Wire Myography Research Articles

TMEM16A Antagonism: Therapeutic Potential with Desensitization of β-agonist Responsiveness in Asthma.

The efficacy of β-agonists in asthma is severely limited by β-adrenoceptor desensitization which results in poorly managed symptoms and refractory bronchoconstriction. Thus, there is a need to identify novel therapeutic pathways and to clarify the relationship between novel therapeutics and functional β-adrenoceptor responsiveness. We have previously demonstrated that acute antagonism of the calcium activated chloride channel, transmembrane member 16A (TMEM16A), relaxes airway smooth muscle (ASM). We sought to determine the efficacy and role of TMEM16A antagonism in the context of desensitization β - adrenoceptor responsiveness. For these studies, we exposed murine tracheal rings on wire myography and precision cut lung slices to contractile mediators in the presence or absence of TMEM16A antagonists and β-agonists with or without prior β-adrenoceptor desensitization. Contractile studies were also performed with human tracheal and bronchial ASM. Finally, the ability of TMEM16A antagonism to prevent desensitization of β2-adrenoceptor-induced cyclic AMP synthesis was measured in human ASM cells. From these studies we demonstrate that acute TMEM16A antagonism is effective in relaxing β-agonist desensitized ASM in central and peripheral murine ASM and human ASM. Furthermore, we demonstrate that chronic pretreatment with TMEM16A antagonists prevents functional desensitization of β-agonist responsiveness in mouse and human upper airways and prevents desensitization of β-agonist-mediated cyclic AMP production in human ASM cells. Taken together, the present study demonstrates a favorable therapeutic profile of TMEM16A antagonism for airway smooth muscle relaxation despite functional desensitization of β-agonist responsiveness which may be a novel therapeutic approach in the face of β-adrenoceptor tachyphylaxis.

Abstract 4134475: Effects of E-cigarette Vaping on Endothelial Function in Diet-Induced Obese Mice

Introduction: Although electronic cigarettes (e-cigs) have been known to increase the risk of vascular complications, little is known about whether e-cig represents an independent risk factor for the development and worsening of obesity and its associated vascular diseases. We hypothesize that a high-fat diet (HFD) enhances e-cig-induced vascular complications. Methods: Male and female eight-week-old C57Bl/6J mice were fed normal chow (NC) or HFD for 15 weeks before being exposed to e-cigs (PG/VG only [PGVG] or PG/VG + 24 mg/ml nicotine [Nic]) or fresh air (FA) for 12 weeks (n=6-8). At the end of the experiments, glucose level and thoracic aortic responses to phenylephrine (PE), acetylcholine (ACh), and sodium nitroprusside (SNP) were assessed via the glucose tolerance test (GTT) and wire myography, respectively. Total RNA from male aortas was extracted and RNA-seq was utilized to identify the transcriptomic changes in our diet-induced obesity mouse model subjected to e-cig vapors (n=4). Results: Compared to NC with FA, HFD alone caused glucose intolerance as shown by the GTT, which was improved only by exposure to nicotine-containing e-cigs (HFD+Nic). Similar improvement in glucose intolerance were found in the NC group with e-cigs (PGVG or Nic). In addition, both NC and HFD-fed male mice exhibited endothelial dysfunction characterized by a decrease in the ACh-dependent (endothelium) relaxation of PE-induced contractions. Importantly, nicotine-containing e-cig exposure (Nic) dramatically reduced the sensitivity of the aorta in the HFD group (HFD+Nic) to both ACh and SNP, while it only affected the sensitivity and maximum relaxation to ACh in the male NC group (NC+Nic). There were no significant differences in the vasoreactivity of female mice exposed to e-cig or HFD. Finally, RNA-seq results suggested that e-cig-treated male mice had altered expression of genes related to endothelial and smooth muscle function and metabolism, particularly fatty acid metabolism, which was also significant with the addition of HFD. Conclusion: Collectively, our results suggest that acute e-cig exposure results in significant vascular dysfunction in male mice, which is worsened in HFD mice. The identification of genes dysregulated in mice exposed to e-cigs provides novel insights into vascular biology and could have a significant impact on our understanding of vascular complications associated with e-cig usage and provide insights into its contributions to obesity and diabetes.

Abstract 4139148: Mechanisms of Maternal Cardiovascular Disease Development in a Novel Mouse Model of Pregnancy-Induced Diabetes

Background: Women with gestational diabetes mellitus (GDM) are more likely to develop cardiovascular disease (CVD) within 3 to 10 years following pregnancy. Endothelial dysfunction is a mechanism implicated in both hyperglycemia and CVDs, and it may constitute the missing link in this interaction. One reason for the lack of progress into the cardiovascular complications of GDM is the need for appropriate preclinical models that mimic human GDM. Also, the exact mechanisms mediating increased future cardiovascular risk in maternal with pregnancy-induced diabetes is unclear. Aim: To determine the mechanisms underlying cardiovascular disease in hormonal model of GDM. Methods: Sixteen week-old C57BL/6J female mice were treated with insulin receptor antagonist (S961) or saline (n=4) from the second trimester of pregnancy until delivery to develop a model of GDM. Glucose tolerance test was determined by glucometer and fasting insulin by colorimetry. Cardiac structure and function were measured by echocardiography (VEVO 3100) and pulse wave velocity was measured by Pulse Wave Doppler. Exercise tolerance test was performed on treadmill. Plasma endothelin-1 and nitrite were measured by ELISA and colorimetric assay. Aortic SIRT-1 was determined by western blot and aortic vascular reactivity was determined by wire myograph. Results: S961-induced GDM mice exhibit hyperglycemia (169.2 ± 3.6 vs. 105.5 ± 10.8mg/dL, P<0.004 ) and hyperinsulinemia (1.9 ± 0.2 vs. 0.8 ± 0.1ng/ml , p<0.01 ) which mimics the condition observed in human GDM. Interestingly, S961-induced GDM mice went on to develop systolic and diastolic dysfunction, vascular stiffness (3.7 ± 0.1 vs. 3.1 ± 0.08m/s, p<0.005 ), exercise intolerance and increased thickness of the cardiac wall at 10 weeks post-delivery. Plasma ET-1 was elevated in our model during pregnancy (28.1 ± 1.7 vs. 5.2 ± 0.2pg/ml, p<0.002 ) and at 10 weeks post-delivery (19.9 ± 2.9 vs. 6.7 ± 0.7pg/ml, p<0.05 ). Circulating NO and aortic expression of sirtuin-1 (SIRT 1), that is involved in endothelial longevity is reduced in GDM mice. Additionally, vascular relaxation to acetylcholine was attenuated in S961-induced GDM mice. Conclusion: We have developed a novel mouse model of GDM similar to that observed in pregnant women by treatment with S961. Moreover, S961 treated mice develop cardiac dysfunction and vascular stiffness at 10 weeks post-delivery. Alterations in ET-1, SIRT 1, and NO levels may be responsible for the development of CVD in this model.

OxLDL/LOX-1 mediated sex, age, stiffness, and endothelial dependent alterations in mouse thoracic aortic vascular reactivity.

Elevated plasma levels of oxidized low-density lipoprotein (oxLDL) are a risk factor and key component that accelerates and worsens cardiovascular disease fueling inflammation, plaque buildup and vascular damage. OxLDL can elicit its detrimental action via lectin-like oxLDL receptor 1 (LOX-1). In this study, we determined whether oxLDL, via LOX-1, alters aortic vascular reactivity and determined if sex and age differences exist. Thoracic aortic endothelium-intact or -denuded ring segments were isolated from 7 to 12months old intact C57BL/6J female and male mice and pre-incubated with oxLDL ex vivo (50ug/dL; 2h). Using wire myography, cumulative concentration-response curves to phenylephrine (PE) were generated to determine contractile responses. From these curves, the EC50 was determined and used to contract rings to assess acetylcholine (ACh) dependent relaxation. Calculated aortic stiffness and remodeling were also assessed. BI-0115 (10 M; selective LOX-1 inhibitor) was used to determine LOX-1 dependence. We observed differential sex, age, endothelial cell, and LOX-1 dependent alterations to the efficacy of PE-induced contractile responses and ACh-mediated vasorelaxation in thoracic aortic rings following oxLDL exposure. Additionally, we observed a distinct sex and age effect on thoracic aortic stiffness following exposure to oxLDL. There was also a sex effect on calculated vessel diameter, as well as an age effect on oxLDL-mediated aortic remodeling that was LOX-1 dependent. Thus, LOX-1 inhibition and the resulting attenuation of oxLDL/endothelial-mediated alterations in aortic function suggests that there are differential sex differences in the role of oxLDL/LOX-1 in the thoracic aorta of middle-aged male and female mice. NEW and NOTEWORTHY. We investigated the effects of oxLDL via the LOX-1 receptor on murine thoracic aortic vasoreactivity, stiffness, and remodeling across age and sex. Acute exposure to oxLDL led to altered vasoreactivity, endothelial dysfunction, and changes in aortic stiffness and remodeling. These effects were in-part age, sex, endothelial, and LOX-1 dependent. This study reveals potential complex interactions in oxLDL/LOX-1-mediated vascular responses that could serve as potential therapeutic intervention for vascular diseases such as atherosclerosis and stroke.

Maternal adiponectin restores cholinergic vasodilation in resistance vessels from adult offspring exposed to maternal obesity in utero.

Maternal obesity increases the risk of cardiovascular and metabolic disease in the offspring both during childhood and adult life. Pregnant women and mice with obesity have lower circulating levels of adiponectin (ADN) compared to lean controls. ADN is an adipokine involved in regulating energy metabolism, vascular function, and placental function. We hypothesized that offspring of obese mice have impaired resistance artery function, which can be prevented by restoration of normal circulating ADN levels in obese dams during late pregnancy. Adult female mice were fed either control or obesogenic diet and mated with control diet-fed males. Control dams received a continuous infusion of phosphate saline buffer (PBS) during late pregnancy whereas obese females received either PBS or ADN. After weaning, offspring were fed a control diet. Mesenteric arteries (MsA) were dissected from adult offspring and mounted in a wire myograph or fixed for histology. MsA responses to vasoconstrictors (phenylephrine and endothelin-1) were not different between infusion groups. However, the vasodilatory responses to acetylcholine were reduced in offspring from obese dams as compared to control-fed dams. ADN supplementation during pregnancy restored the cholinergic vasodilatory responses of resistance vessels in offspring from obese dams. These observations suggest that normalizing circulating adiponectin levels in pregnancies complicated by obesity prevents in utero programming of vascular dysfunction in the offspring.

Acute Chikungunya Infection Induces Vascular Dysfunction by Directly Disrupting Redox Signaling in Endothelial Cells.

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

Lack of AMP-activated protein kinase-α1 reduces nitric oxide synthesis in thoracic aorta perivascular adipose tissue

ObjectivePerivascular adipose tissue (PVAT) releases anti-contractile bioactive molecules including NO. PVAT anti-contractile activity is attenuated in mice lacking AMPKα1 (AMP-activated protein kinase-α1). As AMPK regulates endothelial NO synthase (eNOS) activity in cultured cells, NO synthesis was examined in PVAT from AMPKα1 knockout (KO) mice. Methods and resultsEndothelium-denuded thoracic or abdominal aortic rings were isolated from wild type (WT) and KO mice. NOS inhibition enhanced vasoconstriction in PVAT-intact thoracic aortic rings from mice of either genotype yet had no effect on abdominal rings as assessed by wire myography. Thoracic aorta PVAT exhibited increased NO production, NOS activity and levels of the brown adipose tissue marker uncoupling protein-1 (UCP1) compared to abdominal PVAT. In KO mice, NO production was significantly reduced in thoracic but not abdominal PVAT. Reduced NO production in KO thoracic PVAT was not due to altered levels or phosphorylation of eNOS but was associated with increased caveolin-1:eNOS association and caveolin-1 Tyr14 phosphorylation. A peptide that disrupts eNOS:caveolin-1 association increased NO synthesis and reduced vasoconstriction of PVAT-intact thoracic but not abdominal aortic rings. KO thoracic PVAT also exhibited reduced UCP1 levels. ConclusionsMurine thoracic aorta PVAT exhibits higher NO synthesis and UCP1 levels than abdominal aortic PVAT. Downregulation of AMPK suppresses NO synthesis which may contribute to the reduced anticontractile activity and reduced brown adipose tissue phenotype of KO thoracic PVAT. The mechanism underlying the effect of AMPK downregulation likely results from increased caveolin-1:eNOS association associated with caveolin-1 Tyr14 phosphorylation.

Aged Gut Microbiome Induces Metabolic Impairment and Hallmarks of Vascular and Intestinal Aging in Young Mice.

Aging, an independent risk factor for cardiometabolic diseases, refers to a progressive deterioration in physiological function, characterized by 12 established hallmarks. Vascular aging is driven by endothelial dysfunction, telomere dysfunction, oxidative stress, and vascular inflammation. This study investigated whether aged gut microbiome promotes vascular aging and metabolic impairment. Fecal microbiome transfer (FMT) was conducted from aged (>75 weeks old) to young C57BL/6 mice (8 weeks old) for 6 weeks. Wire myography was used to evaluate endothelial function in aortas and mesenteric arteries. ROS levels were measured by dihydroethidium (DHE) staining and lucigenin-enhanced chemiluminescence. Vascular and intestinal telomere function, in terms of relative telomere length, telomerase reverse transcriptase expression and telomerase activity, were measured. Systemic inflammation, endotoxemia and intestinal integrity of mice were assessed. Gut microbiome profiles were studied by 16S rRNA sequencing. Some middle-aged mice (40-42 weeks old) were subjected to chronic metformin treatment and exercise training for 4 weeks to evaluate their anti-aging benefits. Six-week FMT impaired glucose homeostasis and caused vascular dysfunction in aortas and mesenteric arteries in young mice. FMT triggered vascular inflammation and oxidative stress, along with declined telomerase activity and shorter telomere length in aortas. Additionally, FMT impaired intestinal integrity, and triggered AMPK inactivation and telomere dysfunction in intestines, potentially attributed to the altered gut microbial profiles. Metformin treatment and moderate exercise improved integrity, AMPK activation and telomere function in mouse intestines. Our data highlight aged microbiome as a mechanism that accelerates intestinal and vascular aging, suggesting the gut-vascular connection as a potential intervention target against cardiovascular aging and complications.

The modified 6-chromanol SUL-238 protects against accelerated vascular aging in vascular smooth muscle Ercc1-deficient mice

Introduction: Vascular aging is marked by increased mitochondrial reactive oxygen species (ROS) production, which leads to decreased nitric oxide (NO)-mediated vasodilation. Loss of NO can be partially compensated by endothelium-derived hyperpolarization (EDH), which partly relies on increased mitochondrial Ca2+ release to maintain vascular dilation. Thus, intervention in mitochondria may target both NO and EDH signaling to alleviate aging-related vascular dysfunction. DNA damage by mitochondrial ROS is an important cause of organismal aging. Previous work showed that local vascular Ercc1 knockout dramatically accelerates vascular aging. The aim of the study was to investigate the effect of chronic treatment with the modified 6-chromanol, SUL-238, an inhibitor of mitochondrial reverse electron flux and ROS, in a mouse model of accelerated vascular smooth muscle aging induced by DNA repair endonuclease Ercc1 knockout (SMC-KO). Aim: The aim of the study was to investigate the effect of chronic treatment with the modified 6-chromanol, SUL-238, an inhibitor of mitochondrial reverse electron flux and ROS, in a mouse model of accelerated vascular smooth muscle aging induced by DNA repair endonuclease Ercc1 knockout (SMC-KO). Methods: SMC-KO mice and healthy wild-type littermates received SUL-238 (90 mg/kg/day) in drinking water from 12 to 22 weeks of age. At the age of 21 weeks, arterial stiffness was measured in vivo with echography and they were euthanized at the age of 22 weeks. Ex vivo vascular function was assessed in wire myography setups and mitochondrial function of the thoracic aorta was assessed using a seahorse assay. Results: SMC-KO mice showed reduced EDH-mediated vasodilation, elevated arterial stiffness, and increased elastin breaks at 22 weeks of age compared to their wild-type littermates. SUL-238 improved EDH, thus restoring aortic and mesenteric relaxation in SMC-KO mice. Furthermore, the number of elastin breaks was reduced and arterial stiffness normalized after treating SMC-KO mice with SUL-238. Mitochondrial respiration measured in the aorta was not different between the groups. Conclusion: Chronic treatment with SUL-238 alleviates features of vascular aging, including decreased vasodilation and increased arterial stiffness. SUL-238 seems to have a more general effect on aging rather than involving a direct coupling between mitochondrial function and vascular signaling. SUL-238 is the first small-molecule drug reported to increase EDH after chronic treatment.

Resistance artery vasodilator pathways involved in the antihypertensive effects of cocoa shell extract in rats exposed to fetal undernutrition.

Fetal undernutrition establishes the foundations for hypertension development, with oxidative stress being a key hallmark. A growing interest in nutraceuticals for treating hypertension and environmental waste concerns prompted the present study aiming to evaluate whether supplementation with a polyphenol enriched extract from cocoa shell (CSE), a by-product from the chocolate industry with antioxidant properties, reduces hypertension of developmental origin, thus improving mesenteric resistance artery (MRA) vasodilatation. Adult male and female offspring from rats exposed to 50% food restriction from mid-gestation (maternal undernutrition, MUN) and controls were used. Supplementation was given through a gelatine (vehicle, VEH) or containing CSE (250mgkg-1day-1) 5daysweek-1 for 3weeks. Systolic blood pressure (SBP) was assessed by tail-cuff plethysmography. MRA function was studied by wire myography, and superoxide anion and nitric oxide were investigated by fluorescent indicators and confocal microscopy. Compared to control-VEH, MUN-VEH males showed significantly higher SBP, reduced MRA as well as relaxation to ACh, sodium nitroprusside and the AMPK agonist 5-aminoimidazole-4-carboxamide riboside, but not to isoproterenol. In MUN males, endothelial endothelium-derived hyperpolarizing factor and nitric oxide were unaltered, but MRA released a vasoconstrictor prostanoid and produced higher levels of superoxide anion. CSE normalized blood pressure and improved all above-mentioned MRA alterations in MUN males without an effect on control counterparts, except the reduction of superoxide anion. MUN-VEH females were normotensive and only showed a tendency towards larger superoxide anion production, which was abolished by CSE. CSE supplementation reduces SBP improving endothelium-dependent and independent MRA vasodilatation, related to local superoxide anion reduction, being a potential nutraceutical ingredient to counteract hypertension, in addition to contributing to the circular economy. KEY POINTS: Fetal undernutrition induces hypertension in males associated with deficient resistance artery vasodilatation, being normalized by cocoa shell extract (CSE). Release of a cyclooxygenase-derived contractile factor is the main endothelial alteration, which is abolished by CSE. AMPK and soluble guanylyl cyclase-mediated relaxation are also reduced in smooth muscle cells from maternal undernutrition resistance arteries, being improved by CSE. Vascular oxidative damage caused by excess superoxide anion generation can account for impaired vasodilatation, which is improved by CSE. The capacity of CSE to improve relaxation is probably related to its antioxidant bioactive factors, and thus cocoa shell is a potential food by-product to treat hypertension.

Endothelial Cells Increase Mesenchymal Stem Cell Differentiation in Scaffold-Free 3D Vascular Tissue.

In this study, we present a versatile, scaffold-free approach to create ring-shaped engineered vascular tissue segments using human mesenchymal stem cell-derived smooth muscle cells (hMSC-SMCs) and endothelial cells (ECs). We hypothesized that incorporation of ECs would increase hMSC-SMC differentiation without compromising tissue ring strength or fusion to form tissue tubes. Undifferentiated hMSCs and ECs were co-seeded into custom ring-shaped agarose wells using four different concentrations of ECs: 0%, 10%, 20%, and 30%. Co-seeded EC and hMSC rings were cultured in SMC differentiation medium for a total of 22 days. Tissue rings were then harvested for histology, Western blotting, wire myography, and uniaxial tensile testing to examine their structural and functional properties. Differentiated hMSC tissue rings comprising 20% and 30% ECs exhibited significantly greater SMC contractile protein expression, endothelin-1 (ET-1)-meditated contraction, and force at failure compared with the 0% EC rings. On average, the 0%, 10%, 20%, and 30% EC rings exhibited a contractile force of 0.745 ± 0.117, 0.830 ± 0.358, 1.31 ± 0.353, and 1.67 ± 0.351 mN (mean ± standard deviation [SD]) in response to ET-1, respectively. Additionally, the mean maximum force at failure for the 0%, 10%, 20%, and 30% EC rings was 88.5 ± 36. , 121 ± 59.1, 147 ± 43.1, and 206 ± 0.8 mN (mean ± SD), respectively. Based on these results, 30% EC rings were fused together to form tissue-engineered blood vessels (TEBVs) and compared with 0% EC TEBV controls. The addition of 30% ECs in TEBVs did not affect ring fusion but did result in significantly greater SMC protein expression (calponin and smoothelin). In summary, co-seeding hMSCs with ECs to form tissue rings resulted in greater contraction, strength, and hMSC-SMC differentiation compared with hMSCs alone and indicates a method to create a functional 3D human vascular cell coculture model.

Genetic variation in CCDC93 is associated with elevated central systolic blood pressure, impaired arterial relaxation, and mitochondrial dysfunction.

Genetic studies of blood pressure (BP) traits to date have been performed on conventional measures by brachial cuff sphygmomanometer for systolic BP (SBP) and diastolic BP, integrating several physiologic occurrences. Genetic associations with central SBP (cSBP) have not been well-studied. Genetic discovery studies of BP have been most often performed in European-ancestry samples. Here, we investigated genetic associations with cSBP in a Chinese population and functionally validated the impact of a novel associated coiled-coil domain containing 93 (CCDC93) gene on BP regulation. An exome-wide association study (EWAS) was performed using a mixed linear model of non-invasive cSBP and peripheral BP traits in a Han Chinese population (N = 5,954) from Beijing, China genotyped with a customized Illumina ExomeChip array. We identified four SNP-trait associations with three SNPs, including two novel associations (rs2165468-SBP and rs33975708-cSBP). rs33975708 is a coding variant in the CCDC93 gene, c.535C>T, p.Arg179Cys (MAF = 0.15%), and was associated with increased cSBP (β = 29.3 mmHg, P = 1.23x10-7). CRISPR/Cas9 genome editing was used to model the effect of Ccdc93 loss in mice. Homozygous Ccdc93 deletion was lethal prior to day 10.5 of embryonic development. Ccdc93+/- heterozygous mice were viable and morphologically normal, with 1.3-fold lower aortic Ccdc93 protein expression (P = 0.0041) and elevated SBP as compared to littermate Ccdc93+/+ controls (110±8 mmHg vs 125±10 mmHg, P = 0.016). Wire myography of Ccdc93+/- aortae showed impaired acetylcholine-induced relaxation and enhanced phenylephrine-induced contraction. RNA-Seq transcriptome analysis of Ccdc93+/- mouse thoracic aortae identified significantly enriched pathways altered in fatty acid metabolism and mitochondrial metabolism. Plasma free fatty acid levels were elevated in Ccdc93+/- mice (96±7mM vs 124±13mM, P = 0.0031) and aortic mitochondrial dysfunction was observed through aberrant Parkin and Nix protein expression. Together, our genetic and functional studies support a novel role of CCDC93 in the regulation of BP through its effects on vascular mitochondrial function and endothelial function.

Impaired Peripheral Vascular Function Following Ischemic Stroke in Mice: Potential Insights into Blood Pressure Variations in the Post-Stroke Patient.

High systolic blood pressure and increased blood pressure variability after the onset of ischemic stroke are associated with poor clinical outcomes. One of the key determinants of blood pressure is arteriolar size, determined by vascular smooth muscle tone and vasodilatory and vasoconstrictor substances that are released by the endothelium. The aim of this study is to outline alterations in vasomotor function in isolated peripheral arteries following ischemic stroke. The reactivity of thoracic aortic segments from male C57BL/6 mice to dilators and constrictors was quantified using wire myography. Acetylcholine-induced endothelium-dependent vasodilation was impaired after ischemic stroke (LogIC50 Sham = -7.499, LogIC50 Stroke = -7.350, p = 0.0132, n = 19, 31 respectively). The vasodilatory responses to SNP were identical in the isolated aortas in the sham and stroke groups. Phenylephrine-induced vasoconstriction was impaired in the aortas isolated from the stroke animals in comparison to their sham treatment counterparts (Sham LogEC50= -6.652 vs. Stroke LogEC50 = -6.475, p < 0.001). Our study demonstrates that 24 h post-ischemic stroke, peripheral vascular responses are impaired in remote arteries. The aortas from the stroke animals exhibited reduced vasoconstrictor and endothelium-dependent vasodilator responses, while the endothelium-independent vasodilatory responses were preserved. Since both the vasodilatory and vasoconstrictor responses of peripheral arteries are impaired following ischemic stroke, our findings might explain increased blood pressure variability following ischemic stroke.

Investigating the Role of Cannabinoid Type 1 Receptors in Vascular Function and Remodeling in a Hypercholesterolemic Mouse Model with Low-Density Lipoprotein-Cannabinoid Type 1 Receptor Double Knockout Animals.

Hypercholesterolemia forms the background of several cardiovascular pathologies. LDL receptor-knockout (LDLR-KO) mice kept on a high-fat diet (HFD) develop high cholesterol levels and atherosclerosis (AS). Cannabinoid type 1 receptors (CB1Rs) induce vasodilation, although their role in cardiovascular pathologies is still controversial. We aimed to reveal the effects of CB1Rs on vascular function and remodeling in hypercholesterolemic AS-prone LDLR-KO mice. Experiments were performed on a newly established LDLR and CB1R double-knockout (KO) mouse model, in which KO and wild-type (WT) mice were kept on an HFD or a control diet (CD) for 5 months. The vascular functions of abdominal aorta rings were tested with wire myography. The vasorelaxation effects of acetylcholine (Ach, 1 nM-1 µM) were obtained after phenylephrine precontraction, which was repeated with inhibitors of nitric oxide synthase (NOS) and cyclooxygenase (COX), Nω-nitro-L-arginine (LNA), and indomethacin (INDO), respectively. Blood pressure was measured with the tail-cuff method. Immunostaining of endothelial NOS (eNOS) was carried out. An HFD significantly elevated the cholesterol levels in the LDLR-KO mice more than in the corresponding WT mice (mean values: 1039 ± 162 mg/dL vs. 91 ± 18 mg/dL), and they were not influenced by the presence of the CB1R gene. However, with the defect of the CB1R gene, damage to the Ach relaxation ability was moderated. The blood pressure was higher in the LDLR-KO mice compared to their WT counterparts (systolic/diastolic values: 110/84 ± 5.8/6.8 vs. 102/80 ± 3.3/2.5 mmHg), which was significantly elevated with an HFD (118/96 ± 1.9/2 vs. 100/77 ± 3.4/3.1 mmHg, p < 0.05) but attenuated in the CB1R-KO HFD mice. The expression of eNOS was depressed in the HFD WT mice compared to those on the CD, but it was augmented if CB1R was knocked out. This newly established double-knockout mouse model provides a tool for studying the involvement of CB1Rs in the development of hypercholesterolemia and atherosclerosis. Our results indicate that knocking out the CB1R gene significantly attenuates vascular damage in hypercholesterolemic mice.

Abstract P309: Vasodilating Effects of Macrolides: ex vivo and in vivo study

Background: Anecdotal reports showed a decrease in blood pressure (BP) values in subjects assuming macrolide antibiotics when co-administered with calcium channel blockers. Recently, in the MAPA study, in patients with primary hypertension given a single dose of macrolide a decrease in BP was observed. However, whether macrolides induce per se vasorelaxation remains unknown. Aim: To investigate if macrolides induce vasorelaxation and to identify the underlying mechanisms. Methods: The effects of 3 macrolides (roxithromycin, azithromycin, clarithromycin) were investigated in a wire myograph in aortas isolated from C57BL/6J mice, following standard protocols. Relaxation/contraction was evaluated in phenylephrine pre-contracted aorta segments exposed to different macrolide concentrations [10 -10 M to 10 -5 M]. Longer-term effects of azithromycin (50 mg/Kg/d intraperitoneally for 14 days) were investigated in vivo in a mouse model of Ang II-induced hypertension. BP was measured with the tail-cuff method; functional and structural/mechanical properties of the aorta and mesenteric arteries were evaluated with wire or pressure myograph, as appropriate. Results: After exposure to macrolides, phenylephrine pre-contracted mice aorta segments showed relaxation (maximum effect: -15-20% vs DMSO). The vasodilating effect was abolished by the removal of the endothelium, and was blunted by L-NAME. In the vivo studies treatment with azithromycin lowered BP values and prevented Ang II-induced endothelial dysfunction. Conclusions: Macrolides exert a vasodilatory action that involves endothelium-dependent NO release. In a model of Ang II-mediated hypertension, azithromycin exerted a BP-lowering effect.

Abstract P183: Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus Induce Inflammation and Salt Sensitivity of Blood Pressure in Humanized Mice

Salt sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular disease (CVD), even in normotensive people. Systemic lupus erythematosus (SLE) is also a risk factor for CVD and hypertension. SLE and CVD are on the rise, and it is not known if this is attributable to the increased dietary salt in the modern diet and SSBP. Isolevuglandins (IsoLGs) adduct self-proteins forming neoantigens in antigen-presenting cells (APC), activate T cells, and produce cytokines that promote sodium retention, kidney damage, SLE, and SSBP. We hypothesized the adoptive transfer of peripheral blood mononuclear cells (PBMCs) from patients with SLE into immunodeficient mice (NSG/MHCI/MHCII-/-) would increase inflammation and SSBP after high salt feeding. In vitro studies were performed using PBMCs from patients with SLE and treated with and without high salt, and myeloid cell activation and cytokine expression were measured with flow cytometry. We humanized mice with PBMCs from patients with SLE (n=7) and controls without autoimmune disease (n=7) in animal studies. The mice were fed a 4% high-salt diet for two weeks. Blood pressure was measured with radiotelemetry, immune phenotyping was performed with flow cytometry, and vascular reactivity was assessed in mesenteric arteries using wire myography. A high salt diet significantly increased systolic blood pressure in lupus mice compared to the controls (133.0 vs.116.4 mmHg, p=0.001). APC infiltration (macrophages, monocytes, dendritic cells), proinflammatory markers, and oxidative stress (Isolevuglandins, Nox2) were significantly higher in the kidneys of lupus mice. In lupus mice, classical monocyte and proinflammatory markers were significantly elevated in the spleen lymphocytes, and proinflammatory markers were increased in the kidney, spleen, and aorta in lupus mice. These mice also exhibited mesenteric artery vascular dysfunction (p&lt;0.05). Lupus mice tended to excrete more urinary albumin than the controls (25.89 vs. 17.24 µg/ml p=0.142), suggesting kidney injury. High salt treatment in vitro increased intermediate and nonclassical monocytes with increased CD86, IsoLG, and TNF-alpha expression compared to normal salt-treated cells. In conclusion, our findings suggest that immune cells from patients with SLE, in response to high salt, increase SSBP and the proinflammatory milieu in the kidney, aorta, and spleen; impair vascular function; and worsen albuminuria in immunodeficient mice.

Abstract P448: Follistatin Improves Vascular Function by Regulating Perivascular Adipose Tissue in Essential Hypertension

Introduction: Essential hypertension is characterized by resistance artery remodeling induced by oxidative stress. Most vessels are surrounded by perivascular adipose tissue (PVAT) which regulates vascular tone via release of vasoactive substances. In physiological conditions, PVAT induces an anticontractile effect. In pathological conditions such as hypertension, PVAT exhibit increased oxidative stress resulting in augmented contraction and reduced relaxation. Follistatin, an antioxidant glycoprotein, has been shown to induce adipose tissue browning which is linked to improved vasorelaxation and BP. We hypothesize that follistatin, through ROS inhibition and PVAT browning, lowers BP and improves vessel function in a model of essential hypertension. Methods: Spontaneously hypertensive rats (SHR) were treated with vehicle or follistatin every other day for 8 weeks. Wistar Kyoto (WKY) rats served as normotensive controls. BP was measured weekly by radiotelemetry. Mesenteric arteries and PVAT were used. KCl-induced smooth muscle contraction was assessed using wire myography. ROS was measured by DHE (dihydroethidium) staining. Vasodilator nitric oxide (NO) bioavailability in PVAT was measured using DAF-2 FM. Protein expression of markers for ROS or PVAT browning was measured by immunohistochemistry. Results: Follistatin significantly lowered BP in SHRs and restored anticontractile effect of PVAT. Increased ROS in SHR vessels and PVAT were also inhibited. Follistatin inhibited SHR PVAT-induced vascular oxidative stress. SHR PVAT NO bioavailability was increased by acute follistatin treatment via ROS inhibition. Follistatin also increased expression of brown adipose tissue markers in SHR PVAT suggesting PVAT browning. Limitations: Above studies do not confirm whether PVAT pathology causes hypertension in this model. Vascular dysfunction is an independent risk factor for CVD and our studies do not yet consider effects of follistatin on end-organ damage. Conclusions: Follistatin lowers BP in SHRs likely via improved vascular and perivascular structure and function. Specifically, follistatin inhibited perivascular ROS, increased NO and induced PVAT browning suggesting improved regulation of vascular function and thus, BP. Future work will utilize proteomic assays to identify novel mediators of follistatin effects on PVAT in the SHR.

Sex differences and role of lysyl oxidase-like 2 in angiotensin II-induced hypertension in mice.

Hypertension, a disease with known sexual dimorphism, accelerates aging-associated arterial stiffening, partly because of the activation of matrix remodeling caused by increased biomechanical load. In this study, we tested the effect of biological sex and the role of the matrix remodeling enzyme lysyl oxidase-like 2 (LOXL2) in hypertension-induced arterial stiffening. Hypertension was induced by angiotensin II (ANG II) infusion via osmotic minipumps in 12- to 14-wk-old male and female mice. Blood pressure and pulse wave velocity (PWV) were measured noninvasively. Wire myography and uniaxial tensile testing were used to test aortic vasoreactivity and mechanical properties. Aortic wall composition was examined by histology and Western blotting. Uniaxial stretch of cultured cells was used to evaluate the effect of biomechanical strain. LOXL2's catalytic function was examined using knockout and inhibition. ANG II infusion-induced hypertension in both genotypes and sexes. Wild-type (WT) males exhibited arterial stiffening in vivo and ex vivo. Aortic remodeling with increased wall thickness, intralamellar distance, higher LOXL2, and collagen I and IV content was noted in WT males. Female mice did not exhibit increased PWV despite the onset of hypertension. LOXL2 depletion improved vascular reactivity and mechanics in hypertensive males. LOXL2 depletion improved aortic mechanics but worsened hypercontractility in females. Hypertensive cyclic strain contributed to LOXL2 upregulation in the cell-derived matrix in vascular smooth muscle cells (VSMCs) but not endothelial cells. LOXL2's catalytic function facilitated VSMC alignment in response to biomechanical strain. In conclusion, in males, arterial stiffening in hypertension is driven both by VSMC response and matrix remodeling. Females are protected from PWV elevation in hypertension. LOXL2 depletion is protective in males with improved mechanical and functional aortic properties. VSMCs are the primary source of LOXL2 in the aorta, and hypertension increases LOXL2 processing and shifts to collagen I accumulation. Overall, LOXL2 depletion offers protection in young hypertensive males and females.NEW & NOTEWORTHY We examined the effect of sex on the evolution of angiotensin II (ANG II)-induced hypertension and the role of lysyl oxidase-like 2 (LOXL2), an enzyme that catalyzes matrix cross linking. While ANG II led to hypertension and worsening vascular reactivity in both sexes, aortic remodeling and stiffening occurred only in males. LOXL2 depletion improved outcomes in males but not females. Thus males and females exhibit a distinct etiology of hypertension and LOXL2 is an effective target in males.

Abstract P195: Vascular MAPK/ERK Activation in sFTL-1-Induced Preeclampsia In Vivo

Vascular endothelial growth factor (VEGF) inhibition is linked to the development of hypertension and proteinuria in preeclampsia. This occurs as anti-angiogenic factors from the placenta contribute to pregnancy-related hypertension disorder. Soluble VEGF receptor fms-like tyrosine kinase-1 (sFLT-1) is an anti-angiogenic factor that counteracts VEGF. Levels of sFLT-1 are three times higher in preeclamptic placentas compared to those from normotensive pregnancies, and plasma sFLT-1 levels increase with the severity of preeclampsia. Infusing sFLT-1 in rodents induces hypertension, proteinuria, and glomerular endotheliosis, resembling the human condition of preeclampsia. Currently, there is no effective pharmacological treatment of preeclampsia, and in severe cases, early delivery is often the only effective treatment. In this study, we aim to determine how sFLT-1 exposure affects the activation of the vascular MAPK/ERK pathway and the contractility of the aorta ex-vivo using wire myography, to assess if the MAPK/ERK pathway can be a therapeutic target in preeclampsia. To this end, we treated pregnant female C57BL/6 mice from embryonic day E7.5 to E18.5 with either sFLT-1 (3.7mg/kg/day) or vehicle (phosphate-buffered saline) delivered by osmotic minipumps (intraperitoneal) and carried out non-invasive tail-cuff blood pressure measurements. After sacrifice, we isolated descending thoracic aorta for isometric tension measurements in a wire myograph. Infusion of sFLT-1 resulted in marked systolic blood pressure (SBP) elevation (D increase of 14 mmHg, P=0.0003) and augmented aortic phosphorylated-ERK activation (2-fold increase, P=0.040) by western blot and displayed impaired acetylcholine-induced endothelial relaxation in thoracic aortic segments (P&lt;0.05), providing in vivo evidence of increased vascular p-ERK activation, accompanied by higher SBP and endothelial dysfunction in sFLT-1 - indued mouse model of preeclampsia. Future experiments are aimed to test the effect of pharmacological ERK inhibition in sFLT-1 infusion in vivo . We hypothesize that sFLT-1-induced rise in SBP and vascular p-ERK could be prevented in vivo by treatment with the pharmacological ERK inhibitor.

Abstract P147: Inappropriate Activation of the Leptin-Aldosterone-Endothelial Mineralocorticoid Receptor Axis Promotes Salt Sensitivity of Blood Pressure in Young Female Mice

While compelling clinical evidence indicates that women are more salt-sensitive than men, the mechanisms of the onset of women’s salt-sensitive blood pressure (SSBP) remain largely elusive. Herein, we employed salt-sensitive (SS)-Balb/C and salt-resistant (SR)-C57Bl/6 mice to investigate the etiopathology of SSBP in females. We hypothesized that a high-salt diet alters the leptin-aldosterone-endothelial mineralocorticoid receptor axis (ECMR), leading to SSBP and vascular dysfunction in young SS-female mice. Eleven-week-old female mice were fed a normal (0.4%, NSD) or a high salt diet (4%, HSD) for 7-days. We implanted radiotelemeters for BP monitoring at baseline, HSD, and in response to the leptin receptor (LepR) blockade combined with HSD. Following euthanasia, mesenteric arteries are isolated to measure vascular function via wire myography. HSD elevated mean arterial pressure in Balb/C (NSD: 98.4±2.3 vs. HSD: 105.1±0.9 mmHg) but not in C57 mice (NSD: 99.3±0.9 vs. HSD: 101.3±0.5 mmHg). However, SS-Balb/C mice showed superior natriuretic function in response to salt loading experiments (NSD: 13.3% vs. HSD: 42.3%) as opposed to C57 mice (NSD: 25.8% vs. HSD: 35.1%) (N=3). HSD feeding notably blunted endothelial relaxation to acetylcholine in Balb/c (NSD: 96.5% vs. HSD: 75.4%, N=7), an effect not seen in C57. SS-Balb/C mice failed to suppress aldosterone and adrenal aldosterone synthase (CYP11B2) levels (protein and transcript) compared to C57 mice. This concomitated a 1.8-fold increase in adrenal steroidogenic acute regulatory (StAR) mRNA in SS-Balb/C mice. HSD raised CYP11B2 protein levels in the visceral adipose tissue (VAT) of Balb/C mice. Leptin regulating CYP11B2 expression; we measured fat mass, leptin, and LepR expression. HSD enhanced fat accumulation (C57: +0.7% vs. Balb/c: +26.5%, N=12), leptin levels, and LepR transcript in the adrenal and VAT only in Balb/C mice. LepR blockade reduced BP in Balb/C mice fed a HSD (HSD: 105.1±0.9 vs. HSD+Allo-aca: 100.3±1.9 mmHg). HSD elevated ECMR expression only in Balb/C mice (N=6). To test the potential role of aldosterone-mediated ECMR activation in SSBP, we employed mice with selective overexpression of MR in endothelial cells (ECMR-OE). ECMR-OE young female mice exhibited impaired endothelial function (N=7), reproducing the effects of HSD. In conclusion, SSBP involves an over-activation of the leptin-aldosterone axis in both adrenal and adipose tissue, along with an escalated ECMR expression in females.