Julie K. Freed

The Human Microcirculation: Regulation of Flow and Beyond

The microcirculation is responsible for orchestrating adjustments in vascular tone to match local tissue perfusion with oxygen demand. Beyond this metabolic dilation, the microvasculature plays a critical role in modulating vascular tone by endothelial release of an unusually diverse family of compounds including nitric oxide, other reactive oxygen species, and arachidonic acid metabolites. Animal models have provided excellent insight into mechanisms of vasoregulation in health and disease. However, there are unique aspects of the human microcirculation that serve as the focus of this review. The concept is put forth that vasculoparenchymal communication is multimodal, with vascular release of nitric oxide eliciting dilation and preserving normal parenchymal function by inhibiting inflammation and proliferation. Likewise, in disease or stress, endothelial release of reactive oxygen species mediates both dilation and parenchymal inflammation leading to cellular dysfunction, thrombosis, and fibrosis. Some pathways responsible for this stress-induced shift in mediator of vasodilation are proposed. This paradigm may help explain why microvascular dysfunction is such a powerful predictor of cardiovascular events and help identify new approaches to treatment and prevention.

Ceramide Changes the Mediator of Flow-Induced Vasodilation from Nitric Oxide to Hydrogen Peroxide in the Human Microcirculation

Rationale: Mitochondrial-derived hydrogen peroxide (H2O2) regulates flow-induced dilation (FID) in microvessels from patients with coronary artery disease. The relationship between ceramide, an independent risk factor for coronary artery disease and a known inducer of mitochondrial reactive oxygen species, and FID is unknown. Objective: We examined the hypothesis that exogenous ceramide induces a switch in the mediator of FID from nitric oxide to H2O2. Methods and Results: Internal diameter changes of resistance arterioles from human adipose and atrial tissue were measured by video microscopy. Mitochondrial H2O2 production was assayed in arterioles using mito peroxy yellow 1. Polyethylene glycol–catalase, rotenone, and Mito-TEMPO impaired FID in healthy adipose arterioles pretreated with ceramide, whereas N&ohgr;-nitro-L-arginine methyl ester had no effect. Mitochondrial H2O2 production was induced in response to flow in healthy adipose vessels pretreated with ceramide, and this was abolished in the presence of polyethylene glycol–catalase. Immunohistochemistry demonstrated ceramide accumulation in arterioles from both healthy patients and patients with coronary artery disease. N&ohgr;-nitro-L-arginine methyl ester reduced vasodilation to flow in adipose as well as atrial vessels from patients with coronary artery disease incubated with GW4869, a neutral sphingomyelinase inhibitor, whereas polyethylene glycol–catalase had no effect. Conclusions: Our data indicate that ceramide has an integral role in the transition of the mediator of FID from nitric oxide to mitochondrial-derived H2O2 and that inhibition of ceramide production can revert the mechanism of dilation back to nitric oxide. Ceramide may be an important target for preventing and treating vascular dysfunction associated with atherosclerosis.

Critical Role for Telomerase in the Mechanism of Flow Mediated Dilation in the Human Microcirculation

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PGC-1α (Peroxisome Proliferator–Activated Receptor γ Coactivator 1-α) Overexpression in Coronary Artery Disease Recruits NO and Hydrogen Peroxide During Flow-Mediated Dilation and Protects Against Increased Intraluminal Pressure

Blood flow through healthy human vessels releases NO to produce vasodilation, whereas in patients with coronary artery disease (CAD), the mediator of dilation transitions to mitochondria-derived hydrogen peroxide (mtH2O2). Excessive mtH2O2 production contributes to a proatherosclerotic vascular milieu. Loss of PGC-1&agr; (peroxisome proliferator–activated receptor &ggr; coactivator 1&agr;) is implicated in the pathogenesis of CAD. We hypothesized that PGC-1&agr; suppresses mtH2O2 production to reestablish NO-mediated dilation in isolated vessels from patients with CAD. Isolated human adipose arterioles were cannulated, and changes in lumen diameter in response to graded increases in flow were recorded in the presence of PEG (polyethylene glycol)–catalase (H2O2 scavenger) or L-NAME (NG-nitro-L-arginine methyl ester; NOS inhibitor). In contrast to the exclusively NO- or H2O2-mediated dilation seen in either non-CAD or CAD conditions, respectively, flow-mediated dilation in CAD vessels was sensitive to both L-NAME and PEG-catalase after PGC-1&agr; upregulation using ZLN005 and &agr;-lipoic acid. PGC-1&agr; overexpression in CAD vessels protected against the vascular dysfunction induced by an acute increase in intraluminal pressure. In contrast, downregulation of PGC-1&agr; in non-CAD vessels produces a CAD-like phenotype characterized by mtH2O2-mediated dilation (no contribution of NO). Loss of PGC-1&agr; may contribute to the shift toward the mtH2O2-mediated dilation observed in vessels from subjects with CAD. Strategies to boost PGC-1&agr; levels may provide a therapeutic option in patients with CAD by shifting away from mtH2O2-mediated dilation, increasing NO bioavailability, and reducing levels of mtH2O2. Furthermore, increased expression of PGC-1&agr; allows for simultaneous contributions of both NO and H2O2 to flow-mediated dilation.

Proteomic analysis of shear stress-mediated protection from TNF-alpha in endothelial cells.

Microcirculation (2010) 17, 259–270. doi: 10.1111/j.1549‐8719.2010.00031.x

Mitochondrial Reactive Oxygen Species and Vascular Function: Less Is More

Reactive oxygen species (ROS), conventionally known for causing cellular damage, are now accepted as important signaling molecules both physiologically and in the pathogenesis of cardiovascular disease. Elevated levels of ROS have been linked to the development of disorders, such as diabetes mellitus, hypertension, hypercholesterolemia, obesity, and atherosclerosis.1–4 Conversely, oxidative stress has been shown to stimulate and regulate multiple cell functions, including cell growth and proliferation, apoptosis, host defense, genomic stability, and vascular tone.5–8 An emerging concept is evolving of an optimal physiological range of ROS required to maintain cell homeostasis. Redox balance in the endothelium is a model example where ROS critically regulate vascular smooth muscle tone and tissue perfusion. See accompanying article on page 752 Endothelial cells produce an array of ROS, including superoxide (O2·–), hydrogen peroxide (H2O2), peroxynitrite (ONOO–), hydroxyl radicals (OH·), lipid peroxides, and other radicals. O2·– production in the picomolar range reacts quickly with superoxide dismutase to form H2O2, a freely diffusible and stable form of ROS, with typical characteristics of a cell-signaling molecule that can participate in compensatory vasodilation in disease.2,9 Peroxynitrite, formed as the product of excess O2·– quenching available nitric oxide (NO), adds to both cellular nitrosative and oxidative stress.10 The associated decrease in NO bioavailability with concurrent elevation in oxidative stress impairs endothelium-dependent vasodilation, producing what is referred to as endothelial dysfunction, …

Revealing the Role of Phosphatidylserine in Shear Stress –Mediated Protection in Endothelial Cells

Previous studies have demonstrated that endothelial cells exposed to laminar shear stress are protected from apoptotic stimuli such as tumor necrosis factor (TNF)-alpha. The authors investigated the role of phosphatidylserine (PS) in this phenomenon. Western blot analysis of cleaved caspase 3 was used as an indicator of apoptosis and revealed that in the absence of serine, endothelial cells exposed to laminar shear stress were unable to protect against TNF-alpha-induced apoptosis, in contrast to sheared cells grown in regular medium. It was also found that shear-induced activation of the Akt pathway was significantly decreased in cells grown without serine. In addition, quantitation of PS using a novel isotopic labeling technique involving the use of formalin revealed that stearoyl-oleic PS (18:0/18:1) did not increase during shear treatment. These findings suggest that basal levels of PS are required to activate survival pathways in endothelial cells and thereby contribute to the overall protective mechanism initiated by shear stress.

Communication Is Key: Mechanisms of Intercellular Signaling in Vasodilation.

Abstract: Thirty years ago, Robert F. Furchgott concluded that nitric oxide, a compound traditionally known to be a toxic component of fuel exhaust, is in fact released from the endothelium, and in a paracrine fashion, induces relaxation of underlying vascular smooth muscle resulting in vasodilation. This discovery has helped pave the way for a more thorough understanding of vascular intercellular and intracellular communication that supports the process of regulating regional perfusion to match the local tissue oxygen demand. Vasoregulation is controlled not only by endothelial release of a diverse class of vasoactive compounds such as nitric oxide, arachidonic acid metabolites, and reactive oxygen species, but also by physical forces on the vascular wall and through electrotonic conduction through gap junctions. Although the endothelium is a critical source of vasoactive compounds, paracrine mediators can also be released from surrounding parenchyma such as perivascular fat, myocardium, and cells in the arterial adventitia to exert either local or remote vasomotor effects. The focus of this review will highlight the various means by which intercellular communication contributes to mechanisms of vasodilation. Paracrine signaling and parenchymal influences will be reviewed as well as regional vessel communication through gap junctions, connexons, and myoendothelial feedback. More recent modes of communication such as vesicular and microRNA signaling will also be discussed.

Moderate Aortic Valvular Insufficiency Invalidates Vortex Formation Time as an Index of Left Ventricular Filling Efficiency in Patients With Severe Degenerative Calcific Aortic Stenosis Undergoing Aortic Valve Replacement

OBJECTIVE Transmitral blood flow produces a vortex ring (quantified using vortex formation time [VFT]) that enhances the efficiency of left ventricular (LV) filling. VFT is attenuated in LV hypertrophy resulting from aortic valve stenosis (AS) versus normal LV geometry. Many patients with AS also have aortic insufficiency (AI). The authors tested the hypothesis that moderate AI falsely elevates VFT by partially inhibiting mitral leaflet opening in patients with AS. DESIGN Observational study. SETTING Veterans Affairs medical center. PARTICIPANTS Patients with AS in the presence or absence of moderate AI (n = 8 per group) undergoing aortic valve replacement (AVR) were studied after institutional review board approval. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Under general anesthesia, peak early LV filling (E) and atrial systole (A) blood flow velocities and their corresponding velocity-time integrals were obtained using pulse-wave Doppler transesophageal echocardiography (TEE) to determine E/A and atrial filling fraction (beta). Mitral valve diameter (D) was calculated as the average of major and minor axis lengths obtained in the midesophageal bicommissural (transcommissural anterior-lateral-posterior medial) and LV long-axis (anterior-posterior) TEE imaging planes, respectively. VFT was calculated as 4·(1-beta)·SV/πD(3), where SV = stroke volume measured using thermodilution. Hemodynamics, diastolic function, and VFT were determined during steady-state conditions before cardiopulmonary bypass. The severity of AS (mean and peak pressure gradients, peak transvalvular jet velocity, aortic valve area) and diastolic function (E/A, beta) were similar between groups. Moderate centrally directed AI was present in 8 patients with AS (ratio of regurgitant jet width to LV outflow tract diameter of 36±6%). Pulse pressure and mean pulmonary artery pressure were elevated in patients with versus without AI, but no other differences in hemodynamics were observed. Mitral valve minor and major axis lengths, diameter, and area were reduced in the presence versus the absence of AI. VFT was increased significantly (5.7±1.7 v 3.2±0.6; p = 0.00108) in patients with AS and AI compared with AS alone. CONCLUSION Moderate AI falsely elevates VFT in patients with severe AS undergoing AVR by partially inhibiting mitral valve opening. VFT may be an unreliable index of LV filling efficiency with competitive diastolic flow into the LV.

Advanced Age Attenuates Left Ventricular Filling Efficiency Quantified Using Vortex Formation Time: A Study of Octogenarians With Normal Left Ventricular Systolic Function Undergoing Coronary Artery Surgery

OBJECTIVE Blood flow across the mitral valve during early left ventricular (LV) filling produces a 3-dimensional rotational fluid body, known as a vortex ring, that enhances LV filling efficiency. Diastolic dysfunction is common in elderly patients, but the influence of advanced age on vortex formation is unknown. The authors tested the hypothesis that advanced age is associated with a reduction in LV filling efficiency quantified using vortex formation time (VFT) in octogenarians undergoing coronary artery bypass graft (CABG) surgery. DESIGN Observational study. SETTING Veterans Affairs medical center. PARTICIPANTS After institutional review board approval, octogenarians (n = 7; 82 ± 2 year [mean ± standard deviation]; ejection fraction 56% ± 7%) without valve disease or atrial arrhythmias undergoing CABG were compared with a younger cohort (n = 7; 55 ± 6 year; ejection fraction 57% ± 7%) who were undergoing coronary revascularization. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS All patients were monitored using radial and pulmonary arterial catheters and transesophageal echocardiography. Peak early LV filling (E) and atrial systole (A) blood flow velocities and their corresponding velocity-time integrals were obtained using pulse-wave Doppler echocardiography to determine E/A, atrial filling fraction (β), and E wave deceleration time. Pulse-wave Doppler also was used to measure pulmonary venous blood flow during systole and diastole. Mitral valve diameter (D) was calculated as the average of major and minor axis lengths obtained in the midesophageal LV bicommissural and long-axis transesophageal echocardiography imaging planes, respectively. VFT was calculated as 4 × (1 - β) × SV/(πD3), where SV is the stroke volume measured using thermodilution. Systemic and pulmonary hemodynamics, LV diastolic function, and VFT were determined during steady-state conditions 30 minutes before cardiopulmonary bypass. A delayed relaxation pattern of LV filling (E/A 0.81 ± 0.16 v 1.29 ± 0.19, p = 0.00015; β 0.44 ± 0.05 v 0.35 ± 0.03, p = 0.0008; E wave deceleration time 294 ± 58 v 166 ± 28 ms, p < 0.0001; ratio of peak pulmonary venous systolic and diastolic blood flow velocity 1.42 ± 0.23 v 1.14 ± 0.20, p = 0.0255) was observed in octogenarians compared with younger patients. Mitral valve diameter was similar between groups (2.7 ± 0.2 and 2.6 ± 0.2 cm, respectively, in octogenarians v younger patients, p = 0.299). VFT was reduced in octogenarians compared with younger patients (3.0 ± 0.9 v 4.5 ± 1.2; p = 0.0171). An inverse correlation between age and VFT was shown using linear regression analysis (VFT = -0.0627 × age + 8.24; r2 = 0.408; p = 0.0139). CONCLUSION The results indicate that LV filling efficiency quantified using VFT is reduced in octogenarians compared with younger patients undergoing coronary artery bypass grafting.