Josiane Fernandes Silva

Background: Preeclampsia (PE) is a hypertensive disorder of pregnancy characterized by systemic inflammation, oxidative stress, and endothelial dysfunction. Although maternal vascular dysfunction is well established in PE, the mechanisms underlying fetal vascular injury remain poorly understood. We investigated whether inflammatory signaling activates NADPH oxidase 5 (NOX5) and contributes to oxidative stress and dysfunction in human umbilical arteries from pregnancies complicated by PE. Methods: Umbilical arteries and serum samples were obtained from normotensive pregnant women (NP) and women with PE. Vascular reactivity, nitric oxide (NO) bioavailability, reactive oxygen species (ROS) generation, cytokine levels, and NOX isoform expression were evaluated in human umbilical arteries and EA.hy926 endothelial cells. Pharmacological inhibition of NOX5, TNF-α neutralization, Ca²⁺ channel blockade, and siRNA-mediated NOX5 silencing were used to investigate mechanisms. Results: PE umbilical arteries exhibited increased vasoconstrictor responses, oxidative stress, and NOX5 expression, accompanied by impairment of NO bioavailability. NOX5 inhibition reversed vascular hyperreactivity in PE vessels. Exposure of normotensive umbilical arteries to PE serum reproduced the PE vascular phenotype, characterized by enhanced ROS generation, reduced NO levels, and hypercontractility. In endothelial cells, PE serum induced TNF-α-dependent Ca²⁺ influx, oxidative stress, and reduced NO production. Both pharmacological and genetic inhibition of NOX5 prevented these alterations. Conclusions: PE promotes fetal vascular dysfunction through activation of a TNF-α/Ca ²⁺ /NOX5 signaling pathway that amplifies oxidative stress and impairs NO bioavailability. These findings identify NOX5 as a previously unrecognized mediator of umbilical artery dysfunction in PE and suggest the TNF-α/Ca ²⁺ /NOX5 axis as a potential therapeutic target in hypertensive pregnancies.

INTRODUCTION: Cerebrovascular dysfunction occurs in Alzheimer's disease (AD), impairing hemodynamic regulation. Large conductance Ca ²⁺ ‐activated K ⁺ channels (BK _Ca ) regulate cerebrovascular reactivity and are impaired in AD. BK _Ca activity depends on intracellular Ca ²⁺ (Ca ²⁺ sparks) and nitro‐oxidative post‐translational modifications. However, whether these mechanisms underlie BK _Ca impairment in AD remains unknown. METHODS: Cerebral arteries from 5x‐FAD and wild‐type (WT) littermates were used for molecular biology, electrophysiology, ex vivo, and in vivo experiments. RESULTS: Arterial BK _Ca activity is reduced in 5x‐FAD via sex‐dependent mechanisms: in males, there is lower BK _α subunit expression and less Ca ²⁺ sparks. In females, we observed reversible nitro‐oxidative modification of BK _Ca . Further, BK _Ca is involved in hemodynamic regulation in WT mice, and its dysfunction is associated with vascular deficits in 5x‐FAD . DISCUSSION: Our data highlight the central role played by BK _Ca in cerebral hemodynamic regulation and that molecular mechanisms of its impairment diverge based on sex in 5x‐FAD . Highlights: Cerebral microvascular BK _Ca dysfunction occurs in both female and male 5x‐FAD . Reduction in BK _α subunit protein and Ca ²⁺ sparks drive the dysfunction in males. Nitro‐oxidative stress is present in females, but not males, 5x‐FAD . Reversible nitro‐oxidation of BK _α underlies BK _Ca dysfunction in female 5x‐FAD .

Chikungunya virus (CHIKV) infection is characterized by febrile illness, severe joint pain, myalgia, and cardiovascular complications. Given that CHIKV stimulates reactive oxygen species (ROS) and pro- and anti-inflammatory cytokines, events that disrupt vascular homeostasis, we hypothesized that CHIKV induces arterial dysfunction by directly impacting redox-related mechanisms in vascular cells. Wild-type (WT) and iNOS knockout (iNOS^−/−) mice were administered either CHIKV (1.0 × 10⁶ PFU/µL) or Mock vehicle via the intracaudal route. In vivo, CHIKV infection induced vascular dysfunction (assessed by a wire myograph), decreased systolic blood pressure (tail-cuff plethysmography), increased IL-6 and IFN-γ, but not TNF-α levels (determined by ELISA), and increased protein content by Western blot. Marked contractile hyporesponsiveness to phenylephrine was observed 48 h post-infection, which was restored by endothelium removal. L-NAME, 1400W, Tiron, and iNOS gene deletion prevented phenylephrine hyporesponsiveness. CHIKV infection increased vascular nitrite concentration (Griess reaction) and superoxide anion (O₂^•−) generation (lucigenin chemiluminescence), and decreased hydrogen peroxide (H₂O₂, by Amplex Red) levels 48 h post-infection, alongside increased TBARS levels. In vitro, CHIKV infected endothelial cells (EA.hy926) and upregulated ICAM-1 and iNOS protein expression (determined by Western blot). These data support the conclusion that CHIKV-induced alterations in vascular ROS/NF-kB/iNOS/NO signaling potentially contribute to cardiovascular events associated with Chikungunya infection.

Advanced age in mice (>2 yr, similar to a 70- to 80-yr-old human) impairs P2Y and Kir2 channel-induced dilation. Reductions in endothelial Kir2 activity were associated with lower vasodilatory response to purinergic receptor activation and increased resting myogenic tone of cerebral arterioles. Paradoxically, vasodilation following Kir2 activation was larger in aged cerebral arterioles, and unaffected by endothelium removal, suggesting a possible compensation by smooth muscle Kir2 channels. However, this compensation was not sufficient to restore purinergic dilation.

Background: Cerebral microvascular dysfunction and nitro-oxidative stress are present in patients with Alzheimer’s disease (AD) and may contribute to disease progression and severity. Large conductance Ca ²⁺ -activated K ⁺ channels (BK _Ca ) play an essential role in vasodilatory responses and maintenance of myogenic tone in resistance arteries. BK _Ca impairment can lead to microvascular dysfunction and hemodynamic deficits in the brain. We hypothesized that reduced BK _Ca function in cerebral arteries mediates microvascular and neurovascular responses in the 5x-FAD model of AD. Methods: BK _Ca activity in the cerebral microcirculation was assessed by patch clamp electrophysiology and pressure myography, in situ Ca ²⁺ sparks by spinning disk confocal microscopy, hemodynamics by laser speckle contrast imaging. Molecular and biochemical analyses were conducted by affinity-purification assays, qPCR, Western blots and immunofluorescence. Results: We observed that pial arteries from 5-6 months-old male and female 5x-FAD mice exhibited a hyper-contractile phenotype than wild-type (WT) littermates, which was linked to lower vascular BK _Ca activity and reduced open probability. In males, BK _Ca dysfunction is likely a consequence of an observed lower expression of the pore-forming subunit BK _α and blunted frequency of Ca ²⁺ sparks, which are required for BK _Ca activity. However, in females, impaired BK _Ca function is, in part, a consequence of reversible nitro-oxidative changes in the BK _α subunit, which reduces its open probability and regulation of vascular tone. We further show that BK _Ca function is involved in neurovascular coupling in mice, and its dysfunction is linked to neurovascular dysfunction in the model. Conclusion: These data highlight the central role played by BK _Ca in cerebral microvascular and neurovascular regulation, as well as sex-dependent mechanisms underlying its dysfunction in a mouse model of AD.

Cerebral parenchymal arterioles from menopause mice showed increased myogenic tone. We identified an impairment in smooth muscle cell BK_Ca channel activity, without a reduction in endothelium-dependent dilation or nitric oxide production. Microvascular dysfunction was associated with a reduction in neurovascular responses after somatosensory stimulation. Despite the neurovascular impairment, cognitive abilities were maintained in menopausal mice.

Background and Purpose: Metabolic and vascular dysfunction are common features of obesity. Aryl hydrocarbon receptor (AhR) regulates lipid metabolism and vascular homeostasis, but whether vascular AhR are activated in obesity or have a protective and/or harmful effects on vascular function in obesity are unknown. Our study addresses whether AhR activation contributes to obesity‐associated vascular dysfunction and the mechanisms involved in these AhR effects.Experimental Approach: Male AhR KO (Ahr^−/−) and WT mice were fed either control or a HF (high‐fat) diet for 10 weeks. Metabolic and inflammatory parameters were measured in serum and adipose tissue. Vascular reactivity (isometric force) was evaluated using a myography. Endothelial NOS (eNOS) and AhR protein expression was determined by western blot, Cyp1A1 and Nos3 gene expression by RT‐PCR and.NO production was quantified by DAF fluorescence.Key Results: HF diet increased total serum HDL and LDL, as well as vascular AhR protein expression and proinflammatory cytokines in the adipose tissue. HF diet decreased endothelium‐dependent vasodilation. AhR deletion protected mice from HF diet‐induced dyslipidaemia, weight gain and inflammatory processes. HF diet‐induced endothelial dysfunction was attenuated in Ahr^−/− mice. Vessels from Ahr^−/− mice exhibited a greater NO reserve. In cultured endothelial cells, lysophosphatidylcholine (LPC) a major component of LDL and oxidized LDL [oxLDL]) reduced Nos3 gene expression and NO production. Antagonism of the AhR inhibited LPC effects on endothelial cells and induced decreased endothelium‐dependent vasodilation.Conclusion and Implications: AhR deletion attenuates HF diet‐induced dyslipidaemia and vascular dysfunction by improving eNOS/NO signalling. Targeting AhRs may prevent obesity‐associated vascular dysfunction.

High levels of aldosterone (Aldo) trigger oxidative stress and vascular dysfunction independent of effects on blood pressure. We sought to determine whether Aldo disrupts Nrf2 signaling, the main transcriptional factor involved in antioxidant responses that aggravate cell injury. Thoracic aorta from male C57Bl/6J mice and cultured human endothelial cells (EA.hy926) were stimulated with Aldo (100 nM) in the presence of tiron [reactive oxygen species (ROS) scavenger, eplerenone [mineralocorticoid receptor (MR) antagonist], and L-sulforaphane (SFN; Nrf2 activator). Thoracic aortas were also isolated from mice infused with Aldo (600 μg/kg per day) for 14 days. Aldo decreased endothelium-dependent vasorelaxation and increased ROS generation, effects prevented by tiron and MR blockade. Pharmacological activation of Nrf2 with SFN abrogated Aldo-induced vascular dysfunction and ROS generation. In EA.hy926 cells, Aldo increased ROS generation, which was prevented by eplerenone, tiron, and SFN. At short times, Aldo-induced ROS generation was linked to increased Nrf2 activation. However, after three hours, Aldo decreased the nuclear accumulation of Nrf2. Increased Keap1 protein expression, but not activation of p38 MAPK, was linked to Aldo-induced reduced Nrf2 activity. Arteries from Aldo-infused mice also exhibited decreased nuclear Nrf2 and increased Keap1 expression. Our findings suggest that Aldo reduces vascular Nrf2 transcriptional activity by Keap1-dependent mechanisms, contributing to mineralocorticoid-induced vascular dysfunction.

Cells respond to stress by activating a variety of defense signaling pathways, including cell survival and cell death pathways. Although cell survival signaling helps the cell to recover from acute insults, cell death or senescence pathways induced by chronic insults can lead to unresolved pathologies. Arterial hypertension results from chronic physiological maladaptation against various stressors represented by abnormal circulating or local neurohormonal factors, mechanical stress, intracellular accumulation of toxic molecules, and dysfunctional organelles. Hypertension and aging share common mechanisms that mediate or prolong chronic cell stress, such as endoplasmic reticulum stress and accumulation of protein aggregates, oxidative stress, metabolic mitochondrial stress, DNA damage, stress-induced senescence, and proinflammatory processes. This review discusses common adaptive signaling mechanisms against these stresses including unfolded protein responses, antioxidant response element signaling, autophagy, mitophagy, and mitochondrial fission/fusion, STING (signaling effector stimulator of interferon genes)-mediated responses, and activation of pattern recognition receptors. The main molecular mechanisms by which the vasculature copes with hypertensive and aging stressors are presented and recent advancements in stress-adaptive signaling mechanisms as well as potential therapeutic targets are discussed.

Aldosterone excess aggravates endothelial dysfunction in diabetes and hypertension by promoting the increased generation of reactive oxygen species, inflammation, and insulin resistance. Aldosterone activates the molecular platform inflammasome in immune system cells and contributes to vascular dysfunction induced by the mineralocorticoid hormone. It is unclear as to whether the NLRP3 inflammasome associated with the mineralocorticoid receptor contributes to vascular dysfunction in diabetic conditions. Here, we tested the hypothesis that an excess of aldosterone induces vascular dysfunction in type 2 diabetes, via the activation of mineralocorticoid receptors (MR) and assembly of the NLRP3 inflammasome. Mesenteric resistance arteries from control (db/m) and diabetic (db/db) mice treated with vehicle, spironolactone (MR antagonist) or an NLRP3 selective inhibitor (MCC950) were used to determine whether NLRP3 contributes to diabetes-associated vascular dysfunction. Db/db mice exhibited increased vascular expression/activation of caspase-1 and IL-1β, increased plasma IL-1β levels, active caspase-1 in peritoneal macrophages, and reduced acetylcholine (ACh) vasodilation, compared to db/m mice. Treatment of db/db mice with spironolactone and MCC950 decreased plasma IL-1β and partly restored ACh vasodilation. Spironolactone also reduced active caspase-1-positive macrophages in db/db mice, events that contribute to diabetes-associated vascular changes. These data clearly indicate that MR and NLRP3 activation contribute to diabetes-associated vascular dysfunction and pro-inflammatory phenotype.