João Alfredo de Moraes

Heme modulates smooth muscle cell proliferation and migration via NADPH oxidase: A counter-regulatory role for heme oxygenase system

Accumulation of vascular smooth muscle cells (VSMC) in response to inflammatory stimuli is a key event in atherogenesis, which commonly occurs in sinuous vessels with turbulent blood flow what leads to hemolysis and consequent free heme accumulation, a known pro-oxidant and pro-inflammatory molecule. In this work, we investigated the effects of free heme on VSMC, and the molecular mechanisms underlying this process. Free heme induces a concentration-dependent migration and proliferation of VSMC which depends on the production of reactive oxygen species (ROS) derived from NADPH oxidase (NADPHox) activity. Additionally, heme activates redox-sensitive proliferation-related signaling routes, such as mitogen activated protein kinase (MAPK) and NF-κB, and induces heme oxygenase-1 (HO-1) expression. NADPHox-dependent proliferative effect of heme seems to be endogenously modulated by HO since the pretreatment of VSMC with HO inhibitors potentiates heme-induced proliferation and, in parallel, increases ROS production. These effects were no longer observed in the presence of heme metabolites, carbon monoxide and biliverdin. The data indicate that VSMC proliferation induced by heme is endogenously modulated by a critical counter-regulatory crosstalk between NADPHox and HO systems.

α9β1 integrin engagement inhibits neutrophil spontaneous apoptosis: Involvement of Bcl-2 family members

Integrin signaling is comprised of well-characterized pathways generally involved in cell survival. alpha(9)beta(1) integrin has recently become a target of study and has been shown to present pro-survival effects on neutrophils. However, there are no detailed studies on how alpha(9)beta(1) integrin-coupled signaling pathways interact and how they converge to finally modulate spontaneous apoptosis in neutrophils. In this regard we sought to investigate the main signaling events triggered by alpha(9)beta(1) integrin engagement and how these signaling pathways modulate the apoptotic program of human neutrophils. Using VLO5, a snake venom disintegrin shown to bind to alpha(9)beta(1) integrin in neutrophils, we demonstrate that alpha(9)beta(1) integrin engagement leads to the activation of integrin signaling pathways and potently reduces neutrophil spontaneous apoptosis. These effects are dependent on the activation of PI3K and MAPK pathways, since both LY294002 (PI3K inhibitor) or PD95059 (MEK inhibitor) reverted the effects of VLO5/alpha(9)beta(1) interaction. Moreover we show that VLO5/alpha(9)beta(1) engagement induces NF-kappaB nuclear translocation and increases the ratio between anti- and pro-apoptotic proteins by inducing the degradation of pro-apoptotic protein Bad and increasing the expression of anti-apoptotic protein Bcl-x(L). VLO5 also inhibited the early steps of neutrophil spontaneous apoptosis by preventing Bax translocation to the outer mitochondrial membrane and consequent cytochrome c release. In conclusion, as the mechanistic details of alpha(9)beta(1) integrin signaling pathways in human neutrophils becomes clearer, it should become possible to develop new therapeutic agents for human diseases where neutrophils play a prominent role.

Leukotriene B4 mediates vascular smooth muscle cell migration through αvβ3 integrin transactivation

Vascular injury leads to a local inflammatory response, characterized by endothelial damage, extracellular matrix exposition and aggregation/adhesion of platelets and circulating leukocytes. The release of inflammatory mediators amplifies the process, and can induce vascular smooth muscle cells (SMC) migration and proliferation. Released by leukocytes, leukotriene B4 (LTB4) induces reactive oxygen species production and SMC chemotaxis. This study was conducted to elucidate the molecular mechanisms involved in the effect of LTB4 on SMC migration, and a rat linage of vascular SMC (A7r5) were used throughout. The chemotactic effect of LTB4 was dependent on the concentration used, being comparable to AngII at 100 nM. Migration induced by LTB4 was inhibited in the presence of pertussis toxin, CP-105696, a BLT1 receptor antagonist, and by LY294002 or PD98059, two inhibitors of PI3K and MEK1/2, respectively. Stimulation of SMC with LTB4 triggered integrin-associated signaling pathways, inducing focal adhesion kinase (FAK) phosphorylation, mobilization of actin cytoskeleton, association of FAK to PI3K, ERK-2 phosphorylation and nuclear translocation, and also NFκB pathway activation. Pretreatment of SMC with a selective ligand of αvβ3 integrin, kistrin, inhibited LTB4-induced chemotaxis, FAK phosphorylation, FAK-PI3K association, and also inhibited ERK-2 and NFκB pathways activation. Taken together, the data demonstrated, for the first time, that the effect of LTB4 on SMC migration is modulated by integrin signaling activation, suggesting that these adhesion molecules might be important target for therapeutic intervention in cardiovascular diseases.

Alpha1beta1 and integrin-linked kinase interact and modulate angiotensin II effects in vascular smooth muscle cells.

The effects of angiotensin II (Ang II) on vascular smooth muscle cells (VSMC) are modulated by reactive oxygen species (ROS) and also involve integrin engagement. However, the potential link between alpha1beta1 integrin signaling with NOX system and their combined contribution to Ang II effects on VSMC have not been investigated. We aimed to elucidate the moslecular mechanisms underlying the activation of these two pathways in Ang II effects on VSMC. Ang II-induced VSMC migration (2-fold increase) and proliferation (2.5-fold increase) is modulated by alpha1beta1 integrin, being inhibited by obtustatin, a specific alpha1beta1 integrin blocker. Ang II also stimulates ROS production in VSMC (140%) that is NOX1 dependent, being completely inhibited in NOX1 silenced cells. The ROS production develops in two peaks, and the second peak is maintained by NOX2 activation. Apocynin and obtustatin inhibit the NOX2-associated second peak, but not the first peak of ROS production, which is related to NOX1 activation. Corroborating the involvement of alpha1beta1 integrin, the pretreatment of VSMC with obtustatin impaired Ang II-induced FAK phosphorylation, AKT activation, p21 degradation and the increase of ILK expression. Silencing of ILK blocked cell migration, AKT phosphorylation and the second peak of ROS, but partially inhibits (70%) VSMC proliferation induced by Ang II. The data demonstrate a novel role for NOX2 in Ang II effects on VSMC, and suggest alpha1beta1 integrin and ILK as target molecules to the development of more effective therapeutic interventions in cardiovascular diseases.

Redox modulation of FAK controls melanoma survival--role of NOX4.

Studies have demonstrated that reactive oxygen species (ROS) generated by NADPH oxidase are essential for melanoma proliferation and survival. However, the mechanisms by which NADPH oxidase regulates these effects are still unclear. In this work, we investigate the role of NADPH oxidase-derived ROS in the signaling events that coordinate melanoma cell survival. Using the highly metastatic human melanoma cell line MV3, we observed that pharmacological NADPH oxidase inhibition reduced melanoma viability and induced dramatic cellular shape changes. These effects were accompanied by actin cytoskeleton rearrangement, diminished FAKY397 phosphorylation, and decrease of FAK-actin and FAK-cSrc association, indicating disassembly of focal adhesion processes, a phenomenon that often results in anoikis. Accordingly, NADPH oxidase inhibition also enhanced hypodiploid DNA content, and caspase-3 activation, suggesting activation of the apoptotic machinery. NOX4 is likely to be involved in these effects, since silencing of NOX4 significantly inhibited basal ROS production, reduced FAKY397 phosphorylation and decreased tumor cell viability. Altogether, the results suggest that intracellular ROS generated by the NADPH oxidase, most likely NOX4, transmits cell survival signals on melanoma cells through the FAK pathway, maintaining adhesion contacts and cell viability.

Heme modulates intestinal epithelial cell activation: involvement of NADPHox-derived ROS signaling

In many gut chronic inflammatory conditions, intestinal epithelium (IE) is deprived of the protection of the mucus secreted by IE-specialized cells. In these events, bleeding and subsequent lysis of erythrocytes are common. This may lead to the release of high amounts of heme in the intestinal lumen, which interacts with IE. Previous works from our group have shown that heme itself is a proinflammatory molecule, activating a number of phlogistic signaling events in a nicotinamide adenine dinucleotide phosphate oxidase (NADPHox)-dependent manner. In this study, we aim to evaluate the effects of heme upon a well-established nontransformed small intestine epithelial cell lineage (IEC 6). Our results show that free heme evokes intracellular reactive oxygen species (ROS) production by IEC 6 cells, which is inhibited both by pharmacological inhibition with diphenyleneiodonium (10 μM), a NADPHox inhibitor, and small interfering RNA-mediated suppression of NOX1, a constitutive NADPHox isoform present in intestinal epithelial cells. Focal adhesion kinase phosphorylation and actin cytoskeleton polymerization are also induced by heme in a NADPHox-dependent manner. Heme increases monolayer permeability and redistributes key modulators of cell-cell adhesion as zona occludens-1 and E-cadherin proteins via NADPHox signaling. Heme promotes IEC 6 cell migration and proliferation, phenomena also regulated by NADPHox-derived ROS. Heme, in NADPHox-activating concentrations, is able to induce mRNA expression of IL-6, a cytokine implicated in inflammatory and tumorigenic responses. These data indicate a prominent role for heme-derived signaling in the pathophysiology of intestinal mucosa dysfunction and address an important role of NADPHox activity on the pathogenesis of intestinal inflammatory conditions.

Data in support of alpha1beta1 and integrin-linked kinase interact and modulate angiotensin II effects in vascular smooth muscle cells

The data provides information in support of the research article Moraes et al., Atherosclerosis 243(2) (2015) 477–485 [1]. Here we provide data behind the mechanisms involved in Angiotensin II (Ang II) effects on vascular smooth muscle cells (VSMC). Ang II-induced VSMC ROS production is modulated by alpha1beta1 integrin. Ang II also stimulates ROS production in VSMC via p47phox, a NOX2 subunit. Furthermore, Ang II effect on VSMC migration was also inhibited by NOX2 inhibitor. We showed that obtustatin, alpha1beta1 integrin blocker, inhibited Ang II effect on p47phox activation. Ang II effect on ROS production is also PI3K dependent. Finally we showed that NOX1 and Integrin-Linked-Kinase (ILK) are crucial to NOX2 activation. The research provides information about the sequential events of NOX1/alpha1beta1 integrin/ILK/NOX2 in Ang II effects on VSMC.

Rapid NOS-1-derived nitric oxide and peroxynitrite formation act as signaling agents for inducible NOS-2 expression in vascular smooth muscle cells.

Septic vascular dysfunction is characterized by hypotension and hyporeactivity to vasoconstrictors and nitric oxide (NO), reactive oxygen species and peroxynitrite have a prominent role in this condition. However, the mechanism whereby the vascular dysfunction is initiated is poorly understood. Based on previous studies of our group and the literature,we hypothesize that constitutive nitric oxide synthases (c-NOS) and peroxynitrite may play a role in the development of septic vascular dysfunction. Bacterial lipopolysaccharide (LPS) and interferon-γ (IFN) were used to stimulate rat aorta smooth muscle cells (A7r5) and rat aorta slices. This stimulation led to a rapid (within minutes) production of NO and superoxide anion, which led to peroxynitrite formation. When this rapid initial burst was reduced, through the inhibition of c-NOS and NADPH oxidases (NOX) or the scavenging of NO and superoxide the NF-κB activation, NOS-2 expression and nitrite production were significantly attenuated. Although vascular smooth muscle cells express both c-NOS isoforms, gene knockdown revealed that only NOS-1-dependent NO and peroxynitrite formation are important for the later NOS-2 expression. Similar findings were obtained by knockdown NOX-1 gene, one source of superoxide for peroxynitrite formation. Taking together, we show that smooth muscle cell activation by LPS/IFN leads to a rapid formation of NOS-1-derived NO and NOX-1-derived superoxide, forming peroxynitrite; and that this species act as a trigger for NOS-2 expression through NF-κB activation. Therefore, our findings suggest a critical role for NOS-1 and NOX-1 in the initiation of the vascular dysfunction associated with sepsis and septic shock.

Effects of Lonomia obliqua Venom on Vascular Smooth Muscle Cells: Contribution of NADPH Oxidase-Derived Reactive Oxygen Species

Envenomation caused by human contact with the caterpillar Lonomia is characterized by deleterious effects on coagulation and patency of blood vessels. The cellular effects induced by Lonomia obliqua venom highlights its capacity to activate endothelial cells, leading to a proinflammatory phenotype. Having more knowledge about the mechanisms involved in envenomation may contribute to better treatment. We aimed to evaluate the effects of Lonomia obliqua caterpillar bristle extract (LOCBE) on vascular smooth muscle cells (VSMC). We observed that LOCBE induced VSMC migration, which was preceded by alterations in actin cytoskeleton dynamics and Focal Adhesion Kinase activation. LOCBE also induced Extracellular Signal-Regulated Kinase (ERK) phosphorylation in VSMC, and the inhibition of this pathway impaired cell proliferation. Stimulation of VSMC with LOCBE triggered reactive oxygen species (ROS) production through the activation of NADPH oxidase. The rapid increase in these ROS further induced mitochondrial ROS production, however only NADPH oxidase-derived ROS were involved in ERK activation in VSMC. We that demonstrated the chemotactic and proliferative effects of LOCBE on VSMC were dependent on ROS production, mainly through NADPH oxidase. Together, the data show that Lonomia obliqua venom can interact with and activate VSMC. These effects rely on ROS production, suggesting new potential targets for treatment against vascular damage during envenomation.

Recombinant and Chimeric Disintegrins in Preclinical Research

Disintegrins are a family of small cysteine-rich peptides, found in a wide variety of snake venoms of different phylogenetic origin. These peptides selectively bind to integrins, which are heterodimeric adhesion receptors that play a fundamental role in the regulation of many physiological and pathological processes, such as hemostasis and tumor metastasis. Most disintegrins interact with integrins through the RGD (Arg-Gly-Asp) sequence loop, resulting in an active site that modulates the integrin activity. Some variations in the tripeptide sequence and the variability in its neighborhood result in a different specificity or affinity toward integrin receptors from platelets, tumor cells or neutrophils. Recombinant forms of these proteins are obtained mainly through Escherichia coli, which is the most common host used for heterologous expression. Advances in the study of the structure-activity relationship and importance of some regions of the molecule, especially the hairpin loop and the C-terminus, rely on approaches such as site-directed mutagenesis and the design and expression of chimeric peptides. This review provides highlights of the biological relevance and contribution of recombinant disintegrins to the understanding of their binding specificity, biological activities and therapeutic potential. The biological and pharmacological relevance on the newest discoveries about this family of integrin-binding proteins are discussed.