Single-Cell Analyses Offer Insights into the Different Remodeling Programs of Arteries and Veins.

Arteries and veins develop different types of occlusive diseases and respond differently to injury. The biological reasons for this discrepancy are not well understood, which is a limiting factor for the development of vein-targeted therapies. This study contrasts human peripheral arteries and veins at the single-cell level, with a focus on cell populations with remodeling potential. Upper arm arteries (brachial) and veins (basilic/cephalic) from 30 organ donors were compared using a combination of bulk and single-cell RNA sequencing, proteomics, flow cytometry, and histology. The cellular atlases of six arteries and veins demonstrated a 7.8× higher proportion of contractile smooth muscle cells (SMCs) in arteries and a trend toward more modulated SMCs. In contrast, veins showed a higher abundance of endothelial cells, pericytes, and macrophages, as well as an increasing trend in fibroblasts. Activated fibroblasts had similar proportions in both types of vessels but with significant differences in gene expression. Modulated SMCs and activated fibroblasts were characterized by the upregulation of MYH10, FN1, COL8A1, and ITGA10. Activated fibroblasts also expressed F2R, POSTN, and COMP and were confirmed by F2R/CD90 flow cytometry. Activated fibroblasts from veins were the top producers of collagens among all fibroblast populations from both types of vessels. Venous fibroblasts were also highly angiogenic, proinflammatory, and hyper-responders to reactive oxygen species. Differences in wall structure further explain the significant contribution of fibroblast populations to remodeling in veins. Fibroblasts are almost exclusively located outside the external elastic lamina in arteries, while widely distributed throughout the venous wall. In line with the above, ECM-targeted proteomics confirmed a higher abundance of fibrillar collagens in veins vs. more basement ECM components in arteries. The distinct cellular compositions and transcriptional programs of reparative populations in arteries and veins may explain differences in acute and chronic wall remodeling between vessels. This information may be relevant for the development of antistenotic therapies.

The intricate cellular ecosystem of human peripheral veins as revealed by single-cell transcriptomic analysis.

The venous system has been historically understudied despite its critical roles in blood distribution, heart function, and systemic immunity. This study dissects the microanatomy of upper arm veins at the single cell level, and how it relates to wall structure, remodeling processes, and inflammatory responses to injury. We applied single-cell RNA sequencing to 4 non-diseased human veins (3 basilic, 1 cephalic) obtained from organ donors, followed by bioinformatic and histological analyses. Unsupervised clustering of 20,006 cells revealed a complex ecosystem of endothelial cell (EC) types, smooth muscle cell (SMCs) and pericytes, various types of fibroblasts, and immune cell populations. The venous endothelium showed significant upregulation of cell adhesion genes, with arteriovenous zonation EC phenotypes highlighting the heterogeneity of vasa vasorum (VV) microvessels. Venous SMCs had atypical contractile phenotypes and showed widespread localization in the intima and media. MYH11+DESlo SMCs were transcriptionally associated with negative regulation of contraction and pro-inflammatory gene expression. MYH11+DEShi SMCs showed significant upregulation of extracellular matrix genes and pro-migratory mediators. Venous fibroblasts ranging from secretory to myofibroblastic phenotypes were 4X more abundant than SMCs and widely distributed throughout the wall. Fibroblast-derived angiopoietin-like factors were identified as versatile signaling hubs to regulate angiogenesis and SMC proliferation. An abundant monocyte/macrophage population was detected and confirmed by histology, including pro-inflammatory and homeostatic phenotypes, with cell counts positively correlated with age. Ligand-receptor interactome networks identified the venous endothelium in the main lumen and the VV as a niche for monocyte recruitment and infiltration. This study underscores the transcriptional uniqueness of venous cells and their relevance for vascular inflammation and remodeling processes. Findings from this study may be relevant for molecular investigations of upper arm veins used for vascular access creation, where single-cell analyses of cell composition and phenotypes are currently lacking.

Comparative evaluation of biased agonists Sarcosine1 , d-Alanine8 -Angiotensin (Ang) II (SD Ang II) and Sarcosine1 , Isoleucine8 -Ang II (SI Ang II) and their radioiodinated congeners binding to rat liver membrane AT1 receptors.

Angiotensin II analogue and ß-arrestin biased agonist TRV027 (Sarcosine1 , d-Alanine8 -Angiotensin (Ang) II; SD Ang II), developed by Trevena, Inc. in the early 2010s, brought hopes of a novel treatment for cardiovascular diseases, due to its ability to simultaneously cause signaling through the ß-arrestin signaling pathway, while antagonizing the pathophysiological effects of Ang II mediated by the AT1 receptor G protein signaling cascades. However, a phase II clinical trial of this agent revealed no significant benefit compared to placebo treatment. Using 125 I-Sarcosine1 , Isoleucine8 -Ang II (125 I-SI Ang II) radioligand receptor competition binding assays, we assessed the relative affinity of TRV027 compared to SI Ang II for liver AT1 receptors. We also compared radioiodinated TRV027 (125 I-SD Ang II) binding affinity for liver AT1 receptors with 125 I-SI Ang II. We found that despite its anticipated gain in metabolic stability, TRV027 and 125 I-SD Ang II had reduced affinity for the AT1 receptor compared with SI Ang II and 125 I-SI Ang II. Additionally, male-female comparisons showed that females have a higher AT1 receptor density, potentially attributed to tissue-dependent estrogen and progesterone effects. Peptide drugs have become more popular over the years due to their increased bioavailability, fast onset of action, high specificity, and low toxicity. Even though Trevena®'s biased agonist peptide TRV027 offered greater stability and potency compared to earlier AT1 R biased agonists, it failed its phase II clinical trial in 2016. Further refinements to AT1 R biased agonist peptides to improve affinity, as seen with SI Ang II, with better stability and bioavailability, has the potential to achieve the anticipated biased agonism.

Chronic administration of pharmacological doses of angiotensin 1-7 and iodoangiotensin 1-7 has minimal effects on blood pressure, heart rate, and cognitive function of spontaneously hypertensive rats.

Cardiovascular diseases are the principal cause of death worldwide, with hypertension being the most common cardiovascular disease risk factor. High blood pressure (BP) is also associated with an increased risk of poor cognitive performance and dementia including Alzheimer's disease. Angiotensin 1-7 (Ang 1-7), a product of the renin-angiotensin system (RAS), exhibits central and peripheral actions to reduce BP. Recent data from our lab reveals that the addition of a non-radioactive iodine molecule to the tyrosine in position 4 of Ang 1-7 (iodoAng 1-7) makes it ~1000-fold more potent than Ang 1-7 in competing for the 125 I-Ang 1-7 binding site (Stoyell-Conti et al., 2020). Moreover, the addition of the non-radioactive iodine molecule increases (~4-fold) iodoAng 1-7's ability to bind to the AT1 receptor (AT1R), the primary receptor for Ang II. Preliminary data indicates that iodoAng 1-7 can also compete for the 125 I-Ang IV binding site with a low micromolar IC50. Thus, our aims were to compare the effects of chronic treatment of the Spontaneously Hypertensive Rat (SHR) with iodoAng 1-7 (non-radioactive iodine isotope) and Ang 1-7 on arterial pressure, heart rate, and cognitive function. For this study, male SHRs were divided into three groups and treated with Saline, Ang 1-7, or iodoAng 1-7 administrated subcutaneously using a 28-day osmotic mini pump. Systolic BP was measured non-invasively by the tail-cuff technique. Cognitive function was assessed by Y-Maze test and novel object recognition (NOR) test. We have demonstrated in SHRs that subcutaneous administration of high doses of iodoAng 1-7 prevented the increase in heart rate with age, while Ang 1-7 showed a trend toward preventing the increase in heart rate, possibly by improving baroreflex control of the heart. Conversely, neither Ang 1-7 nor iodoAng 1-7 administered subcutaneously affected BP nor cognitive function.

125I-Angiotensin 1-7 binds to a different site than angiotensin 1-7 in tissue membrane preparations.

PURPOSE: To study the receptor for Angiotensin (Ang) 1-7 using a radioligand (125I-Ang 1-7)-binding assay. For more than a decade, Mas has been viewed as the receptor for Ang 1-7; however, Ang 1-7 binding has not been pharmacologically characterized in tissue membrane preparations. METHODS: Radioligand-binding assays were carried out using tissue membrane preparations using radioiodinated Angiotensin 1-7 (125I-Ang 1-7) to characterize its binding site. Non-radioactive 127I-Ang 1-7 was used to test if the addition of an iodine to the tyrosine4 moiety of Ang 1-7 changes the ability of Ang 1-7 to competitively inhibit 125I-Ang 1-7 binding. RESULTS: 125I-Ang 1-7 binds saturably, with moderately high affinity (10-20 nM) to a binding site in rat liver membranes that is displaceable by 127I-Ang 1-7 at nanomolar concentrations (IC50 = 62 nM) while Ang 1-7 displaces at micromolar concentrations (IC50 = 80 µM) at ~22 °C. This binding was also displaceable by inhibitors of metalloproteases at room temperature. This suggests that 125I-Ang 1-7 binds to MMPs and/or ADAMs as well as other liver membrane elements at ~ 22 °C. However, when 125I-Ang 1-7-binding assays were run at 0-4 °C, the same MMP inhibitors did not effectively compete for 125I-Ang 1-7. CONCLUSIONS: The addition of an iodine molecule to the tyrosine in position 4 of Ang 1-7 drastically changes the binding characteristics of this peptide making it unsuitable for characterization of Ang 1-7 receptors.

Aerobic or resistance training improves autonomic control of circulation in oophorectomized rats with cardiometabolic dysfunctions: Impact on renal oxidative stress.

Cardiovascular autonomic dysfunction is associated with end organ damage and increased risk of mortality. Menopause and metabolic syndrome increase the risk for cardiorenal complications. In this study, we investigated the effects of aerobic or resistance exercise training on autonomic control of circulation and renal oxidative stress in a model of menopause and metabolic syndrome. Female Wistar rats and spontaneously hypertensive rats (SHR) were divided into 5 groups (n = 8): control (C), hypertensive (H), and sedentary (FHO), aerobic trained (FHOTa) and resistance trained (FHOTr) oophorectomized hypertensive treated with fructose (100 mg/mL drink water for 19 weeks). The FHO group presented increased vascular sympathetic modulation (LF-SBP), impaired baroreflex sensitivity (BRS) associated with increased blood pressure (BP) when compared to the H group. Aerobic exercise training enhanced tachycardic responses, while resistance training improved bradycardic responses to BP changes, thus ameliorating BRS. Moreover, despite unchanged BP, both exercise training protocols were effective in preventing increase in LF-SBP, reduction in systemic nitric oxide bioavailability (NOx), and increase in oxidative stress in the renal tissue, by decreasing lipid and protein oxidation in renal tissue. Positive correlation between LF-SBP and renal lipoperoxidation (r = 0.63, p < 0.05), as well as a negative correlation between NOx and renal lipoperoxidation (r = -0.66, p < 0.05) were observed. In conclusion, both aerobic and resistance exercise training were effective in improving autonomic control of circulation and reducing renal oxidative stress, thus attenuating the deleterious effects induced by arterial hypertension and fructose overload in female rats after ovarian hormone deprivation.

Correction: Impact of combined exercise training on the development of cardiometabolic and neuroimmune complications induced by fructose consumption in hypertensive rats.

[This corrects the article DOI: 10.1371/journal.pone.0233785.].

Impact of combined exercise training on the development of cardiometabolic and neuroimmune complications induced by fructose consumption in hypertensive rats.

This study evaluated the impact of combined exercise training on the development of cardiovascular and neuroimmune complications induced by fructose consumption (10% in the drinking water) in hypertensive rats (SHR). After weaning, SHR were divided into 3 groups: SHR (H), SHR+fructose (HF) and SHR+fructose+combined exercise training (treadmill+ladder, 40-60% of maximum capacity) (HFTC). Metabolic, hemodynamic, autonomic, inflammatory and oxidative stress parameters were evaluated in the subgroups (n = 6 group/time) at 7, 15, 30 and 60 days of protocol. Fructose consumption (H vs. HF groups) decreased spontaneous baroreflex sensitivity and total variance of pulse interval at day 7 (7 to 60); increased IL-6 and TNFα in the heart (at day 15, 30 and 60) and NADPH oxidase activity and cardiac lipoperoxidation (LPO) (day 60); increased white adipose tissue weight, reduced insulin sensitivity and increased triglycerides (day 60); induced an additional increase in mean arterial pressure (MAP) (days 30 and 60). Combined exercise training prevented such dysfunctions and sustained increased cardiac IL-10 (day 7) and glutathione redox balance (GSH/GSSG) for the entire protocol. In conclusion, combined exercise training performed simultaneously with exacerbated fructose consumption prevented early cardiovascular autonomic dysfunction, probably trigging positive changes in inflammation and oxidative stress, resulting in a better cardiometabolic profile in rats genetically predisposed to hypertension.

Aerobic Training Is Better Than Resistance Training on Cardiac Function and Autonomic Modulation in Female ob/ob Mice.

Objective: This study evaluated the effects of aerobic, resistance, and combined exercise training on cardiac function and autonomic modulation in female ob/ob mice. Methods: Four-week-old female wild type and obese (ob/ob) mice were divided into five groups (n = 8): control (WT), obese (OB) obese + aerobic training (OBA), obese + resistance training (OBR), and obese + combined training (OBC). The exercise training was performed on treadmill and/or ladder at 40-60% maximum test during 8 weeks. Cardiac function was measured using echo machine. Heart rate variability (HRV) was evaluated in the time and frequency domain. Results: OB group presented higher body weight gain (~600%), glycemia (~44%) and glucose intolerance (~150%), reduction of cardiac vagal modulation, evidenced by a lower RMMSD (~56%), total power and high frequency band, and a higher isovolumic relaxation time (IVRT) (~24%) in relation to the WT group. Aerobic and combined training led to a lower IVRT (OBA: ~14%; OBC: ~14%) and myocardial global index (OBA: ~37%; OBC: ~44%). The OBA group presented an increased in vagal indexes of HRV than the other ob/ob groups. A negative correlation was observed between the delta of aerobic exercise capacity and MPI (r = 0.45; p = 0.002) and exercise capacity and body weight gain (r = 0.39; p = 0.002). Conclusion: Only the obese females underwent to aerobic exercise training showed improvement in cardiac function and HRV. Moreover, the aerobic exercise capacity as well as a greater responsivity to aerobic exercise training is intimately associated with these improvements, reinforcing the importance of aerobic exercise training to this population.

Exercise benefits the cardiac, autonomic and inflammatory responses to organophosphate toxicity.

The organophosphate, diisopropyl fluorophosphate (DFP), may impair cardiovascular, autonomic and immune function while exercise training is thougt to be restorative. Experiments determined effects of wheel exercise in C57B1 male mice, testing cardiovascular and autonomic function and characterization of the immunological profile. Sedentary (S) and exercise (ET) groups were treated with corticosterone (CORT) followed by injection of DFP. This model was associated with systolic and diastolic dysfunction in the S group, measured using echocardiography (ECHO). Chronic exercise ameliorated the cardiac deficit. Autonomic balance, accessed by heart rate variability (HRV), showed increased sympathetic and decreased parasympathetic modulation in S group. Autonomic balance in ET mice was not affected by DFP. Our DFP model resulted in mild neuroinflammation seen by increased IL5, IL12 and MIP2 in brain and plasma IL6 and IL1a. DFP had a negative impact on cardiac/autonomic function and inflammatory markers, effects reduced by exercise. Data suggest a beneficial effect of exercise training on the cardiovascular and autonomic responses to DFP/CORT.

Exercise training initiated at old stage of lifespan attenuates aging-and ovariectomy-induced cardiac and renal oxidative stress: Role of baroreflex.

BACKGROUND: The association of aging and menopause is a potent risk factor for cardiometabolic disease. We studied the impact of aerobic exercise training (ET) initiated in the old stage of lifespan in hemodynamics, metabolic, autonomic and oxidative stress. METHODS: Aged (18 months old) female Wistar rats were divided into: ovariectomized and untrained (AG-OVX), and ovariectomized and trained (AG-OVXt, ET for 8 weeks). Intact aged (AG) and young female rats (3 months old; Y) were also studied. Blood pressure and metabolic parameters were measured. Baroreflex sensitivity (BRS) was studied by bradycardic (BR) and tachycardic (TR) responses to vasoactive drugs. Cardiac and renal lipid peroxidation (LPO), catalase (CAT), superoxide dismutase (SOD) and gluthatione peroxidase (GPx), and gluthatione redox balance (GSH/GSSG) were analyzed. RESULTS: AG-OVXt group increased aerobic performance in 35%, decreased adipose tissue and triglycerides in 36% and 27%, respectively, and improved insulin tolerance in 50% in comparison to AG-OVX. AG-OVX presented hypertensive levels of blood pressure (systolic: 155 ±â€¯5, diastolic: 111 ±â€¯3 mmHg). In contrast, AG-OVXt presented blood pressure values similar to Y rats (systolic: 129 ±â€¯3, diastolic: 112 ±â€¯3 mmHg). TR and BR were reduced by 70% and 46%, respectively, in AG-OVX vs. Y. Once more, AG-OVXt presented similar results to Y. ET decreased LPO in the heart and kidney. In the latter, renal CAT and SOD were corrected by ET, while cardiac redox balance was partially recovered. Improved BRS was correlated with improved oxidative stress markers. CONCLUSIONS: Even when initiated after aging and ovariectomy deleterious effects, ET is able to normalize BRS and highly improve cardiac and renal oxidative stress.

Measurement of Mouse Heart Rate Variability using Echocardiographic System.

AIM: We employed an echocardiographic (ECHO) system as the backbone for the collection of electrocardiogram (ECG) and heart rate variability (HRV) data. The system was tested using an exercise model in which C57 male mice were exposed to sham or forced wheel running. METHODS: Peak/peak (RR) interval was recorded over a 3 min period using the ECG platform of the ECHO system. Isoflurane-anesthetized male mice were divided into two groups (n = 8/group): sedentary (S) and forced wheel trained (T). HRV was analyzed in time and frequency domains (Fast Fourier Transform). Exercise training (T) was performed on a motorized wheel at low intensity 1 h/day, 5 days/week, 8 weeks duration. Cardiac morphometry and function were analyzed using ECHO while ECG was the basis to measure HRV. The sampling rate was 8000 Hz. Results show that the trained mice presented a reduction in heart rate as compared to the sedentary group. This was associated with lower cardiac sympathetic and higher parasympathetic modulation leading to an improved sympathetic/parasympathetic ratio (low-frequency band/high-frequency band). The trained group showed a reduction in isovolumetric relaxation time, reduced myocardial performance index, increased relative wall thickness, and left ventricle mass when compared to the sedentary group. CONCLUSION: Results document the utility of combining the ECHO and the ECG platform, allowing for the dual measurement of autonomic and cardiac function in mice.

Baroreflex Impairment Precedes Cardiometabolic Dysfunction in an Experimental Model of Metabolic Syndrome: Role of Inflammation and Oxidative Stress.

This study analyzes whether autonomic dysfunction precedes cardiometabolic alterations in spontaneously hypertensive rats (SHR) with fructose overload. Animals were randomly distributed into three groups: control, hypertensive and hypertensive with fructose overload. Fructose overload (100 g/L) was initiated at 30 days old, and the animals (n = 6/group/time) were evaluated after 7, 15, 30 and 60 days of fructose consumption. Fructose consumption reduced baroreflex sensitivity by day 7, and still induced a progressive reduction in baroreflex sensitivity over the time. Fructose consumption also increased TNFα and IL-6 levels in the adipose tissue and IL-1ß levels in the spleen at days 15 and 30. Fructose consumption also reduced plasmatic nitrites (day 15 and 30) and superoxide dismutase activity (day 15 and 60), but increased hydrogen peroxide (day 30 and 60), lipid peroxidation and protein oxidation (day 60). Fructose consumption increased arterial pressure at day 30 (8%) and 60 (11%). Fructose consumption also induced a late insulin resistance at day 60, but did not affect glucose levels. In conclusion, the results show that baroreflex sensitivity impairment precedes inflammatory and oxidative stress disorders, probably by inducing hemodynamic and metabolic dysfunctions observed in metabolic syndrome.