Vascular dysfunction occurs prior to the onset of amyloid pathology and Aß plaque deposits colocalize with endothelial cells in the hippocampus of female APPswe/PSEN1dE9 mice.

Increasing evidence shows that cardiovascular diseases (CVDs) are associated with an increased risk of cognitive impairment and Alzheimer's diseases (AD). It is unknown whether systemic vascular dysfunction occurs prior to the development of AD, if this occurs in a sex-dependent manner, and whether endothelial cells play a role in the deposition of amyloid beta (Aß) peptides. We hypothesized that vascular dysfunction occurs prior to the onset of amyloid pathology, thus escalating its progression. Furthermore, endothelial cells from female mice will present with an exacerbated formation of Aß peptides due to an exacerbated pressure pulsatility. To test this hypothesis, we used a double transgenic mouse model of early-onset AD (APPswe/PSEN1dE9). We evaluated hippocampus-dependent recognition memory and the cardiovascular function by echocardiography and direct measurements of blood pressure through carotid artery catheterization. Vascular function was evaluated in resistance arteries, morphometric parameters in the aortas, and immunofluorescence in the hippocampus and aortas. We observed that endothelial dysfunction occurred prior to the onset of amyloid pathology irrespective of sex. However, during the onset of amyloid pathology, only female APP/PS1 mice had vascular stiffness in the aorta. There was elevated Aß deposition which colocalized with endothelial cells in the hippocampus from female APP/PS1 mice. Overall, these data showed that vascular abnormalities may be an early marker, and potential mediator of AD, but exacerbated aortic stiffness and pressure pulsatility after the onset of amyloid pathology may be associated with a greater burden of Aß formation in hippocampal endothelial cells from female but not male APP/PS1 mice.

A Single-Short Partial Reprogramming of the Endothelial Cells decreases Blood Pressure via attenuation of EndMT in Hypertensive Mice.

Background: Small artery remodeling and endothelial dysfunction are hallmarks of hypertension. Growing evidence supports a likely causal association between cardiovascular diseases and the presence of endothelial-to-mesenchymal transition (EndMT), a cellular transdifferentiation process in which endothelial cells (ECs) partially lose their identity and acquire additional mesenchymal phenotypes. EC reprogramming represents an innovative strategy in regenerative medicine to prevent deleterious effects induced by cardiovascular diseases. Methods: Using a partial reprogramming of ECs, via overexpression of Oct-3/4, Sox-2, and Klf-4 (OSK) transcription factors, we aimed to bring ECs back to a youthful phenotype in hypertensive mice. Primary ECs were infected with lentiviral vectors (LV) containing the specific EC marker cadherin 5 (Cdh5) and the fluorescent reporter enhanced green fluorescence protein (EGFP) with empty vector (LVCO) or with OSK (LV-OSK). Confocal microscopy and western blotting analysis were used to confirm the OSK overexpression. Cellular migration, senescence, and apoptosis were evaluated. Human aortic ECs (HAoECs) from male and female normotensive and hypertensive patients were analyzed after OSK or control treatments for their endothelial nitric oxide synthase (eNOS) levels, nitric oxide (NO), and genetic profile. Male and female normotensive (BPN/3J) and hypertensive (BPH/2J) mice were treated with an intravenous (i.v.) injection of LVCO or LV-OSK and evaluated 10 days post-infection. The blood pressure, cardiac function, vascular reactivity of small arteries, in vivo EGFP signal and EndMT inhibition were analyzed. Results: OSK overexpression induced partial EC reprogramming in vitro , and these cells showed endothelial progenitor cell (EPC)-like features with lower migratory capability. OSK treatment of hypertensive BPH/2J mice normalized blood pressure and resistance arteries hypercontractility, via the attenuation of EndMT and elastin breaks. EGFP signal was detected in vivo in the prefrontal cortex of both BPN/3J and BPH/2J-treated mice, but OSK induced angiogenesis only in male BPN/3J mice. OSK-treated human ECs from hypertensive patients showed high eNOS activation and NO production, with low ROS formation. Single-cell RNA analysis showed that OSK alleviated EC senescence and EndMT, restoring their phenotypes in human ECs from hypertensive patients. Conclusion: Overall, these data indicate that OSK treatment and EC reprogramming can decrease blood pressure and reverse hypertension-induced vascular damage.

The O-GlcNAc dichotomy: when does adaptation become pathological?

O-Linked attachment of ß-N-acetylglucosamine (O-GlcNAc) on serine and threonine residues of nuclear, cytoplasmic, and mitochondrial proteins is a highly dynamic and ubiquitous post-translational modification that impacts the function, activity, subcellular localization, and stability of target proteins. Physiologically, acute O-GlcNAcylation serves primarily to modulate cellular signaling and transcription regulatory pathways in response to nutrients and stress. To date, thousands of proteins have been revealed to be O-GlcNAcylated and this number continues to grow as the technology for the detection of O-GlcNAc improves. The attachment of a single O-GlcNAc is catalyzed by the enzyme O-GlcNAc transferase (OGT), and their removal is catalyzed by O-GlcNAcase (OGA). O-GlcNAcylation is regulated by the metabolism of glucose via the hexosamine biosynthesis pathway, and the metabolic abnormalities associated with pathophysiological conditions are all associated with increased flux through this pathway and elevate O-GlcNAc levels. While chronic O-GlcNAcylation is well associated with cardiovascular dysfunction, only until recently, and with genetically modified animals, has O-GlcNAcylation as a contributing mechanism of cardiovascular disease emerged. This review will address and critically evaluate the current literature on the role of O-GlcNAcylation in vascular physiology, with a view that this pathway can offer novel targets for the treatment and prevention of cardiovascular diseases.

Specialized Pro-resolving Mediator Improves Vascular Relaxation via Formyl Peptide Receptor-2.

BACKGROUND: The resolution of inflammation is an active phenomenon important for switching off inflammatory processes once the harmful stimuli are removed and facilitate the return to homeostasis. Specialized pro-resolving mediators (SPMs), such as lipoxin A4, resolvin D1, and resolvin E1, derived from ω-3 or ω-6 polyunsaturated fatty acids, are crucial for the resolution of inflammation. We hypothesized that SPMs are decreased in hypertension which contributes to the acetylcholine-induced contraction in resistance arteries, which are well known to be mediated by leukotrienes and prostaglandins. Moreover, treatment with SPMs will decrease this contraction via formyl peptide receptor-2 (FPR-2) in resistance arteries from spontaneously hypertensive rats (SHR). METHODS AND RESULTS: We performed a comprehensive eicosanoid lipid panel analysis, and our data showed for the first time that precursors of SPMs are decreased in SHR, limiting the production of SPMs and resolution of inflammation in vivo. This phenomenon was associated with an increase in lipid peroxidation in resistance arteries. Although SPMs did not abolish acetylcholine-induced contraction, these lipid mediators improved endothelial function in arteries from SHR via FPR-2 activation at nanomolar concentrations. SPMs also buffered TNF-α-induced reactive oxygen species generation in endothelial cells from C57Bl/6 mice. CONCLUSIONS: We suggest that FPR-2 and SPMs could be revealed as a new target or therapeutic agent to improve vascular function in arteries from hypertensive rats.

Soluble and insoluble protein aggregates, endoplasmic reticulum stress, and vascular dysfunction in Alzheimer's disease and cardiovascular diseases.

Dementia refers to a particular group of symptoms characterized by difficulties with memory, language, problem-solving, and other thinking skills that affect a person's ability to perform everyday activities. Alzheimer's disease (AD) is the most common form of dementia, affecting about 6.2 million Americans aged 65 years and older. Likewise, cardiovascular diseases (CVDs) are a major cause of disability and premature death, impacting 126.9 million adults in the USA, a number that increases with age. Consequently, CVDs and cardiovascular risk factors are associated with an increased risk of AD and cognitive impairment. They share important age-related cardiometabolic and lifestyle risk factors, that make them among the leading causes of death. Additionally, there are several premises and hypotheses about the mechanisms underlying the association between AD and CVD. Although AD and CVD may be considered deleterious to health, the study of their combination constitutes a clinical challenge, and investigations to understand the mechanistic pathways for the cause-effect and/or shared pathology between these two disease constellations remains an active area of research. AD pathology is propagated by the amyloid ß (Aß) peptides. These peptides give rise to small, toxic, and soluble Aß oligomers (SPOs) that are nonfibrillar, and it is their levels that show a robust correlation with the extent of cognitive impairment. This review will elucidate the interplay between the effects of accumulating SPOs in AD and CVDs, the resulting ER stress response, and their role in vascular dysfunction. We will also address the potential underlying mechanisms, including the possibility that SPOs are among the causes of vascular injury in CVD associated with cognitive decline. By revealing common mechanistic underpinnings of AD and CVD, we hope that novel experimental therapeutics can be designed to reduce the burden of these devastating diseases. Graphical abstract Alzheimer's disease (AD) pathology leads to the release of Aß peptides, and their accumulation in the peripheral organs has varying effects on various components of the cardiovascular system including endoplasmic reticulum (ER) stress and vascular damage. Image created with BioRender.com.

Carotid dysfunction in senescent female mice is mediated by increased α1A-adrenoceptor activity and COX-derived vasoconstrictor prostanoids.

α-Adrenergic receptors are crucial regulators of vascular hemodynamics and essential pharmacological targets for cardiovascular diseases. With aging, there is an increase in sympathetic activation, which could contribute to the progression of aging-associated cardiovascular dysfunction, including stroke. Nevertheless, there is little information directly associating adrenergic receptor dysfunction in the blood vessels of aged females. This study determined the role of a-adrenergic receptors in carotid dysfunction of senescent female mice (accelerated-senescence prone, SAMP8), compared with a nonsenescent (accelerated-senescence prone, SAMR1). Vasoconstriction to phenylephrine (Phe) was markedly increased in common carotid artery of SAMP8 [area under the curve (AUC), 527 ± 53] compared with SAMR1 (AUC, 334 ± 30, P = 0.006). There were no changes in vascular responses to the vasoconstrictor agent U46619 or the vasodilators acetylcholine (ACh) and sodium nitroprusside (NPS). Hyperactivity to Phe in female SAMP8 was reduced by cyclooxygenase-1 and cyclooxygenase-2 inhibition and associated with augmented ratio of TXA2/PGI2 release (SAMR1, 1.1 ± 0.1 vs. SAMP8, 2.1 ± 0.3, P = 0.007). However, no changes in cyclooxygenase expression were seen in SAMP8 carotids. Selective α1A-receptor antagonism markedly reduced maximal contraction, whereas α1D antagonism induced a minor shift in Phe contraction in SAMP8 carotids. Ligand binding analysis revealed a threefold increase of α-adrenergic receptor density in smooth muscle cells (VSMCs) of SAMP8 vs. SAMR1. Phe rapidly increased intracellular calcium (Cai2+) in VSMCs via the α1A-receptor, with a higher peak in VSMCs from SAMP8. In conclusion, senescence intensifies vasoconstriction mediated by α1A-adrenergic signaling in the carotid of female mice by mechanisms involving increased Cai2+ and release of cyclooxygenase-derived prostanoids.NEW & NOTEWORTHY The present study provides evidence that senescence induces hyperreactivity of α1-adrenoceptor-mediated contraction of the common carotid. Impairment of α1-adrenoceptor responses is linked to increased Ca2+ influx and release of COX-derived vasoconstrictor prostanoids, contributing to carotid dysfunction in the murine model of female senescence (SAMP8). Increased reactivity of the common carotid artery during senescence may lead to morphological and functional changes in arteries of the cerebral microcirculation and contribute to cognitive decline in females. Because the elderly population is growing, elucidating the mechanisms of aging- and sex-associated vascular dysfunction is critical to better direct pharmacological and lifestyle interventions to prevent cardiovascular risk in both sexes.

The janus face of ketone bodies in hypertension.

Hypertension is the most important risk factor for the development of terminal cardiovascular diseases, such as heart failure, chronic kidney disease, and atherosclerosis. Lifestyle interventions to lower blood pressure are generally desirable prior to initiating pharmaceutical drug treatments, which may have undesirable side effects. Ketogenic interventions are popular but the scientific literature supporting their efficacy is specific to certain interventions and outcomes in animal models and patient populations. For example, although caloric restriction has its own inherent difficulties (e.g. it requires high levels of motivation and adherence is difficult), it has unequivocally been associated with lowering blood pressure in hypertensive patients. On the other hand, the antihypertensive efficacy of ketogenic diets is inconclusive, and this is surprising, given that these diets have been largely helpful in mitigating metabolic syndrome and promoting longevity. It is possible that side effects associated with ketogenic diets (e.g. dyslipidemia) aggravate the hypertensive phenotype. However, given the recent data from our group, and others, reporting that the most abundant ketone body, ß-hydroxybutyrate, can have positive effects on endothelial and vascular health, there is hope that ketone bodies can be harnessed as a therapeutic strategy to combat hypertension. Therefore, we conclude this review with a summary of the type and efficacy of ketone supplements. We propose that ketone supplements warrant investigation as low-dose antihypertensive therapy that decreases total peripheral resistance with minimal adverse side effects.

Low-dose 1,3-butanediol reverses age-associated vascular dysfunction independent of ketone body ß-hydroxybutyrate.

With an aging global population, identifying novel therapeutics are necessary to increase longevity and decrease the deterioration of essential end organs such as the vasculature. Secondary alcohol, 1,3-butanediol (1,3-BD), is commonly administered to stimulate the biosynthesis of the most abundant ketone body ß-hydroxybutyrate (ßHB), in lieu of nutrient deprivation. However, suprapharmacological concentrations of 1,3-BD are necessary to significantly increase systemic ßHB, and 1,3-BD per se can cause vasodilation at nanomolar concentrations. Therefore, we hypothesized that 1,3-BD could be a novel antiaging therapeutic, independent of ßHB biosynthesis. To test this hypothesis, we administered a low-dose (5%) 1,3-BD to young and old Wistar-Kyoto (WKY) rats via drinking water for 4 wk and measured indices of vascular function and metabolism posttreatment. We observed that low-dose 1,3-BD was sufficient to reverse age-associated endothelial-dependent and -independent dysfunction, and this was not associated with increased ßHB bioavailability. Further analysis of the direct vasodilator mechanisms of 1,3-BD revealed that it is predominantly an endothelium-dependent vasodilator through activation of potassium channels and nitric oxide synthase. In summary, we report that 1,3-BD, at a concentration that does not stimulate ßHB biosynthesis, could be a nutraceutical that can reverse the age-associated decline in vascular function. These results emphasize that 1,3-BD has multiple, concentration-dependent mechanisms of action. Therefore, we suggest alternative approaches to study the physiological and cardiovascular effects of ßHB.NEW & NOTEWORTHY 1,3-Butanediol (1,3-BD) is often administered to stimulate the biosynthesis of the most abundant ketone body, ß-hydroxybutyrate (ßHB), and its purported salubrious effects. Here, we report that a low dose of 1,3-BD (5%) is sufficient to reverse age-associated vascular dysfunction, independent of ßHB. Therefore, low-dose 1,3-BD could be a novel therapeutic to increase blood flow and improve the quality of life in the elderly.

Ketone body ß-hydroxybutyrate is an autophagy-dependent vasodilator.

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, ß-hydroxybutyrate (ßHB), 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 ßHB can be impaired by preventing autophagy. Subsequently, ßHB caused potent vasodilation via potassium channels but not Gpr109a. Finally, we observed that chronic consumption of a high-salt diet negatively regulates both ßHB biosynthesis and hepatic autophagy and that reconstitution of ßHB 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.

Opioids Cause Sex-Specific Vascular Changes via Cofilin-Extracellular Signal-Regulated Kinase Signaling: Female Mice Present Higher Risk of Developing Morphine-Induced Vascular Dysfunction than Male Mice.

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.

Physiologic, Metabolic, and Toxicologic Profile of 1,3-Butanediol.

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. ß-hydroxybutyrate (ßHB) is the most abundant ketone body. The secondary alcohol, 1,3-butanediol (1,3-BD), is commonly administered to raise ßHB bioavailability in vivo and in the absence of nutrient deprivation. However, the concentration of 1,3-BD that yields a systemic concentration of ßHB 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 ßHB concentration, physiologic, metabolic, and toxicologic parameters were measured. We report that only 20% 1,3-BD significantly elevates the systemic and urinary concentrations of ßHB. 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 ßHB 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, ß-hydroxybutyrate (ßHB), and its purported salubrious effects. This article reports that suprapharmacological concentrations of 1,3-BD are necessary to yield a systemic concentration of ßHB 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 ßHB.

Ethanol: striking the cardiovascular system by harming the gut microbiota.

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.

Guidelines for the measurement of vascular function and structure in isolated arteries and veins.

The measurement of vascular function in isolated vessels has revealed important insights into the structural, functional, and biomechanical features of the normal and diseased cardiovascular system and has provided a molecular understanding of the cells that constitutes arteries and veins and their interaction. Further, this approach has allowed the discovery of vital pharmacological treatments for cardiovascular diseases. However, the expansion of the vascular physiology field has also brought new concerns over scientific rigor and reproducibility. Therefore, it is appropriate to set guidelines for the best practices of evaluating vascular function in isolated vessels. These guidelines are a comprehensive document detailing the best practices and pitfalls for the assessment of function in large and small arteries and veins. Herein, we bring together experts in the field of vascular physiology with the purpose of developing guidelines for evaluating ex vivo vascular function. By using this document, vascular physiologists will have consistency among methodological approaches, producing more reliable and reproducible results.

Intrinsic exercise capacity induces divergent vascular plasticity via arachidonic acid-mediated inflammatory pathways in female rats.

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.

FPR-1 (Formyl Peptide Receptor-1) Activation Promotes Spontaneous, Premature Hypertension in Dahl Salt-Sensitive Rats.

[Figure: see text].

Macrophage-Specific Toll Like Receptor 9 (TLR9) Causes Corpus Cavernosum Dysfunction in Mice Fed a High Fat Diet.

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.