Lobe-Specific Calcium Binding in Calmodulin Regulates Endothelial Nitric Oxide Synthase Activation

BACKGROUND: Human endothelial nitric oxide synthase (eNOS) requires calcium-bound calmodulin (CaM) for electron transfer but the detailed mechanism remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using a series of CaM mutants with E to Q substitution at the four calcium-binding sites, we found that single mutation at any calcium-binding site (B1Q, B2Q, B3Q and B4Q) resulted in ∼2-3 fold increase in the CaM concentration necessary for half-maximal activation (EC50) of citrulline formation, indicating that each calcium-binding site of CaM contributed to the association between CaM and eNOS. Citrulline formation and cytochrome c reduction assays revealed that in comparison with nNOS or iNOS, eNOS was less stringent in the requirement of calcium binding to each of four calcium-binding sites. However, lobe-specific disruption with double mutations in calcium-binding sites either at N- (B12Q) or at C-terminal (B34Q) lobes greatly diminished both eNOS oxygenase and reductase activities. Gel mobility shift assay and flavin fluorescence measurement indicated that N- and C-lobes of CaM played distinct roles in regulating eNOS catalysis; the C-terminal EF-hands in its calcium-bound form was responsible for the binding of canonical CaM-binding domain, while N-terminal EF-hands in its calcium-bound form controlled the movement of FMN domain. Limited proteolysis studies further demonstrated that B12Q and B34Q induced different conformational change in eNOS. CONCLUSIONS: Our results clearly demonstrate that CaM controls eNOS electron transfer primarily through its lobe-specific calcium binding

Collateral circulation: Past and present

Following an arterial occlusion outward remodeling of pre-existent inter-connecting arterioles occurs by proliferation of vascular smooth muscle and endothelial cells. This is initiated by deformation of the endothelial cells through increased pulsatile fluid shear stress (FSS) caused by the steep pressure gradient between the high pre-occlusive and the very low post-occlusive pressure regions that are interconnected by collateral vessels. Shear stress leads to the activation and expression of all NOS isoforms and NO production, followed by endothelial VEGF secretion, which induces MCP-1 synthesis in endothelium and in the smooth muscle of the media. This leads to attraction and activation of monocytes and T-cells into the adventitial space (peripheral collateral vessels) or attachment of these cells to the endothelium (coronary collaterals). Mononuclear cells produce proteases and growth factors to digest the extra-cellular scaffold and allow motility and provide space for the new cells. They also produce NO from iNOS, which is essential for arteriogenesis. The bulk of new tissue production is carried by the smooth muscles of the media, which transform their phenotype from a contractile into a synthetic and proliferative one. Important roles are played by actin binding proteins like ABRA, cofilin, and thymosin beta 4 which determine actin polymerization and maturation. Integrins and connexins are markedly up-regulated. A key role in this concerted action which leads to a 2-to-20 fold increase in vascular diameter, depending on species size (mouse versus human) are the transcription factors AP-1, egr-1, carp, ets, by the Rho pathway and by the Mitogen Activated Kinases ERK-1 and -2. In spite of the enormous increase in tissue mass (up to 50-fold) the degree of functional restoration of blood flow capacity is incomplete and ends at 30% of maximal conductance (coronary) and 40% in the vascular periphery. The process of arteriogenesis can be drastically stimulated by increases in FSS (arterio-venous fistulas) and can be completely blocked by inhibition of NO production, by pharmacological blockade of VEGF-A and by the inhibition of the Rho-pathway. Pharmacological stimulation of arteriogenesis, important for the treatment of arterial occlusive diseases, seems feasible with NO donors

Dobutamine stress echocardiography: a review and update

Lauren Gray Gilstrap,1 R Sacha Bhatia,2 Rory B Weiner,3 David M Dudzinski3 1Division of Cardiology, Brigham and Women's Hospital, Boston, MA, USA; 2Institute for Health Systems Solutions, Women's College Hospital, Toronto, ON, Canada; 3Cardiology Division, Massachusetts General Hospital, Boston, MA, USA Abstract: Stress echocardiography is a noninvasive cardiovascular diagnostic test that provides functional and hemodynamic information in the assessment of a number of cardiac diseases. Performing stress echocardiography with a pharmacologic agent such as dobutamine allows for simulation of increased heart rate and increased myocardial physiologic demands in patients who may be unable to exercise due to musculoskeletal or pulmonary comorbidities. Dobutamine stress echocardiography (DSE), like exercise echocardiography, has found its primary application in ischemic heart disease, with roles in identification of obstructive epicardial coronary artery disease, detection of viable myocardium, and assessment of the efficacy of anti-ischemic medical therapy in patients with known coronary artery disease. DSE features prominently in the evaluation and management of valvular heart disease by helping to assess the effects of mitral and aortic stenoses, as well as a specific use in differentiating true severe valvular aortic stenosis from pseudostenosis that may occur in the setting of left ventricular systolic dysfunction. DSE is generally well tolerated, and its side effects and contraindications generally relate to consequences of excess inotropic and/or chronotropic stimulation of the heart. The aim of this paper is to review the indications, contraindications, advantages, disadvantages, and risks of DSE. Keywords: stress echocardiography, dobutamine, coronary artery disease, myocardial ischemi

Multidisciplinary approach to the management of pulmonary embolism patients: the pulmonary embolism response team (PERT)

Christopher W Root,1 David M Dudzinski,2 Bishoy Zakhary,3 Oren A Friedman,4 Akhilesh K Sista,5 James M Horowitz6 1Icahn School of Medicine at Mount Sinai, New York, NY, USA; 2Division of Cardiology, Department of Internal Medicine, Massachusetts General Hospital, Boston, MA, USA; 3Department of Internal Medicine, Oregon Health & Science University, Portland, OR, USA; 4Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA; 5Division of Vascular and Interventional Radiology, Department of Radiology, NYU Langone Health, New York, NY, USA; 6Leon H. Charney Division of Cardiology, Department of Medicine, NYU Langone Health, New York, NY, USA Abstract: Pulmonary embolism (PE) is a potentially fatal disease with a broad range of treatment options that spans multiple specialties. The rapid evolution and expansion of novel therapies to treat PE make it a disease process that is well suited to a multidisciplinary approach. In order to facilitate a rapid, robust response to the diagnosis of PE, some hospitals have established multidisciplinary pulmonary embolism response teams (PERTs). The PERT model is based on existing multidisciplinary teams such as heart teams and rapid response teams. A PERT is composed of clinicians from the range of specialties involved in the treatment of PE, including pulmonology critical care, interventional radiology, cardiology, and cardiothoracic surgery among others. A PERT is a 24/7 consult service that is able to provide expert advice on the initial management of PE patients and convene in real time to develop a consensus treatment plan specifically tailored to the needs of a particular patient and consistent with the capabilities of the institution. In this review, we discuss the rationale for establishing a PERT and its potential benefits. We discuss considerations in forming a PERT and present case studies of several PERTs currently in operation at different institutions. We also discuss potential difficulties in forming a PERT and review evidence that has been generated by some of the PERTs that have been in operation the longest. Keywords: pulmonary embolism, pulmonary embolism response team, thrombosis, thrombolysis, venous thromboembolis

Yes (again) to local NO

A pair of fluorescent indicator-tagged DNA-duplex scaffolds permit assessments of nitric oxide (NO) production on cell surfaces and in intracellular networks. The application of these nanoprobes indicates formations of local NO signals that might conserve cancer cell integrity