Inhalation of ultrafine particles (UFP) mediates systemic vascular impairment which is, in part, driven by elevated rates of oxidant generation. One significant source of oxidant production in the vascular compartment is the purine catabolizing enzyme, xanthine oxidoreductase (XOR). However, mechanisms linking XOR and/or endothelial glycosaminoglycan (GAG)-sequestered XOR to vessel dysfunction allied to UFP inhalation remain underexplored. Based on known interactions between UFP and the liver, we hypothesized that exposure could lead to hepatic release of XOR to the circulation which subsequently contributes to vascular impairment. Utilizing our murine hepatocyte-specific XOR knockout (XORHep-/-) model (loss of function) in conjunction with reintroducing exogenous XOR (restoration of function) we demonstrate a specific role for liver-derived XOR in the pathogenesis of UFP-induced vascular impairment. Exposure of mice as well as in vitro exposure of hepatocytes to our model UFP, nano titanium dioxide (nTiO2) results in the upregulation and active release of XOR. Drinking water supplemented with the XOR inhibitor febuxostat or nitrite ( NaNO 2 - ) partially prevented nTiO2-induced impairment of vascular reactivity. Interestingly, nitrite appears to cause a down-regulation of hepatic XOR. XORHep-/- mice were partially protected against both impairment of endothelial dependent dilation and augmented angiotensin II constriction. To further demonstrate the role of circulating XOR in nTiO2-induced impairment of vessel reactivity, XORHep-/- mice had circulating XOR restored by i.v. injection prior to exposure, which eliminated the protection of the hepatic knockout. It is important to note that acute restoration of intraluminal XOR in isolated vessels did not alter endothelial-dependent dilation or angiotensin II constriction. As such, we interrogated potential downstream mediators of XOR effects on endothelial function and found a decrease in the repressive trimethylation of lysine 9 on histone 3. Together these findings demonstrate that circulating XOR is a key contributor to endothelial dysfunction caused by UFP exposure. However, the impairment is not acute in nature and might involve epigenetic-mediated alterations in gene expression.

Background and Purpose: The mitochondrial unfolded protein response (UPR mt ), is a highly conserved, evolutionary stress response, which maintains mitochondrial homeostasis. UPR mt signaling has been shown to be cytoprotective in various cardiovascular pathologies. However, whether the UPR mt attenuates doxorubicin (DOX) induced cardiotoxicity, is unknown. This study sought to determine whether DOX induces UPR mt activation, and whether this activation exerts protective effects during DOX treatment. Experimental approach: Human cardiac (AC-16) cells were treated with increasing concentrations (vehicle, 0.5μM, 1µM) of DOX in a time-course manner, to assess UPR mt induction. In parallel, 8-week-old male-mice of a C57BL/6 background were treated with either 10mg/kg doxorubicin or vehicle, and hearts were collected 24 hrs. or seven days post-treatment to assess UPR mt signaling in-vivo . Further, to enhance UPR mt induction, the transcription factor ATF5 was overexpressed in AC-16 cells prior to DOX treatment. Key Results: DOX induced UPR mt signaling in a time-dependent manner. Upregulation of this pathway occurred prior to cell death in AC-16 cells, or structural changes within the mouse heart. Compared to controls, AC-16 cells overexpressing ATF5 showed increased transcript and protein levels of UPR mt associated genes, both at baseline and following DOX treatment. Importantly, ATF5 mediated of UPR mt signaling significantly reduced the apoptotic index, and improved cell viability in AC-16 cells treated with DOX. Conclusion and Implications: These findings support the UPR mt , and ATF5 mediated UPR mt activation, as promising targets by which to attenuate doxorubicin induced cardiotoxicity.

BACKGROUND AND PURPOSE:The pathogenesis of type 2 diabetes mellitus (T2DM)-induced cardiomyopathy involves cardiac fibrosis that leads to diastolic dysfunction. We established that replacement of lost substance P (SP) that occurs in T2DM reduces cardiac fibrosis and decreases inflammation in T2DM mice and non-human primates. This study aimed to identify the specific anti-fibrotic SP receptor. EXPERIMENTAL APPROACH:Age-matched male wild type (WT) and Leprdb/db mice at 12 weeks of age were treated with either saline or the neurokinin-1 receptor (NK-1R) agonist, GR73632 (300 μg·kg-1·day-1) for 4 weeks. The left ventricles were assessed for cardiac function, fibrosis, mast cells and macrophage phenotype. Mouse cardiac fibroblast and bone marrow-derived macrophage cultures were exposed to high glucose and treated with GR73632 to assess collagen release, signalling pathways and cytokine release respectively. Proteomics analysis was conducted to assess the left ventricular proteomic profile between WT and Leprdb/db mice, and the effects of GR73632. KEY RESULTS:NK-1R activation decreased cardiac fibrosis, improved diastolic function, decreased mast cell numbers and promoted an anti-inflammatory macrophage phenotype in Leprdb/db mice. NK-1R activation reduced collagen I production by high glucose treated mouse cardiac fibroblasts. NK-1R activation decreased P65 phosphorylation (NF-κB) and CCL2 chemokine release. Proteomic analysis revealed a distinct proteome profile between WT and Leprdb/db mouse hearts. CONCLUSION AND IMPLICATIONS:The NK-1R is the anti-fibrotic SP receptor and improves diastolic function in the diabetic heart. This likely involves direct effects on cardiac fibroblasts and macrophages. This study provides a potential target for treatment of diabetic cardiomyopathy.

Doxorubicin (DOX), a first-line chemotherapeutic agent, has been linked to severe off-target cardiotoxicity in the clinic. Previous works suggest that mitochondria are key mediators of this cardiotoxicity. Leakage of mitochondrial contents after DOX treatment, including mitochondrial DNA (mtDNA), is thought to activate apoptotic and inflammatory signaling pathways implicated in cardiomyocyte cell death. Whether the master mitochondrial protease, LonP1, can dampen these pathways and improve cardiomyocyte viability following DOX treatment remains unknown. Human cardiac cells (AC-16) and primary (1°) human cardiomyocytes were subjected to DOX treatment, followed by bulk RNA-Seq, RT-qPCR, qPCR, and immunoblotting to assess apoptotic signaling, inflammatory signaling, mtDNA release, and LonP1 expression, respectively. Lentivirus transduction of AC-16 cells was used to generate both knockdown (KD) and overexpression (OE) LonP1 cell lines to determine the effects of altered LonP1 levels on DOX-induced apoptosis and mtDNA release. Further, levels of mitochondrial DNA (mtDNA) were measured using qPCR from serum samples obtained from patients undergoing DOX treatment to assess the clinical relevance of released mtDNA as a potential biomarker for the development of DOX cardiotoxicity. DOX treatment of AC-16 cells, as well as 1° human cardiomyocytes, upregulated both apoptotic and inflammatory signaling in both cell models. Increased LonP1 levels were also observed under DOX treatment in AC-16 cells and 1° human cardiomyocytes. Likewise, DOX increased mtDNA release from both cell lines, both prior to, and as a sequel to cell death. Decreasing LonP1 levels exacerbated DOX-mediated apoptotic signaling and mtDNA release, whereas overexpression of LonP1 attenuated these effects. Furthermore, DOX treatment in cancer patients increases plasma mtDNA levels. These findings suggest LonP1 plays a protective role in the heart following DOX treatment, supporting LonP1 as a potential novel therapeutic target for prevention of DOX cardiotoxicity. Patterns of mtDNA release within patients undergoing DOX treatment also highlight the potential of mtDNA as a potential biomarker and target for prevention of DOX cardiotoxicity, justifying the need for more extensive, prospectively monitored cohort studies to expand upon these findings and statistically model mtDNA release patterns.

Background This study uses the Unpredictable Chronic Mild Stress (UCMS) model to investigate the effects of mid-life stress (MLS) on vascular and neurobiological changes in triple transgenic Alzheimer's disease mice (3xTg-AD) during critical developmental stages. Objective To investigate how mid-life stress (MLS) affects cerebrovascular function and AD progression. We hypothesize that chronic stress in 3xTg-AD mice will accelerate cerebrovascular dysfunction and, subsequently, the associated AD pathology. Methods Wild-type (WT) and 3xTg-AD mice were subjected to UCMS for 8 weeks at 4 months of age, with physiological, vascular, and molecular outcomes assessed at 6 and 9 months of age. We evaluated cerebrovascular function in the middle cerebral artery (MCA) and measured the expression of key mRNA and protein alterations associated with amyloid-β (Aβ) and tau pathology, which drive AD progression. Results Both WT and 3xTg-AD mice exposed to UCMS had significant MCA endothelial dysfunction. Additionally, UCMS accelerated the expression of key AD-related genes, and we observed increased oxidative stress, characterized by higher pro-oxidants and lower antioxidants. Elevated APP and BACE protein levels further suggest that MLS accelerated AD progression. Conclusions This study highlights the harmful effects of MLS on cerebrovascular health and the neurobiological mechanisms underlying AD progression. Our findings emphasize the critical link between chronic stress, oxidative dysfunction, and the acceleration of AD progression, offering important insights into potential therapeutic targets for alleviating the impacts of mid-life environmental stressors on AD development.

BACKGROUND TEAD1, the mammalian Hippo pathway-regulated transcription factor, plays a critical and non-redundant role in maintaining cardiomyocyte (CM) homeostasis. However, the specific cellular pathways regulated by TEAD1 in CMs remain poorly defined. We hypothesized that TEAD1 has an essential, cell-autonomous role in the CM oxidative stress response by directly regulating the transcription of NRF2, the master regulator of oxidative stress response. METHODS AND RESULTS Tamoxifen-induced conditional CM-specific TEAD1 deletion in adult mice leads to acute heart failure (HF) and altered expression of antioxidant genes. In silico analysis of publicly available RNA-seq data from human hearts with end-stage dilated (DCM) and ischemic (ICM) cardiomyopathy revealed significant downregulation of TEAD1 transcript levels and a positive correlation between TEAD1 and NRF2 gene expression. ChIP-seq and ATAC-seq in adult mouse hearts confirmed TEAD1 occupancy at promoter/enhancer elements within open chromatin regions of multiple antioxidant genes, including NRF2 and its targets. Ex vivo and in vitro TEAD1 knockout in primary neonatal and adult murine CMs, as well as in H9C2 cells, resulted in significantly increased cellular and mitochondrial ROS le, accompanied by a marked decrease in NRF2 expression and promoter-luciferase activity, under both basal and oxidative stress conditions. Mosaic, conditional deletion of TEAD1 in ∼40–50% of murine heart CMs provided a novel in vivo model for studying TEAD1-regulated pathways in the heart, independent of the confounding effects of HF. This model demonstrated reduced NRF2 expression and heightened oxidative stress in neonatal and adult TEAD1 mosaic knockout hearts. Notably, 8OHdG staining identified oxidative DNA damage in TEAD1-deficient CMs compared to TEAD1-expressing CMs within the mosaic knockout hearts. Upon in vivo AngII infusion, TEAD1 mosaic knockout hearts showed a significant increase in oxidative stress markers and an impaired NRF2 response. Overexpression of human TEAD1 restored NRF2 activity and mitigated ROS accumulation in TEAD1 knockout CMs in vitro. Furthermore, TEAD1 deletion in human iPSC-derived CMs resulted in increased oxidative stress and downregulation of NRF2 expression and functional activity, confirming the requirement of TEAD1 in NRF2-mediated oxidative stress response in human CMs. Collectively, these findings establish that TEAD1 is essential for NRF2 expression and activity under both basal and AngII-induced conditions and plays a crucial role in the oxidative stress response in CMs. CONCLUSIONS TEAD1 is a cell-autonomous, direct transcriptional regulator of NRF2 and the cardiomyocyte (CM) oxidative stress response. Its gene expression, which directly correlates with NRF2 transcript levels in the human myocardium, is significantly downregulated in human end-stage heart failure, potentially compromising the oxidative stress response in the failing heart. ### Competing Interest Statement The authors have declared no competing interest.

The emergence of glucagon-like peptide-1 agonists represents a notable advancement in the pharmacological treatment of obesity, yet complementary approaches are essential. Through phenotypic drug discovery, we developed promising nitroalkene-containing small molecules for obesity-related metabolic dysfunctions. Here, we present SANA, a nitroalkene derivative of salicylate, demonstrating notable efficacy in preclinical models of diet-induced obesity. SANA reduces liver steatosis and insulin resistance by enhancing mitochondrial respiration and increasing creatine-dependent energy expenditure in adipose tissue, functioning effectively in thermoneutral conditions and independently of uncoupling protein 1 and AMPK activity. Finally, we conducted a randomized, double-blind, placebo-controlled phase 1A/B clinical trial, which consisted of two parts, each with four arms: (A) single ascending doses (200–800 mg) in healthy lean volunteers; (B) multiple ascending doses (200–400 mg per day for 15 days) in healthy volunteers with overweight or obesity. The primary endpoint assessed safety and tolerability. Secondary and exploratory endpoints included pharmacokinetics, tolerability, body weight and metabolic markers. SANA shows good safety and tolerability, and demonstrates beneficial effects on body weight and glucose management within 2 weeks of treatment. Overall, SANA appears to be a first-in-class activator of creatine-dependent energy expenditure and thermogenesis, highlighting its potential as a therapeutic candidate for ‘diabesity’. Australian New Zealand Clinical Trials Registry registration: ACTRN12622001519741 . In this study, the authors describe SANA, a nitroalkene derivative of salicylate, as a potential activator of creatine-dependent energy expenditure and thermogenesis in adipose tissue. Preclinical and clinical data from this paper also suggest that SANA improves glucose homeostasis and promotes weight loss in mice and humans.

Pregnancy requires metabolic and endocrine changes that must occur to support fetal growth and development. Aberrations in these necessary modifications can impact maternal health and fetal growth. Exposure to toxicants during pregnancy can negatively affect fetal health and development, but studies on electronic cigarette (e-cig) exposure is limited. We hypothesized that maternal e-cig exposure during gestation leads to hormonal and redox imbalance and negatively impacts fetal development. Pregnant Sprague–Dawley rats were exposed to e-cig aerosols (1227 ± 131 mg/m3) or HEPA-filtered air for 90 min from gestational day (GD) 10–19 for a total of 6 days. Dams were euthanized on GD 20 and dam serum, liver, lung, ovaries, and placental tissue were collected for analysis. Fetal mass, placental mass, and sex ratios were assessed. Fetal and placental mass were significantly decreased in e-cig exposed compared to sham-control (2.61 ± 0.19 g vs. 3.37 ± 0.09 g and 0.62 ± 0.03 g vs. 0.70 ± 0.02 g, respectively). Placental xanthine oxidase (XO) activity was significantly increased in e-cig exposed compared to sham-control (5.29 ± 0.27 µU/mL vs. 4.28 ± 0.36 µU/mL). Circulating prolactin (PRL) levels of e-cig exposed dams were significantly decreased compared to sham-control (2.40 ± 0.06 ng/mL of plasma vs. 3.83 ± 0.64 ng/mL of plasma). Maternal e-cig inhalation exposure during gestation negatively impacted fetal growth, increased placental XO activity, and decreased circulating PRL levels. These data demonstrate two potential mechanisms that could lead to the observed reduction in fetal growth following maternal exposure: potential redox imbalance within the placenta and/or hormonal imbalance directly affects fetal growth and potentially influences growth later in life.

Alarmins are classified by their release from damaged or ruptured cells. Many alarmins have been found to increase vascular tone and oppose endothelium-dependent dilatation (EDD). Interleukin (IL)-33 plays a prominent role in lung injury and can be released during vascular injury and in chronic studies found to be cardioprotective. Our recent work has implicated IL-33 in acute vascular dysfunction following inhalation of engineered nanomaterials (ENM). However, the mechanisms linking IL-33 to vascular tone have not been interrogated. We therefore aimed to determine whether IL-33 directly influenced microvascular tone and endothelial function. Isolated feed arteries and in vivo arterioles from male and female Sprague-Dawley rats were used to determine direct vascular actions of IL-33. Mesenteric feed arteries and arterioles demonstrated reduced intraluminal diameters when treated with increasing concentrations of recombinant IL-33. IL-33 activated extracellular signal regulated kinase (ERK)1/2 of rat aortic smooth muscle cells but not phosphorylation of myosin light chain kinase. This suggested IL-33 may sensitize arterioles to Ca2+-mediated responses. Indeed, IL-33 augmented the myogenic- and phenylephrine-induced vasoconstriction. Additionally, incubation of arterioles with 1 ng IL-33 attenuated ACh-mediated EDD. Mechanistically, in human aortic endothelial cells, we demonstrate that IL-33-mediated ERK1/2 activation leads to inhibitory phosphorylation of serine 602 on endothelial nitric oxide synthase. Finally, we demonstrate that IL-33-ERK1/2 contributes to vascular tone following two known inducers of IL-33; ENM inhalation and the rupture endothelial cells. The present study provides novel evidence that IL-33 increases vascular tone via canonical ERK1/2 activation in microvascular smooth muscle and endothelium. Altogether, it is suggested IL-33 plays a critical role in microvascular homeostasis following barrier cell injury. KEY POINTS: Interleukin (IL)-33 causes a concentration-dependent reduction in feed artery diameter. IL-33 acts on vascular smooth muscle cells to augment Ca2+-mediated processes. IL-33 causes inhibitory phosphorylation of endothelial nitric oxide synthase and opposes endothelium-dependent dilatation. Engineered nanomaterial-induced lung injury and endothelial cell rupture in part act through IL-33 to mediate increased vascular tone.

Nano-titanium dioxide (nano-TiO2) is an engineered nanomaterial (ENM), which is widely utilized in diverse products like surface coatings, cosmetics, medical devices, and filters. Inhaled nano-TiO2 has been demonstrated to induce pulmonary inflammation, impair microvascular function, and hinder generational reproductive endpoints. These outcomes indicate why it is important to explore gestation, as it requires timely vascular adaptations to support maternal and fetal health and development. Therefore, the central hypothesis of my dissertation is: maternal nano-TiO2 inhalation exposure will impact maternal microvascular function and fetoplacental hemodynamics and function in a sexually dimorphic manner.

Introduction: Poor air quality has become a growing health concern. Ultrafine particles (UFP), a component of air pollution, has been associated with cardiovascular disease/ events, establishing a critical need to understand the cellular mechanisms involved. Using a model UFP we have shown elevated vascular oxidant production, and inflammation. However, the underlying mechanisms were unexplored. Xanthine oxidoreductase (XOR), increases in circulation following various insults, which we recently established is actively derived from hepatocytes. The binding of endothelial cells (EC) and the mechanisms by which EC bound XOR mediates impairment are not fully understood. Bound XOR produces almost exclusively H 2 O 2 , which can across the membrane and influence cellular signaling and gene expression. We hypothesize that UFP inhalation elevates hepatic XOR release into circulation and XOR binds to EC initiating redox signaling pathways leading to epigenetic-mediated inflammatory gene upregulation. Methods & Results: Male (M) and female (F) rats and mice (8 wk) were exposed to sham air or UFP for 3 days. Plasma XOR activity increased 5-fold M and 3-fold F exposed to UFP (both p<0.05). We then determined that UFP activation of PKC (rat liver 2.4-fold, mouse liver 2.3-fold, AML12 cells 1.7-fold, all p<0.05) leads to G9a-dependent upregulation and release of XOR in hepatocytes (Control 2± 0.3 vs PMA 8± 1 vs PMA G9a siRNA 2± 2 μU/ml, p<0.05). In arterioles, UFP arterioles showed greater XOR binding when perfused with Alexaflur tagged XOR (69±6 vs 51±4 AFU, p<0.05) and impaired dilation, which was prevented in hepatocyte XOR knockouts mice (KO) (max dilation sham 62± 3 vs UFP 35± 4 vs UFP KO 50± 4, p<0.05). In culture EC were treated with XOR for 1h (for binding) then washed with PBS and cultured for 24 h (XOR-EC). XOR-EC increased demethylase KDM4a expression (1.7-fold, p<0.05) and inflammatory markers ICAM, VCAM, TNF, and TLR4 (p<0.05), leading to enhanced leukocyte adhesion (~2-fold, p<0.05). KDM4a loss of function (iKDM4a) prevented ICAM expression (XOR-EC 8-fold vs iKDM4a 2-fold, p<0.05) and diminished leukocyte adhesion (p<0.05). Conclusion: These findings suggest that vascular inflammation caused by UFP inhalation is mediated in part by an EC bound XOR- KDM4a pathway.

Xanthine oxidase (XO) catalyzes the catabolism of hypoxanthine to xanthine and xanthine to uric acid, generating oxidants as a byproduct. Importantly, XO activity is elevated in numerous hemolytic conditions including sickle cell disease (SCD); however, the role of XO in this context has not been elucidated. Whereas long-standing dogma suggests elevated levels of XO in the vascular compartment contribute to vascular pathology via increased oxidant production, herein, we demonstrate, for the first time, that XO has an unexpected protective role during hemolysis. Using an established hemolysis model, we found that intravascular hemin challenge (40 μmol/kg) resulted in a significant increase in hemolysis and an immense (20-fold) elevation in plasma XO activity in Townes sickle cell phenotype (SS) sickle mice compared to controls. Repeating the hemin challenge model in hepatocyte-specific XO knockout mice transplanted with SS bone marrow confirmed the liver as the source of enhanced circulating XO as these mice demonstrated 100% lethality compared to 40% survival in controls. In addition, studies in murine hepatocytes (AML12) revealed hemin mediates upregulation and release of XO to the medium in a toll like receptor 4 (TLR4)-dependent manner. Furthermore, we demonstrate that XO degrades oxyhemoglobin and releases free hemin and iron in a hydrogen peroxide-dependent manner. Additional biochemical studies revealed purified XO binds free hemin to diminish the potential for deleterious hemin-related redox reactions as well as prevents platelet aggregation. In the aggregate, data herein reveals that intravascular hemin challenge induces XO release by hepatocytes through hemin-TLR4 signaling, resulting in an immense elevation of circulating XO. This increased XO activity in the vascular compartment mediates protection from intravascular hemin crisis by binding and potentially degrading hemin at the apical surface of the endothelium where XO is known to be bound and sequestered by endothelial glycosaminoglycans (GAGs).

We recently reported a previously unknown salutary role for xanthine oxidoreductase (XOR) in intravascular heme overload whereby hepatocellular export of XOR to the circulation was identified as a seminal step in affording protection. However, the cellular signaling and export mechanisms underpinning this process were not identified. Here, we present novel data showing hepatocytes upregulate XOR expression/protein abundance and actively release it to the extracellular compartment following exposure to hemopexin-bound hemin, hemin or free iron. For example, murine (AML-12 cells) hepatocytes treated with hemin (10 μM) exported XOR to the medium in the absence of cell death or loss of membrane integrity (2.0 ± 1.0 vs 16 ± 9 μU/mL p < 0.0001). The path of exocytosis was found to be noncanonical as pretreatment of the hepatocytes with Vaculin-1, a lysosomal trafficking inhibitor, and not Brefeldin A inhibited XOR release and promoted intracellular XOR accumulation (84 ± 17 vs 24 ± 8 hemin vs 5 ± 3 control μU/mg). Interestingly, free iron (Fe2+ and Fe3+) induced similar upregulation and release of XOR compared to hemin. Conversely, concomitant treatment with hemin and the classic transition metal chelator DTPA (20μM) or uric acid completely blocked XOR release (p < 0.01). Our previously published time course showed XOR release from hepatocytes likely required transcriptional upregulation. As such, we determined that both Sp1 and NF-kB were acutely activated by hemin treatment (∼2-fold > controls for both, p < 0.05) and that silencing either or TLR4 with siRNA prevented hemin-induced XOR upregulation (p < 0.01). Finally, to confirm direct action of these transcription factors on the Xdh gene, chromatin immunoprecipitation was performed indicating that hemin significantly enriched (∼5-fold) both Sp1 and NF-kB near the transcription start site. In summary, our study identified a previously unknown pathway by which XOR is upregulated via SP1/NF-kB and subsequently exported to the extracellular environment. This is, to our knowledge, the very first study to demonstrate mechanistically that XOR can be specifically targeted for export as the seminal step in a compensatory response to heme/Fe overload.

Nano-titanium dioxide (nano-TiO2) is a widely used nanomaterial found in several industrial and consumer products, including surface coatings, paints, sunscreens and cosmetics, among others. Studies have linked gestational exposure to nano-TiO2 with negative maternal and fetal health outcomes. For example, maternal pulmonary exposure to nano-TiO2 during gestation has been associated not only with maternal, but also fetal microvascular dysfunction in a rat model. One mediator of this altered vascular reactivity and inflammation is oxylipid signaling. Oxylipids are formed from dietary lipids through several enzyme-controlled pathways as well as through oxidation by reactive oxygen species. Oxylipids have been linked to control of vascular tone, inflammation, pain and other physiological and disease processes. In this study, we use a sensitive UPLC-MS/MS based analysis to probe the global oxylipid response in liver, lung, and placenta of pregnant rats exposed to nano-TiO2 aerosols. Each organ presented distinct patterns in oxylipid signaling, as assessed by principal component and hierarchical clustering heatmap analysis. In general, pro-inflammatory mediators, such as 5-hydroxyeicosatetraenoic acid (1.6 fold change) were elevated in the liver, while in the lung, anti-inflammatory and pro-resolving mediators such as 17-hydroxy docosahexaenoic acid (1.4 fold change) were elevated. In the placenta the levels of oxylipid mediators were generally decreased, both inflammatory (e.g. PGE2, 0.52 fold change) and anti-inflammatory (e.g. Leukotriene B4, 0.49 fold change). This study, the first to quantitate the levels of these oxylipids simultaneously after nano-TiO2 exposure, shows the complex interplay of pro- and anti-inflammatory mediators from multiple lipid classes and highlights the limitations of monitoring the levels of oxylipid mediators in isolation.

Xanthine oxidase (XO) mediates vascular function. Chronic stress impairs cerebrovascular function and increases the risk of stroke and cognitive decline. Our study determined the role of XO on stress-induced cerebrovascular dysfunction and cognitive decline. We measured middle cerebral artery (MCA) function, free radical formation, and working memory in 6-month-old C57BL/6 mice who underwent 8 weeks of control conditions or unpredictable chronic mild stress (UCMS) with or without febuxostat (50 mg/L), a XO inhibitor. UCMS mice had an impaired MCA dilation to acetylcholine vs. controls (p < 0.0001), and increased total free radical formation, XOR protein levels, and hydrogen peroxide production in the liver compared to controls. UCMS increased hydrogen peroxide production in the brain and cerebrovasculature compared to controls. Working memory, using the y-maze test, was impaired (p < 0.05) in UCMS mice compared to control mice. However, blocking XO using febuxostat prevented the UCMS-induced impaired MCA response, while free radical production and hydrogen peroxide levels were similar to controls in the liver and brain of UCMS mice treated with febuxostat. Further, UCMS + Feb mice did not have a significant reduction in working memory. These data suggest that the cerebrovascular dysfunction associated with chronic stress may be driven by XO, which leads to a reduction in working memory.

Pulmonary arterial hypertension (PAH) is a rare yet devastating and incurable disease with few treatment options. The underlying mechanisms of PAH appear to involve substantial cellular proliferation and vascular remodeling, causing right ventricular overload and eventual heart failure. Recent evidence suggests a significant seminal role of the pulmonary endothelium in the initiation and promotion of PAH. Our previous work identified elevated reactive oxygen species (ROS)-producing enzyme NADPH oxidase 1 (NOX1) in human pulmonary artery endothelial cells (HPAECs) of PAH patients promoting endothelial cell proliferation in vitro. In this study, we interrogated chemokine CXCL12′s (aka SDF-1) role in EC proliferation under the control of NOX1 and specificity protein 1 (Sp1). We report here that NOX1 can drive hypoxia-induced endothelial CXCL12 expression via the transcription factor Sp1 leading to HPAEC proliferation and migration. Indeed, NOX1 drove hypoxia-induced Sp1 activation, along with an increased capacity of Sp1 to bind cognate promoter regions in the CXCL12 promoter. Sp1 activation induced elevated expression of CXCL12 in hypoxic HPAECs, supporting downstream induction of expression at the CXCL12 promoter via NOX1 activity. Pathological levels of CXCL12 mimicking those reported in human PAH patient serum restored EC proliferation impeded by specific NOX1 inhibitor. The translational relevance of our findings is highlighted by elevated NOX1 activity, Sp1 activation, and CXCL12 expression in explanted lung samples from PAH patients compared to non-PAH controls. Analysis of phosphofructokinase, glucose-6-phosphate dehydrogenase, and glutaminase activity revealed that CXCL12 induces glutamine and glucose metabolism, which are foundational to EC cell proliferation. Indeed, in explanted human PAH lungs, demonstrably higher glutaminase activity was detected compared to healthy controls. Finally, infusion of recombinant CXCL12 into healthy mice amplified pulmonary arterial pressure, right ventricle remodeling, and elevated glucose and glutamine metabolism. Together these data suggest a central role for a novel NOX1-Sp1-CXCL12 pathway in mediating PAH phenotype in the lung endothelium.

Cardiovascular toxicology is the study of chemicals that cause untoward effects on the heart or vasculature. This is an ongoing course of study because the toxicants that humans are exposed to is constantly changing, however many of the mechanisms of toxicity remain largely unchanged over hundreds of years. Therefore, this chapter introduces the concept of cardiovascular homeostasis, and how fundamental anatomical and physiological elements make this possible. Lists of common toxicants that target cardiac and vascular mechanisms, and examples of how such agents alter cardiovascular health are discussed. Upon completion of this chapter, the reader should have a general understanding of how certain toxicants influence cardiovascular health, and to distinguish between therapeutic dose/benefit vs untoward outcome.