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

 

Curator and Investigator Initiated Study: Aviva Lev-Ari, PhD, RN

Agent Alternative #1: Niacin (Vitamin B₃), Fibrates and Genistein

Low HDL levels predict an increased risk of coronary artery disease independently of LDL levels, and 60–70% of major cardiovascular events cannot be prevented with current approaches focused on LDL, such as statin therapy (Werner et al., 2003), (Vasa et al., 2001a), (Walter et al., 2002), (Dimmeler et al., 2001), (Llevadot et al., 2001), (Spyridopoulos et al., 2004). In addition, low HDL levels are particularly common in males with early-onset atherosclerosis (Wilson et al., 1988). Based on these observations, prevention trials have been performed with agents such as niacin and fibrates, which raise HDL, and they indicate that modest increases in HDL independently yield a significant reduction in cardiovascular events (Rubins et al., 1999), (Brown et al., 2001), (Boden, 2000). Thus, there is compelling evidence that HDL is not solely a marker of lower risk of cardiovascular disease but instead is a mediator of vascular health.

Genistein – Phytoestrogens have received widespread attention over the past few years because of their potential for preventing some highly prevalent chronic diseases, including cardiovascular disease, osteoporosis, and hormone-related cancers. Genistein, the primary soy-derived phytoestrogen, has various biological actions (Liu et al., 2004), including a weak estrogenic effect and inhibition of tyrosine kinases. Genistein acutely stimulates Nitric Oxide synthesis in vascular Eendothelial cells by a cyclic adenosine 5′-monophosphate-dependent mechanism (Liu et al., 2004). The intracellular signaling pathways for activation of eNOS by genistein were independent of PI3K/Akt or ERK/MAPK but depended on the cAMP/PKA cascade. In addition, the genistein action on eNOS was not inhibited by an ER antagonist and was unrelated to tyrosine kinase inhibition.

Studies demonstrate that genistein has antiatherogenic effects and inhibits proliferation of vascular endothelial and smooth muscle cells. Data from animal and in vitro studies suggest a protective role of genistein in the vasculature. Studies investigating its effect on plasma lipid profiles show either a moderate positive effect or a neutral effect. Some human intervention studies suggest a beneficial effect on atherosclerosis (Anthony et al., 1998), markers of cardiovascular risk (van der Schouw et al., 2000), vasomotor tone (Walker et al., 2001), vascular endothelial function (Squadrito et al., 2003), and systemic arterial compliance (Nestel et al., 1997). Genistein also inhibits human platelet aggregation in vitro (Dobrydneva et al., 2002), (Gottstein et al., 2003) and decreases TNF-induced monocyte chemoattractant protein-1 secretion in human vascular endothelial cells (Gottstein et al., 2003). Other studies suggest that genistein may induce vascular relaxation by cAMP-dependent mechanisms (Satake and Shibata, 1999) or inhibition of tyrosine kinases (Duarte et al., 1997). In vitro studies elucidating the cellular or molecular mechanisms of the genistein action on vascular cells are lacking.

NO produced is a potent vasodilator and also has anti-inflammatory (Yu et al., 2002), antiatherogenic (Shin et al., 1996), antithrombotic (Alonso and Radomski, 2003), and antiapoptotic properties (Kotamraju, 2001). Liu et al., (2004), hypothesized that genistein directly regulates vascular function through stimulation of eNOS and NO synthesis from vascular endothelial cells. To test this hypothesis, they focused on the acute effects of genistein on eNOS and the cellular signaling related to this effect. They specifically tested the protein kinase A and tyrosine kinase pathways because these have been proposed in previous vascular studies (Satake and Shibata, 1999), (Duarte et al., 1997).

In Liu et al., (2004) study, genistein acted directly on BAECs and HUVECs to activate eNOS and NO production through nongenomic mechanisms in whole vascular endothelial cells. The intracellular signaling pathways for activation of eNOS by genistein were independent of PI3K/Akt or ERK/MAPK but depended on the cAMP/PKA cascade. In addition, the genistein action on eNOS was not inhibited by an ER antagonist and was unrelated to tyrosine kinase inhibition. The findings suggest a molecular mechanism that may underlie some of the beneficial cardiovascular effects that have been proposed for genistein.

Agent Alternative #2: Serotonin, 5-HT

5-hydroxytryptamine evokes endothelial nitric oxide synthase activation

eNOS activation in microvascular endothelial bEnd.3 cell. NO plays an important role in the dynamic regulation of the intercellular junctions of the endothelium. They have shown that eNOS is enriched at these junctions, which is a prerequisite for its activation by agonists. At the junctions, eNOS co-localizes with PECAM-1, but not with VE-cadherin and plakoglobin. The nature of the molecular mechanisms that lead to the enrichment of eNOS at intercellular junctions, and which allow these junctions to be regulated by NO, remains to be determined. Data from three experiments are presented as means±S.D. ‘D’ represents l-NAME-dependent (i.e. NOS-mediated) nitrite formation (Grovers et al., 2002).

Comparative analysis of eNOS efficacy on NO production. 5-HT is second in effectiveness.

Agonist Nitrite (nmol·mg of protein-1) -l-NAME +l-NAME D None 0.31±0.05 0.08±0.05 0.23±0.07

A23187 (5µM) 1.44±0.06 0.35±0.06* 1.09±0.08†

Acetylcholine (1µM) 0.83±0.12 0.06±0.09 0.77±0.15†

5-Hydroxytryptamine (1µM) 0.94±0.07 0.05±0.05 0.88±0.08†

VEGF (20ng/ml) 0.60±0.03 0.10±0.03 0.50±0.05†

Bradykinin (1µM) 0.28±0.06 0.04±0.05 0.24±0.07

Histamine (10µM) 0.36±0.04 0.08±0.05 0.28±0.06

Activation of endothelial nitric oxide synthase (eNOS) resulted in the production of nitric oxide (NO) that mediates the vasorelaxing properties of endothelial cells. The goal of this project was to address the possibility that 5-hydroxytryptamine (5-HT) stimulates eNOS activity in bovine aortic endothelial cell (BAEC) cultures. McDuffie et al., (1999, 2000) tested the hypothesis that 5-HT receptors mediate eNOS activation by measuring agonist-stimulated [3H]L-citrulline ([3H]L-Cit) formation in BAEC cultures. They found that 5-HT stimulated the conversion of [3H]L- arginine ([3H]L-Arg) to [3H]L-Cit, indicating eNOS activation. The high affinity 5-HT1B receptor agonist, 5-nonyloxytryptamine (5-NOT)- stimulated [3H]L-Cit turnover responses were concentration-(0.01 nM to 100 microM) and time-dependent. Maximal responses were observed within 10 min following agonist exposures. These responses were effectively blocked by the 5-HT1B receptor antagonist, isamoltane, the 5-HT1B/5-HT2 receptor antagonist, methiothepin, and the eNOS selective antagonists (0.01-10 microM): L-Nomega -monomethyl-L-arginine (L-NMMA) and L-N omega-iminoethyl-L-ornithine (L-NIO). Their findings lend evidence of a 5-HT1B receptor/eNOS pathway, accounting in part for the activation of eNOS by 5-HT.

3 orpholinosyndnonimine inhibits 5-hydroxytryptamine induced phosphorylation of nitric oxide synthase in endothelial cells.

5-Hydroxytryptamine (5-HT) is a vasoactive substance that is taken up by endothelial cells to activate endothelial nitrite oxide synthase (eNOS). The activation of eNOS results in the production of nitric oxide (NO), which is responsible for vasodilation of blood vessels. NO also interacts with superoxide anion (O2*-) to form peroxynitrite (ONOO-), a potent oxidant that has been shown to induce vascular endothelial dysfunction (Richardson et al., 2003). They examined the ability of 3-morpholinosyndnonimine (SIN-1), an ONOO- generator, to inhibit 5-HT-induced phosphorylation of eNOS in cultured bovine aortic endothelial cells (BAECs). They observed that 5-HT phosphorylates Ser1179 eNOS in a time- and concentration-dependent manner. Maximum phosphorylation occurred at 30 sec using a concentration of 1.0 microM 5-HT. BAECs treated with SIN-1 (1-1000 microM) for 30 min showed no significant increase in eNOS phosphorylation. However, 5-HT-induced eNOS phosphorylation was inhibited in cells treated with various concentrations of SIN-1 for 30 min and stimulated with 5-HT. These data suggest that an increase in ONOO- as a result of an increase in the production of O2*-, may feedback to inhibit 5-HT-induced eNOS phosphorylation at Ser1179 and therefore, contribute to endothelial dysfunction associated with cardiovascular diseases.

Agent Alternative #3: Nebivolol

A Third-Generation ß-Blocker that Augments Vascular Nitric Oxide Release. (Broeders et al., 2000), (Brugada et al., 2001), (Dessy et al., 2005), (Iaccarino et al., 2002), (Jordan et al., 2001), (Kalinowski et al., 2003), (Mason et al., 2005), (McEniery et al., 2004), (Mollnau et al., 2003), (Mukherjee et al., 2004), (Ritter et al., 2006).

In vivo metabolized nebivolol increases vascular NO production. This phenomenon involves endothelial ß2-adrenergic receptor ligation, with a subsequent rise in endothelial free [Ca2+]i and endothelial NO synthase–dependent NO production. This may be an important mechanism underlying the nebivolol-induced, NO-mediated arterial dilation in humans. Nebivolol is a ß1-selective adrenergic receptor antagonist with proposed nitric oxide (NO)–mediated vasodilating properties in humans. In this study, they explored whether nebivolol indeed induces NO production and, if so, by what mechanism. they hypothesized that not nebivolol itself but rather its metabolites augment NO production (Broeders et al., 2000).

Dose and Time Concentration for Agents affecting endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production 

• time concentration dependence on eNOS reuptake
• dose concentration dependence on NO production

In the literature we found evidence for dose concentration dependence manner

Ach, Histamine, Genistein, ACEI, Fenofibrates, NEBIVOLOL, Calcium channel blocker, Enzyme S-nitrosylation

In the literature we found evidence for time concentration dependence manner:

Ach, BRL37344, a 3-adrenoceptor agonist

In the literature we found evidence for time and dose concentration dependence manner:

Histamine

NO, eNOS AgonistsStimulate phosphorylation of eNOS at serine 1177, 1179, 116 Conversion of L-arginine toL-citrulline	time concentration dependence manner	dose concentration dependencemanner	time and dose	dose (nmol·mg of protein-1)Grovers et al., (2002)
A23187				(5µM)
Acetylcholine	Xu et al., (2002)	Sanchez et al., (2006)		(1µM)
5-Hydroxytryptamine				(1µM)
VEGF (				(20ng/ml)
Bradykinin				(1µM)
Histamine		McDuffie et al., (1999)	McDuffie et al., (2000)	(10µM)
genistein		Liu et al., (2004)		(1µM)
ACEI		Skidgel et al., (2006)
Fenofibrates		Asai et al., (2006)
BRL37344, a 3-adrenoceptor agonist	Pott et al., (2005)
NEBIVOLOLß1-selective adrenergic receptor antagonist with nitric oxide (NO)–mediation for vasodilation		Ritter et al., (2006)
Calcium channel blocker		Church and Fulton, (2006),
Enzyme S-nitrosylation		Erwin et al., (2006)

Proposed integration plan of Nebivolol with CVD patients’ current medication regimen for selective medical diagnoses

Blood Pressure Medicine:

Beta blockers, Verapamil (Calan), Reserpine (Hydropes), Clonidine (Catapres), Methyldopa (Aldomet)

Diuretics:

Thiazides, Spironolactone (Aldactone), Hydralazine

Antidepressants:

Prozac, Lithium, MOA’s, Tricyclics

Stomach Medicine:

Tagamet and Zantac, plus other compounds containing Cimetidine and Ranitidine or associated compounds in Anticholesterol Drugs

Antipsychotics:

Chlorpromazine (Thorazine), Pimozide (Orap), Thiothixine (Navane), Thiordazine (Mellaril), Sulpiride, Haloperidol (haldol), Fluphenazine (Modecate, Prolixin)

Heart Medicine:

Clofibrate (Atromid), Gemfibrozil, Diagoxin

Hormones:

Estrogen, Progesterone, Proscar, Casodex, Eulexin, Corticosteroids Gonadotropin releasing antagonists: Zoladex and Lupron

Cytotoxic agents:

Cyclophosphamide, Methotrexate, Roferon Non-steroidal anti-inflammatories

Others-

Alprazolam, Amoxapine, Chlordiazepoxide, Sertraline, Paroxetine, Clomipramine, Fluvoxamine, Fluoxetine, Imipramine, Doxepine, Desipramine, Clorprothixine, Bethanidine, Naproxen, Nortriptyline, Thioridazine, Tranylcypromine, Venlafaxine, Citalopram.

INTERACTIONS for Nebivolol

Calcium Antagonists:

Caution should be exercised when administering beta-blockers with calcium antagonists of the verapamil or diltiazem type because of their negative effect on contractility and atrio-ventricular conduction. Exaggeration of these effects can occur particularly in patients with impaired ventricular function and/or SA or AV conduction abnormalities. Neither medicine should therefore be administered intravenously within 48 hours of discontinuing the other.

Anti-arrhythmics:

Caution should be exercised when administering beta-blockers with Class I anti-arrhythmic drugs and amiodarone as their effect on atrial conduction time and their negative inotropic effect may be potentiated. Such interactions can have life threatening consequences.

Clonidine:

Beta-blockers increase the risk of rebound hypertension after sudden withdrawal of chronic clonidine treatment.

Digitalis:

Digitalis glycosides associated with beta-blockers may increase atrio-ventricular conduction times. Nebivolol does not influence the kinetics of digoxin & clinical trials have not shown any evidence of an interaction.

Special note: Digitalisation of patients receiving long term beta-blocker therapy may be necessary if congestive cardiac failure is likely to develop. The combination can be considered despite the potentiation of the negative chronotropic effect of the two medicines. Careful control of dosages and of individual patient’s response (notably pulse rate) is essential in this situation.

Insulin & Oral Antidiabetic drugs:

Glucose levels are unaffected, however symptoms of hypoglycemia may be masked.

Anaesthetics:

Concomitant use of beta-blockers & anaesthetics e.g. ether, cyclopropane & trichloroethylene may attenuate reflex tachycardia & increase the risk of hypotension

CVD patients’ current medication regimen for selective medical diagnoses

Medical Diagnoses	Current medication regiment	eNOS agonists &production stimulation of NO	PPAR-gamma agonist (TZD) as eNOS stimulant
CAD patients	Beta blockers, ACEI, ARB, CCB, Diagoxin, Coumadin	yes
Endothelial Dysfunction in DM patients with or without Erectile Dysfunction	Insulin	yes	yes
Atherosclerosis patients: Arteries and or veins	AntihypertensiveCoumadin	yes	yes
pre-stenting treatment phase	Beta blockers, Verapamil (Calan), Reserpine (Hydropes), Clonidine (Catapres), Methyldopa (Aldomet)	yes
post-stenting treatment phase	Antiplatelets		yes
if stent is a Bare Mesh stent (BMS)	CoumadinBeta blockers		yes
if stent is Drug Eluting stent (DES)	antibiotics
if stent is EPC antibody coated			yes
post CABG patients	CoumadinBeta blockers, Verapamil (Calan), Reserpine (Hydropes), Clonidine (Catapres), Methyldopa (Aldomet)		yes
CVD patients on blood thinner drugs	Coumadin	yes

Conclusions

•  Most favorable and unexpected to us was finding in the literature new indications for TDZs as stimulators of eNOS, in addition to the new indication for atherosclerosis besides the classic indication in pharmacology books, being in the reduction of insulin resistance.

•  Most favorable and unexpected to us was finding in the literature new indications for beta blockers as NO stimulant, nebivolol, a case in point, thus, fulfilling two indications in one drug along the direction of the study to identify eNOS agonists. Nebivolol is a vasodilator, thus functions as an antihypertensive.

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Nebivolol is a long-acting, cardioselective beta-blocker currently licensed for the treatment of hypertension.

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Other aspects of Nitric Oxide involvement in biological systems in humans are covered in the following posts on this site:

 

Nitric Oxide in bone metabolism July 16, 2012 

Author: Aviral Vatsa PhD, MBBS

http://pharmaceuticalintelligence.com/2012/07/16/nitric-oxide-in-bone-metabolism/?goback=%2Egde_4346921_member_134751669 

Nitric Oxide production in Systemic sclerosis July 25, 2012 

Curator: Aviral Vatsa, PhD, MBBS

http://pharmaceuticalintelligence.com/2012/07/25/nitric-oxide-production-in-systemic-sclerosis/?goback=%2Egde_4346921_member_138370383 

Nitric Oxide Signalling Pathways August 22, 2012 

Curator/ Author: Aviral Vatsa, PhD, MBBS

http://pharmaceuticalintelligence.com/2012/08/22/nitric-oxide-signalling-pathways/?goback=%2Egde_4346921_member_151245569

Nitric Oxide: a short historic perspective August 5, 2012 

Author/Curator: Aviral Vatsa PhD, MBBS

http://pharmaceuticalintelligence.com/2012/08/05/nitric-oxide-a-short-historic-perspective-7/

Nitric Oxide: Chemistry and function August 10, 2012 

Curator/Author: Aviral Vatsa PhD, MBBS

http://pharmaceuticalintelligence.com/2012/08/10/nitric-oxide-chemistry-and-function/?goback=%2Egde_4346921_member_145137865 

 

Nitric Oxide and Platelet Aggregation August 16, 2012 

Author: Dr. Venkat S. Karra, Ph.D.

http://www.tginnovations.wordpress.com/ 

http://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/?goback=%2Egde_4346921_member_147475405 

 

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure August 20, 2012 

Author: Larry Bernstein, MD

http://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/  

Nitric Oxide: The Nobel Prize in Physiology or Medicine 1998 Robert F. Furchgott, Louis J. Ignarro, Ferid Murad August 16, 2012 

Reporter: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/08/16/nitric-oxide-the-nobel-prize-in-physiology-or-medicine-1998-robert-f-furchgott-louis-j-ignarro-ferid-murad/ 

 

Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012 

Author: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/08/13/coronary-artery-disease-medical-devices-solutions-from-first-in-man-stent-implantation-via-medical-ethical-dilemmas-to-drug-eluting-stents/

Nano-particles as Synthetic Platelets to Stop Internal Bleeding Resulting from Trauma

August 22, 2012 

Reporter: Dr. V. S. Karra, Ph.D.

http://www.tginnovations.wordpress.com/ 

http://pharmaceuticalintelligence.com/2012/08/22/nano-particles-as-synthetic-platelets-to-stop-internal-bleeding-resulting-from-trauma/ 

 

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012 

Curator and Investigator Initiated Study: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/ 

Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery July 2, 2012

Curator: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/07/02/macrovascular-disease-therapeutic-potential-of-cepcs-reduction-methods-for-cv-risk/  

Bone remodelling in a nutshell June 22, 2012

Author: Aviral Vatsa, Ph.D., MBBS

http://pharmaceuticalintelligence.com/2012/06/22/bone-remodelling-in-a-nutshell/