novel therapeutic target for vascular pathologies with a significant VSMC migratory component, such as restenosis and atherosclerosis.

Objective Vascular NADPH oxidases (Noxes) have been implicated in cardiovascular diseases; however, the importance of individual Nox homologues remains unclear. Here, the role of the vascular smooth muscle cell (VSMC) Nox1 in neointima formation was studied using genetically modified animal models. Methods and results Wire injury-induced neointima formation in the femoral artery, along with proliferation and apoptosis, was reduced in Nox1y/- mice, but there was little difference in TgSMCnox1 mice compared with wild type (WT) mice. Proliferation and migration were reduced in cultured Nox1y/- VSMCs and increased in TgSMCnox1 cells. TgSMCnox1 cells exhibited increased fibronectin secretion, but neither collagen I production nor cell adhesion was affected by alteration of Nox1. Using antibody microarray and Western blotting analysis, increased cofilin phosphorylation and mDia1 expression and decreased PAK1 expression were detected in Nox1y/- cells. Overexpression of S3A, a constitutively active cofilin mutant, partially recovered reduced migration of Nox1y/- cells, suggesting that reduction in cofilin activity contributes to impaired migration of Nox1y/- VSMCs. Conclusions These results indicate that Nox1 plays a critical role in neointima formation by mediating VSMC migration, proliferation and extracellular matrix production, and that cofilin is a major effector of Nox1-mediated migration. Inhibition of Nox1 may be an efficient strategy to suppress neointimal formation.

Objective Insulin resistance of vascular smooth muscle cells (VSMCs) has been linked to accelerated atherosclerosis in diabetes; however, the effects of insulin on VSMCs remain controversial. Most VSMC insulin receptors are sequestered into insulin-insensitive hybrids with insulin-like growth factor-1 receptors (IGF1R). Thus we hypothesized that regulation of IGF1R expression may impact cellular insulin sensitivity. Methods and Results IGF1R expression was increased in aortas from diabetic mice. IGF1R overexpression in VSMCs impaired insulin-induced Akt phosphorylation. Conversely, IGF1R downregulation by siRNA allowed assembly of insulin holoreceptors, enhanced insulin-induced phosphorylation of its receptor, Akt, Erk1/2 and further augmented insulin-induced glucose uptake. IGF1R downregulation uncovered an insulin-induced reduction in activation of NF-κB and inhibition of MCP-1 upregulation in response to TNF-α. Conclusions Downregulation of IGF1R increases the fraction of insulin receptors organized in holoreceptors, which leads to enhanced insulin signaling and unmasks potential anti-inflammatory properties of insulin in VSMCs. Therefore, IGF1R, which is susceptible to feedback regulation by its own ligand, may represent a novel target for interventions designed to treat insulin resistance in the vasculature.

Reactive oxygen species (ROS) are ubiquitous signaling molecules in biological systems. Four members of the NADPH oxidase (Nox) enzyme family are important sources of ROS in the vasculature: Nox1, Nox2, Nox4 and Nox5. Signaling cascades triggered by stresses, hormones, vasoactive agents and cytokines control the expression and activity of these enzymes and of their regulatory subunits, among which p22phox, p47phox, Noxa1 and p67phox are present in blood vessels. Vascular Nox enzymes are also regulated by Rac, ClC-3, Poldip2 and PDI. Multiple Nox subtypes, simultaneously present in different subcellular compartments, produce specific amounts superoxide, some of which is rapidly converted to hydrogen peroxide. The identity and location of these ROS, and of the enzymes that degrade them, determine their downstream signaling pathways. Nox enzymes participate in a broad array of cellular functions including differentiation, fibrosis, growth, proliferation, apoptosis, cytoskeletal regulation, migration and contraction. They are involved in vascular pathologies such as hypertension, restenosis, inflammation, atherosclerosis and diabetes. As our understanding of the regulation of these oxidases progresses, so will our ability to alter their functions and associated pathologies.

Several mammalian enzymes are capable of transferring electrons to molecular oxygen, sequentially forming the 1 electron-reduction product superoxide (O2•−) and the 2 electron-reduction product hydrogen peroxide (H2O2). These serve as progenitors for other reactive oxygen species (ROS), including peroxynitrite (ONOO−), hypochlorous acid, the hydroxyl radical, lipid peroxides, lipid peroxy- radicals, and lipid alkoxyl radicals. Another relevant group of molecules is the reactive nitrogen species, including NO, the nitrogen dioxide radical, and the nitrosonium cation. In the cardiovascular system, the most important enzymes that produce ROS are the Nox-based reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases, xanthine oxidase, the mitochondrial electron transport system and, under certain circumstances, NO synthase. Conditions such as hypertension, atherosclerosis, hypercholesterolemia, diabetes, and insulin resistance increase either the activity or the expression of these enzymes, leading to elevated ROS production. ROS, in turn, contribute to these disorders. As examples, virtually every aspect of atherosclerotic lesion formation is augmented by oxidative events. Via several mechanisms involving vessels, the kidney, and the central nervous system, ROS augment hypertension. ROS have been implicated in causing insulin resistance and pancreatic damage leading to diabetes. Moreover, ROS, when produced in specific subcellular compartments in controlled amounts, can act as signaling molecules to regulate normal cellular functions. These reactions have been reviewed in depth elsewhere recently.