Nitric Oxide Bioavailability Research Articles

Nicotine-mediated signaling in glomerular podocytes

Introduction: Although nicotine’s harmful effects on renal function are established, the precise cellular mechanisms of smoking-related damage are understudied. Smoking-Related Glomerulopathy (SRG) is a renal disease phenotype associated with smoking. This condition histologically mimics diabetic nephropathy, however, SRG patients present with proteinuria and renal insufficiency without diabetes. Here we investigated the acute and chronic nicotine-related oxidative and nitrosative stress in glomerular podocytes. We hypothesized that nicotine may promote nitrosative stress in the kidney cells through the rapid production of peroxynitrite (ONOO - ) and a decline in nitric oxide (NO) bioavailability. Methods: To test the hypothesis, we used a conditionally immortalized human podocyte line and confocal imaging to detect the production of ONOO - (Hydroxyl Radical and Peroxynitrite Sensor; HPF), intracellular Ca 2+ (Fluo-8), and NO (DAF-FM). The presence of nicotinic acetylcholine receptors (nAChR) receptor subunits in podocytes was confirmed with immunocytochemistry and live imaging using commercially available pharmacology. NOS activity was analyzed in response to Ang II with the specific blockers for NOS1 (NΩ-Propyl-L-arginine hydrochloride) and NOS2 (L-NIL) subunits. One-way ANOVA (OriginPro) was used for statistical analysis. Results: Immunostaining indicated that human and rat kidneys express nicotinic acetylcholine receptors (nAChR). Notably, we detected specific expression of α7 nAChR in glomerular podocytes. In podocytes, acute nAChR activation promoted the mobilization of intracellular Ca 2+ controlled by intracellular store activation, and fast ONOO - transients. Multiple consecutive applications of nicotine resulted in repeated intracellular Ca 2+ and ONOO - transients. Nicotine-mediated ONOO - response was efficiently blocked in the presence of superoxide dismutase (SOD). The application of specific α7 or α4β2, α2β4, α4β4 and α3β4 nAChR agonists elicited Ca 2+ transients but did not reproduce the ONOO - response to nicotine, suggesting that nitrosative processes may occur independently from Ca 2+ influx or nAChR function. Chronic exposure to nicotine (12-hrs) resulted in a significant decline in podocytes’ NO bioavailability (p<0.05). While under normal conditions, NO is produced primarily by NOS1 (70%), and the rest is attributed to NOS2, chronic nicotine exposure led to an elevation of NOS2 (75%) and a decline in NOS1 activity (p<0.001). We observed similar NOS remodeling under hyperglycemic conditions, suggesting similar nitrosative processes in response to nicotine and high glucose. Conclusions: Nicotine promotes superoxide-stimulated nitrosative stress and peroxynitrite formation in podocytes leading to decline in NO bioavailability and pathological activation of NOS2. R01 NIDDK DK126720 (OP), DK129227 (OP), HL148114 (DVI), NIH/NCATS/SCTR UL1TR001450/SCTR 2214 (OP). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chronic mitochondria-targeted antioxidant supplementation in older adults alters circulating factors to improve endothelial cell function

Age-related vascular endothelial dysfunction, characterized by reduced nitric oxide (NO) bioavailability, is mediated in part by excess production of mitochondrial reactive oxygen species (mtROS). Excess mtROS also oxidizes circulating lipids (i.e., low-density lipoprotein [LDL]) to promote endothelial dysfunction with aging. We recently showed that 6-weeks of treatment with the mitochondria-targeted antioxidant MitoQ (MQ) (20 mg/day) improved endothelial function in vivo in older adults. The improvements in endothelial function with MQ were mediated by reduced mtROS and associated with lower plasma levels of oxidized LDL (oxLDL). However, the complete mechanisms by which MQ improves endothelial function, and if MQ-related decreases in oxLDL are mechanistically involved, remain unknown. PURPOSE. To determine if chronic MQ supplementation-mediated changes in the circulating milieu contribute to improvements in endothelial cell (EC) function and the mechanistic role of reductions in oxLDL. METHODS. Plasma samples were obtained 24 h after the last dose from 19 older adults (mean±SEM: 67±1 yrs; 11 women) who completed our crossover design pilot trial to assess the efficacy of 6-weeks of MQ vs. placebo (PLA) treatment for improving endothelial function. Using an ex vivo model of endothelial function, acetylcholine (Ach)-stimulated NO bioavailability (DAR-4M-AM) was quantified in human aortic ECs (HAECs) following exposure to plasma collected after each phase of the study (MQ and PLA). The role of mtROS production (MitoSOX) in plasma-mediated changes in HAEC function was also assessed. To elucidate the role of lower oxLDL with MQ treatment on HAEC function, physiological levels of oxLDL were ‘added back’ to plasma collected from subjects after MQ treatment at a concentration corresponding to the average reduction after MQ (14 ng/mL). To determine if lower oxLDL with MQ treatment improves HAEC function through a reduction in binding to the oxLDL receptor LOX-1, HAECs were subjected to LOX-1 blocking antibodies prior to plasma exposure under the 3 experimental conditions (PLA; MQ; MQ + oxLDL [‘add back’]) in a subset of subjects (N=9; 67±1 yrs; 5 women). RESULTS. Ach-stimulated NO bioavailability was higher (PLA: 1.0±0.05; MQ: 1.24±0.08; P=0.0002) and mtROS was lower (PLA: 1.0±0.09; MQ: 0.76±0.08; P=0.003) in HAECs exposed to plasma collected from subjects after MQ treatment compared to PLA. Normalizing oxLDL (‘add back’) abolished the effects of MitoQ treatment. LOX-1 blockade modulated Ach-stimulated NO and mtROS production to levels comparable to (P>0.98) or lower than (P<0.047) MQ in all 3 groups, respectively. CONCLUSIONS. Chronic MQ supplementation in older adults alters circulating factors by decreasing plasma oxLDL to reduce oxLDL-LOX-1 signaling-mediated mtROS production and improve EC function. These findings extend insight into the mechanisms by which mitochondria-targeted antioxidant treatment improves endothelial function with aging. 1R21AG049451-01A1 (DRS); 5T32DK007135-46 (KOM); K01DK115524-01A1 (MJR) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ethanol induces endothelial dysfunction via angiotensin II: Role of mitochondrial ROS

Introduction: Excessive ethanol consume is a public health problem and it is associated with increased cardiovascular risk. We and other have previously demonstrated a key participation of renin angiotensin system (RAS) in ethanol-induced vascular abnormalities by leading to excessive reactive oxygen species (ROS) production via redox unbalance. However, whether ethanol consumption leads to vascular dysfunction by altering mitochondrial function and dynamic are unknown. We tested the hypothesis that ethanol consumption increases angiontensin II (ANG II) levels triggering mitochondria dysfunction and ROS production, which in turn promotes endothelial dysfunction. Methods: 6–8-weeks old male C57/BL6J mice were randomized in 4 groups: 1) Control; 2) Ethanol (20%, vol./vol.); 3) Losartan (10 mg/kg/feed); 4) Ethanol (20%, vol./vol.) + Losartan (10 mg/kg/feed). After 12 weeks of treatment, aortae were harvested for studies of vascular function. Results: Ethanol increased phenylephrine-induced contractility in an endothelium (Endo)-dependent manner [Maximal response (mN): Endo (+): Control: 6.1 ± 2.9, n=6; Ethanol: 11.2 ± 3.2*, n=6; Endo (-): Control: 10.8±1.4, n=5; Ethanol: 10.1±1.3, n=8). Therefore, for the next findings, we only focused on endothelium intact arteries. Remarkably, losartan treatment prevented ethanol-induced hypercontractility (Ethanol+losartan: 4.3±1.2, n=5). To study whether ethanol consumption leads to endothelial dysfunction by impairing mitochondrial function and increasing O 2 ∙- production in aorta, we used MnTMPyP (a membrane permeant Mn‐SOD mimetic, 3×10−5 M) or CCCP (a mitochondrial uncoupler, 10−6 M), respectively. Both, partially prevented the increase in contraction induced by ethanol consume. Furthermore, L-NAME (a non-specific NOS inhibitor, 10-4 M) blunted the difference between control and ethanol groups, whereas EUK 134 (a synthetic superoxide dismutase and catalase mimetic, 10-4 M) did not affect the vascular response in ethanol group, but it increased the contractility in all other groups, suggesting that nitric oxide (NO) and H2O2 bioavailability might participate of vascular dysfunctionin ethanol group. Conclusion: Our preliminary findings indicate that ethanol induces endothelial dysfunction via regulating endothelial ANGII signaling, which seems to be dependent on mitochondrial dysfunction and reduction in NO and H2O2 bioavailability. Our study may benefit the understanding of molecular and cellular mechanisms associated with ethanol consumption and cardiovascular risk and it helps advancing on treatment for cardiovascular diseases in ethanol consumers. FAPESP (2021/13075-5), NHLBI-R00 (R00HL14013903), AHA-CDA (CDA857268), Vascular Medicine Institute, the Hemophilia Center of Western Pennsylvania Vitalant, and Children's Hospital of Pittsburgh of the UPMC Health System. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Shear stress facilitates Akt activation in human pulmonary microvascular endothelial cells

Introduction: Pulmonary arterial obstruction due to acute thromboembolism causes local reduction or cessation of blood flow. Under normal conditions, pulmonary blood flow exerts shear stress on endothelial cells, known to play an important role in maintaining vascular homeostasis. We hypothesized that pulmonary microvascular endothelial cells exposed to abrupt reductions in shear stress would demonstrate attenuated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and phosphorylation of downstream effectors Akt and endothelial nitric oxide synthase (eNOS). Methods: We tested our hypothesis in primary cultures of human lung microvascular endothelial cells (hLMVECs). We cultured early passage (p≤8) hLMVECs from 5 unique donor lots in standard 6-well culture plates and exposed cells to shear stress using an orbital shaker. Using a fixed depth (volume) and viscosity of tissue culture media, we adjusted the magnitude of shear stress exerted on hLMVECs by changing the angular velocity of orbital rotation. We acclimatized hLMVECs to physiologically-relevant shear stress (~12 dyn/cm 2 ) for 24 hours, then exposed cells to low shear stress (~3 dyn/cm 2 ) or no shear stress (0 dyn/cm 2 ) for 30 minutes. Control cells were maintained at 12 dyn/cm 2 of shear stress. In some experiments, PI3K activity was inhibited throughout the acclimatization and shear stress manipulation period by including LY294002 (10 μM) or vehicle (DMSO, 0.1% v/v) in culture media. We prepared whole cell lysates and used western blot to evaluate the expression and phosphorylation of Akt (ser473) and eNOS (ser1177). Results: We found that phosphorylation of Akt, but not Akt expression, decreased significantly (p<0.05) when shear stress was acutely reduced (~3 dyn/cm 2 ) or removed (0 dyn/cm 2 ). Inhibition of PI3K activity with LY294002 further diminished shear-mediated changes in Akt phosphorylation in all 3 groups. Conversely, eNOS phosphorylation was not modified by acute reductions in shear stress, but eNOS expression was markedly attenuated by PI3K inhibition under all conditions. Conclusions: Our results indicate that phosphorylation of Akt, but not its downstream target eNOS, is rapidly attenuated by abrupt reductions in shear stress in hLMVECs. However, shear stress appears to regulate eNOS expression in a PI3K-dependent manner. Taken together, these findings suggest that acute disruption of flow, as seen in pulmonary embolism, may disrupt human lung microvascular endothelial cell homeostasis and nitric oxide bioavailability via multiple PI3K-dependent signaling pathways. This work is supported by HL126514 and HL159906. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Analysis of dietary nitrate incorporation in porcine ocular tissues after supplementing 15N-labeled sodium nitrate

The mechanisms for the generation of nitric oxide (NO) include reduction of nitrate (NO 3 - ) and nitrite (NO 2 - ) as well as the classic nitric oxide synthase (NOS) pathways. In the reductive pathways, ingested nitrate is reduced to nitrite, mainly by oral bacteria and to some extent by tissue enzymes, then these nitrite ions can be reduced to NO by several endogenous proteins or, in stomach, by acid disproportionation. Dietary nitrate, rich in green leafy vegetables and beets, has beneficial effects in lowering blood pressure and improving vascular health by enhancing NO bioavailability. Previously, we showed that porcine eye components contain substantial amounts of nitrate and nitrite ions that are similar to those in blood. More importantly, cornea and sclera exhibited a nitrate reduction capability. To extend our knowledge on nitrate metabolism in the eye, in the present study, we fed pigs (Yorkshire domestic cross swine weighing 35-40kg, housed in an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care)-accredited facility with environmental enrichment) with either NaCl or Na 15 NO 3 and assessed the amount of 15 NO 3 - and 15 NO 2 - in various parts of the eye by LC-MS/MS at 3 or 24h after supplementation. We also measured the total concentrations of nitrate and nitrite ions using a standard chemiluminescence nitric oxide analyzer. At 3h, there was a marked increase in both 15 NO 3 - and 15 NO 2 - in all parts of the eye; in particular, aqueous and vitreous humor showed very high 15 NO 3 - contents (77.5 and 74.5% respectively), similar to plasma (77.1%) and even higher 15 NO 2 - contents (39.9% and 35.3% respectively) than plasma (19.8%). Cornea (64.4%) and sclera (63.4%) also showed notable increases in 15 NO 3 - content at 3h, then retina (48.8%) and lens (44.8%) followed. In 15 NO 2 - percent analysis at 3h, except aqueous and vitreous humor, other eye parts showed similar levels to plasma. At 24h, 15 NO 3 - contents decreased significantly compared to the 3h point, but it was still higher than the baseline in all ocular components. The 15 NO 2 - contents at 24h were almost back to the baseline. Absolute values of nitrate and nitrite ions in these tissues show similar patterns as shown in 15 N analysis. These results indicate that dietary nitrate supplementation has a vast impact on the levels of nitrate and nitrite and likely NO as well in the eye. NO generation via the nitrate-nitrite reductive pathway may contribute to various physiological and pathophysiological processes in the eye and may be useful therapeutically. This work was supported by intramural NIDDK/NIH grant ZIA DK 025104-15 to Alan N. Schechter. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endothelial Nrf2 modulates blood pressure and vasoconstrictor responses in conscious mice

Introduction: Excessive oxidant stress has been shown to mediate a variety of cardiovascular pathological conditions, including hypertension. The level of reactive oxygen species (ROS) in tissues is regulated by the balance between the production of ROS and the ability to scavenge superoxide and hydrogen peroxide by a myriad of antioxidant enzymes and small molecules. One of the most important regulators of antioxidant activity is the ubiquitous transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 binds to antioxidant response elements of multiple genes following its release from its inhibitory protein Keap1 and part of the E3 ubiquitin ligase complex. In this study we hypothesized that endothelial Nrf2 is a critical factor in blood pressure regulation following vasoconstrictor stimuli. Methods: Transgenic mice were created using the endothelial Tie2 promoter expressing Cre recombinase (Tie2-Cre) crossed with either Nrf2 floxed or Keap1 floxed mice so that Nrf2 would be deleted or upregulated respectively, and selectively in the endothelium. Cre negative mice were used as wild type (WT) controls. Mice were instrumented with radiotelemetry transducers to chronically record arterial blood pressure and heart rate in the conscious state. Blood pressure and heart rate were recorded for 24 hours once per week. After baseline recordings, animals were challenged with an osmotic minipump infusion of Angiotensin II (Ang II) (600ng/Kg/min) for 7 days or L-nitroarginine methyl ester (L-NAME) (100 mg/kg/d in drinking water). Results: Baseline mean arterial pressure (MAP) was 101.6 ± 2.5 mm Hg (mean±SEM; n=7) in WT mice (daytime =95.4±1.5; nighttime =106.1±1.0). In mice with endothelial Nrf2 deletion baseline MAP was significantly higher(p<0.01) during nighttime (112.1±1.1). In addition, the response to Ang II was significantly augmented (p < 0.001) in Nrf2 KO mice compared to WT mice (148.9±5.3 vs 116.8±6.7 mm Hg). Both day and nighttime values were higher in Nrf2 KO mice. Furthermore, Nrf2 KO mice exhibited a delayed return to baseline upon pump emptying. In contrast, in one Keap1 KO mouse there was a response to Ang II that was similar to WT (92.1 mm Hg baseline to 116.3 mmHg post Ang II). In Nrf2 deleted mice, the hypertensive response to L-NAME was no different than that of WT mice. Conclusion: In summary, these preliminary data suggest that endothelial Nrf2 plays an important role in modulation of blood pressure at rest and during oxidative stress induced by Ang II. The L-NAME data suggests that upregulation of Nrf2 increases the bioavailability of nitric oxide to blunt Ang II mediated vasoconstriction. Supported by NHLBI P01-62222 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The effects of standard and soy-free chow on cerebral artery function in ovariectomized female mice

As females age, they transition through menopause, experiencing a decrease in estrogen and an increase in cardiovascular and neurodegenerative disease risk. When studying the effects of menopause in mice, modeled by ovariectomy, it is important to consider the effects of diet. Most standard rodent chows contain phytoestrogen-rich soybean meal, which can mimic the effects of estrogen. Understanding the impact of this soybean meal on outcomes is crucial to proper experimental design. Thus, the aim of this study was to compare the effects of standard and soy-free chows on cerebral artery endothelial function and cognitive function. We studied young female C57Bl/6J mice (n=43; ~6 months) divided into three groups: sham-controls on a soy-free diet (sham-SF), ovariectomized on a soy-free diet (OVX-SF), or OVX on a standard chow diet (OVX-SC). We assessed endothelium-dependent and -independent vasodilation in isolated pressurized posterior cerebral arteries (PCAs). We measured gene expression in cerebral arteries by real-time PCR. To assess behavioral function, we measured instinctual behavior via nest building. Data are presented as mean±SD. Compared with sham-SF, OVX-SF mice had impaired PCA endothelium-dependent vasodilation as evidenced by a maximal response that was 21% lower to acetylcholine (ACh, 60±14% vs. 47±14%, p=0.002) and 27% lower to insulin (52±17% vs. 38±8%, p=0.005). OVX-SC mice had a 27% greater PCA maximal vasodilation to insulin compared with OVX-SF mice (52±11% vs. 38±8%, p=0.001). The PCA responses to ACh and insulin did not differ between sham-SF and OVX-SC (p>0.05). Additionally, the PCA response to ACh and insulin in the presence of nitric oxide synthase inhibitor L-NAME did not differ between groups (p>0.05). Endothelium-independent vasodilation, measured as the PCA response to sodium nitroprusside, also did not differ between groups (p>0.05). There was a trend for lower gene expression of superoxide dismutase 2 ( Sod2) in cerebral arteries from OVX-SF mice (0.7±0.2 AU) compared with OVX-SC (1.0±0.4 AU, p=0.14) but this difference did not reach statistical significance. Cerebral artery gene expression was not different between groups for Esr1 (estrogen receptor α), Insr (insulin receptor), Slca2 (GLUT1), Sod1, or Sod3 (all p>0.05). Lastly, nest building scores were higher in the OVX-SC (4.3±0.5 AU) compared with sham-SF (3.2±1.2 AU, p=0.02) and OVX-SF (3.2±1.0 AU, p=0.04) mice. In summary, we found that ovariectomy impairs endothelial function in cerebral arteries by reducing nitric oxide bioavailability, but a diet containing soy mitigates these effects of ovariectomy. In addition, a diet containing soy leads to better instinctual nest-building behavior in mice. These findings highlight the importance of considering dietary soy when performing vascular and behavior tests in mice, particularly when interested in the effects of sex hormones. Luvaas Family Fund This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Improving nitric oxide bioavailability to treat endothelial dysfunction in women with type 1 diabetes: impact of race

BACKGROUND: Type 1 diabetes (T1D) is associated with a greater risk of cardiovascular disease (CVD), mediated in part, by impaired nitric oxide (NO) bioavailability and endothelial dysfunction. In fact, women with T1D have a greater risk of CVD compared to men, and although the incidence of T1D is greater in Caucasians (Cs), African Americans (AAs) with T1D have significantly worse health outcomes. In healthy women, both resveratrol (RES) and antioxidants have been used to improve NO bioavailability and endothelial function by reducing oxidative stress. Accordingly, the present investigation sought to determine the role of resveratrol and antioxidants on vascular function in C and AA women with T1D. METHODS: 15 C women with T1D and 6 AA women with T1D were enrolled. Participants were randomized to either an acute dose of RES or an antioxidant cocktail (AOC) containing Vitamin C, Vitamin E, and Alpha-Lipoic Acid. The flow-mediated dilation (FMD) test, a bioassay of NO bioavailability, was utilized to assess endothelial function normalized for shear (FMD/Shear) before and after treatment. In addition, a venous blood sample was collected to assess baseline clinical laboratory values and plasma concentrations of oxidative stress (8-isoprostane (8-iso) and superoxide dismutase (SOD)) before and after treatment. Data are reported as mean ± SD. RESULTS: No differences in baseline demographics were observed between Cs and AAs. Clinical laboratory values were similar between groups, except HbA1c was higher ( p=0.013) in AAs (9.6±1.2%) compared to Cs (7.7±1.5%). Using a mixed model repeated measures design, a significant race x treatment x time interaction ( p=0.050) was observed for FMD/Shear. Treatment with AOC in AAs resulted in a significant increase ( p=0.002) in FMD/Shear. In addition, FMD/Shear was higher ( p=0.019) in AAs following treatment with AOC (0.370±0.128) compared to RES (0.212±0.049). Further, the % increase in FMD/Shear following treatment with AOC was greater ( p<0.001) in AAs (48±32%) compared to Cs (-1±42%). Independent of treatment, SOD was higher at both baseline ( p<0.001) and after treatment ( p=0.015) in AAs compared to Cs. Additionally, there was a decrease in SOD over time in AAs, collapsing treatment ( p=0.005). No differences in 8-iso were observed. CONCLUSION: Findings from the present investigation have identified that treatment to improve endothelial function in T1D can be race dependent. Compared to RES, treatment with AOC in AA women with T1D may be more effective at improving endothelial function. Neither treatment was effective at improving endothelial function in C women with T1D. Differences in oxidative stress may play a role; however, future studies are needed to understand how race impacts the treatment response in women with T1D. 1R01HL137087 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Increasing Nitric Oxide Bioavailability attenuates Vascular Dysfunction and rescues Fetal Growth in female rats with history of acute kidney injury prior to pregnancy

Clinical data report a 3-5-fold increase in adverse pregnancy outcomes in women with a history of acute kidney injury (AKI), despite clinical recovery of renal function (creatinine clearance) before pregnancy. We recently showed that female Sprague-Dawley (SD) rats recovered from AKI develop increased blood pressure, uterine artery resistance and decreased fetal growth in pregnancy. Healthy pregnancies are characterized by robust nitric oxide (NO) production and reduction in total peripheral vascular resistance, however, our lab has shown decreased renal nitric oxide synthase (NOS) expression in pregnant rats post-AKI. We, therefore, tested the hypothesis that AKI prior to pregnancy decreases NO which induces endothelial dysfunction and fetal growth restriction. Female SD rats received either 45-min bilateral renal ischemia-reperfusion (AKI) or sham surgery. We utilized sildenafil to increase NO bioavailability. Sham and AKI rats were given vehicle or sildenafil chow 15 days (n=8/grp) post-AKI or sham surgery and mated 15 days later. A separate group of rats (prolonged AKI/Sham) (n=3-6/grp) were mated for pregnancy 6 months post-AKI or sham surgery. Gestational day 1 (GD1) was identified through vaginal smear. On GD19, rats were placed in metabolic cages for urine collection and euthanized on GD20. Pups were weighed and vascular function was assessed via wire myography on 3rd order mesenteric arteries. AKI prior to pregnancy reduced urinary nitrate/nitrite levels assessed via ELISA (3.2±0.9 AKI vs 8.9±1.6 μM/day Sham, *P<0.05) and trended to decrease aortic protein expression of endothelial NOS (eNOS; 0.5±0.1 AKI phospho-/total-eNOS vs 0.8±0.1 Sham, n=3/group, P=0.1) compared to sham pregnant rats. Pup weight decreased in post-AKI pregnant rats (2.14±0.02 g Sham Vehicle vs 2.02±0.05 AKI Vehicle, P=0.06), however, sildenafil restored pup weights in post-AKI rats (2.17±0.04 Sham Sildenafil vs 2.08±0.06 AKI Sildenafil). Pre-pregnancy AKI reduced endothelial-dependent relaxation to acetylcholine (ACh) compared to sham (concentration-response, 1 nM-10 μM, 2-way ANOVA, RM, *P<0.05), indicating endothelial dysfunction. Sildenafil restored endothelial relaxation responses to ACh in post-AKI pregnant rats (P>0.05). None of our groups demonstrated differences in vascular responses to the constrictor phenylephrine or relaxation to sodium nitroprusside. Interestingly, the prolonged 6-month post AKI/Sham group showed increased constrictor responses to phenylephrine (*P<0.05) and KCl (*P<0.05) in post-AKI pregnant rats compared to sham, suggesting prolonged time removed from AKI increases vascular stiffness in pregnancy. Taken together, these data indicate AKI prior to pregnancy induces endothelial dysfunction and that sildenafil, potentially by increasing NO, rescues endothelial function and fetal growth in post-AKI pregnant rats. In addition, our data show that increased recovery time does not ablate vascular dysfunction in pregnancies with history of AKI. R00 HL146948-03 to DM; AHA CDA858380 to JLF; AHA DIVSUP0224732 to DM This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sevoflurane and isoflurane anesthesia induce redox imbalance, but only sevoflurane impairs vascular contraction.

Volatile anesthetics may cause vascular dysfunction; however, underlying effects are unclear. The aim of the present study was to investigate whether sevoflurane and isoflurane affect vascular function, nitric oxide (NO) bioavailability, and biomarkers of oxidative stress and inflammation. Wistar rats were divided into three experimental groups: Not anesthetized (control group) or submitted to anesthesia with isoflurane (Iso group) or sevoflurane (Sevo group). Hemodynamic parameters were monitored during anesthesia, and blood gas values and biochemical determinants were analyzed. Isometric contractions were recorded in aortic rings. Vasoconstriction induced by potassium chloride (KCl) and phenylephrine (Phe) were measured. No differences in hemodynamic parameters and blood gasses variables were observed. Impaired KCl and Phe-induced contractions were observed in endothelium-intact aorta of Sevo compared to Iso and Control groups. Redox imbalance was found in Sevo and Iso groups. Reduced NO bioavailability and increased activity of matrix metalloproteinase 2 (MMP-2) were observed in Sevo, but not in the Iso group. While reduced IL-10 and IL-1β were observed in Sevo, increases in IL-1β in the Iso group were found. Sevoflurane, but not isoflurane, anesthesia impairs vasocontraction, and reduced NO and cytokines and increased MMP-2 activity may be involved in vascular dysfunction after sevoflurane anesthesia.

Acute beetroot juice reduces blood pressure in young Black and White males but not females

A complex interplay of social, lifestyle, and physiological factors contribute to Black Americans having the highest blood pressure (BP) in America. One potential contributor to Black adult's higher BP may be reduced nitric oxide (NO) bioavailability. Therefore, we sought to determine whether augmenting NO bioavailability with acute beetroot juice (BRJ) supplementation would reduce resting BP and cardiovascular reactivity in Black and White adults, but to a greater extent in Black adults. A total of 18 Black and 20 White (∼equal split by biological sex) young adults completed this randomized, placebo-controlled (nitrate (NO3−)-depleted BRJ), crossover design study. We measured heart rate, brachial and central BP, and arterial stiffness (via pulse wave velocity) at rest, during handgrip exercise, and during post-exercise circulatory occlusion. Compared with White adults, Black adults exhibited higher pre-supplementation resting brachial and central BP (Ps ≤0.035; e.g., brachial systolic BP: 116(11) vs. 121(7) mmHg, P = 0.023). Compared with placebo, BRJ (∼12.8 mmol NO3−) reduced resting brachial systolic BP similarly in Black (Δ-4±10 mmHg) and White (Δ-4±7 mmHg) adults (P = 0.029). However, BRJ supplementation reduced BP in males (Ps ≤ 0.020) but not females (Ps ≥ 0.299). Irrespective of race or sex, increases in plasma NO3− were associated with reduced brachial systolic BP (ρ = −0.237, P = 0.042). No other treatment effects were observed for BP or arterial stiffness at rest or during physical stress (i.e., reactivity); Ps ≥ 0.075. Despite young Black adults having higher resting BP, acute BRJ supplementation reduced systolic BP in young Black and White adults by a similar magnitude, an effect that was driven by males.

Inflammaging and Immunosenescence as Part of Skin Aging-A Narrative Review.

Inflammaging and immunosenescence are associated with aging of the human body, but there are key differences between them. Immunosenescence aims to adapt the body systems to aging, while inflammaging is considered a consequence of immunosenescence. There has been much research in the area of immunosenescence and inflammaging recently, yet our understanding of aging and the ability to develop interventions to decrease the harmful effect of aging on the human body is insufficient. This review is focused on immunosenescence and inflammaging processes in the skin. We aimed to identify factors that influence inflammaging, skin aging, and their mechanisms. We discussed the role of triggering factors (e.g., UV radiations, changes in bioavailability of nitric oxide, senescence-associated secretory phenotype factors, and reactive oxygen species) and inhibiting factors that can potentially be used as anti-aging treatments, as well as the idea of geroprotectors and senotherapeutics. We concluded that while knowledge on external factors can help people to improve their health conditions, knowledge on biochemical factors can help researchers to understand inflammaging process and develop interventions to minimize the impact of aging on the human body. Further research is needed to better understand the role of factors that can slow down or accelerate inflammaging.

The rationale and study design of two phase II trials examining the effects of BI 685509, a soluble guanylyl cyclase activator, on clinically significant portal hypertension in patients with compensated cirrhosis

BackgroundClinically significant portal hypertension (CSPH) drives cirrhosis-related complications (i.e. hepatic decompensation). Impaired nitric oxide (NO) bioavailability promotes sinusoidal vasoconstriction, which is the initial pathomechanism of CSPH development. Activation of soluble guanylyl cyclase (sGC), a key downstream effector of NO, facilitates sinusoidal vasodilation, which in turn may improve CSPH. Two phase II studies are being conducted to assess the efficacy of the NO-independent sGC activator BI 685509 in patients with CSPH due to various cirrhosis aetiologies.MethodsThe 1366.0021 trial (NCT05161481) is a randomised, placebo-controlled, exploratory study that will assess BI 685509 (moderate or high dose) for 24 weeks in patients with CSPH due to alcohol-related liver disease. The 1366.0029 trial (NCT05282121) is a randomised, open-label, parallel-group, exploratory study that will assess BI 685509 (high dose) alone in patients with hepatitis B or C virus infection or non-alcoholic steatohepatitis (NASH) and in combination with 10 mg empagliflozin in patients with NASH and type 2 diabetes mellitus for 8 weeks. The 1366.0021 trial will enrol 105 patients, and the 1366.0029 trial will enrol 80 patients. In both studies, the primary endpoint is the change from baseline in hepatic venous pressure gradient (HVPG) until the end of treatment (24 or 8 weeks, respectively). Secondary endpoints include the proportion of patients with an HVPG reduction of >10% from baseline, the development of decompensation events and the change from baseline in HVPG after 8 weeks in the 1366.0021 trial. In addition, the trials will assess changes in liver and spleen stiffness by transient elastography, changes in hepatic and renal function and the tolerability of BI 685509.DiscussionThese trials will enable assessment of the short-term (8 weeks) and longer-term (24 weeks) safety of BI 685509, and the effect of sGC activation by BI 685509 on CSPH due to various cirrhosis aetiologies. The trials will use central readings of the diagnostic gold standard HVPG for the primary endpoint, and changes in established non-invasive biomarkers, such as liver and spleen stiffness. Ultimately, these trials will provide key information for developing future phase III trials.Trial registration1366.0021: EudraCT no. 2021–001285-38; ClinicalTrials.gov NCT05161481. Registered on 17 December 2021, https://www.clinicaltrials.gov/ct2/show/NCT05161481.1366.0029: EudraCT no. 2021–005171-40; ClinicalTrials.gov NCT05282121. Registered on 16 March 2022, https://www.clinicaltrials.gov/ct2/show/NCT05282121.

Low Grade Endotoxemia and Oxidative Stress in Offspring of Patients with Early Myocardial Infarction.

Background and aims: Offspring of patients with early myocardial infarction are at higher cardiovascular risk, but the underlying physio-pathological mechanism is unclear. NADPH oxidase-type 2 (NOX-2) plays a pivotal role as mediator of oxidative stress and could be involved in activating platelets in these patients. Furthermore, altered intestinal permeability and serum lipopolysaccharide (LPS) could be a trigger to promote NOX-2 activation and platelet aggregation. This study aims to evaluate the behavior of low grade endotoxemia, oxidative stress and platelet activation in offspring of patients with early myocardial infarction. Methods: We enrolled, in a cross-sectional study, 46 offspring of patients with early myocardial infarction and 86 healthy subjects (HS). LPS levels and gut permeability (assessed by zonulin), oxidative stress (assessed by serum NOX-2-derived peptide (sNOX2-dp) release, hydrogen peroxide (H2O2) production and isoprostanes), serum nitric oxide (NO) bioavailability and platelet activation (by serum thromboxane B2 (TXB2) and soluble P-Selectin (sP-Selectin)) were analyzed. Results: Compared to HS, offspring of patients with early myocardial infarction had higher values of LPS, zonulin, serum isoprostanes, sNOX2-dp H2O2, TXB2, p-selectin and lower NO bioavailability. Logistic regression analysis showed that the variables associated with offspring of patients with early myocardial infarction were LPS, TXB2 and isoprostanes. The multiple linear regression analysis confirmed that serum NOX-2, isoprostanes, p-selectin and H2O2 levels were significantly associated to LPS. Furthermore, serum LPS, isoprostanes and TXB2 levels were significantly associated with sNOX-2-dp. Conclusions: Offspring of patients with early myocardial infarction have a low grade endotoxemia that could generate oxidative stress and platelet activation increasing their cardiovascular risk. Future studies are needed to understand the role of dysbiosis in this population.

Eicosapentaenoic acid (EPA) reduces pulmonary endothelial dysfunction and inflammation due to changes in protein expression during exposure to particulate matter air pollution

AimsInhalation of air pollution small particle matter (PM) is a leading cause of cardiovascular (CV) disease. Exposure to PMs causes endothelial cell (EC) dysfunction as evidenced by nitric oxide (NO) synthase uncoupling, vasoconstriction and inflammation. Eicosapentaenoic acid (EPA) has been shown to mitigate PM-induced adverse cardiac changes in patients receiving omega-3 fatty acid supplementation. We set out to determine the pro-inflammatory effects of multiple PMs (urban and fine) on pulmonary EC NO bioavailability and protein expression, and whether EPA restores EC function under these conditions. Methods and resultsWe pretreated pulmonary ECs with EPA and then exposed them to urban or fine air pollution PMs. LC/MS-based proteomic analysis to assess relative expression levels. Expression of adhesion molecules was measured by immunochemistry. The ratio of NO to peroxynitrite (ONOO–) release, an indication of eNOS coupling, was measured using porphyrinic nanosensors following calcium stimulation. Urban/fine PMs also modulated 9/12 and 13/36 proteins, respectively, linked to platelet and neutrophil degranulation pathways and caused > 50% (p < 0.001) decrease in the stimulated NO/ONOO– release ratio. EPA treatment altered expression of proteins involved in these inflammatory pathways, including a decrease in peroxiredoxin-5 and an increase in superoxide dismutase-1. EPA also increased expression of heme oxygenase-1 (HMOX1), a cytoprotective protein, by 2.1-fold (p = 0.024). EPA reduced elevations in sICAM-1 levels by 22% (p < 0.01) and improved the NO/ONOO– release ratio by > 35% (p < 0.05). ConclusionThese cellular changes may contribute to anti-inflammatory, cytoprotective and lipid changes associated with EPA treatment during air pollution exposure.

Dipeptidyl peptidase 4 inhibition rescues PKA-eNOS signaling and suppresses aortic hypercontractility in male rats with heart failure

AimsVascular dysfunction and elevated circulating dipeptidyl peptidase 4 (DPP4) activity are both reported to be involved in the progression of heart failure (HF). While the cardiac benefits of DPP4 inhibitors (DPP4i) have been extensively studied, little is known about the effects of DPP4i on vascular dysfunction in nondiabetic HF. This study tested the hypothesis that vildagliptin (DPP4i) mitigates aortic hyperreactivity in male HF rats. Materials and methodsMale Wistar rats were subjected to left ventricle (LV) radiofrequency ablation to HF induction or sham operation (SO). Six weeks after surgery, radiofrequency-ablated rats who developed HF were treated with vildagliptin (120 mg⸱kg−1⸱day−1) or vehicle for 4 weeks. Thoracic aorta reactivity, dihydroethidium fluorescence, immunoblotting experiments, and enzyme-linked immunosorbent assays were performed. Key findingsDPP4i ameliorated the hypercontractility of HF aortas to the α-adrenoceptor agonist phenylephrine towards SO levels. In HF, the reduced endothelium and nitric oxide (NO) anticontractile effect on phenylephrine response was restored by DPP4i. At the molecular level, this vasoprotective effect of DPP4i was accompanied by (i) reduced oxidative stress and NADPH oxidase 2 (Nox2) expression, (ii) enhanced total endothelial nitric oxide synthase (eNOS) expression and phosphorylation at Ser1177, and (iii) increased PKA activation, which acts upstream of eNOS. Additionally, DPP4i restored the higher serum angiotensin II concentration towards SO. SignificanceOur data demonstrate that DPP4i ameliorates aortic hypercontractility, most likely by enhancing NO bioavailability, showing that the DPP4i-induced cardioprotection in male HF may arise from effects not only in the heart but also in conductance arteries.

Tobacco smoking and vascular biology and function: evidence from human studies

Tobacco cigarette smoking is among the most complex and least understood health risk factors. A deeper insight into the pathophysiological actions of smoking exposure is of special importance as smoking is a major cause of chronic non-communicable diseases, in particular of cardiovascular disease as well as risk factors such as atherosclerosis and arterial hypertension. It is well known that smoking exerts its negative effects on cardiovascular health through various interdependent pathophysiological actions including hemodynamic and autonomic alterations, oxidative stress, inflammation, endothelial dysfunction, thrombosis, and hyperlipidemia. Importantly, impaired vascular endothelial function is acknowledged as an early key event in the initiation and progression of smoking-induced atherosclerosis. Increasing evidence from human studies indicates that cigarette smoke exposure associates with a pathological state of the vascular endothelium mainly characterized by reduced vascular nitric oxide bioavailability due to increased vascular superoxide production. In the present overview, we provide compact evidence on the effects of tobacco cigarette smoke exposure on vascular biology and function in humans centered on main drivers of adverse cardiovascular effects including endothelial dysfunction, inflammation, and oxidative stress.

Endothelial Autophagy Dysregulation in Diabetes.

Diabetes mellitus is a major public health issue that affected 537 million people worldwide in 2021, a number that is only expected to increase in the upcoming decade. Diabetes is a systemic metabolic disease with devastating macro- and microvascular complications. Endothelial dysfunction is a key determinant in the pathogenesis of diabetes. Dysfunctional endothelium leads to vasoconstriction by decreased nitric oxide bioavailability and increased expression of vasoconstrictor factors, vascular inflammation through the production of pro-inflammatory cytokines, a loss of microvascular density leading to low organ perfusion, procoagulopathy, and/or arterial stiffening. Autophagy, a lysosomal recycling process, appears to play an important role in endothelial cells, ensuring endothelial homeostasis and functions. Previous reports have provided evidence of autophagic flux impairment in patients with type I or type II diabetes. In this review, we report evidence of endothelial autophagy dysfunction during diabetes. We discuss the mechanisms driving endothelial autophagic flux impairment and summarize therapeutic strategies targeting autophagy in diabetes.

Erythrocyte-Derived microRNAs: Emerging Players in Cardiovascular and Metabolic Disease.

Recent studies have demonstrated a novel function of red blood cells (RBCs) beyond their classical role as gas transporters, that is, RBCs undergo functional alterations in cardiovascular and metabolic disease, and RBC dysfunction is associated with hypertension and the development of cardiovascular injury in type 2 diabetes, heart failure, preeclampsia, familial hypercholesterolemia/dyslipidemia, and COVID-19. The underlying mechanisms include decreased nitric oxide bioavailability, increased arginase activity, and reactive oxygen species formation. Of interest, RBCs contain diverse and abundant micro (mi)RNAs. microRNA expression pattern in RBCs reflects the expression in the whole blood, serum, and plasma. microRNA levels in RBCs have been found to be altered in various cardiovascular and metabolic diseases, which contributes to the development of cardiovascular complications. Evidence has shown that RBC-derived miRNAs interact with the cardiovascular system via extracellular vesicles and argonaute RISC catalytic component 2 as carriers. Alteration of RBC-to-vascular communication via miRNAs may serve as potential disease mechanism for vascular complications. The present review summarizes RBCs and their released miRNAs as potential mediators of cardiovascular injury. We further focus on the possible mechanisms by which RBC-derived miRNAs regulate cardiovascular function. A better understanding of the function of RBC-derived miRNAs will increase insights into the disease mechanism and potential targets for the treatment of cardiovascular complications.

Overview and New Insights into the Metabolic Syndrome: Risk Factors and Emerging Variables in the Development of Type 2 Diabetes and Cerebrocardiovascular Disease.

Metabolic syndrome (MetS) is considered a metabolic disorder that has been steadily increasing globally and seems to parallel the increasing prevalence of obesity. It consists of a cluster of risk factors which traditionally includes obesity and hyperlipidemia, hyperinsulinemia, hypertension, and hyperglycemia. These four core risk factors are associated with insulin resistance (IR) and, importantly, the MetS is known to increase the risk for developing cerebrocardiovascular disease and type 2 diabetes mellitus. The MetS had its early origins in IR and syndrome X. It has undergone numerous name changes, with additional risk factors and variables being added over the years; however, it has remained as the MetS worldwide for the past three decades. This overview continues to add novel insights to the MetS and suggests that leptin resistance with hyperleptinemia, aberrant mitochondrial stress and reactive oxygen species (ROS), impaired folate-mediated one-carbon metabolism with hyperhomocysteinemia, vascular stiffening, microalbuminuria, and visceral adipose tissues extracellular vesicle exosomes be added to the list of associated variables. Notably, the role of a dysfunctional and activated endothelium and deficient nitric oxide bioavailability along with a dysfunctional and attenuated endothelial glycocalyx, vascular inflammation, systemic metainflammation, and the important role of ROS and reactive species interactome are discussed. With new insights and knowledge regarding the MetS comes the possibility of new findings through further research.