Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease.

A nitric oxide synthase (NOS)-like activity has been demonstrated in human red blood cells (RBCs), but doubts about its functional significance, isoform identity and disease relevance remain. Using flow cytometry in combination with the nitric oxide (NO)-imaging probe DAF-FM we find that all blood cells form NO intracellularly, with a rank order of monocytes > neutrophils > lymphocytes > RBCs > platelets. The observation of a NO-related fluorescence within RBCs was unexpected given the abundance of the NO-scavenger oxyhemoglobin. Constitutive normoxic NO formation was abolished by NOS inhibition and intracellular NO scavenging, confirmed by laser-scanning microscopy and unequivocally validated by detection of the DAF-FM reaction product with NO using HPLC and LC-MS/MS. Using immunoprecipitation, ESI-MS/MS-based peptide sequencing and enzymatic assay we further demonstrate that human RBCs contain an endothelial NOS (eNOS) that converts L-(3)H-arginine to L-(3)H-citrulline in a Ca(2+)/calmodulin-dependent fashion. Moreover, in patients with coronary artery disease, red cell eNOS expression and activity are both lower than in age-matched healthy individuals and correlate with the degree of endothelial dysfunction. Thus, human RBCs constitutively produce NO under normoxic conditions via an active eNOS isoform, the activity of which is compromised in patients with coronary artery disease.

Assessment of tissue perfusion and vascular function in mice by scanning laser Doppler perfusion imaging.

Post-occlusive reactive hyperemia (PORH) is a key feature of physiological vasomotion to appropriately match the supply/demand ratio of tissues. This adaptive mechanism is severely disturbed in endothelial dysfunction with a reduced flow-mediated dilation (FMD). Reduced PORH and FMD are powerful prognostic risk factors in cardiovascular diseases. While these parameters are frequently determined in human beings, comparable methods applicable to mouse models are sparse. We aimed to evaluate the applicability and accuracy of scanning laser Doppler perfusion imaging (LDPI) to measure PORH in the mouse hindlimb. Changes in mean perfusion in response to vasoactive drugs and PORH (assessed by scanning LDPI) were compared with changes in diameter and blood flow in the femoral artery, as assessed by high-resolution ultrasound. We found that the measured LDPI signal significantly correlated with changes of inflow into the femoral artery. Vasodilation induced by administration of nitroglycerine and acetylcholine increased vessel diameter, blood flow and mean perfusion, while vasoconstriction following administration of epinephrine decreased all three parameters. PORH was induced by temporal occlusion of the femoral artery with an external cuff. During occlusion, mean perfusion decreased to a condition of zero-perfusion and release of the cuff induced an immediate increase in blood flow that was followed by femoral artery dilation driving PORH/perfusion. Surgical removal of the femoral artery decreased mean perfusion to a zero-perfusion level and fully abolished PORH. Importantly, the measurement of the PORH response by scanning LDPI is highly reproducible as determined by repeated measurements and intra/interobserver variation analysis. Last, we found that the PORH response was dependent on nitric oxide synthase and cyclooxygenase and declined with age. Thus, we here provide novel and robust non-invasive methods to serially measure tissue perfusion at baseline and during physiological and pharmacological modulation of vasomotor tone in the hindlimb of mice. The application of these LDPI scanning and ultrasound-based methods may be useful for testing the effects of drugs affecting vasomotor function or future elucidation of mechanisms leading to vasomotor dysfunction in mice in vivo.

Left ventricular diastolic dysfunction in Nrf2 knock out mice is associated with cardiac hypertrophy, decreased expression of SERCA2a, and preserved endothelial function.

Increased production of reactive oxygen species and failure of the antioxidant defense system are considered to play a central role in the pathogenesis of cardiovascular disease. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key master switch controlling the expression of antioxidant and protective enzymes, and was proposed to participate in protection of vascular and cardiac function. This study was undertaken to analyze cardiac and vascular phenotype of mice lacking Nrf2. We found that Nrf2 knock out (Nrf2 KO) mice have a left ventricular (LV) diastolic dysfunction, characterized by prolonged E wave deceleration time, relaxation time and total diastolic time, increased E/A ratio and myocardial performance index, as assessed by echocardiography. LV dysfunction in Nrf2 KO mice was associated with cardiac hypertrophy, and a downregulation of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) in the myocardium. Accordingly, cardiac relaxation was impaired, as demonstrated by decreased responses to ß-adrenergic stimulation by isoproterenol ex vivo, and to the cardiac glycoside ouabain in vivo. Surprisingly, we found that vascular endothelial function and endothelial nitric oxide synthase (eNOS)-mediated vascular responses were fully preserved, blood pressure was decreased, and eNOS was upregulated in the aorta and the heart of Nrf2 KO mice. Taken together, these results show that LV dysfunction in Nrf2 KO mice is mainly associated with cardiac hypertrophy and downregulation of SERCA2a, and is independent from changes in coronary vascular function or systemic hemodynamics, which are preserved by a compensatory upregulation of eNOS. These data provide new insights into how Nrf2 expression/function impacts the cardiovascular system.

DLK-1 as a marker to distinguish unrestricted somatic stem cells and mesenchymal stromal cells in cord blood.

In addition to hematopoietic stem cells, cord blood (CB) also contains different nonhematopoietic CD45-, CD34- adherent cell populations: cord blood mesenchymal stromal cells (CB MSC) that behave almost like MSC from bone marrow (BM MSC) and unrestricted somatic stem cells (USSC) that differentiate into cells of all 3 germ layers. Distinguishing between these populations is difficult due to overlapping features such as the immunophenotype or the osteogenic and chondrogenic differentiation pathway. Functional differences in the differentiation potential suggest different developmental stages or different cell populations. Here we demonstrate that the expression of genes and the differentiation toward the adipogenic lineage can discriminate between these 2 populations. USSC, including clonal-derived cells lacking adipogenic differentiation, strongly expressed δ-like 1/preadipocyte factor 1 (DLK-1/PREF1) correlating with high proliferative potential, while CB MSC were characterized by a strong differentiation toward adipocytes correlating with a weak or negative DLK-1/PREF1 expression. Constitutive overexpression of DLK-1/PREF1 in CB MSC resulted in a reduced adipogenic differentiation, whereas silencing of DLK-1 in USSC resulted in adipogenic differentiation.