Posts Tagged ‘Vascular health’

Curated/reported by : Aviral Vatsa PhD, MBBS

Based on : S Moncada et al

It was in 1980 that Furchgott & Zawadzki first described endothelium- dependent relaxation of the blood vessels by acetylcholine. Further studies in 1984 revealed that other factors such as bradykinin, histamine and 5-hydroxytryptamine release endothelium derived relaxing factor (EDRF), which can modulate vessel tone. EDRF was shown to stimulate soluble guanylate cyclase and was inhibited by haemoglobin. In 1986 it was demonstrated that superoxide (O2) anions mediated EDRF inactivation and that the inhibitors of EDRF generated superoxide (O2) anions in solution as a mean to inhibit EDRF. It was later established that all compounds that inhibit EDRF have one property in common, redox activity, which accounted for their inhibitory action on EDRF. One exception was haemoglobin, which inactivates EDRF by binding to it. In 1987 Furchgott proposed that EDRF might be nitric oxide (NO) based on a study of the transient relaxations of endothelium-denuded rings of rabbit aorta to ‘acidified’ inorganic nitrite (NO) solutions and the observations that superoxide dismutase (SOD, which removes O2) protected EDRF. Till then NO was not known to be produced in mammalian cells. In 1988 Palmer et al could detect NO production both biologically and chemically by chemiluminescence. The following year in 1989 the enzyme responsible for NO production, NO synthase, was discovered and L-arginine:NO pathway was proposed.

Roles of L-arginine:NO pathway

By 1987 it was proposed that NO is generated in tissues other than endothelium. Hibbs et al and Marletta et al proposed that NO was generated by macrophages. Moreover release of EDRF was demonstrated in cerebellar cells following activation with N-methyl-D- aspartate (NMDA ). Both noradrenergic and cholinergic responses are ‘controlled’ by the nitrergic system so that the release of NO (e.g., during electrical field stimulation) counteracts and dominates the response to the noradrenergic or cholinergic stimulus (Cellek & Moncada, 1997). Mechanism of penile erection was unveiled by the studies on nitrergic neurotransmission that led to therapeutic intervention. Selective damage of nitrergic nerves in disease states was proposed as a potent mechanism of pathophysiology. Broadly three areas of research based on three isoforms of NOS came into being;

  • cardiovascular
  • nervous
  • immunology

Identification of NG-monomethyl-L-arginine (L-NMMA) as an inhibitor of the synthesis of NO lay the basis of future research into investigating the role of NO in biological systems. In 1989 it was demonstrated that intravenous infusion of L-NMMA resulted in increase in blood pressure that was reversible by infusing L-arginine. NO was thus implicated in constantly maintaining blood vessel tone. eNOS knockout studies showed a hypertensive phenotypes in the animal models and over expression of eNOS led to lowering of the blood pressure. Furthermore, eNOS activation was attributed to phosphorylation of a specific tyrosine residue in the enzyme.

NO and Mitochondria

NO reacts with some of the complexes of the respiratory chain, and inhibits mitochondrial respiration – this is a well accepted notion. Initially it was believed that the target for NO was soluble guanylate cyclase, which in vasculature would lead to elevation of cGMP that eventually results in NO mediated vasodilatation and platelet aggregation inhibition. In 1994, another potential target, cytochrome c oxidase, for inhibitory effects of NO was discovered. This was a reversible effect, in competition with oxygen concentrations. Increases in NO production were also shown to inhibit cellular respiration irreversibly by selectively inhibiting complex I . Hence in 2002 it was proposed that this might be a mechanism through which cell pathology was initiated in certain conditions. Furthermore, NO was proposed to be implicated in the activation of the grp78-dependent stress response , via modulating calcium-related interaction between mitochondria and endoplasmic reticulum . This host defence mechanism might also have role in vasculature. Further evidence was provided in 2003 to link the role of NO in mitochondrogenesis and thus indicating that NO might be involved in the regulation of the balance between glycolysis and oxidative phosphorylation in cells.

NO and Pathophysiology

Lack of NO: By 2000, NO was established as a haemostatic regulator in the vasculature. Its absence was implicated in pathological states such as hypertension and vasospasm. These pathophysiological states share a common beginning of endothelial dysfunction, which has low NO production as one of its characterstic features. This dysfunction has been observed prior to the appearance of cardiovascular disease in predisposed subjects with family history of essential hypertension and atherosclerosis. The most likely mechanism for endothelial dysfunction is that of a reduced bioavailability of NO . The mechanism of this aspect is discussed elsewhere on this site. Protection against reduction of NO bio-availability in the vasculature is a vital therapeutic target and is extensively explored. This can be achieved by the use of antioxidants and/or augmentation of eNOS expression. In 2003 statins were shown to increase the production of endothelial NO in endothelial cell cultures and in animals by the reduction of oxidative stress or by increasing the coupling of the eNOS. It was way back in 1994 that oestrogen was shown to increase both the activity and expression of eNOS. In addition, more recently in 2003, oestrogen was shown to reduce the breakdown of available NO.

Excess of NO: In 2000 it was shown that NO produced from iNOS in vasculature is involved in extensive vasodilatation in septic shock. Later it was demonstrated that inhibition of mitochondrial respiration is an important component of the NO-induced tissue damage. This inhibition of respiration, which is initially NO-dependent and reversible, becomes persistent with time as a result of oxidative stress . Such metabolic hypoxic states where in tissues cannot utilise available oxygen due to NO, could also contribute to other inflammatory and degenerative conditions. An obvious therapeutic target for reducing NO production in such conditions would be L-NMMA. L-NMM was tested in a clinical trial for septic shock in 2004. The results were however disappointing probably due to the blanket reduction in NO production from other NOS enzymes there by having deleterious effects on the treatment group. More specific inhibitors for NOS forms are being investigated for in different disease states.

In conclusion, the L-arginine: NO pathway has had a major impact in many areas of research, specially vascular biology. A lot has been understood about this pathway and its interactions, therapeutic targets are being aggressively investigated, but further investigations are required to delineate further the role of NO in human health and disease.

Further Reading

Nitric Oxide and Platelet Aggregation

Inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

Nitric Oxide in bone metabolism

Nitric oxide and signalling pathways

Rationale of NO use in hypertension and heart failure

Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function


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Reporter: Sudipta Saha, Ph.D.

Assessment of the propensity for vascular events has been based on measurement of risk factors predisposing one to vascular injury. These assessments are based on the strong associations between risk factors such as hypertension, cholesterol levels, smoking, and diabetes which were first described almost a half century ago. The more recent discovery of the relationship between ongoing inflammation and clinical outcomes has led to a variety of blood-based assays which may impart additional knowledge about an individual’s propensity for future cardiovascular events. Vascular health is now better represented as a balance between ongoing injury and resultant vascular repair, mediated at least in part by circulating endothelial progenitor cells ( Accurate enumeration of circulating endothelial progenitor cells is essential for their potential application as biomarkers of angiogenesis. Different stem cell markers (CD34, CD133) and endothelial cell antigens (KDR/VEGFR-2, CD31) in different flow cytometric protocols are assessed for the purpose of circulating progenitor endothelial cell quantification ( Enumeration of circulating progenitor endothelial cells are used in the assessment of various diseases and physiological states, such as: type 2 diabetes patients with peripheral vascular disease, certain phases during congestive heart failure, acute myocardial infarction, atherosclerosis, cardiovascular disease, physical training, cessation of smoking. Two modern instruments used now-a-days to measure the circulating progenitor endothelial cells are discussed below:

MACSQuant® Analyzer:

Circulating progenitor endothelial cells are defined by co-expression of the markers CD34, CD309 (VEGFR-2/KDR), and CD133, though CD133 expression is lost during maturation to endothelial cells.8-10 Since circulating progenitor endothelial cells are rare in peripheral blood, EPC enumeration protocols are rather extensive and laborious. To obtain reliable enumeration results for these rare cells, the sensitivity of flow cytometric analysis needs to be increased. This has been achieved by magnetic enrichment of circulating progenitor endothelial cells prior to flow cytometric analysis, which reduces the number of events that have to be analyzed. The circulating progenitor endothelial cell Enrichment and Enumeration Kit have been designed for enumeration of circulating progenitor endothelial cells from peripheral blood, cord blood, bone marrow, or leukapheresis products. In combination with magnetic pre-enrichment and flow cytometric analysis on the MACSQuant® Analyzer, this kit overcomes some of the limitations of circulating progenitor endothelial cell analysis and offers a simple and time effective solution for EPC enumeration. The circulating progenitor endothelial cell Enrichment and Enumeration Kit in combination with pre-enrichment and flow cytometric analysis on the MACSQuant Analyzer is an effective method to enumerate circulating progenitor endothelial cells in 10 mL of whole blood. Based on the calculated starting number of cells, the circulating progenitor endothelial cell Express Mode analysis template automatically calculates the absolute number and concentration of circulating progenitor endothelial cells in 10 mL of starting material, i.e., whole blood, bone marrow, cord blood, or leukapheresis products. The MACSQuant Analyzer has the ability to enrich cells using MACS technology. This capability makes the enumeration of circulating progenitor endothelial cells fast and easy. The entire process takes less than 2 hours to perform from blood draw to analyzed data and drastically reduces the time and difficulty of such a protocol by combining magnetic enrichment and flow cytometric analysis in one streamlined experiment (

Attune® Acoustic Focusing Cytometer:

In cancer research, circulating progenitor endothelial cells have been suggested as a noninvasive biomarker for angiogenic activity, providing insight into tumor regrowth, resistance to chemotherapy, early recurrence, and metastasis during or after chemotherapy. In healthy individuals, circulating progenitor endothelial cells are reported to be present in very low numbers: 0.01%–0.0001% of all peripheral blood mononuclear cells. Flow cytometry offers the necessary collection and analysis capabilities for detection of circulating progenitor endothelial cells, but is subject to numerous technical challenges. In comparison to traditional hydrodynamic focusing cytometers, the Attune® Acoustic Focusing Cytometer, with its fast acquisition times and increased precision, overcomes the technological hurdles involved in analyzing circulating progenitor endothelial cells. The method includes a number of conventional ways to improve rare-event detection: a blocking step, a viability stain (SYTOX® AADvanced™ Dead Cell Stain), and the use of a dump channel to eliminate unwanted cells and decrease background fluorescence. The challenge of collecting a large enough number of events in a reasonable amount of time is met by using a collection rate of 1,000 μL/min with the Attune® cytometer. This setting enables the collection of more than 4,000,000 live white blood cell (WBC) events in just 35 minutes; the acquisition time using a traditional hydrodynamic focusing cytometer would be 10–12 times longer, close to 6 hours. Furthermore, this method delivers additional time savings by eliminating wash steps to avoid sample loss and employing a simpler sample preparation method. (

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