Abstract 1421: In Vivo Measurement of Flow-Mediated Vasodilation in Living Rats using High Resolution Ultrasound: Age-Dependent Endothelial Dysfunction

In humans, endothelial function serves as a surrogate marker for cardiovascular health and is measured as changes in arterial diameter after temporary ischemia (flow-mediated dilation; FMD). We developed an FMD-related approach to study conduit artery vasodilation in living rats, and demonstrate a reduction in FMD in older versus younger animals consistent with age-related endothelial dysfunction. Diameter and Doppler-flow measurements were obtained from the femoral artery using high-resolution ultrasound (35 MHz). We observed dose-dependent vasodilation using both endothelium-dependent and endothelium-independent pharmacologic vasodilators (acetylcholine and nitroglycerine). Flow-dependent vasodilation was observed in response to flow increase induced both by adenosine and local saline infusion. Transient hindlimb ischemia led to reactive hyperemia with sequential flow velocity increase and femoral artery dilation, the latter of which was completely abolished by NO-synthase (NOS) inhibition with L-NMMA. To demonstrate its applicability in a model of endothelial dysfunction, we show that FMD is significantly reduced in older versus younger animals. While FMD was completely NOS-dependent in younger animals, NOS-dependent mechanisms accounted for only half of the FMD in older animals, with the remainder being blocked by charybdotoxin (CTx) and apamin suggesting contribution of endothelium-derived-hyperpolarizing-factor. Using this new integrative physiologic model to reproducibly study FMD in living rats, we show that age-dependent endothelial dysfunction is accompanied by a shift in mechanisms underlying vasodilatory endothelial function.

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In vivo measurement of flow-mediated vasodilation in living rats using high-resolution ultrasound

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00811.2007 ◽

2008 ◽

Vol 294 (2) ◽

pp. H1086-H1093 ◽

Cited By ~ 28

Author(s):

Christian Heiss ◽

Richard E. Sievers ◽

Nicolas Amabile ◽

Tony Y. Momma ◽

Qiumei Chen ◽

...

Keyword(s):

Shear Stress ◽

High Resolution ◽

Wall Shear Stress ◽

Cardiovascular Health ◽

Reactive Hyperemia ◽

Surrogate Marker ◽

Automated Analysis ◽

Physiological Model ◽

Wall Shear

In humans, endothelial vasodilator function serves as a surrogate marker for cardiovascular health and is measured as changes in conduit artery diameter after temporary ischemia [flow-mediated dilation (FMD)]. Here we present an FMD-related approach to study femoral artery (FA) vasodilation in anesthetized rats. Diameter and Doppler flow were monitored in the FA. Using high-resolution ultrasound (35 MHz) and automated analysis software, we detected dose-dependent vasodilation using established endothelium-independent [intravenous nitroglycerin EC50 = 3.3 × 10−6 mol/l, peak 21Δ% (SD 4)] and endothelium-dependent [intra-arterial acetylcholine EC50 = 1.3 × 10−6 mol/l, peak 27Δ% (SD 4)] pharmacological vasodilators. Wall shear stress induced by intra-aortic injection of adenosine and infusion of saline at increasing rates (1.5–4.5 ml/min) led to vasodilation at 1 to 2 min. Transient hindlimb ischemia by common iliac occlusion (5 min) led to reactive hyperemia with flow velocity and wall shear stress increase and was followed by FA dilation [16Δ% (SD 2)], the latter of which was completely abolished by nitric oxide synthase (NOS) inhibition with NG-monomethyl-l-arginine [1Δ% (SD 2)]. FMD was significantly reduced in adult 20–24-wk-old animals compared with 9- to 10-wk-old animals, consistent with age-dependent endothelial dysfunction [16Δ% (SD 3) vs. 10Δ% (SD 3), P < 0.05]. Whereas FMD was completely NOS dependent in 9- to 10-wk-old animals, NOS-dependent mechanisms accounted for only half of the FMD in 20–24-wk-old animals, with the remainder being blocked by charybdotoxin and apamin, suggesting a contribution of endothelium-derived hyperpolarizing factor. To our knowledge, this is the first integrative physiological model to reproducibly study FMD of conduit arteries in living rats.

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Erythrocytes induce vascular dysfunction in COVID-19

European Heart Journal ◽

10.1093/eurheartj/ehab724.3364 ◽

2021 ◽

Vol 42 (Supplement_1) ◽

Author(s):

A Mahdi ◽

A Collado ◽

J Tengbom ◽

T Jiao ◽

T Wodaje ◽

...

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Endothelial Dysfunction ◽

Endothelial Function ◽

Vascular Injury ◽

Healthy Subjects ◽

Vascular Dysfunction ◽

Reactive Hyperemia ◽

Oxygen Species

Abstract Background Vascular injury has been implicated as a major cause of clinical complications in patients with coronavirus disease 2019 (COVID-19). Autopsy studies have revealed destruction of the endothelial cell lining, which might explain cardiovascular alterations arising from the infection. However, data demonstrating endothelial dysfunction during ongoing infection are sparse, and the underlying mechanisms are still largely unknown. Red blood cells (RBCs) are affected by COVID-19 with alterations in their structure and function, possibly contributing to vascular injury via increased oxidative stress. Purpose To determine the presence of endothelial dysfunction in patients with COVID-19 and to explore the RBC as a possible mediator of such dysfunction. Methods The study was performed on 17 patients hospitalized for moderate COVID-19 infection and age- and sex-matched healthy subjects. Inclusion criteria of the COVID-19 patients were PCR-verified SARS-CoV2 infection, pulmonary infiltrates on x-ray, oxygen demand during hospital stay and ≤ one cardiovascular co-morbidity. Microvascular endothelial function in vivo was assessed with a pulse amplitude tonometry device on each index finger at baseline and during reactive hyperemia and expressed as reactive hyperemia index (RHI). RBCs from COVID-19 patients (C19-RBCs) and healthy subjects (H-RBCs) were incubated with isolated rat aortic segments for evaluation of endothelium-dependent and -independent relaxation. Results COVID-19 patients displayed profound impairment in endothelial function in vivo with RHI 1.56 (1.30–1.81, median and interquartile range) compared to healthy subjects 2.36 (1.97–2.79, p<0.001). C19-RBCs induced severe impairment in both endothelium-dependent (27% maximal relaxation) and -independent relaxations (54%) compared to H-RBCs (67% and 95% relaxation, respectively). Further, C19-RBCs induced upregulation of vascular arginase 1 (∼2 fold increase compared to H-RBCs) and markers of oxidative stress (∼6 fold). Consequently, inhibition of vascular arginase or superoxide attenuated the impairment in endothelial function induced by C19-RBCs. C19-RBCs were characterized by increased production of reactive oxygen species (∼1.4 fold) and reduced export of the nitric oxide metabolite nitrate. Following pre-incubation with interferon-γ, but not interleukin-6 or tumor necrosis factor-α, H-RBCs induced impairment in endothelial function. Conclusions This study demonstrates the presence of marked endothelial dysfunction in an otherwise mainly healthy patient group hospitalized for COVID-19, and clearly implicates a central role of the RBC as a mediator of endothelial injury through enhancement of reactive oxygen species and arginase. These data shed light on a new pathological mechanism underlying vascular dysfunction in COVID-19 patients and may lay the foundation for future therapeutic developments. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung foundationSwedish Research Council

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Age-Dependent Demethylation ofSod2Promoter in the Mouse Femoral Artery

Oxidative Medicine and Cellular Longevity ◽

10.1155/2016/8627384 ◽

2016 ◽

Vol 2016 ◽

pp. 1-6 ◽

Cited By ~ 7

Author(s):

Albert Nguyen ◽

François Leblond ◽

Maya Mamarbachi ◽

Steve Geoffroy ◽

Eric Thorin

Keyword(s):

Endothelial Function ◽

Femoral Artery ◽

Promoter Methylation ◽

Transcriptional Activity ◽

Manganese Superoxide Dismutase ◽

Defense Mechanism ◽

Age Related ◽

Manganese Superoxide ◽

Age Dependent

We studied the age-dependent regulation of the expression of the antioxidant enzyme manganese superoxide dismutase (MnSOD encoded bySod2) through promoter methylation. C57Bl/6 mice were either (i) sedentary (SED), (ii) treated with the antioxidant catechin (CAT), or (iii) voluntarily exercised (EX) from weaning (1-month old; mo) to 9 mo. Then, all mice aged sedentarily and were untreated until 12 mo.Sod2promoter methylation was similar in all groups in 9 mo but decreased (p<0.05) in 12 mo SED mice only, which was associated with an increased (p<0.05) transcriptional activityin vitro. At all ages, femoral artery endothelial function was maintained; this was due to an increased (p<0.05) contribution of eNOS-derived NO in 12 mo SED mice only. CAT and EX prevented these changes in age-related endothelial function. Thus, a ROS-dependent epigenetic positive regulation ofSod2gene expression likely represents a defense mechanism prolonging eNOS function in aging mouse femoral arteries.

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Circulating biologic markers of endothelial dysfunction in cerebral small vessel disease: A review

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2015.116 ◽

2015 ◽

Vol 36 (1) ◽

pp. 72-94 ◽

Cited By ~ 98

Author(s):

Anna Poggesi ◽

Marco Pasi ◽

Francesca Pescini ◽

Leonardo Pantoni ◽

Domenico Inzitari

Keyword(s):

Endothelial Dysfunction ◽

Endothelial Function ◽

Small Vessel Disease ◽

Brain Magnetic Resonance Imaging ◽

Cerebral Small Vessel Disease ◽

Small Vessel ◽

Perivascular Spaces ◽

Vessel Disease ◽

Brain Changes

The term cerebral small vessel disease (SVD) refers to a group of pathologic processes with various etiologies that affect small arteries, arterioles, venules, and capillaries of the brain. Magnetic resonance imaging (MRI) correlates of SVD are lacunes, recent small subcortical infarcts, white-matter hyperintensities, enlarged perivascular spaces, microbleeds, and brain atrophy. Endothelial dysfunction is thought to have a role in the mechanisms leading to SVD-related brain changes, and the study of endothelial dysfunction has been proposed as an important step for a better comprehension of cerebral SVD. Among available methods to assess endothelial function in vivo, measurement of molecules of endothelial origin in peripheral blood is currently receiving selective attention. These molecules include products of endothelial cells that change when the endothelium is activated, as well as molecules that reflect endothelial damage and repair. This review examines the main molecular factors involved in both endothelial function and dysfunction, and the evidence linking endothelial dysfunction with cerebral SVD, and gives an overview of clinical studies that have investigated the possible association between endothelial circulating biomarkers and SVD-related brain changes.

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Arginase inhibition restores NOS coupling and reverses endothelial dysfunction and vascular stiffness in old rats

Journal of Applied Physiology ◽

10.1152/japplphysiol.91393.2008 ◽

2009 ◽

Vol 107 (4) ◽

pp. 1249-1257 ◽

Cited By ~ 159

Author(s):

Jae Hyung Kim ◽

Lukasz J. Bugaj ◽

Young Jun Oh ◽

Trinity J. Bivalacqua ◽

Sungwoo Ryoo ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Dysfunction ◽

Endothelial Function ◽

Vascular Stiffness ◽

Arginase Activity ◽

Young Rats ◽

Arginase 1 ◽

Enos Uncoupling ◽

Old Rats

There is increasing evidence that upregulation of arginase contributes to impaired endothelial function in aging. In this study, we demonstrate that arginase upregulation leads to endothelial nitric oxide synthase (eNOS) uncoupling and that in vivo chronic inhibition of arginase restores nitroso-redox balance, improves endothelial function, and increases vascular compliance in old rats. Arginase activity in old rats was significantly increased compared with that shown in young rats. Old rats had significantly lower nitric oxide (NO) and higher superoxide (O2−) production than young. Acute inhibition of both NOS, with NG-nitro-l-arginine methyl ester, and arginase, with 2( S)-amino- 6-boronohexanoic acid (ABH), significantly reduced O2− production in old rats but not in young. In addition, the ratio of eNOS dimer to monomer in old rats was significantly decreased compared with that shown in young rats. These results suggest that eNOS was uncoupled in old rats. Although the expression of arginase 1 and eNOS was similar in young and old rats, inducible NOS (iNOS) was significantly upregulated. Furthermore, S-nitrosylation of arginase 1 was significantly elevated in old rats. These findings support our previously published finding that iNOS nitrosylates and activates arginase 1 (Santhanam et al., Circ Res 101: 692–702, 2007). Chronic arginase inhibition in old rats preserved eNOS dimer-to-monomer ratio and significantly reduced O2− production and enhanced endothelial-dependent vasorelaxation to ACh. In addition, ABH significantly reduced vascular stiffness in old rats. These data indicate that iNOS-dependent S-nitrosylation of arginase 1 and the increase in arginase activity lead to eNOS uncoupling, contributing to the nitroso-redox imbalance, endothelial dysfunction, and vascular stiffness observed in vascular aging. We suggest that arginase is a viable target for therapy in age-dependent vascular stiffness.

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Endothelial Function in Patients with Severe and Moderate Haemophilia A and B

Hämostaseologie ◽

10.1055/s-0038-1673415 ◽

2018 ◽

Vol 39 (02) ◽

pp. 195-202

Author(s):

Stephanie Böhmert ◽

Ralf Schubert ◽

Stephan Fichtlscherer ◽

Sonja Alesci ◽

Wolfgang Miesbach

Keyword(s):

Endothelial Function ◽

Adhesion Molecule ◽

Reactive Hyperemia ◽

Intercellular Adhesion Molecule ◽

Control Group ◽

Low Grade ◽

Haemophilia A ◽

Intercellular Adhesion Molecule 1 ◽

Age Related ◽

Artery Disease

AbstractThe life expectancy of patients with haemophilia has increased and therefore the interest in age-related comorbidities has grown. The aim of this study was to determine whether haemophilia patients have a different endothelial function compared with the general population. A total of 26 patients with severe or moderate haemophilia A or B, 14 controls and 36 patients with coronary artery disease (CAD) were included in this study. Five markers of endothelial dysfunction (MOEDs) were determined. Moreover, the endothelial function was examined using the Itamar Endo-PAT, and the reactive hyperemia index (RHI) was calculated from the results. The MOEDs soluble intercellular adhesion molecule-1 (p = 0.0095) and interleukin-6 (p = 0.010) were significantly higher for patients with haemophilia compared with the control group. The presence of increased adhesion molecule levels and low-grade inflammation is suggestive of a decreased endothelial function. RHI is impaired in CAD patients (1.862), whereas haemophilia patients have an RHI of 1.958 in comparison with 2.112 in controls (p = 0.127). Therefore, laboratory and functional measurements imply a possible higher risk for CAD in haemophilia patients.

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The Relationship Between the Impairment of Endothelial Function and Thyroid Antibodies in Hashimoto’s Thyroiditis Patients with Euthyroidism

Hormone and Metabolic Research ◽

10.1055/a-1178-5882 ◽

2020 ◽

Vol 52 (09) ◽

pp. 642-646

Author(s):

Yanjin Hu ◽

Zhi Yao ◽

Guang Wang

Keyword(s):

Endothelial Dysfunction ◽

Endothelial Function ◽

Hashimoto’S Thyroiditis ◽

Reactive Hyperemia ◽

Density Lipoprotein ◽

Hashimoto Thyroiditis ◽

Hashimoto's Thyroiditis ◽

Thyroid Peroxidase ◽

Healthy Controls ◽

Negatively Associated

AbstractEndothelial dysfunction is the important early step in the development of atherosclerosis. Hypothyroidism caused by Hashimoto’s thyroiditis and other thyroid disease is one of the risk factors of endothelial dysfunction. The present study tried to investigate the endothelial function and its associated factors in Hashimoto thyroiditis with euthyroidism. A total of 95 newly diagnosed Hashimoto’s thyroiditis patients with euthyroidism and 45 healthy controls were studied. Hashimoto’s patients were divided into 3 subgroups namely, single thyroglobulin antibody (TGAb) positive subgroup, single thyroid peroxidase antibody (TPOAb) positive subgroup, and both TGAb and TPOAb positive subgroup. Endothelial function was tested by the reactive hyperemia index (RHI). Hashimoto’s thyroiditis patients had lower RHI than healthy controls (1.73±0.42 vs 1.96±0.51, p<0.05). Hashimoto’s thyroiditis with single TGAb positive patients had higher RHI than single TPOAb positive (1.98±0.57 vs. 1.69±0.33, p<0.05) and TGAB + TPOAb positive patients (1.98±0.57 vs. 1.68±0.42, p<0.05). RHI were negatively associated with total cholesterol (TC, r=−0.215, p<0.05), low density lipoprotein cholesterol (LDL-C, r=−0.268, p<0.05), triglyceride (TG, r=−0.192, p<0.05), and TPOAb (r=−0.288, p<0.05). In the regression analysis, LDL-C (β=−0.146, p<0.05), TG (β=−0.034, p<0.05) and TPOAb (β=−0.001, p<0.05) were independently associated with RHI. Hashimoto’s patients had poor endothelial function. TPOAb levels were negatively associated with endothelial function.

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Early adaptations in collateral and microvascular resistances after ligation of the rat femoral artery

Journal of Applied Physiology ◽

10.1152/jappl.1995.79.1.73 ◽

1995 ◽

Vol 79 (1) ◽

pp. 73-82 ◽

Cited By ~ 32

Author(s):

J. L. Unthank ◽

J. C. Nixon ◽

J. M. Lash

Keyword(s):

Femoral Artery ◽

Reactive Hyperemia ◽

Pressure Measurements ◽

Total Resistance ◽

Perfusion Technique ◽

Vascular Conductance ◽

Collateral Vessels ◽

Microvascular Resistance

Collateral and microvascular (including feed artery) resistances in the rat hindlimb were determined immediately or 1 wk after ligation of the femoral artery. Collateral-to-microvascular resistance ratios were determined from in vivo pressure measurements proximal and distal to the ligation. Microvascular resistance was 32 +/- 2.5 and 41 +/- 1.5% of the total collateral-dependent vasculature in acutely and chronically ligated limbs, respectively, and decreased 20% in both groups during reactive hyperemia. Minimum resistances of collateral vessels and the microcirculation arising from arterial branches proximal and distal to the ligation were determined by using a modification of the standard hindquarter perfusion technique for determining maximum vascular conductance. One week postligation, minimum total hindquarter resistance was decreased by a reduction in the resistance of the collaterals (approximately 50%) and microcirculation (approximately 33%) proximal to the ligation. The results suggest that the microvasculature distal to the occlusion is able to increase flow by dilation both initially and at 1 wk postligation but that collateral adaptations are primarily responsible for decreases in the minimum total resistance of the collateral-dependent region.

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Impaired nitric oxide bioavailability and l-arginine–reversible endothelial dysfunction in adults with falciparum malaria

Journal of Experimental Medicine ◽

10.1084/jem.20070819 ◽

2007 ◽

Vol 204 (11) ◽

pp. 2693-2704 ◽

Cited By ~ 208

Author(s):

Tsin W. Yeo ◽

Daniel A. Lampah ◽

Retno Gitawati ◽

Emiliana Tjitra ◽

Enny Kenangalem ◽

...

Keyword(s):

Endothelial Dysfunction ◽

Endothelial Function ◽

Falciparum Malaria ◽

Reactive Hyperemia ◽

Severe Disease ◽

Microvascular Endothelium ◽

Tissue Ischemia ◽

Exhaled No ◽

No Bioavailability ◽

Severe Falciparum Malaria

Severe falciparum malaria (SM) is associated with tissue ischemia related to cytoadherence of parasitized erythrocytes to microvascular endothelium and reduced levels of NO and its precursor, l-arginine. Endothelial function has not been characterized in SM but can be improved by l-arginine in cardiovascular disease. In an observational study in Indonesia, we measured endothelial function using reactive hyperemia–peripheral arterial tonometry (RH-PAT) in 51 adults with SM, 48 patients with moderately severe falciparum malaria (MSM), and 48 controls. The mean RH-PAT index was lower in SM (1.41; 95% confidence interval [CI] = 1.33–1.47) than in MSM (1.82; 95% CI = 1.7–2.02) and controls (1.93; 95% CI = 1.8–2.06; P < 0.0001). Endothelial dysfunction was associated with elevated blood lactate and measures of hemolysis. Exhaled NO was also lower in SM relative to MSM and controls. In an ascending dose study of intravenous l-arginine in 30 more patients with MSM, l-arginine increased the RH-PAT index by 19% (95% CI = 6–34; P = 0.006) and exhaled NO by 55% (95% CI = 32–73; P < 0.0001) without important side effects. Hypoargininemia and hemolysis likely reduce NO bioavailability. Endothelial dysfunction in malaria is nearly universal in severe disease, is reversible with l-arginine, and likely contributes to its pathogenesis. Clinical trials in SM of adjunctive agents to improve endothelial NO bioavailability, including l-arginine, are warranted.

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Age-dependent changes in protein incorporation into collagen-rich tissues of mice by in vivo pulsed SILAC labelling

eLife ◽

10.7554/elife.66635 ◽

2021 ◽

Vol 10 ◽

Author(s):

Yoanna Ariosa-Morejon ◽

Alberto Santos ◽

Roman Fischer ◽

Simon Davis ◽

Philip Charles ◽

...

Keyword(s):

Skeletal Maturity ◽

Tissue Level ◽

Skeletal Maturation ◽

Healthy Life ◽

Adult Mice ◽

Age Related ◽

Age Dependent ◽

Protein Incorporation ◽

New Protein

Collagen-rich tissues have poor reparative capacity that predisposes to common age-related disorders such as osteoporosis and osteoarthritis. We used in vivo pulsed SILAC labelling to quantify new protein incorporation into cartilage, bone, and skin of mice across the healthy life course. We report dynamic turnover of the matrisome, the proteins of the extracellular matrix, in bone and cartilage during skeletal maturation, which was markedly reduced after skeletal maturity. Comparing young adult with older adult mice, new protein incorporation was reduced in all tissues. STRING clustering revealed changes in epigenetic modulators across all tissues, a decline in chondroprotective growth factors such as FGF2 and TGFβ in cartilage, and clusters indicating mitochondrial dysregulation and reduced collagen synthesis in bone. Several pathways were implicated in age-related disease. Fewer changes were observed for skin. This methodology provides dynamic protein data at a tissue level, uncovering age-related molecular changes that may predispose to disease.

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