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Ketone bodies are essential energy substrates in the absence of exogenous nutrients, and more recently, they have been suggested to prevent disease and improve longevity. β-hydroxybutyrate (βHB) is the most abundant ketone body. The secondary alcohol, 1,3-butanediol (1,3-BD), is commonly administered to raise βHB bioavailability in vivo and in the absence of nutrient deprivation. However, the concentration of 1,3-BD that yields a systemic concentration of βHB similar to that observed after a 24-hour fast has yet to be determined. To evaluate this knowledge gap, we administered 5%, 10%, or 20% 1,3-BD via the drinking water to adult, male Wistar-Kyoto rats for four weeks. In addition to systemic and excreted βHB concentration, physiologic, metabolic, and toxicologic parameters were measured. We report that only 20% 1,3-BD significantly elevates the systemic and urinary concentrations of βHB. Rats treated with 20% 1,3-BD had a rapid and sustained reduction in body mass. All concentrations of 1,3-BD decreased food consumption, but only the 20% concentration decreased fluid consumption. Urine volume, red blood cell count, and hematocrit suggested dehydration in the 10% and 20% 1,3-BD–treated rats. Finally, 20% 1,3-BD–treated rats presented with indicators of metabolic acidosis and sinusoidal dilation, but no evidence of fatty liver or hepatotoxicity. In summary, we report that 20% 1,3-BD, but not 5% or 10%, produces a systemic concentration of βHB similar to that observed after a 24-hour fast. However, this concentration is associated with deleterious side effects such as body mass loss, dehydration, metabolic acidosis, and sinusoidal dilation. SIGNIFICANCE STATEMENT 1,3-Butanediol (1,3-BD) is often administered to stimulate the biosynthesis of the most abundant ketone body, β-hydroxybutyrate (βHB), and its purported salubrious effects. This article reports that suprapharmacological concentrations of 1,3-BD are necessary to yield a systemic concentration of βHB similar to that observed after a 24-hour fast, and this is associated with undesirable side effects. On the other hand, low concentrations of 1,3-BD were better tolerated and may improve health independent of its conversion into βHB.

Recent studies have shown that chronic use of prescription or illicit opioids leads to an increased risk of cardiovascular events and pulmonary arterial hypertension. Indices of vascular age and arterial stiffness are also shown to be increased in opioid-dependent patients, with the effects being more marked in women. There are currently no studies investigating sex-specific vascular dysfunction in opioid use, and the mechanisms leading to opioid-induced vascular damage remain unknown. We hypothesized that exposure to exogenous opioids causes sex-specific vascular remodeling that will be more pronounced in female. Acknowledging the emerging roles of cofilins and extracellular signal-regulated kinases (ERKs) in mediating actin dynamics, we investigated the effects of morphine on these molecules. Twenty-four hour exposure to morphine increased inactivated cofilin and activated ERKs in resistance arteries from female mice, which may promote stress fiber over-assembly. We also performed continuous intraluminal infusion of morphine in pressurized resistance arteries from male and female mice using culture pressure myographs. We observed that morphine reduced the vascular diameter in resistance arteries from female, but not male mice. These results have significant implications for the previously unexplored role of exogenous opioids as a modifiable cardiovascular risk factor, especially in women.

Commensal gut microbiota are strongly correlated with host hemodynamic homeostasis, but only broadly associated with cardiovascular health. This includes a general correspondence of quantitative and qualitative shifts in intestinal microbial communities found in hypertensive rat models and human patients. However, the mechanisms by which gut microbes contribute to the function of organs important for blood pressure control remain unanswered. To examine the direct effects of microbiota on blood pressure, we conventionalized germ-free (GF) rats with specific pathogen free rats for a short-term period of 10 days, which served as a model system to observe the dynamic responses when reconstituting the holobiome. The absence of microbiota in GF rats resulted with relative hypotension compared to their conventionalized counterparts, suggesting an obligatory role of microbiota in blood pressure homeostasis. Hypotension observed in GF rats was accompanied by a marked reduction in vascular contractility. Both blood pressure and vascular contractility were restored by the introduction of microbiota to GF rats, indicating that microbiota could be impacting blood pressure through a vascular-dependent mechanism. This is further supported by the decrease in actin polymerization in arteries from GF rats. Improved vascular contractility in conventionalized GF rats, as indicated through stabilized actin filaments, was associated with an increase in cofilin phosphorylation. These data indicate that the vascular system senses the presence (or lack of) microbiota to maintain vascular tone via actin polymerization. Taken together, these results constitute a fundamental discovery of the essential nature of microbiota in blood pressure regulation.

For over 100 years, essential hypertension has been researched from different perspectives ranging from genetics, physiology, and immunology to more recent ones encompassing microbiology (microbiota) as a previously underappreciated field of study contributing to the cause of hypertension. Each field of study in isolation has uniquely contributed to a variety of underlying mechanisms of blood pressure regulation. Even so, clinical management of essential hypertension has remained somewhat static. We, therefore, asked if there are any converging lines of evidence from these individual fields that could be amenable for a better clinical prognosis. Accordingly, here we present converging evidence which support the view that metabolic dysfunction underlies essential hypertension.

Hypertension has been described as a condition of premature vascular aging, relative to actual chronological age. In fact, many factors that contribute to the deterioration of vascular function as we age are accelerated and exacerbated in hypertension. Nonetheless, the precise mechanisms that underlie the aged phenotype of arteries from hypertensive patients and animals remain elusive. Classically, the aged phenotype is the buildup of cellular debris and dysfunctional organelles. One means by which this can occur is insufficient degradation and cellular recycling. Mitophagy is the selective catabolism of damaged mitochondria. Mitochondria are organelles that contribute importantly to the determination of cellular age via their production of reactive oxygen species (ROS; Harman’s free radical theory of aging). Therefore, the accumulation of dysfunctional and ROS-producing mitochondria could contribute to the acceleration of vascular age in hypertension. This review will address and critically evaluate the current literature on mitophagy in vascular physiology and hypertension.

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Insufficient autophagy has been proposed as a mechanism of cellular aging, as this leads to the accumulation of dysfunctional macromolecules and organelles. Premature vascular aging occurs in hypertension. In fact, many factors that contribute to the deterioration of vascular function as we age are accelerated in clinical and experimental hypertension. Previously, we have reported decreased autophagy in arteries from spontaneously hypertensive rats (SHRs); however, the effects of restoring autophagic activity on blood pressure and vascular function are currently unknown. We hypothesized that reconstitution of arterial autophagy in SHRs would decrease blood pressure and improve endothelium-dependent relaxation. We treated 14- to 18-wk-old Wistar rats ( n = 7 vehicle and n = 8 trehalose) and SHRs ( n = 7/group) with autophagy activator trehalose (2% in drinking water) for 28 days. Blood pressure was measured by radiotelemetry, and vascular function and structure were measured in isolated mesenteric resistance arteries (MRAs) using wire and pressure myographs, respectively. Treatment with trehalose had no effect on blood pressure in SHRs; however, isolated MRAs presented enhanced relaxation to acetylcholine, in a cyclooxygenase- and reactive oxygen species-dependent manner. Similarly, trehalose treatment shifted the relaxation to the Rho kinase (ROCK) inhibitor Y-27632 to the right, indicating reduced ROCK activity. Finally, trehalose treatment decreased arterial stiffness as indicated by the slope of the stress-strain curve. Overall these data indicate that reconstitution of arterial autophagy in SHRs improves endothelial and vascular smooth muscle function, which could synergize to prevent stiffening. As a result, restoration of autophagic activity could be a novel therapeutic for premature vascular aging in hypertension. NEW & NOTEWORTHY This work supports the concept that diminished arterial autophagy contributes to premature vascular aging in hypertension and that therapeutic reconstitution of autophagic activity can ameliorate this phenotype. As vascular age is a new clinically used index for cardiovascular risk, understanding this mechanism may assist in the development of new drugs to prevent premature vascular aging in hypertension. Listen to this article’s corresponding podcast at https://ajpheart.podbean.com/e/behind-the-bench-episode-one-cam-squared/ .

Resolution of inflammation is an active phenomenon to switch off the inflammatory processes andfacilitate the return to homeostasis. Increasing the levels of pro-resolving lipid mediators topromote the resolution of inflammation is emerging as a novel therapeutic approach. Arachidonicacid (AA) and docosahexaenoic acid (DHA) are substrates to produce the pro-resolving lipidmediators lipoxin A4 (LXA4) and resolvin D1 (RvD1), respectively. However, it is unknown if thesemediators can ameliorate dysfunction in arteries from hypertensive animals. Therefore, wehypothesized that pro-resolving lipid mediators decrease acetylcholine-induced contractions inarteries from spontaneously hypertensive rats (SHR). Mesenteric resistance arteries from maleSHR and Wistar Kyoto (WKY) (14-weeks old, n=6-8) were used for vascular function via wiremyograph. MRA were incubated with either RvD1 or LXA4 (10 nM, 1 hour) or vehicle prior toconcentration response curves to acetylcholine or phenylephrine (1 nM - 3 μM). We alsoperformed lipidomic analysis on plasma from WKY and SHR. As expected, low concentrations (≤100 nM) of acetylcholine induced relaxation in arteries from both groups, however highconcentrations (≥ 1 μM) of acetylcholine induced contraction in arteries from SHR, but not in WKY[Relaxation to acetylcholine [Area Under the curve (AUC)]: WKY: 396.6 ± 17.7 vs. SHR-control:296.0 ± 26.7*, t-test *vs. WKY, p=0.03). Treatment with the mediators did not change relaxationin arteries from WKY (AUC: Vehicle: 396.6 ±17.7 vs. RvD1: 402.8 ± 18.0; LXA4: 399.0 ± 18.4,p>0.05). However, incubation with RvD1 or LXA4 reduced acetylcholine induced-contraction inarteries from SHR (AUC: Vehicle: 296.0 ± 26.7 vs. RvD1: 372.5 ± 18.5*; LXA4: 376.8 ± 23.6*, t-test *vs. vehicle, p

Amyloid β proteins, including toxic soluble oligomers (SPOs) are not only found in the brain duringAlzheimer’s, but also in the peripheral vascular system. The precise mechanism linking increasedcirculating levels of SPOs and vascular dysfunction remains unknown. We hypothesized that SPOslead to endoplasmic reticulum (ER) stress, further release of SPOs and vascular injury. Mesentericresistance arteries (MRAs) from 14 weeks old, male and female C57BL/6 mice were used forvascular function. Agonists were acetylcholine and phenylephrine (1nM-10mM). In acuteconditions, SPOs (0.1μM) caused pathologically exacerbated endothelium-dependent vasodilationcompared to vehicle (F12 media) [Male: EC50: SPOs: -7.0 ± 0.1 (n=4), vs. Vehicle -6.6 ± 0.1 (n=7)p=0.03; Female: EC50: SPOs: -7.3 ± 0.06 (n=5) vs. Vehicle -6.7 ± 0.1 (n=6), p=0.001]. Thisphenotype was similar to the positive control tunicamycin (5mg/ml) [Male: EC50: Tunicamycin: -7.3(n=4), vs. Vehicle -6.6 (n=7) p=0.2; Female: EC50: Tunicamycin: -7.7 (n=4) vs. Vehicle -6.8 (n=5)p=0.04]. To determine whether SPO’s cause ER stress, arteries were treated with ER stressinhibitor 4-Phenylbutyric acid (2mM). The ER stress inhibitor prevented the exacerbatedvasodilation induced by SPOs showing SPOs trigger ER stress in acute conditions independent ofsex. To determine whether SPOs are a consequence of ER stress, arteries were incubated withtunicamycin in the presence of the SPO inhibitor K01-162 (10mM). Interestingly, K01-162 did notprevent the tunicamycin-induced exacerbated vasodilation in arteries from male mice. However,this response was decreased in arteries from female mice showing that inducing ER stress leadsto the release of SPOs, escalating a feed-forward mechanism of further SPO release. There wereno changes in vascular contraction with tunicamycin or SPOs irrespective of sex. ER stress wasconfirmed with anti-KDEL antibody staining, specific for ER resident chaperones Grp78/94 andvisualized with multiphoton fluorescent confocal microscopy. These results demonstrate that SPO’sexacerbate endothelium-dependent vasodilation acutely and may contribute to brain and peripheralvascular edema and loss of autoregulation observed during cardiovascular and Alzheimer’sdisease.

Metabolic syndrome prevalence has increased among US adults, particularly among non-hispanic white and black women. Sedentary behavior often leads to chronic inflammation, a triggering factor of metabolic syndrome. Given that intrinsic exercise capacity is genetically inherited, we questioned if low-grade chronic inflammation would be present in a female rat model of low intrinsic exercise capacity-induced metabolic syndrome, while beneficial increase of resolution of inflammation would be present in a female rat model of high intrinsic exercise capacity. In the vascular system, two primary markers for inflammation and resolution of inflammation are cyclooxygenase (COX) and lipoxygenase (LOX), respectively. Our study focused on the novel hypothesis that untrained, inherited exercise capacity induces divergent vascular plasticity via changes in the delicate balance between COX and LOX inflammatory mediators. We used divergent rat strains with low (LCR) and high (HCR) aerobic running capacity. By using animals with contrasting intrinsic exercise capacities, it is possible to determine the exact triggers that lead to inherited vascular plasticity in female rats. We observed that female LCR displayed increased periovarian fat pad and body weight, which is congruent with their obesity-presenting phenotype. Furthermore, LCR presented with vascular hypocontractility and increased COX and LOX-derived pro-inflammatory factors. On the other hand, HCR presented with a "shutdown" of COX-induced vasoconstriction and enhanced resolution of inflammation to maintain vascular tone and homeostasis. In conclusion, LCR display low-grade chronic inflammation via increased COX activity. These results provide mechanistic clues as to why lower intrinsic aerobic capacity correlates with a predisposition to risk of vascular disease. Conversely, being born with higher intrinsic aerobic capacity is a significant factor for improved vascular physiology in female rats.

Cell death has long been a characteristic phenotype of organ damage in hypertension, and recently, leaky gut has been revealed as a novel hypertensive phenotype. However, despite the increase in bacterial and damaged mitochondrial products in the circulation of hypertensive patients and animals, the mechanistic contribution of these two phenomena to hypertension pathophysiology is unknown. Mitochondria and bacteria both start protein translation with an N-formyl methionine residue and thus are the only sources of NFPs (N-formyl peptides), which activate the FPR-1 (formyl peptide receptor-1). We hypothesized that the synergistic action of bacterial and mitochondrial NFPs would cause the spontaneous elevation of blood pressure and vascular remodeling in male Dahl salt-sensitive rats via FPR-1. We observed that mitochondria-derived peptides originating from cell death in the kidneys are responsible for FPR-1–induced vascular hypercontractility and remodeling and premature elevation of BP in Dahl salt-sensitive rats fed a low-salt diet. However, a high-salt diet leads to gut barrier disruption and, subsequently, a synergistic action of mitochondria, and bacteria-derived leaky gut NFPs lead to a severe and established hypertension. Administration of an FPR-1 antagonist lowered blood pressure in Dahl salt-sensitive rats on a low-salt diet but amoxicillin administration did not. These results reveal for the first time that cell death can be a cause of hypertensive pathophysiology, whereas leaky gut is a consequence.

Autophagy has long been associated with longevity, and it is well established that autophagy reverts and prevents vascular deterioration associated with aging and cardiovascular diseases. Currently, our understanding of how autophagy benefits the vasculature is centered on the premise that reduced autophagy leads to the accumulation of cellular debris, resulting in inflammation and oxidative stress, which are then reversed by reconstitution or upregulation of autophagic activity. Evolutionarily, autophagy also functions to mobilize endogenous nutrients in response to starvation. Therefore, we hypothesized that the biosynthesis of the most physiologically abundant ketone body, β-hydroxybutyrate (βHB), would be autophagy dependent and exert vasodilatory effects via its canonical receptor, Gpr109a. To the best of our knowledge, we have revealed for the first time that the biosynthesis of βHB can be impaired by preventing autophagy. Subsequently, βHB caused potent vasodilation via potassium channels but not Gpr109a. Finally, we observed that chronic consumption of a high-salt diet negatively regulates both βHB biosynthesis and hepatic autophagy and that reconstitution of βHB bioavailability prevents high-salt diet-induced endothelial dysfunction. In summary, this work offers an alternative mechanism to the antiinflammatory and antioxidative stress hypothesis of autophagy-dependent vasculoprotection. Furthermore, it reveals a direct mechanism by which ketogenic interventions (e.g., intermittent fasting) improve vascular health.

Uncontrolled immune system activation amplifies end-organ injury in hypertension. Nonetheless, the exact mechanisms initiating this exacerbated inflammatory response, thereby contributing to further increases in blood pressure (BP), are still being revealed. While participation of lymphoid-derived immune cells has been well described in the hypertension literature, the mechanisms by which myeloid-derived innate immune cells contribute to T cell activation, and subsequent BP elevation, remains an active area of investigation. In this article, we critically analyze the literature to understand how monocytes, macrophages, dendritic cells, and polymorphonuclear leukocytes, including mast cells, eosinophils, basophils, and neutrophils, contribute to hypertension and hypertension-associated end-organ injury. The most abundant leukocytes, neutrophils, are indisputably increased in hypertension. However, it is unknown how (and why) they switch from critical first responders of the innate immune system, and homeostatic regulators of BP, to tissue-damaging, pro-hypertensive mediators. We propose that myeloperoxidase-derived pro-oxidants, neutrophil elastase, neutrophil extracellular traps (NETs), and interactions with other innate and adaptive immune cells are novel mechanisms that could contribute to the inflammatory cascade in hypertension. We further posit that the gut microbiota serves as a set point for neutropoiesis and their function. Finally, given that hypertension appears to be a key risk factor for morbidity and mortality in COVID-19 patients, we put forth evidence that neutrophils and NETs cause cardiovascular injury post-coronavirus infection, and thus may be proposed as an intriguing therapeutic target for high-risk individuals. © 2021 American Physiological Society. Compr Physiol 11:1575-1589, 2021.

Ethanol consumption represents a significant public health problem, and excessive ethanol intake is a risk factor for cardiovascular disease (CVD), one of the leading causes of death and disability worldwide. The mechanisms underlying the effects of ethanol on the cardiovascular system are complex and not fully comprehended. The gut microbiota and their metabolites are indispensable symbionts essential for health and homeostasis and therefore, have emerged as potential contributors to ethanol-induced cardiovascular system dysfunction. By mechanisms that are not completely understood, the gut microbiota modulates the immune system and activates several signaling pathways that stimulate inflammatory responses, which in turn, contribute to the development and progression of CVD. This review summarizes preclinical and clinical evidence on the effects of ethanol in the gut microbiota and discusses the mechanisms by which ethanol-induced gut dysbiosis leads to the activation of the immune system and cardiovascular dysfunction. The cross talk between ethanol consumption and the gut microbiota and its implications are detailed. In summary, an imbalance in the symbiotic relationship between the host and the commensal microbiota in a holobiont, as seen with ethanol consumption, may contribute to CVD. Therefore, manipulating the gut microbiota, by using antibiotics, probiotics, prebiotics, and fecal microbiota transplantation might prove a valuable opportunity to prevent/mitigate the deleterious effects of ethanol and improve cardiovascular health and risk prevention.

Recent studies have shown that chronic use of prescription opioids leads to increased risk of cardiovascular events and pulmonary arterial hypertension. Indices of vascular age and arterial stiffness are increased in opioid dependent patients and the effects are more marked in women. We hypothesized that chronic exposure to exogenous opioids causes sex-specific vascular remodeling via suppression of actin depolymerization. Using culture pressure myographs, we performed continuous intraluminal infusion of morphine (10μM for 24 hours) in pressurized mesenteric resistance arteries (MRA, fifth order) from male and female mice (C57BL/6NTac, 10 weeks old; n=4). Intraluminal pressure curve showed that morphine reduced lumen diameter by 23% (without morphine: 185 ± 10μm vs. morphine: 143 ± 14μm*) in MRA from female mice, whereas little to no change was observed in MRA from male mice (Fig. 1). Cofilin is a key molecule in preventing stress fiber overassembly and is inactivated by extracellular signal-regulated kinases (ERK). Here, we observed that after 24-hour exposure, morphine (10μM) phosphorylated and inactivated cofilin 1.6 fold [AU: control: 0.59 ± 0.14 vs. morphine: 0.95 ± 0.10*, *p

Elevated blood pressure or hypertension is the single largest risk factor for cardiovascular diseases which are the leading cause of human deaths. Current clinical management of blood pressure is focused on restoring homeostasis of the host alone, without accounting for commensal gut microbiota. Recent evidence from the CARDIA study in humans and multiple studies using animal models suggest that development of hypertension in the host is associated with alterations in microbiotal communities. Here we examined whether microbiota is necessary for blood pressure and vascular homeostasis by functional evaluation of the gut homeostasis, hemodynamic, and vascular function of gnotobiotic rats reconstituted with microbiota to represent the complete holobiont. Gnotobiotic rats were used to represent incomplete holobionts. To reconstitute complete holobionts, gnotobiotic rats were co-housed with conventionally-raised rats. Acquisition of microbiota was evaluated through monitoring of gross ceca and fecal samples by metagenomic 16S sequencing. BP was recorded and vascular, renal, hepatic, cardiac and gut features were assessed using histology and ex vivo myography. Markers of innate immune effectors (Immune cell population, level of Lcn2, Gut permeability) were used to examine the nature and extent of host immune cell processes concomitantly occurring along with observations of host hemodynamics. Compared to the reconstituted holobiont represented by the animals exposed to microbiota, the incomplete-holobiont represented by gnotobiotic rats, had significantly lower BP (SBP of germ free:109±8 mmHg, SBP of conventionalized:138±10mmHg * ) and vascular contractility responses to phenylephrine (Emax (mN): germ-free: 6.9±1.3, GFC: 11.7±0.7*). Acute exposure of the host to microbiota reconstituted gut microbiotal communities, significantly boosted their gut epithelial cell proliferation, innate immune function and restored vascular contractility. These data indicate that in addition to the dependency of the host on microbiota for essential bodily functions such as digestion of plant-derived complex carbohydrates, the host is also dependent on microbiota for maintaining blood pressure and vascular function

Overconsumption of dietary salt is unequivocally linked with end organ damage. We have recently observed that a high salt (HS) diet induces liver dysfunction in the form of impaired synthesis of ketone body, β-hydroxybutyrate (βOHB), in Dahl salt-sensitive rats. However, the molecular mechanisms as to why βOHB is decreased after consuming a HS diet remains unknown, as well as if it contributes to vascular dysfunction. We hypothesized that a HS diet (2%) for 8 weeks would decrease systemic levels of βOHB in Dahl salt-resistant (R) rats and this would be associated with endothelial dysfunction. Furthermore, reconstitution of βOHB bioavailability by giving 1,3-Butanediol (1,3-BD; 20% v/v ) in the drinking water would improve endothelial function in HS fed Dahl R. As hypothesized, a HS diet decreased serum concentration (mM) of βOHB [Low salt (LS), non-fasting (NF): 0.31±0.02 vs. fasting (F): 0.96±0.04*; HS, NF: 0.33±0.02 vs. F: 0.77±0.04*#, *p0.05). Evolutionarily, autophagy functions to mobilize nutrients in times of starvation, including ketone bodies such as βOHB. Therefore, we hypothesized that inhibition of autophagy would decrease βOHB. Treatment with lysosomal alkalizer chloroquine (CQ), impeded the starvation-induced increase in βOHB (NF: 0.38±0.02 vs. F: 1.11±0.09* vs. F+CQ: 0.78±0.19*#, *p

Hypertension is the single prominent risk factor of epidemic proportions leading to cardiovascular disease and stroke, which comprise the top two reasons for mortality of humans in the modern age. Much of the attention for the unknown causes of hypertension was focused on genetics and dietary salt, but in recent years, host-microbiotal interaction is gaining importance. Host-microbiotal partnership is key for the generation of many bioactive molecules including bile acid (BA) metabolites. Primary bile acids are synthesized and conjugated by the host but deconjugated and further modified to secondary BA by gut commensal bacteria. BA metabolites serve as important ligands for host nuclear receptors and/or G-protein-coupled receptors. These receptors have pivotal roles in blood pressure regulation. However, the effect of the host-microorganism biliary network on blood pressure (BP) remains poorly characterized. Here we report that both dietary salt and genetic factors rewire the composition of bile acids and BP. Specific reductions in conjugated bile acids were noted in human hypertensives as well as in rats with hypertension. Conjugation of bile acids by the host alone, devoid of the deconjugation step by microbiota, was sufficient to decrease BP of germ-free rats compared to germ-free conventionalized rats. Nutritional restoration of the conjugation of bile acids with Taurine increased the availability of circulating conjugated bile acids as ligands and ameliorated host susceptibility to hypertension via BA nuclear receptors and G-protein-coupled receptors. Thus, hosts and their bacterial symbionts can control host BP homeostasis via the resulting pool of bile acid metabolites. Sources of funding: National Institutes of Health (R01HL143082).

Hypertension is an important clinical symptom of metabolic syndrome (MetS). Rats selectively bred for low intrinsic aerobic capacity (LCR) are animal models for MetS, and present with increased blood pressure and vascular dysfunction. In contrast, rats selected for high intrinsic aerobic capacity (HCR) display reduced vascular inflammation and no metabolic abnormalities. Two important enzymes for vascular inflammation and the resolution of inflammation are cyclooxygenase (COX) and lipoxygenase (LOX), respectively; however, it is unknown whether COX and LOX play a role in the vascular function of LCR and HCR. We hypothesized that mesenteric resistance arteries (MRA) from untrained LCR present increased COX activity, while arteries from HCR show decreased COX and increased LOX activity. Female (18-38 weeks old) LCR, HCR, and high response trained (HRT) rats, control, were used. HRT rats present higher intrinsic aerobic capacity than LCR, but lower than HCR. MRA were mounted onto a wire myograph. One-way ANOVA: p# vs. HCR; & vs. absence of indomethacin (INDO), a COX inhibitor. LCR rats showed increased periovarian fat pad [HRT: 0.95±0.1 (n=7) vs. LCR: 1.80±0.1* # (n=7) vs. HCR: 1.18±0.1 (n=7) (g)]. No significant differences were observed in the KCl (120 mM), acetylcholine, and sodium-nitroprusside-induced responses. However, LCR presented a decrease in phenylephrine (PE)-induced contraction [PE: E max %: HRT: 103±3 (n=8); LCR: 74±9* # (n=11); HCR: 112±5 (n=9)]. Inhibiting COX [INDO, 10 μM] decreased contraction in HRT arteries, but had little effect on HCR arteries. Contrarily, INDO abolished contraction in MRA from LCR [PE+INDO: E max %: HRT: 31±18 & (n=7); LCR: 2±0.9 & (n=8); HCR: 77±9 (n=8)]. Lipoxin (LXA4), a LOX-derived mediator for resolution of inflammation, induced contraction in MRA from HCR, but relaxation in LCR and HRT arteries [LXA4: E max %: HRT: -69±19 (n=4); LCR: -18±9 (n=3); HCR: 11±5 (n=4)*]. Thus, HCR are unresponsive to COX inhibition, suggesting a change from a normal inflammatory state to a higher resolution state. LCR display low-grade chronic inflammation via increased COX activity. These data reveal novel, inherited mechanisms for vascular physiology in high vs. low intrinsic aerobic capacity.

Background: Recent evidence supports that metabolic dysfunction underlies hypertension. Injection of kynurenate, a metabolite of tryptophan pathway, into the paraventricular nucleus of the hypothalamus (PVN) lowers blood pressure (BP). Intestinal absorption and metabolism of tryptophan are impacted by gut microbiota. Since gut-brain axis contributes to gut dysbiosis-inducd hypertension, we hypothesized that gut microbiota modulates the levels of kynurenic pathway metabolites that have central impact on BP regulation. Methods: We, for the first time, used 7 weeks old male Germ-free (GF) Spague Dawley (SD) rats (n=5) and GF rats co-housed with conventional SD rats for 10 days (GFC) (n=6). BP was measured by tail-cuff. Serum metabolites were quantified by 6495 triple quandrople mass spectrometryand data was normalized using isotoplic labelled compounds. The nucleus of the solitary tract (NTS), the principal sensory nucleus for peripheral changes, and the PVN, a relay center projecting sympathetic output based on the integrated afferent inputs from brain regions including NTS, were analyzed by microarray hybridization for mRNA expression. Results: Compared to the GF rats, GFC rats had significantly higher systolic (139 mmHg vs 115 mmHg, p p p p Cd74, Il1b, Cxcl1, Mmp14 ) in the PVN (gene ontology analysis, p Sox11, Tp53, Cdk6, Hoxb4, Foxo4, Cyr61 ) in the NTS (gene ontology analysis, p Conclusion: Colonization of gut microbiota in GF rats induced increased cell differentiation and synaptic plasticity in the NTS and immune responses in the PVN, indicating the restructured sensory neurons of the NTS and enhanced sympathetic output from the PVN. These are in line with increased levels of kynurenic acid and 3-hydroxy kynurenine, and BP, respectively, suggesting that BP regulation by the gut-brain axis may be mediated by kynurenic pathway.

Background Erectile dysfunction (ED) has been shown to be related with inflammatory markers in humans. Chronic infusion of TNF-α caused ED in mice while TNF-α knockout mice exhibited improvement in the relaxation of the corpus cavernosum (CC). Aim Since obesity triggers an inflammatory process, we aimed to investigate the hypothesis that in obesity, Toll-like receptor 9 (TLR9) activation leads to increased TNF-α levels and impairment in CC reactivity. Methods Four-week old male C57BL6 (WT) and TLR9 mutant (TLR9MUT) mice were fed a standard chow or high fat diet (HFD) for 12 weeks. Body weight and nonfasting blood glucose were analyzed. Contractile and relaxation responses of the CC were evaluated by electrical field stimulation and concentration response curves to phenylephrine and acetylcholine. Protein expression of nNOS, TNF-α, TNF-R1, TLR9 and MyD88 were measured by western blot. Plasma levels of TNF-α were measured by ELISA. Outcome In obesity, impaired cavernosal relaxation is associated with the activation of the innate immune system, by increasing the production of TNF-α through the activation of TLR9 in the macrophages. Results After 12 weeks of HFD both WT and TLR9MUT mice had increased body weight and nonfasting blood glucose compared to standard chow. In the CC, acetylcholine-induced relaxation was not changed. A trend to increased contraction to phenylephrine and KCl was seen in WT HFD only. electrical field stimulation-induced relaxation of the CC was decreased in WT HFD as well as nNOS expression in the CC of WT HFD, but not in TLR9MUT HFD. In the CC, protein expression of TLR9 and MyD88 was similar in all groups. While circulating levels of TNF-α presented only a trend to increase in mice fed HFD, the CC expression of TNF-α was increased only in WT HFD mice. Clinical Translation The innate immune system can be a target for the treatment of erectile complications in obesity. Strengths and Limitations This is the first study demonstrating that activation of TLR9 expressed in macrophages leads to impaired cavernosal relaxation. The main limitation of the study is the lack of understanding about the source/expression of the macrophages in the cavernous tissue. Further, herein, the experiments were performed only in isolated cavernous tissue (in vitro), thus the lack of knowledge on how the TLR9 modulates the in vivo response of the erectile tissue is another limitation of this study. Conclusion Our findings indicate that CC dysfunction observed in obesity is at least in part mediated by the production of TNF-α upon activation of TLR9 expressed in the macrophages. Priviero F, Calmasini F, Dela Justina V, et al. Macrophage-Specific Toll Like Receptor 9 (TLR9) Causes Corpus Cavernosum Dysfunction in Mice Fed a High Fat Diet. J Sex Med 2021;18:723-731.

Morphological and physiological changes in the vasculature have been described in the evolution and maintenance of hypertension. Hypertension-induced vascular dysfunction may present itself as a contributing, or consequential factor, to vascular remodeling caused by chronically elevated systemic arterial blood pressure. Changes in all vessel layers, from the endothelium to the perivascular adipose tissue (PVAT), have been described. This mini-review focuses on the current knowledge of the structure and function of the vessel layers, specifically muscular arteries: intima, media, adventitia, PVAT, and the cell types harbored within each vessel layer. The contributions of each cell type to vessel homeostasis and pathophysiological development of hypertension will be highlighted.

Mitochondrial damage-associated molecular patterns (mtDAMPs), such as mitochondrial DNA and N-formylated peptides, are endogenous molecules released from tissue after traumatic injury. mtDAMPs are potent activators of the innate immune system. They have similarities with bacteria, which allow mtDAMPs to interact with the same pattern recognition receptors and mediate the development of systemic inflammatory response syndrome (SIRS). Current recommendations for management of an open abdomen include returning to the operating room every 48 hours for peritoneal cavity lavage until definitive procedure. These patients are often critically ill and develop SIRS. We hypothesized that mitochondrial DAMPs are present in the peritoneal cavity fluid in this setting, and that they accumulate in the interval between washouts.We conducted a prospective pilot study of critically ill adult patients undergoing open abdomen management in the surgical and trauma intensive care units. Peritoneal fluid was collected daily from 10 open abdomen patients. Specimens were analyzed via quantitative polymerase chain reaction (qPCR) for mitochondrial DNA (mtDNA), via enzyme immunoassay for DNAse activity and via Western blot analysis for the ND6 subunit of the NADH: ubiquinone oxidoreductase, an N-formylated peptide.We observed a reduction in the expression of ND6 the day after lavage of the peritoneal cavity, that was statistically different from the days with no lavage (% change in ND6 expression, postoperative from washout: -50 ± 11 vs. no washout day, 42 ± 9; p0.05). Contrary to expectation, the mtDNA levels remained relatively constant from sample to sample. We then hypothesized that DNAse present in the effluent may be degrading mtDNA.These results indicate that the peritoneal cavity irrigation reduces the presence of mitochondrial DAMPs in the open abdomen. It is possible that increased frequency of peritoneal cavity lavage may lead to decreased systemic absorption of mtDAMPs, thereby reducing the risk of SIRS.Prospective study, Case Series, Level V.

Background Pediatric hypertension is recognized as an emerging global health concern. Although new guidelines are developed for facilitating clinical management, the reasons for the prevalence of hypertension in children remain unknown. Genetics and environmental factors do not fully account for the growing incidence of pediatric hypertension. Because stable bacterial flora in early life are linked with health outcomes later in life, we hypothesized that reshaping of gut microbiota in early life affects blood pressure ( BP ) of pediatric subjects. Methods and Results To test this hypothesis, we administered amoxicillin, the most commonly prescribed pediatric antibiotic, to alter gut microbiota of young, genetically hypertensive rats (study 1) and dams during gestation and lactation (study 2) and recorded their BP . Reshaping of microbiota with reductions in Firmicutes/Bacteriodetes ratio were observed. Amoxicillin treated rats had lower BP compared with untreated rats. In young rats treated with amoxicillin, the lowering effect on BP persisted even after antibiotics were discontinued. Similarly, offspring from dams treated with amoxicillin showed lower systolic BP compared with control rats. Remarkably, in all cases, a decrease in BP was associated with lowering of Veillonellaceae, which are succinate‐producing bacteria. Elevated plasma succinate is reported in hypertension. Accordingly, serum succinate was measured and found lower in animals treated with amoxicillin. Conclusions Our results demonstrate a direct correlation between succinate‐producing gut microbiota and early development of hypertension and indicate that reshaping gut microbiota, especially by depleting succinate‐producing microbiota early in life, may have long‐term benefits for hypertension‐prone individuals.

The endothelium produces many substances that can regulate vascular tone. Acetylcholine is a widely used pharmacological tool to assess endothelial function. In general, acetylcholine binds to G-protein coupled muscarinic receptors that mediate a transient elevation in intracellular, free calcium. This intracellular rise in calcium is responsible for triggering several cellular responses, including the synthesis of nitric oxide, endothelium- derived hyperpolarizing factor, and eicosanoids derived from arachidonic acid. Endothelial arachidonic acid metabolism is also an important signaling pathway for mediating inflammation. Therefore, in conditions with sustained and excessive inflammation such as hypertension, arachidonic acid serves as a substrate for the synthesis of several vasoconstrictive metabolites, predominantly via the cyclooxygenase and lipoxygenase enzymes. Cyclooxygenase and lipoxygenase products can then activate G-protein coupled receptors expressed on vascular smooth muscle cells to causes contractile responses. As a result, acetylcholine-induced contraction due to arachidonic acid is a commonly observed feature of endothelial dysfunction and vascular inflammation in hypertension. In this review, we will critically analyze the literature supporting this concept, as well as address the potential underlying mechanisms, including the possibility that arachidonic acid signaling is diverted away from the synthesis of pro-resolving metabolites in conditions such as hypertension.

The United States of America is currently undergoing an opioid crisis. According to the 2018 CDC Annual Surveillance Report of Drug Related Risks and Outcomes there were 78,840 hospitalizations and an estimated 140,077 emergency room visits for opioid related poisonings in 2015 alone. It has been shown that opioid exposure increases vascular age relative to chronological age. Further, long-term abuse of opioids leads to changes in regional cerebral blood flow. However, there is a lack of understanding about acute and chronic opioid abuse and how it affects microvasculature structure and function; specifically on myogenic tone, a fundamental mechanism to maintain blood flow to important tissues including the brain, kidneys, and more. We hypothesize that opioids cause an increase in proliferation of vascular smooth muscle cells (VSMC) leading to an increase in myogenic tone in mesenteric resistance arteries. To test this hypothesis, aortic vascular smooth muscle cells were cultured and treated with the endogenous opioid (ENO) beta-endorphin or the exogenous opioid (EXO) morphine. ENO treatment increased cellular proliferation with chronic exposure (greater than 72 hours). We measured a maximum proliferation at 120 hours and performed 2 way ANOVA with Bonferroni posttests [confluence (%): DMSO vehicle 20.4 ± 4.8, 1 nM 34.9 ± 7.4*, and 1 μM 29.9 ± 7.0; * p

According to the new AHA guidelines, over half of Americans have hypertension. Multiple factors contribute to hypertension including genetics, diet, stress, and infection. N-formyl peptides (NFPs) are a potent chemoattractant that bind formyl peptide receptors (FPR), an innate immune receptor. Interestingly, the only source of NFPs are bacteria and mitochondria, which evolved from bacteria. We have observed that FPR is expressed in arteries and its activation with bacterial (fMLPs) or mitochondrial (FMIT) NFPs induces vascular remodeling via actin polymerization. Given that increased gut permeability is present in hypertension, we questioned if bacterial NFPs derived from a leaky gut also play a role in hypertension. We hypothesized that bacterial NFPs activate FPR leading to vascular dysfunction and remodeling. We used male (6 weeks) Dahl Resistant (DR) and Sensitive (DS) rats on a low (0.3 %) or high salt (2 %) diet for 9 weeks. After 6 weeks some animals on high salt received water + antibiotic [neomycin (NEO), 0.5 g/L or amoxicillin (AMO), 50 mg/kg/day) for 3 weeks to decrease bacterial levels. Statistics: t-test *p2 ): DS 45,624 ± 1370 vs. DS + NEO 28,700 ± 8370* and DS + AMO 23,276 ± 9775*]. We concluded that low-grade chronic infection plays a role in vascular dysfunction and remodeling observed in hypertension.

Background and Purpose Erectile dysfunction (ED) is frequently encountered in patients with arterial hypertension and there is a recently uncovered functional correlation between the expression of ...

It has been suggested that hypertension results from a loss of immunological tolerance and the resulting autoimmunity may be an important underlying factor of its pathogenesis. This stems from the observations that many of the features involved in autoimmunity are also implicated in hypertension. Furthermore, the underlying presence of hypertension and cardiovascular disease are frequently observed in patients with autoimmune diseases. Antimalarial agents such as chloroquine are generally among the first line treatment options for patients with autoimmune diseases; however, whether they can improve a hypertensive phenotype in a genetic model of essential hypertension remains to be clarified. Therefore, we hypothesized that chloroquine treatment would improve endothelial function and lower blood pressure in spontaneously hypertensive rats (SHR). We treated adult SHR and Wistar-Kyoto rats (12 weeks old), as well as a group of young SHR (5 weeks old), with chloroquine (40 mg/kg/day via intraperitoneal injection) for 21 days. Chloroquine lowered blood pressure in adult SHR, but did not impede the development of high blood pressure in young SHR. In isolated mesenteric resistance arteries from SHR of both ages, chloroquine treatment inhibited cyclooxygenase-dependent contraction to acetylcholine, lowered vascular and systemic generation of reactive oxygen species, and improved nitric oxide bioavailability. Overall, these data reveal the anti-hypertensive mechanisms of chloroquine in the vasculature, which may be important for lowering risk of cardiovascular disease in patients with autoimmune diseases. Furthermore, it adds to the growing body of evidence suggesting that autoimmunity underlies hypertension.

Background Innate immune system responses to damage-associated molecular patterns (DAMPs) are involved in hypertension. However, the mechanisms of this contribution are not well understood. Circulating mitochondrial DNA is a DAMP that activates Toll-like receptor (TLR) 9 and is elevated in spontaneously hypertensive rats (SHR). Therefore, we hypothesized that lysosomotropic agent chloroquine (CQ) would impair TLR9 signaling, as well as prevent the development of hypertension and immune cell recruitment to the vasculature, in SHR. Methods Initially, adult SHR and Wistar-Kyoto (WKY) rats (12 weeks old), as well as a group of young SHR (5 weeks old), were treated with CQ (40mg/kg/day) or vehicle (saline) via intraperitoneal injections for 21 days and then TLR9-myeloid differentiation primary response protein (MyD88) signaling proteins were assessed in mesenteric resistance arteries (MRA) via western blot. Subsequently, young SHR and WKY were treated from 5-8 weeks of age and then were allowed to mature without further treatment. Blood pressure was measured pretreatment, posttreatment, and after maturation, and immune cell recruitment to the vasculature was measured via flow cytometry after maturation. Results In MRA from adult SHR, CQ increased the expression of MyD88-dependent proteins, whereas young SHR MRA exhibited a decrease. This inhibition was subsequently associated with suppression of blood pressure, as well as decreased counts of circulating T cells and vascular infiltrating leukocytes in SHR, when CQ was administered during the prehypertensive phase. Conclusions These data bring into question the participation of TLRs during the maintenance phase of hypertension and promote the exploration of innate immune system therapy during the critical developmental phase.

Introduction Erectile dysfunction (ED) is frequently encountered in patients with arterial hypertension and there is a recent functional correlation between the expression of thermoreceptor channels TRPM8 (melastatin 8) and alterations in blood pressure in hypertension. The aim of this study was to investigate the function of cold-sensing TRPM8 channel in internal pudendal artery (IPA) in both normotensive and hypertensive rats. Methods We performed experiments integrating physiological, pharmacological, biochemical and cellular techniques. Results TRPM8 channels are expressed in the IPA and in vascular smooth muscle cells from IPA. In addition, TRPM8 activation, by both a cooling compound icilin (82.1 ± 3.0%, n = 6) and cold temperature [thermal stimulus, basal tone (25 °C, 41.2 ± 3.4%, n = 5) or pre-contracted tone induced by phenylephrine (25 °C, 87.0 ± 3.6%, n = 7)], induced relaxation in IPA. Furthermore, the results showed that the concentration-response curve to icilin was significantly shifted to the right in different conditions, such as: the absence of the vascular endothelium, in the presence of L-NAME (10−4 M), or indomethacin (10−5 M) or by a combination of charybdotoxin (10-7 M) and apamin (5 × 10-6 M), and Y27632 (10-6 M). Interestingly, icilin-induced vasodilation was significantly higher in IPA from spontaneously hypertensive (SHR, E10−4M = 75.3 ± 1.7%) compared to wistar rats (E10−4M = 56.4 ± 2.6%), despite no changes in the TRPM8 expression in IPA between the strains, suggesting that the sensitivity of TRPM8 channels is higher in SHR. Conclusions These data demonstrate for the first time, the expression and function of TRPM8 channels in the IPA involving, at least in part, endothelium-derived relaxing factors and ROCK inhibition. Overall, this channel could potentially be a new target for the treatment of hypertension associated-ED.

Hypertension has been described as a condition of premature vascular aging, relative to actual chronological age. In fact, many factors that contribute to the deterioration of vascular function as we age are accelerated in hypertension. Nonetheless, the precise mechanisms that underlie the aged phenotype of arteries from hypertensive patients and animals remain elusive. Cellular senescence is an age-related physiologic process in which cells undergo irreversible growth arrest. Although controlled senescence negatively regulates cell proliferation and promotes tissue regeneration, uncontrolled senescence can contribute to disease pathogenesis by presenting the senescence-associated secretory phenotype, in which molecules such as proinflammatory cytokines, matrix metalloproteases, and reactive oxygen species are released into tissue microenvironments. This review will address and critically evaluate the current literature on the role of cellular senescence in hypertension, with particular emphasis on cells types that mediate and modulate vascular function and structure.

Hypertension (HTN) is associated with increased renal sodium (Na + ) reabsorption, vascular and endothelial dysfunction and an increase in cytokines, such as interleukin 6 (IL-6). We have previously shown that IL-6 increases blood pressure (BP), and increases the expression/activity of distal nephron Na + transport proteins via the mineralocorticoid receptor. We hypothesized that IL-6 increases Na + reabsorption, and induces endothelial dysfunction, leading to HTN. All mice were fed high salt food, and osmotic minipumps were implanted containing IL-6 (16 ng/hr, 0.01% BSA/saline), or vehicle/sham surgery (Ctl). To allow for early urine collection, IL-6 infusion was delayed by adding catheters to the minipumps during metabolic cage studies. Tail cuff plethysmography was used to measure systolic BP. Concentration response curves (CRC) to phenylephrine (Phe) and acetylcholine (ACh) were performed on aorta (Ao) and mesenteric arteries (Mes) from IL-6 or Ctl mice. IL-6 infused mice had increased BP by day 3 (D3), as compared to Ctl. Total urine volume (U Vol ) on D1 of infusion was reduced in IL-6 mice (U Vol 17.3±4 vs. 29.5±3 mL/day/per 100g body weight (BW); p+ excretion (U Na 81±19 IL-6 vs . 139±13 mg/day/BW Ctl; pNa were observed, and by D3 U Na levels were increasing in IL-6 infused mice. Similar trends for U Vol were observed. These results corresponded with our observed increases in BP. CRCs (D3) to Phe were not changed in Ao. However, Mes from IL-6 mice exhibited a surprising reduction in Phe-mediated contraction (136±7% vs. 155±5%; p50 -7.5±0.12 vs . -6.9±0.15; pNa /U Vol , followed by steady state recovery and pressure natriuresis (D3). Additionally, the increased relaxation and reduced contractility observed in the resistance arteries may suggest a compensatory response to the increased BP. Together, our studies suggest that in the early stages of HTN, cytokines may cause changes in renal Na + reabsorption via activation of distal Na + transporters.

Cardiovascular disease is the leading cause of death in cancer survivors. The short-term and long-term cardiotoxic effects of targeted chemotherapeutics is not well known. Everolimus, an inhibitor to mammalian target of rapamycin (mTOR) complex 1, is a targeted therapy approved for the treatment of metastatic breast, colon, renal cell and pancreatic cancer. Clinical retrospective reviews suggest heart failure and hypertension as cardiotoxic effects of everolimus. Our lab previously showed that everolimus exposure on normotensive vessels increased sensitivity to adrenergic stimuli in a time and concentration dependent manner. We hypothesized that everolimus leads to enhanced contractility and impaired relaxation in hypertensive vessels. We studied the effects of everolimus on the contractility and relaxation of mesenteric resistance arteries (MRA) of male Wistar rats (12-15 weeks old, n=4) and spontaneously hypertensive rats (SHR) (15-18 weeks old, n=2) on a wire myograph. Two concentrations of everolimus were evaluated, 0.1μM and 0.1nM, during a one-hour incubation. Normotensive MRA exposed to everolimus (0.1μM) showed a decreased contractile response to phenylephrine (LogEC50 + SEM, Ctrl:-5.627 + 0.07 vs Drug: -5.390 + 0.10) but no difference when exposed to 0.1nM. SHR MRA showed no difference in PE-induced contraction at 0.1μM (LogEC50 + SEM, Ctrl:-5.638 + 0.08 vs Drug:-6.286 + 0.36) or 0.1nM (LogEC50 + SEM, Ctrl:-5.638 + 0.08 vs Drug:-5.332 + 0.16). No differences were observed in endothelium dependent relaxation response to acetylcholine (ACh) for either drug concentration in the normotensive vessels; however, SHR MRA exhibited enhanced sensitivity to ACh with everolimus exposure (LogEC50 + SEM, Ctrl: -6.851 + 0.15 vs Drug:-9.233 + 0.37 at 0.1nM; LogEC50 + SEM, Ctrl: -6.851 + 0.15 vs Drug:-8.244 + 0.34 at 0.1μM). Our data show that everolimus affects contractility in normotensive vessels, while affecting the dilatory response in SHR vessels with an improvement in endothelium-dependent relaxation. Everolimus may be a good therapeutic agent for hypertensive cancer patients. However, long term treatments with this targeted therapy need to be evaluated for vascular dysfunction induction, which could be associated with cardiotoxicity.

Controversial evidence indicates that endogenous ouabain, a cardiotonic steroid can be synthesized in the adrenal cortex and hypothalamus and secreted as a hormone reaching nanomolar concentrations in the blood. The steroid has pathophysiological significance since its blood levels are positively correlated with blood pressure elevation. Inflammation of the vascular wall and endothelial dysfunction contribute to vascular stiffening in hypertension and it has been proposed that the endothelium is a site of action of ouabain. We hypothesized that ouabain would increase pro-inflammatory events in the endothelium. We observed that a low concentration ouabain (10nM; 24hr) sharply increased mRNA levels of the pro-inflammatory cytokines compared to control group (vehicle; 24h), TNF-α (59.9 fold vs con), IL-1β (3.23 fold vs con), COX2 (309.08 fold vs con), IL-6 (5.76 fold vs con) in cultured, human aortic endothelial cell (HAEC) and increased cyclooxygenase (COX2) expression (1.78 fold vs con). Simultaneously, a burst of nitric oxide (NO) was produced (1.2 fold vs con). Thus, low nanomolar concentrations of ouabain induced endothelial cell inflammation and dysfunction. Additionally, we observed that ouabain (10nM; 24hr) decreased mRNA expression of epidermal growth factor receptor (EGFR) (0.31 fold decrease vs con), and decreased the protein expression of EGFR (0.23 fold decrease vs con). But we found that ouabain induced the expression of non-receptor tyrosine kinase Src (2.27 fold vs con) and Toll-like receptor 4 (TLR4) (2.03 fold vs con), and activated the downstream signaling molecules, Myd88 (1.69 fold vs con), Akt (P-AKT/AKT, 4.31 fold vs con). Overall, the results provide a mechanism for ouabain-induced endothelial cell inflammation involving the inhibition of EGFR and the activation of Src and TLR4 to induce Akt signaling leading to secretion of pro-inflammatory cytokines.