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Posts Tagged ‘Atherosclerosis’


Carotid Ultrasound more sensitive for Detecting Subclinical Atherosclerosis in patients with rheumatoid arthritis (RA) than CT with calculation of Coronary Artery Calcification Scores

Reporter: Aviva Lev-Ari, PhD, RN

Ultrasound Predicts CVD Risk in Arthritis

Published: Oct 8, 2013 | Updated: Oct 8, 2013

By Nancy Walsh, Staff Writer, MedPage Today
Reviewed by Zalman S. Agus, MD; Emeritus Professor, Perelman School of Medicine at the University of Pennsylvania and Dorothy Caputo, MA, BSN, RN, Nurse Planner

 

Carotid ultrasound was more sensitive for detecting subclinical atherosclerosis in patients with rheumatoid arthritis (RA) than CT with calculation of coronary artery calcification scores, Spanish researchers found.

Among a group of 60 patients classified as being at moderate cardiovascular risk on a conventional scoring system, the presence of severe abnormalities on ultrasound reclassified 51 as being at high or very high risk, according to Miguel A. Gonzalez-Gay, MD, of Universitario Marques de Valdecilla in Santander, and colleagues.

And of those 51 reclassified patients, only 12 would have been reclassified as being at high or very high cardiovascular risk using a coronary artery calcification score,the researchers reported in the NovemberAnnals of the Rheumatic Diseases.

Patients with RA are at markedly increased risk for cardiovascular disease (CVD), both from conventional risk factors and the ongoing systemic inflammation associated with RA.

Comprehensive management of these patients therefore should include risk assessment and appropriate interventions, but “adequate stratification of the CV risk in patients with RA is still far from being completely established,” Gonzalez-Gay and colleagues noted.

The insensitivity of conventional risk assessments such as the Systematic Coronary Risk Evaluation (SCORE), even when modified by the European League Against Rheumatism(mSCORE) to account for the increased background risk in RA, has been confirmed byreports of ischemic heart disease among patients not considered to be at elevated risk on these measures.

These researchers previously suggested that carotid ultrasonography be added to the overall risk assessment of RA patients, particularly those with moderate SCORE risk, but whether other noninvasive approaches such as coronary artery calcification also could be useful has been uncertain.

Therefore, they enrolled 95 rheumatoid arthritis patients with no history of cardiovascular events and no diabetes or chronic renal disease.

Most were women, mean age was 59, and mean disease duration was 11 years.

Rheumatoid factor and/or anticyclic citrullinated peptide was present in 72%, and extra-articular manifestations in 16%.

All patients had carotid ultrasonography to assess for plaque and multi-detector CT scanning to detect coronary artery calcification.

Carotid intima-media thickness of 0.90 or the presence of plaque was considered predictive of CVD on ultrasound.

A coronary artery calcification score of zero was considered normal, and a score over 100 indicated a high likelihood of coronary artery disease.

Patients also were given conventional SCORE ratings, based on factors such as age, sex, smoking, blood pressure, and atherogenic index, as well as mSCORE ratings, to estimate the 10-year risk for a fatal cardiovascular event.

The mean SCORE was 2.30, and the mean mSCORE was 2.78.

Cardiovascular risk according to mSCORE was low in 21, moderate in 60, and high or very high in 14.

Most patients with low mSCOREs also had scores of zero for coronary artery calcification, and none of the low mSCORE patients had calcification scores above 100.

But 57% of patients with calcification scores of zero had carotid plaques identified on ultrasound, as did 76.3% of patients with calcification scores between 1 and 100.

While calcification scores above 100 weren’t much more sensitive than mSCOREs for detection of high risk (23.6% versus 19.4%), almost all (70 of 72) patients with high or very high risk were identified with carotid ultrasound, for a sensitivity of 97.2% (95% CI 90.3-99.7).

And when the ultrasound model of intima-media thickness above 0.9 mm and/or carotid plaque also included mSCOREs above 5%, all 72 were correctly identified, for a sensitivity of 100% (95% CI 95-100).

This lack of sensitivity for calcification scores likely reflects the finding that arterial calcification is a later vascular development, and its absence doesn’t rule out the presence of the more vulnerable noncalcified plaques, the researchers explained.

“These results support the use of carotid ultrasonography as the imaging technique of choice for detection of high/very high CV risk in RA patients with moderate mSCORE,” they said.

In an editorial accompanying the study, Patrick H. Dessein, MD, of the University of Witwatersrand in Johannesburg, South Africa, and Anne G. Semb, MD, of Diakonhjemmet Hospital in Oslo, Norway, noted that the use of ultrasound more than tripled the number of patients considered to be at high risk.

If only mSCORE was used for risk stratification, they pointed out, many patients “in routine clinical settings” would be unlikely to receive preventive treatments, “with the serious consequences this has.”

Dessein and Semb also noted that there were certain limitations to this study, including its cross-sectional design and inclusion of patients with long disease duration.

“It remains to be clarified whether carotid ultrasound is as helpful among patients with early disease versus those with longstanding disease in enhancing CVD risk stratification,” the editorialists wrote.

The authors reported no conflicting interests.

From the American Heart Association:

http://www.medpagetoday.com/Rheumatology/Arthritis/42138?xid=nl_mpt_DHE_2013-10-09&utm_content=&utm_medium=email&utm_campaign=DailyHeadlines&utm_source=WC&eun=g99985d0r&userid=99985&email=avivalev-ari@alum.berkeley.edu&mu_id=5099207

 

 

 

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Drug Eluting Stents: On MIT’s Edelman Lab’s Contributions to Vascular Biology and its Pioneering Research on DES


Drug Eluting Stents: On MIT‘s Edelman Lab’s Contributions to Vascular Biology and its Pioneering Research on DES

Author: Larry H Bernstein, MD, FACP

and 

Curator: Aviva Lev-Ari, PhD, RN
http://PharmaceuticalIntelligence.com/2013/04/25/Contributions
-to-vascular-biology/

This is the first of a three part series on the evolution of vascular biology and the studies of the effects of biomaterials in vascular reconstruction and on drug delivery, which has embraced a collaboration of cardiologists at Harvard Medical School , Affiliated Hospitals, and MIT,
requiring cardiovascular scientists at the PhD and MD level, physicists, and computational biologists working in concert, and
an exploration of the depth of the contributions by a distinguished physician, scientist, and thinker.

The first part – Vascular Biology and Disease – will cover the advances in the research on

  • vascular biology,
  • signaling pathways,
  • drug diffusion across the endothelium and
  • the interactions with the underlying muscularis (media),
  • with additional considerations for type 2 diabetes mellitus.

The second part – Stents and Drug Delivery – will cover the

  • purposes,
  • properties and
  • evolution of stent technology with
  • the acquired knowledge of the pharmacodynamics of drug interactions and drug distribution.

The third part – Problems and Promise of Biomaterials Technology – will cover the shortcomings of the cardiovascular devices, and opportunities for improvement

Vascular Biology and Cardiovascular Disease

Early work on endothelial injury and drug release principles

The insertion of a catheter for the administration of heparin is not an innocuous procedure. Heparin is infused to block coagulation, lowering the risk of a dangerous

  • clot formation and
  • dissemination.

It was shown experimentally that the continuous infusion of heparin

  • suppresses smooth muscle proliferation after endothelial injury. It may lead to
  • hemorrhage as a primary effect.

The anticoagulant property of heparin was removed by chemical modification without loss of the anti-proliferative effect.

In this study, MIT researches placed ethylene-vinyl acetate copolymer matrices containing standard and modified heparin adjacent to rat carotid arteries at the time of balloon deendothelialization.

Matrix delivery of both heparin compounds effectively diminished this proliferation in comparison to controls without producing systemic anticoagulation or side effects.

This mode of therapy appeared more effective than administering the agents by either

  • intravenous pumps or
  • heparin/polymer matrices placed in a subcutaneous site distant from the injured carotid artery

This indicated that the site of placement at the site of injury is a factor in the microenvironment, and is a preference for avoiding restenosis after angioplasty and other interventions.

This raised the question of why the proliferation of vascular muscle occurs in the first place.
 Edelman, Nugent and Karnovsky  (1) showed that the proliferation required first the denudation of vascular surface endothelium. This exposed the underlayer to the effect of basic fibroblast growth factor, which stimulates mitogenesis of the exposed cell, explained by the endothelium as a barrier from circulating bFGF.

To answer this question, they compared the effect of

  • 125I-labelled bFGF intravenously given with perivascular controlled bFGF release.
  • Polymeric controlled release devices delivered bFGF to the extravascular space without transendothelial transport. 
Deposition within the blood vessel wall was rapidly distributed circumferentially and was substantially greater than that observed following intravenous injection.

The amount of bFGF deposited in arteries adjacent to the release devices was 40 times that deposited in similar arteries in animals who received a single intravenous bolus of bFGF.

The presence of intimal hyperplasia increased deposition of perivascularly released bFGF 2.4-fold but decreased the deposition of intravenously injected bFGF by 67%.

  • bFGF was 5- to 30-fold more abundant in solid organs after intravenous injection than it was following perivascular release, and
  • bFGF deposition was greatest in the kidney, liver, and spleen and was substantially lower in the heart and lung.

This result indicated that vascular deposition of bFGF is independent of endothelium, and

  • bFGF delivery is effectively perivascular. (2)

Drug activity studies have to be done in well controlled and representative conditions.
 Edelsman’s Lab researchers studied the

  • dose response of injured arteries to exogenous heparin in vivo by providing steady and predictable arterial levels of drug.
  • Controlled-release devices were fabricated to direct heparin uniformly and at a steady rate to the adventitial surface of balloon-injured rat carotid arteries.

Researchers predicted the distribution of heparin throughout the arterial wall using computational simulations and correlated these concentrations with the biologic response of the tissues.

Researchers determined from this process that an in vivo arterial concentration of 0.3 mg/ml of heparin is required to maximallyinhibit intimal hyperplasia after injury.

This estimation of the required tissue concentration of a drug is

  • independent of the route of administration and
  • applies to all forms of drug release.

In this way the Team was able to

  • evaluate the potential of  widely disparate forms of drug release and, to finally
  • create some rigorous criteria by which to guide the development of particular delivery strategies for local diseases. (3)

Chiefly, the following three effects:

(1) Effect of controlled adventitial heparin delivery on smooth muscle cell proliferation following endothelial injury. ER Edelman, DH Adams, and MJ Karnovsky. PNAS May 1990; 87: 3773-3777.


(2) Perivascular and intravenous administration of basic fibroblast growth factor: Vascular and solid organ deposition. ER Edelman, MA Nugent, and MJ Karnovsky. PNAS Feb 1993; 90: 1513-1517.


(3) Tissue concentration of heparin, not administered dose, correlates with the biological response of injured arteries in vivo. MA Lovich and ER Edelman. PNAS Sep 1999; 96: 11111–11116.

Vascular Injury and Repair

Perlecan is a heparin-sulfate proteoglycan that might be critical for regulation of vascular repair by inhibiting the binding and mitogenic activity of basic fibroblast growth factor-2 (bFGF-2) in vascular smooth muscle cells .

The Team generated

  • Clones of endothelial cells expressing an antisense vector targeting domain III of perlecan. The transfected cells produced significantly less perlecan than parent cells, and they had reduced bFGF in vascular smooth muscle cells.
  • Endothelial cells were seeded onto three-dimensional polymeric matrices and implanted adjacent to porcine carotid arteries subjected to deep injury.
  • The parent endothelial cells prevented thrombosis, but perlecan deficient cells were ineffective.

The ability of endothelial cells to inhibit intimal hyperplasia, however, was only in part suppressed by perlecan. The differential regulation by perlecan of these aspects of vascular repair may clarify why control of clinical clot formation does not lead to full control of intimal hyperplasia.

The use of genetically modified tissue engineered cells provides a new approach for dissecting the role of specific factors within the blood vessel wall.(1) Successful implementation of local arterial drug delivery requires transmural distribution of drug. The physicochemical properties of the applied compound govern its transport and tissue binding.

  • Hydrophilic compounds are cleared rapidly.
  • Hydrophobic drugs bind to fixed tissue elements, potentially prolonging tissue residence and biological effect.

Local vascular drug delivery provides

  • elevated concentrations of drug in the target tissue while
  • minimizing systemic side effects.

To better characterize local pharmacokinetics the Team examined the arterial transport of locally applied dextran and dextran derivatives in vivo.

Using a two-compartment pharmacokinetic model to correct

  • The measured transmural flux of these compounds for systemic
  • Redistribution and elimination as delivered from a photo-polymerizable hydrogel.
  • The diffusivities and the transendothelial permeabilities were strongly dependent on molecular weight and charge
  • For neutral dextrans, the diffusive resistance increased with molecular weightapproximately 4.1-fold between the molecular weights of 10 and 282 kDa.
  • Endothelial resistance increased 28-fold over the same molecular weight range.
  • The effective medial diffusive resistance was unaffected by cationic charge as such molecules moved identically to neutral compounds, but increased approximately 40% when dextrans were negatively charged.

Transendothelial resistance was 20-fold lower for the cationic dextrans, and 11-fold higher for the anionic dextrans, when both were compared to neutral counterparts.

These results suggest that, while

  • low molecular weight drugs will rapidly traverse the arterial wall with the endothelium posing a minimal barrier,
  • the reverse is true for high molecular weight agents.

The deposition and distribution of locally released vascular therapeutic compounds might be predicted based upon chemical properties, such as molecular weight and charge. (2)

Paclitaxel is hydrophobic and has therapeutic potential against proliferative vascular disease.
 The favorable preclinical data with this compound may, in part, result from preferential tissue binding.
 The complexity of Paclitaxel pharmacokinetics required in-depth investigation if this drug is to reach its full clinical potential in proliferative vascular diseases.

Equilibrium distribution of Paclitaxel reveals partitioning above and beyond perfusate concentration and a spatial gradient of drug across the arterial wall.

The effective diffusivity (Deff) was estimated from the Paclitaxel distribution data to

  • facilitate comparison of transport of Paclitaxel through arterial parenchyma with that of other vasoactive agents and to
  • characterize the disparity between endovascular and perivascular application of drug.

This transport parameter described the motion of drug in tissues given an applied concentration gradient and includes, in addition to diffusion,

  • the impact of steric hindrance within the arterial interstitium;
  • nonspecific binding to arterial elements; and, in the preparation used here,
  • convective effects from the applied transmural pressure gradient.

At all times, the effective diffusivity for endovascular delivery exceeded that of perivascular delivery. The arterial transport of Paclitaxel was quantified through application ex vivo and measurement of the subsequent transmural distribution.

  • Arterial Paclitaxel deposition at equilibrium varied across the arterial wall.
  • Permeation into the wall increased with time, from 15 minutes to 4 hours, and
  • varied with the origin of delivery.

In contrast to hydrophilic compounds, the concentration in tissue exceeded the applied concentration and the rate of transport was markedly slower. Furthermore, endovascular and perivascular Paclitaxel application led to differences in deposition across the blood vessel wall.

This leads to a conclusion that Paclitaxel interacts with arterial tissue elements  as it moves under the forces of

  • diffusion and
  • convection and
  • can establish substantial partitioning and spatial gradients across the tissue. (3)

Endovascular drug-eluting stents have changed the practice of  cardiovascular vascularization, and yet it is unclear how they so dramatically reduce restenosis

We don’t know how to distinguish between the different formulations available.
 Researchers are now questioning whether individual properties of different drugs beyond lipid avidity effect arterial transport and distribution.

In bovine internal carotid segments, tissue-loading profiles for

  • Hydrophobic Paclitaxel and Rapamycin are indistinguishable, reaching load steady state after 2 days.
  • Hydrophilic dextran reaches equilibrium in hours.

Paclitaxel and Rapamycin bind to the artery at 30–40 times bulk concentration, and bind to specific tissue elements.

Transmural drug distribution profiles are markedly different for the two compounds.

  • Rapamycin binds specifically to FKBP12 binding protein and it distributes evenly through the artery,
  • Paclitaxel binds specifically to microtubules, and remains primarily in the subintimal space.

The binding of Rapamycin and Paclitaxel to specific intracellular proteins plays an essential role in

  • determining arterial transport and distribution and in
  • distinguishing one compound from another.

These results offer further insight into the

  • mechanism of local drug delivery and the
  • specific use of existing drug-eluting stent formulations. (4)

The Role of Amyloid beta (A) in Creation of Vascular Toxic Plaque

Amyloid beta (A) is a peptide family produced and deposited in neurons and endothelial cells (EC).
It is found at subnanomolar concentrations in the plasma of healthy individuals.
 Simple conformational changes produce a form of A-beta , A-beta 42, which creates toxic plaque in the brains of Alzheimer’s patients.

Oxidative stress induced blood brain barrier degeneration has been proposed as a key factor for A-beta 42 toxicity.

This cannot account for lack of injury from the same peptide in healthy tissues.
Researchers hypothesized that cell state mediates A-beta’s effect.
 They examined the viability in the presence of A-beta secreted from transfected
Chinese hamster ovary cells (CHO) of

  • aortic Endothelial Cells (EC),
  • vascular smooth muscle cells (SMC) and
  • epithelial cells (EPI) in different states

A-beta was more toxic to all cell types when they were subconfluent.
 Subconfluent EC sprouted and SMC and EPI were inhibited by A-beta.
Confluent EC were virtually resistant to A-beta and suppressed A-beta production by A-beta +CHO.

Products of subconfluent EC overcame this resistant state, stimulating the production and toxicity of A-beta 42. Confluent EC overgrew >35% beyond their quiescent state in the presence of A-beta conditioned in media from subconfluent EC.

These findings imply that A-beta 42 may well be even more cytotoxic to cells in injured or growth states and potentially explain the variable and potent effects of this protein.

One may now need to consider tissue and cell state in addition to local concentration of and exposure duration to A-beta.

The specific interactions of A-beta and EC in a state-dependent fashion may help understand further the common and divergent forms of vascular and cerebral toxicity of A-beta and the spectrum of AD. (5)

(1) Perlecan is required to inhibit thrombosis after deep vascular injury and contributes
to endothelial cell-mediated inhibition of intimal hyperplasia. MA Nugent, HM Nugent,
RV Iozzoi, K Sanchack, and ER Edelman. PNAS Jun 2000; 97(12): 6722-6727


(2) Correlation of transarterial transport of various dextrans with their physicochemical properties.
O Elmalak, MA Lovich, E Edelman. Biomaterials 2000; 21: 2263-2272


(3) Arterial Paclitaxel Distribution and Deposition. CJ Creel, MA Lovich, ER Edelman. Circ Res. 2000;86:879-884


(4) Specific binding to intracellular proteins determines arterial transport properties for rapamycin and Paclitaxel.
AD Levin, N Vukmirovic, Chao-Wei Hwang, and ER Edelman. PNAS Jun 2004; 101(25): 9463–9467.
www.pnas.org/cgi/doi/10.1073/pnas.0400918101

(5) Amyloid beta toxicity dependent upon endothelial cell state. M Balcells, JS Wallins, ER Edelman.
Neuroscience Letters 441 (2008) 319–322

Endothelial Damage as an Inflammatory State

Autoimmunity may drive vascular disease through anti-endothelial cell (EC) antibodies. This raises a question about whether an increased morbidity of cardiovascular diseases in concert with systemic illnesses may involve these antibodies.

Matrix-embedded ECs act as powerful regulators of vascular repair accompanied by significant reduction in expected systemic and local inflammation.

The Lab researchers compared the immune response against free and matrix-embedded ECs in naive mice and mice with heightened EC immune reactivity. Mice were presensitized to EC with repeated subcutaneous injections of saline-suspended porcine EC (PAE) (5*10^5 cells).

On day 42, both naive mice (controls) and mice with heightened EC immune reactivity received 5*10^5 matrix-embedded or free PAEs. Circulating PAE-specific antibodies and effector T-cells were analyzed 90 days after implantation for –

  • PAE-specific antibody-titers,
  • frequency of CD4+-effector cells, and
  • xenoreactive splenocytes

These were 2- to 4-fold lower (P<0.0001) when naıve mice were injected with matrix-embedded instead of saline-suspended PAEs.

Though basal levels of circulating antibodies were significantly elevated after serial PAE injections (2210+341 mean fluorescence intensity, day 42) and almost doubled again 90 days after injection of a fourth set of free PAEs, antibody levels declined by half in recipients of matrix-embedded PAEs at day 42 (P<0.0001), as did levels of CD4+-effector cells and xenoreactive splenocytes.

A significant immune response to implantation of free PAE is elicited in naıve mice, that is even more pronounced in mice with pre-developed anti-endothelial immunity.

Matrix-embedding protects xenogeneic ECs against immune reaction in naive mice and in mice with heightened immune reactivity.

Matrix-embedded EC might offer a promising approach for treatment of advanced cardiovascular disease. (1)

Researchers examined the molecular mechanisms through which

mechanical force and hypertension modulate

endothelial cell regulation of vascular homeostasis.

Exposure to mechanical strain increased the paracrine inhibition of vascular smooth muscle cells (VSMCs) by endothelial cells.

Mechanical strain stimulated the production by endothelial cells of perlecan and heparan-sulfate glycosaminoglycans. By inhibiting the expression of perlecan with an antisense vector researchers demonstrated that perlecan was essential to the strain-mediated effects on endothelial cell growth control.

Mechanical regulation of perlecan expression in endothelial cells was

  • governed by a mechano-transduction pathway
  • requiring transforming growth factor (TGF-β) signaling and
  • intracellular signaling through the ERK pathway.

Immunohistochemical staining of the aortae of spontaneously hypertensive rats
demonstrated strong correlations between

  • endothelial TGF-β,
  • phosphorylated signaling intermediates, and
  • arterial thickening.

Studies on ex vivo arteries exposed to varying levels of pressure demonstrated that

ERK and TGF-beta signaling were required for pressure-induced upregulation of endothelial HSPG.

The Team’s findings suggest a novel feedback control mechanism in which

  • net arterial remodeling to hemodynamic forces is controlled by a dynamic interplay between growth stimulatory signals from vSMCs and
  • growth inhibitory signals from endothelial cells. (2)

Heparan-sulfate proteoglycans (HSPGs) are potent regulators of vascular remodeling and repair.
 The major enzyme capable of degrading HSPGs is heparanase, which led us to examine
the role of heparanase in controlling

  • arterial structure,
  • mechanics, and
  • remodeling.

In vitro studies suggested heparanase expression in endothelial cells serves as a negative regulator of endothelial inhibition of vascular smooth muscle cell (vSMC) proliferation.

ECs inhibit vSMC proliferation through the interplay between

  • growth stimulatory signals from vSMCs and
  • growth inhibitory signals from ECs.

This would be expected if ECs had HSPGs that are degraded by heparanase.
Arterial structure and remodeling to injury is modified by heparanase expression.
Transgenic mice overexpressing heparanase had

  • increased arterial thickness,
  • cellular density, and
  • mechanical compliance.

Endovascular stenting studies in Zucker rats demonstrated increased heparanase expression in the neointima of obese, hyperlipidemic rats in comparison to lean rats.

The extent of heparanase expression within the neointima strongly correlated with the neointimal thickness following injury. To test the effects of heparanase overexpression on arterial repair, researchers developed a novel murine model of stent injury using small diameter self-expanding stents.

Using this model, researchers found that increased

  • neointimal formation and
  • macrophage recruitment occurs in transgenic mice overexpressing heparanase.
  • Taken together, these results support a role for heparanase in the regulation of arterial structure, mechanics, and repair. (3)

The first host–donor reaction in transplantation occurs at the blood–tissue interface.
When the primary component of the implant (donor) is the endothelial cells, it incites an immunologic reaction. Injections of free endothelial cell implants elicit a profound major histocompatibility complex (MHC) II dominated immune response.

Endothelial cells embedded within three-dimensional matrices behave like quiescent endothelial cells.

Perivascular implants of such embedded ECs cells are the most potent inhibitor of intimal hyperplasia and thrombosis following controlled vascular injury, but without any immune reactivity.

Allo- and even exenogenic endothelial cells evoke no significant humoral or
cellular immune response in immune-competent hosts when embedded within matrices.
 Moreover,  endothelial implants are immune-modulatory, reducing the extent of the memory response to previous free cell implants.

Attenuated immunogenicity results in muted activation of adaptive and innate immune cells. These findings point toward a pivotal role of matrix–cell-interconnectivity for

  • the cellular immune phenotype and might therefore assist in the design  of
  • extracellular matrix components for successful tissue engineering. (4)

Because changes in subendothelial matrix composition are associated with alterations of the endothelial immune phenotype, researchers sought to understand if

  • cytokine-induced NF-κB activity and
  • downstream effects depend on substrate adherence of endothelial cells (EC).

The team compared the upstream

  • phosphorylation cascade,
  • activation of NF-ĸβ, and
  • expression/secretion

of downstream effects of EC grown on tissue culture polystyrene plates (TCPS) with EC embedded within collagen-based matrices (MEEC).

Adhesion of natural killer (NK) cells was quantified in vitro and in vivo.

  • NF-κβ subunit p65 nuclear levels were significantly lower and
  • p50 significantly higher in cytokine-stimulated MEEC than in EC-TCPS.

Despite similar surface expression of TNF-α receptors, MEEC had significantly decreased secretion and expression of IL-6, IL-8, MCP-1, VCAM-1, and ICAM-1.

Attenuated fractalkine expression and secretion in MEEC (two to threefold lower than in EC-TCPS; p < 0.0002) correlated with 3.7-fold lower NK cell adhesion to EC (6,335 ± 420 vs. 1,735 ± 135 cpm; p < 0.0002).

Furthermore, NK cell infiltration into sites of EC implantation in vivo was significantly reduced when EC were embedded within matrix.

Matrix embedding enables control of EC substratum interaction.

This in turn regulates chemokine and surface molecule expression and secretion, in particular – of those compounds within NF-κβ pathways,

  • chemoattraction of NK cells,
  • local inflammation, and
  • tissue repair. (5)

Monocyte recruitment and interaction with the endothelium is imperative to vascular recovery.

Tie2 plays a key role in endothelial health and vascular remodeling.
Researchers studied monocyte-mediated Tie2/angiopoietin signaling following interaction of primary monocytes with endothelial cells and its role in endothelial cell survival.

The direct interaction of primary monocytes with subconfluent endothelial cells

resulted in transient secretion of angiopoietin-1 from monocytes and

the activation of endothelial Tie2. This effect was abolished by preactivation of monocytes with tumor necrosis factor-α (TNFα).

Although primary monocytes contained high levels of

  • both angiopoietin 1 and 2,
  • endothelial cells contained primarily angiopoietin 2.

Seeding of monocytes on serum-starved endothelial cells reduced caspase-3 activity by 46+5.1%, and 52+5.8% after TNFα treatment, and it decreased single-stranded DNA levels by 41+4.2% and 40+ 3.5%, respectively.

This protective effect of monocytes on endothelial cells was reversed by Tie2 silencing with specific short interfering RNA.

The antiapoptotic effect of monocytes was further supported by the

  • activation of cell survival signaling pathways involving phosphatidylinositol 3-kinase,
  • STAT3, and
  • AKT.

Monocytes and endothelial cells form a unique Tie2/angiopoietin-1 signaling system that affects endothelial cell survival and may play critical a role in vascular remodeling and homeostasis. (6)

(1) Cell–Matrix Contact Prevents Recognition and Damage of Endothelial Cells in States of Heightened Immunity.
H Methe, ER Edelman. Circulation. 2006;114[suppl I]:I-233–I-238.
http://www.circulationaha.org/DOI/10.1161/CIRCULATIONAHA.105.000687

(2) Endothelial Cells Provide Feedback Control for Vascular Remodeling Through a Mechanosensitive Autocrine
TGFβ Signaling Pathway. AB Baker, DS Ettenson, M Jonas, MA Nugent, RV Iozzo, ER Edelman.
Circ. Res. 2008;103;289-297   http://dx.doi.org/10.1161/CIRCRESAHA.108.179465http://circres.ahajournals.org/cgi/content/full/103/3/289

(3) Heparanase Alters Arterial Structure, Mechanics, and Repair Following Endovascular Stenting in Mice.
AB Baker, A Groothuis, M Jonas, DS Ettenson…ER Edelman.   Circ. Res. 2009;104;380-387;
http://dx.doi.org/10.1161/CIRCRESAHA.108.180695  http://circres.ahajournals.org/cgi/content/full/104/3/380

(4) The effect of three-dimensional matrix-embedding of endothelial cells on the humoral and cellular immune response.
H Methe, S Hess, ER Edelman. Seminars in Immunology 20 (2008) 117–122. http://dx.doi.org/10.1016/j.smim.2007.12.005

(5) NF-kB Activity in Endothelial Cells Is Modulated by Cell Substratum Inter-actions and Influences Chemokine-Mediated
Adhesion of Natural Killer Cells.  S Hess, H Methe, Jong-Oh Kim, ER Edelman.
Cell Transplantation 2009; 18: 261–273


(6) Primary Monocytes Regulate Endothelial Cell Survival Through Secretion of Angiopoietin-1 and Activation of Endothelial Tie2.
SY Schubert, A Benarroch, J Monter-Solans and ER Edelman. Arterioscler Thromb Vasc Biol 2011;31;870-875
http://dx.doi.org/10.1161/ATVBAHA.110.218255

Neointimal Formation, Shear Stress, and Remodelling with Reference to Diabetes

Innate immunity is of major importance in vascular repair. The present study evaluated whether

  • systemic and transient depletion of monocytes and macrophages with
  • liposome-encapsulated bisphosphonates inhibits experimental in-stent neointimal formation.

The Experiment

Rabbits fed on a hypercholesterolemic diet underwent bilateral iliac artery balloon denudation and stent deployment.

Liposomal alendronate (3 or 6 mg/kg) was given concurrently with stenting.

  • Monocyte counts were reduced by 90% 24 to 48 hours aftera single injection of liposomal alendronate, returning to basal levels at 6 days.

This treatment significantly reduced

  • intimal area at 28 days, from 3.88+0.93 to 2.08+0.58 and 2.16 +0.62 mm2.
  • Lumen area was increased from 2.87+0.44 to 3.57­+0.65 and 3.45+0.58 mm2, and
  • arterial stenosis was reduced from 58 11% to 37 8% and 38 7% in controls, in rabbits treated with 3 mg/kg, and with 6 mg/kg, respectively (mean+SD, n=8 rabbits/group, P< 0.01 for all 3 parameters).

No drug-related adverse effects were observed.
Reduction in neointimal formation was associated with

  • reduced arterial macrophage infiltration and proliferation at 6 days and with an
  • equal reduction in intimal macrophage and smooth muscle cell content at 28 days after injury.

Conversely, drug regimens ineffective in reducing monocyte levels did not inhibit neointimal formation.
Researchers have shown that a

  • single liposomal bisphosphonates injection concurrent with injury reduces in-stent neointimal formation and
  • arterial stenosis in hypercholesterolemic rabbits, accompanied by systemic transient depletion of monocytes and macrophages. (1)

Diabetes and insulin resistance are associated with increased disease risk and poor outcomes from cardiovascular interventions.

Even drug-eluting stents exhibit reduced efficacy in patients with diabetes.
Researchers reported the first study of vascular response to stent injury in insulin-resistant and diabetic animal models.

Endovascular stents were expanded in the aortae of

  • obese insulin-resistant and
  • type 2 diabetic Zucker rats,
  • in streptozotocin-induced type 1 diabetic Sprague-Dawley rats, and
  • in matched controls.

Insulin-resistant rats developed thicker neointima (0.46+0.08 versus 0.37+0.06 mm2, P 0.05), with  decreased lumen area (2.95+0.26 versus 3.29+0.15 mm2, P 0.03) 14 days after stenting compared with controls, but without increased vascular inflammation (tissue macrophages).

Insulin-resistant and diabetic rat vessels did exhibit markedly altered signaling pathway activation 1 and 2 weeks after stenting, with up to a 98% increase in p-ERK (anti-phospho ERK) and a 54% reduction in p-Akt (anti-phospho Akt) stained cells. Western blotting confirmed a profound effect of insulin resistance and diabetes on Akt and ERK signaling in stented segments. p-ERK/p-Akt ratio in stented segments uniquely correlated with neointimal response (R2 = 0.888, P< 0.04) , but not in lean controls.

Transfemoral aortic stenting in rats provides insight into vascular responses in insulin resistance and diabetes.

Shifts in ERK and Akt signaling related to insulin resistance may reflect altered tissue repair in diabetes accompanied by a

  • shift in metabolic : proliferative balance.

These findings may help explain the increased vascular morbidity in diabetes and suggest specific therapies for patients with insulin resistance and diabetes. (2)

Researchers investigated the role of Valsartan (V) alone or in combination with Simvastatin (S) on coronary atherosclerosis and vascular remodeling, and tested the hypothesis that V or V/S attenuate the pro-inflammatory effect of low endothelial shear stress (ESS).

Twenty-four diabetic, hyperlipidemic swine were allocated into Early (n = 12) and Late (n=12) groups.
Diabetic swine in each group were treated with Placebo (n=4), V (n = 4) and V/S (n = 4) and  followed for 8 weeks in the Early group and 30 weeks in the Late group.

Blood pressure, serum cholesterol and glucose were similar across the treatment subgroups.
ESS was calculated in plaque-free subsegments of interest (n = 109) in the Late group at week 23.
Coronary arteries of this group were harvested at week 30, and the subsegments of interest were identified, and analyzed histopathologically.

Intravascular geometrically correct 3-dimensional reconstruction of the coronary arteries of 12 swine was performed 23 weeks after initiation of diabetes mellitus and a hyperlipidemic diet. Local endothelial shear stress was calculated

  • in plaque-free subsegments of interest (n=142) with computational fluid dynamics, and
  • the coronary arteries (n=31) were harvested and the same subsegments were identified at 30 weeks.

V alone or with S

  • reduced the severity of inflammation in high-risk plaques.
Both regimens attenuated the severity of enzymatic degradation of the arterial wall, reducing the severity of expansive remodeling.
  • attenuated the pro-inflammatory effect of low ESS.
V alone or with S
  • exerts a beneficial effect of reducing and stabilizing high-risk plaque characteristics independent of a blood pressure- and lipid-lowering effect. (3)

This study tested the hypothesis that low endothelial shear stress  augments the

  • expression of matrix-degrading proteases, promoting the
  • formation of thin-capped atheromata.

Researchers assessed the messenger RNA and protein expression, and elastolytic activity of selected elastases and their endogenous inhibitors.

Subsegments with low endothelial shear stress at week 23 showed

  • reduced endothelial coverage,
  • enhanced lipid accumulation, and
  • intense infiltration of activated inflammatory cells at week 30.

These lesions showed increased expression of messenger RNAs encoding

  • matrix metalloproteinase-2, -9, and -12, and cathepsins K and S
  • relative to their endogenous inhibitors and
  • increased elastolytic activity.

Expression of these enzymes correlated positively with the severity of internal elastic lamina fragmentation.

Thin-capped atheromata in regions with

  • lower preceding endothelial shear stress had
  • reduced endothelial coverage,
  • intense lipid and inflammatory cell accumulation,
  • enhanced messenger RNA expression and
  • elastolytic activity of MMPs and cathepsins with
  • severe internal elastic lamina fragmentation.

Low endothelial shear stress induces endothelial discontinuity and

  • accumulation of activated inflammatory cells, thereby
  • augmenting the expression and activity of elastases in the intima and
  • shifting the balance with their inhibitors toward matrix breakdown.

Team’s results provide new insight into the mechanisms of regional formation of plaques with thin fibrous caps. (4)

Elevated CRP levels predict increased incidence of cardiovascular events and poor outcomes following interventions. There is the suggestion that CRP is also a mediator of vascular injury.

Transgenic mice carrying the human CRP gene (CRPtg) are predisposed to arterial thrombosis post-injury.

Researchers examined whether CRP similarly modulates the proliferative and hyperplastic phases of vascular repair in CRPtg when thrombosis is controlled with daily aspirin and heparin at the time of trans-femoral arterial wire-injury.

Complete thrombotic arterial occlusion at 28 days was comparable for wild-type and CRPtg mice (14 and 19%, respectively). Neointimal area at 28d was 2.5 fold lower in CRPtg (4190±3134 m2, n = 12) compared to wild-types (10,157±8890 m2, n = 11, p < 0.05).

Likewise, neointimal/media area ratio was 1.10±0.87 in wild-types and 0.45±0.24 in CRPtg (p < 0.05).

  • Seven days post-injury, cellular proliferation and apoptotic cell number in the intima were both less pronounced in CRPtg than wild-type.
  • No differences were seen in leukocyte infiltration or endothelial coverage.
CRPtg mice had significantly reduced p38 MAPK signaling pathway activation following injury.

The pro-thrombotic phenotype of CRPtg mice was suppressed by aspirin/heparin, revealing CRP’s influence on neointimal growth after trans-femoral arterial wire-injury.

  • Signaling pathway activation,
  • cellular proliferation, and
  • neointimal formation

were all reduced in CRPtg following vascular injury.
 Increasingly the Team was aware of CRP multipotent effects.
 Once considered only a risk factor, and recently a harmful agent, CRP is a far more complex regulator of vascular biology. (5)

(1) Liposomal Alendronate Inhibits Systemic Innate Immunity and Reduces In-Stent Neointimal
Hyperplasia in Rabbits. HD Danenberg, G Golomb, A Groothuis, J Gao…, ER Edelman.
Circulation. 2003;108:2798-2804


(2) Vascular Neointimal Formation and Signaling Pathway Activation in Response to Stent Injury
in Insulin-Resistant and Diabetic Animals. M Jonas, ER Edelman, A Groothuis, AB Baker, P Seifert, C Rogers.
Circ. Res. 2005;97;725-733.        http://dx.doi.org/10.1161/01.RES.0000183730.52908.C6
http://circres.ahajournals.org/cgi/content/full/97/7/725

(3) Attenuation of inflammation and expansive remodeling by Valsartan alone or in combination with
Simvastatin in high-risk coronary atherosclerotic plaques. YS Chatzizisis, M Jonas, R Beigel, AU Coskun…
ER Edelman, CL Feldman, PH Stone.  Atherosclerosis 203 (2009) 387–394


(4) Augmented Expression and Activity of Extracellular Matrix-Degrading Enzymes in Regions of Low
Endothelial Shear Stress Colocalize With Coronary Atheromata With Thin Fibrous Caps in Pigs.
YS Chatzizisis, AB Baker, GK Sukhova,…P Libby, CL Feldman, ER Edelman, PH Stone
Circulation 2011;123;621-630     http://dx.doi.org/10.1161/CIRCULATIONAHA.110.970038
http://circ.ahajournals.org/cgi/content/full/123/6/621


(5) Neointimal formation is reduced after arterial injury in human crp transgenic mice
HD Danenberg, E Grad, RV Swaminathan, Z Chenc,…ER Edelman
Atherosclerosis 201 (2008) 85–91

A Rattle Bag of Science and the Art of Translation

Science Translational Medicine – A rattle bag of science and the art of translation
E. R. Edelman, G. A. FitzGerald.
Sci.Transl. Med. 3, 104ed3 (2011). http://dx.doi.org/10.1126/scitranslmed.3002131

Elazer R. Edelman is the Thomas D. and Virginia W. Cabot Professor of Health Sciences and Technology at MIT,
Professor of Medicine at Harvard Medical School, a coronary care unit cardiologist at the Brigham and Women’s
Hospital, and Director of the Harvard-MIT Biomedical Engineering Center. E-mail: ere@mit.edu

Garret A. FitzGerald is the McNeil Professor in Translational Medicine and Therapeutics, Chair of the Department of
Pharmacology, and Director of the Institute for Translational Medicine & Therapeutics, University of Pennsylvania.
E-mail: garret@upenn.edu

In 2011, the American Association for the Advancement of Science (AAAS)  founded Science Translational Medicine (STM)
to disseminate interdisciplinary science integrating basic and clinical research that defines and fosters new therapeutics, devices, and diagnostics.

Conceived and nourished under the creative vision of Elias Zerhouni and Katrina Kelner, the journal has attracted widespread attention.
Now, as we assume the mantle of co-chief scientific advisors, we look back on the journal’s early accomplishments, restate our mission, and make clear the kinds of manuscripts we seek and accept for publication.

STM’s mission, as articulated by Elias and Katrina, was to

“promote human health by providing a forum for communication and cross-fertilization among basic, translational, and clinical research practitioners and trainees from all relevant established and emerging disciplines.”

This statement remains relevant and accurate today.
 With this mission on our masthead, STM now receives ~25 manuscripts (full-length research articles) per week and publishes ~10% of them. Roughly half of the submissions are deemed inappropriate for the journal and are returned without review within 8 to 10 days of receipt.

Of those papers that undergo full peer review,

decisions to reject are made within 48 days and

the mean time to acceptance (including the revision period) is 125 days.

There is now an average wait of only 24 days between acceptance and publication.

Defining TRANSLATIONAL Medicine

In accord with the journal’s broad readership, the ideal manuscript meets five criteria: It
(i) reports a discovery of translational relevance with high-impact potential;
(ii) has a conceptual focus with interdisciplinary appeal;
(iii) elucidates a biological mechanism;
(iv) is innovative and novel; and
(v) is presented in clear, broadly accessible language.
 STM seeks to publish research that describes

  • how innovative concepts drive the creative biomedical science
  • that ultimately improves the quality of people’s lives—

This is the broadest of our journal’s criteria but is the one that sets us apart as well.
Translational relevance does not require demonstration of benefit in humans but does require the evident potential to advance clinical medicine, thus impacting the direction of our culture and the welfare of our communities. Conceptual focus and mechanistic emphasis discriminate our papers from those that contain observational descriptions of technical findings for which value is restricted to a specific discipline.

However, innovation and novelty may apply to a fundamental scientific discovery or to the nature of its application and relevance to the translational process. Criteria enable the journal to consider versatile technological advances that apply new and creative thinking but may not necessarily offer fresh insights into biological mechanisms. Finally, while the subsequent additional efforts of the STM editorial staff are not to be discounted, the clarity of writing and coherence of argument presented within a submitted manuscript are likely to facilitate its progress through the challenge of peer review.

On Causes – Hippocrates, Aristotle, Robert Koch, and the Dread Pirate Roberts

Elazer R. Edelman
Circulation 2001;104:2509-2512

The idea of risk factors for vascular disease has evolved

  • from a dichotomous to continuous hazard analysis and
  • from the consideration of a few factors to
  • mechanistic investigation of many interrelated risks.

However, confusion still abounds regarding issues of association and causation. Originally, the simple presence of

  • tobacco abuse, hypertension, and/or hypercholesterolemia were tallied, and
  • the cumulative score was predictive of subsequent coronary artery disease.

Since then, dose responses have been defined for these and other factors and it has been suggested that almost 300 factors place patients at risk; these factors include elevations in plasma homocysteine.
 Recent studies shed interesting light on the mechanism of this potentially causal relationship, which was first noted in 1969.

Aside from putative effects on vessel wall dynamics, there is now direct evidence that homocysteine is atherogenic. Twenty-fold increases in plasma homocysteine achieved by dietary manipulation of apoE–/– mice increased aortic root lesion size 2-fold and produced a prolonged chronic inflammatory mural response accompanied by elevations in vascular cell adhesion molecule-1 (VCAM) and tumor necrosis factor-a (TNF-a).

In long term followup, homocysteine levels elevated by

  • dietary supplementation with methionine or homocysteine
  • promoted lesion size and plaque fibrosis in these
  • atherosclerosis-prone mice early in life, but without influencing ultimate plaque burden as the animals aged.

A number of mechanisms were proposed by which homocysteine achieved this effect, including

  • promotion of inflammation,
  • regulation of lipoprotein metabolism, and
  • modification of critical biochemical pathways and
  • metabolites including nitric oxide (NO).

See p 2569
In the present issue of Circulation,

Stühlinger et al 7 advance these mechanistic insights one critical step further by defining homocysteine’s effects at an enzymatic level.

The group led by Lentz published an association between levels of the

  • endogenous inhibitor of Nirtic Oxide synthase,
  • asymmetric dimethyl arginine (ADMA), and
  • homocysteine in cultured endothelial cells and in the serum of cynomolgus monkeys.

Such an association is interesting because the L-arginine–NO synthase pathway seems to be a critical component in the full range of endothelial cell biology and vascular dysfunction.

Stühlinger et al 7  now show that increased cultured endothelial cell elaboration of ADMA by homocysteine and its precursor L-methionine is associated with a dose-dependent impairment of the activity of endothelial dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that degrades ADMA. Homocysteine directly inhibited DDAH activity in a cell-free system by targeting a critical sulfhydryl group on this enzyme.

Thus, one could envision that the balance of cardiovascular health and disease could well be determined by the ability of an intact Nirtic Oxide synthase system to overcome environmental, dietary, and even genetic factors.

In patients with altered enzymatic defense systems,

  • elevated homocysteine,
  • oxidized lipoproteins,
  • inflammation, and other
  • vasotoxins

may dominate even the most potent defense mechanisms.
These studies raise a number of issues.
Do we need to add to our list of established cardiovascular risk factors to accommodate new findings and associations?
Is there a final common pathway for all risk factors or perhaps even a unified factor theory into which all potential risks can be grouped?
And, as always, should we consider Nirtic Oxide at the core of this universality?
Finally, should we change our focus altogether and speak not of risk factors but of

  • genetic predisposition,
  • extent of biochemical aberration, and
  • degree of physical damage?

Some would view these remarkable success stories and the repeated association of hyperhomocyst(e)inemia with coronary, cerebral, and peripheral vascular disease and simply advocate for increased folic acid intake for all.

Indeed, this intervention of negligible cost and

  • insignificant side effect is already partially in place;
  • many foods are fortified with folate to prevent congenital neural tube defects.

This reader considers the seminal work by Vernon Young and Yves Ingenbleek on the relationship between

  • S8 and regions distant from lava flows in Asia and Indian subcontinents,
  • where they have determined hyperhomocysteinemia and the consequence associated with:
  • veganism (not voluntary)
  • impaired methyl donor reactions and transsulfuration pathways (not corrected by B12, folate)
  • loss of lean body mass due to the constant relationship of S:N (insufficient from plant sources)

What happens, when we fail to continue to pursue causality,

  • the linkage of biological significance or scientific plausibility with
  • epidemiologically or statistically significant association?

In medicine, risk becomes the likelihood that people without a disease will acquire the disease through contact with factors thought to increase disease risk.

All of these risk factors are then, by nature, imprecise and nonspecific.
 They are stochastic measures of what will happen to normal people who fall into particular measures of these parameters.

The daring may be willing to accept these risks, citing friend and foe who live well beyond or for far lesser times than anticipated by risk alone. Such concerns may well become moot if we can simultaneously identify patients at risk

  • by linking phenotype with genotype,
  • gene expression with protein elaboration, and
  • environmental exposures with the biochemical consequences and
  • direct anatomic aberrations they induce.

This kind of characterization may well replace a family history of arterial disease as a rough estimate of

  • genotype,
  • serum cholesterol as an indirect measure of the health of lipoprotein metabolism,
  • serum glucose as a crude determinant of the ravages of diabetes mellitus,
  • blood pressure measurement as a marker of long-standing endogenous exposure to altered flow, and
  • tobacco abuse as a maker of long-standing exposure to exogenous toxins.

Rather than identifying patients on the basis of their serum cholesterol, we will have a direct measure of their

  • LDL receptor number,
  • internalization rate,
  • macrophage content in the blood vessel wall,
  • metalloproteinase activity, etc.
  • insulin receptor metabolism,
  • oxidative state, and
  • glycated burden.
  • Serum glucose will similarly give way to these tests

Evaluating a new way to open clogged arteries: Computational model offers insight into mechanisms of drug-coated balloons.

A new study from MIT analyzes the potential usefulness of a new treatment that combines the benefits of angioplasty balloons and drug-releasing stents, but may pose fewer risks. With this new approach, a balloon is inflated in the artery for only a brief period, during which it releases a drug that prevents cells from accumulating and clogging the arteries over time.
While approved for limited use in Europe, these drug-coated balloons are still in development in the United States and have not received FDA approval. The MIT study, which models the behavior of the balloons, should help scientists optimize their performance and aid regulators in evaluating their effectiveness and safety.
“Until now, people who evaluate such technology could not distinguish hype from promise,” says Elazer Edelman, the Thomas D. and Virginia W. Cabot Professor of Health Sciences and Technology and senior author of the paper describing the study, which appeared online recently in the journal Circulation.
Lead author of the paper is Vijaya Kolachalama, a former MIT postdoc who is now a principal member of the technical staff at the Charles Stark Draper Laboratory.
Edelman’s lab is investigating a possible alternative to the current treatments: drug-coated balloons. “We’re trying to understand how and when this therapy could work and identify the conditions in which it may not,” Kolachalama says. “It has its merits; it has some disadvantages.”

Modeling drug release

The drug-coated balloons are delivered by a catheter and inflated at the narrowed artery for about 30 seconds, sometimes longer. During that time, the balloon coating, containing a drug such as Zotarolimus, is released from the balloon. The properties of the coating allow the drug to be absorbed in the body’s tissues. Once the drug is released, the balloon is removed.
In their new study, Kolachalama, Edelman and colleagues set out to rigorously characterize the properties of the drug-coated balloons. After performing experiments in tissue grown in the lab and in pigs, they developed a computer model that explains the dynamics of drug release and distribution. They found that factors such as the size of the balloon, the duration of delivery time, and the composition of the drug coating all influence how long the drug stays at the injury site and how effectively it clears the arteries.
One significant finding is that when the drug is released, some of it sticks to the lining of the blood vessels. Over time, that drug is slowly released back into the tissue, which explains why the drug’s effects last much longer than the initial 30-second release period.
“This is the first time we can explain the reasons why drug-coated balloons can work,” Kolachalama says. “The study also offers areas where people can consider thinking about optimizing drug transfer and delivery.”

http://circ.ahajournals.org/content/127/20/2047.short  
http://www.mit.edu/people/vbk/Circulation_2013.pdf 
http://www.sciencedaily.com/…13/05/130521121513.ht…    
Circulation, 2013; 127 (20): 2047 – 2055
http://dx.doi.org/10.1161/CIRCULATIONAHA.113.002051;

 

Conclusion

MIT’s Edelman’s Lab conducted the pioneering work in Vascular biology, animal models of drug eluting stents and was at the forefront of Empirical Molecular Cardiology in its studies in vascular physiology, biology and biomaterials for medical devices.

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The Heart Revolution By Kilmer McCully, Martha McCully

HarperCollinsPublishers, 1969

http://books.google.com/books?id=iYLbuZFxEt8C&pg=PR20&dq=New+York+Times+homocysteine+and+Cholesterol&hl=en&sa=X&ei=_0F7UfDRA8zB4APozIHQAQ&ved=0CEMQ6AEwAg

 

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Aviva Lev-Ari, PhD, RN 7/19/2012

https://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/

Resident-cell-based Therapy in Human Ischaemic Heart Disease: Evolution in the PROMISE of Thymosin beta4 for Cardiac Repair

Aviva Lev-Ari, PhD, RN 4/30/2012

https://pharmaceuticalintelligence.com/2012/04/30/93/

Triple Antihypertensive Combination Therapy Significantly Lowers Blood Pressure in Hard-to-Treat Patients with Hypertension and Diabetes

Aviva Lev-Ari, PhD, RN 5/29/2012

https://pharmaceuticalintelligence.com/2012/05/29/445/

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

Aviva Lev-Ari, PhD, RN 7/2/2012

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

Mitochondria Dysfunction and Cardiovascular Disease – Mitochondria: More than just the “powerhouse of the cell”

Aviva Lev-Ari, PhD, RN 7/9/2012

https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Proginetor Cells endogenous augmentation

Aviva Lev-Ari, PhD, RN 7/16/2012

https://pharmaceuticalintelligence.com/2012/07/16/bystolics-generic-nebivolol-positive-effect-on-circulating-endothilial-progrnetor-cells-endogenous-augmentation/

Arteriogenesis and Cardiac Repair: Two Biomaterials – Injectable Thymosin beta4 and Myocardial Matrix Hydrogel

Aviva Lev-Ari, PhD, RN 2/27/2013

https://pharmaceuticalintelligence.com/2013/02/27/arteriogenesis-and-cardiac-repair-two-biomaterials-injectable-thymosin-beta4-and-myocardial-matrix-hydrogel/

Cardiac Surgery Theatre in China vs. in the US: Cardiac Repair Procedures, Medical Devices in Use, Technology in Hospitals, Surgeons’ Training and Cardiac Disease Severity”

Aviva Lev-Ari, PhD, RN 1/8/2013

https://pharmaceuticalintelligence.com/2013/01/08/cardiac-surgery-theatre-in-china-vs-in-the-us-cardiac-repair-procedures-medical-devices-in-use-technology-in-hospitals-surgeons-training-and-cardiac-disease-severity/

Heart Remodeling by Design – Implantable Synchronized Cardiac Assist Device: Abiomed’s Symphony

Aviva Lev-Ari, PhD, RN 7/23/2012

https://pharmaceuticalintelligence.com/2012/07/23/heart-remodeling-by-design-implantable-synchronized-cardiac-assist-device-abiomeds-symphony/

Acute Chest Pain/ER Admission: Three Emerging Alternatives to Angiography and PCI

Aviva Lev-Ari, PhD, RN 3/10/2013

https://pharmaceuticalintelligence.com/2013/03/10/acute-chest-painer-admission-three-emerging-alternatives-to-angiography-and-pci/

Dilated Cardiomyopathy: Decisions on implantable cardioverter-defibrillators (ICDs) using left ventricular ejection fraction (LVEF) and Midwall Fibrosis: Decisions on Replacement using late gadolinium enhancement cardiovascular MR (LGE-CMR)

Aviva Lev-Ari, PhD, RN 3/10/2013
https://pharmaceuticalintelligence.com/2013/03/10/dilated-cardiomyopathy-decisions-on-implantable-cardioverter-defibrillators-icds-using-left-ventricular-ejection-fraction-lvef-and-midwall-fibrosis-decisions-on-replacement-using-late-gadolinium/

The Heart: Vasculature Protection – A Concept-based Pharmacological Therapy including THYMOSIN

Aviva Lev-Ari, PhD, RN 2/28/2013
https://pharmaceuticalintelligence.com/2013/02/28/the-heart-vasculature-protection-a-concept-based-pharmacological-therapy-including-thymosin/

FDA Pending 510(k) for The Latest Cardiovascular Imaging Technology

Aviva Lev-Ari, PhD, RN 1/28/2013
https://pharmaceuticalintelligence.com/2013/01/28/fda-pending-510k-for-the-latest-cardiovascular-imaging-technology/

PCI Outcomes, Increased Ischemic Risk associated with Elevated Plasma Fibrinogen not Platelet Reactivity

Aviva Lev-Ari, PhD, RN 1/10/2013
https://pharmaceuticalintelligence.com/2013/01/10/pci-outcomes-increased-ischemic-risk-associated-with-elevated-plasma-fibrinogen-not-platelet-reactivity/

The ACUITY-PCI score: Will it Replace Four Established Risk Scores — TIMI, GRACE, SYNTAX, and Clinical SYNTAX

Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/01/03/the-acuity-pci-score-will-it-replace-four-established-risk-scores-timi-grace-syntax-and-clinical-syntax/

Coronary artery disease in symptomatic patients referred for coronary angiography: Predicted by Serum Protein Profiles

Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2012/12/29/coronary-artery-disease-in-symptomatic-patients-referred-for-coronary-angiography-predicted-by-serum-protein-profiles/

Heart Renewal by pre-existing Cardiomyocytes: Source of New Heart Cell Growth Discovered

Aviva Lev-Ari, PhD, RN 12/23/2012
https://pharmaceuticalintelligence.com/2012/12/23/heart-renewal-by-pre-existing-cardiomyocytes-source-of-new-heart-cell-growth-discovered/

Cardiovascular Risk Inflammatory Marker: Risk Assessment for Coronary Heart Disease and Ischemic Stroke – Atherosclerosis.

Aviva Lev-Ari, PhD, RN 10/30/2012
https://pharmaceuticalintelligence.com/2012/10/30/cardiovascular-risk-inflammatory-marker-risk-assessment-for-coronary-heart-disease-and-ischemic-stroke-atherosclerosis/

To Stent or Not? A Critical Decision

Aviva Lev-Ari, PhD, RN 10/23/2012
https://pharmaceuticalintelligence.com/2012/10/23/to-stent-or-not-a-critical-decision/

New Definition of MI Unveiled, Fractional Flow Reserve (FFR)CT for Tagging Ischemia

Aviva Lev-Ari, PhD, RN 8/27/2012
https://pharmaceuticalintelligence.com/2012/08/27/new-definition-of-mi-unveiled-fractional-flow-reserve-ffrct-for-tagging-ischemia/

Ethical Considerations in Studying Drug Safety — The Institute of Medicine Report

Aviva Lev-Ari, PhD, RN 8/23/2012
https://pharmaceuticalintelligence.com/2012/08/23/ethical-considerations-in-studying-drug-safety-the-institute-of-medicine-report/

New Drug-Eluting Stent Works Well in STEMI

Aviva Lev-Ari, PhD, RN 8/22/2012
https://pharmaceuticalintelligence.com/2012/08/22/new-drug-eluting-stent-works-well-in-stemi/

Expected New Trends in Cardiology and Cardiovascular Medical Devices

Aviva Lev-Ari, PhD, RN 8/17/2012
https://pharmaceuticalintelligence.com/2012/08/17/expected-new-trends-in-cardiology-and-cardiovascular-medical-devices/

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

Aviva Lev-Ari, PhD, RN 8/13/2012

https://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/

Percutaneous Endocardial Ablation of Scar-Related Ventricular Tachycardia

Aviva Lev-Ari, PhD, RN 7/18/2012

https://pharmaceuticalintelligence.com/2012/07/18/percutaneous-endocardial-ablation-of-scar-related-ventricular-tachycardia/

Competition in the Ecosystem of Medical Devices in Cardiac and Vascular Repair: Heart Valves, Stents, Catheterization Tools and Kits for Open Heart and Minimally Invasive Surgery (MIS)

Aviva Lev-Ari, PhD, RN 6/22/2012

https://pharmaceuticalintelligence.com/2012/06/22/competition-in-the-ecosystem-of-medical-devices-in-cardiac-and-vascular-repair-heart-valves-stents-catheterization-tools-and-kits-for-open-heart-and-minimally-invasive-surgery-mis/

Global Supplier Strategy for Market Penetration & Partnership Options (Niche Suppliers vs. National Leaders) in the Massachusetts Cardiology & Vascular Surgery Tools and Devices Market for Cardiac Operating Rooms and Angioplasty Suites

Aviva Lev-Ari, PhD, RN 6/22/2012

https://pharmaceuticalintelligence.com/2012/06/22/global-supplier-strategy-for-market-penetration-partnership-options-niche-suppliers-vs-national-leaders-in-the-massachusetts-cardiology-vascular-surgery-tools-and-devices-market-for-car/

Blood_Vessels

Blood_Vessels (Photo credit: shoebappa)

Visceral Myopathy in Statins

Visceral Myopathy in Statins (Photo credit: Snipergirl)

Medical science has advanced significantly sin...

Medical science has advanced significantly since 1507, when Leonardo da Vinci drew this diagram of the internal organs and vascular systems of a woman. (Photo credit: Wikipedia)

English: Lee Hood, MD, PhD, President and Co-f...

English: Lee Hood, MD, PhD, President and Co-found of the Institute for Systems Biology (Photo credit: Wikipedia)

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Approach to Controlling Pathogenic Inflammation in Arthritis

Curator: Larry H Bernstein, MD, FCAP

A network approach to controlling pathogenic inflammation: Sequence sharing pattern peptides downregulate experimental arthritis

a new approach to network regulation of inflammation based on

Chai Ezerzer, Raanan Margalit and Irun R. Cohen

Aberrant inflammation probably results from aberrant regulation of the molecules that mediate inflammation; the actual molecules mediating inflammation –

  • chemokines,
  • cytokines, and
  • growth factors and their receptors –
    • would appear to be normal in their chemical structure.

If faulty regulation is indeed the problem,

  • a reasonable approach to alleviating inflammatory diseases might be to influence the interactions
  • within the network of connectivity of the disease-associated proteins (DAPs).
Aberrant inflammation appears to be a pathogenic factor in autoimmune diseases and other noxious inflammatory
conditions in which the inflammatory process
  1. is misapplied,
  2. exaggerated,
  3. recurrent or chronic.
The protein molecules involved in pathogenic inflammation—
disease-associated proteins (DAP )
  1. chemokines,
  2. cytokines, and
  3. growth factors and their receptors,
  • appear normal; their networks of interaction are at fault.

These researchers asked the question – 

  • whether shared amino acid sequence motifs among DAPs
  • might identify novel peptide treatments for regulating inflammation.

We aligned the sequences of 37 DAPs previously discovered to be associated with arthritis

  • to uncover shared sequence motifs.

We focused on chemokine receptor molecules because

  • chemokines and chemokine receptors play important roles in directing the migration of inflammatory cells into sites of tissue inflammation.
  •  different chemokine receptors shared amino acid sequence motifs in their extra-cellular loop domains (ECL2);
  • the ECL2 loop is outside of the known ligand binding site.

These shared sequence motifs established what we term a sequence-sharing network (SSN). SSN motifs exhibited very low E-values,

  • indicating their preservation during evolution.
This study demonstrates a new
  • approach to network regulation of inflammation based on peptide sequence motifs
  • shared by the second extra-cellular loop (EC L2) of different chemokine receptors;
  • previously known chemokine receptor binding sites have not involved the EC L2 loop.
These motifs of 9 amino acids, which were detected by sequence alignment, manifest very low E-values
  • compared with slightly modified sequence variations,
  • indicating that they were not likely to have evolved by chance.
To test whether this shared sequence network (SSN) might serve a regulatory function,
  • theysynthesized 9-amino acid SSN peptides from the EC L2 loops of three different chemokine receptors.
Theye administered these peptides to rats during the
Two of the peptides significantly downregulated the arthritis; one of the peptides
  • synergized with non-specific anti-inflammatory treatment with dexamethasone.
These findings suggest that
  • the SSN peptide motif reported here is likely to have adaptive value in controlling inflammation.
  • detection of SSN motif peptides could provide a network-based approach to immune modulation.
administering a highly connected chemokine receptor peptide motif , as done here, induced
  • the downregulation of inflammation in a rat model of arthritis.
Thus, study of the SSN provides a new network approach toward modulating inflammation
English: Typical chemokine receptor structure ...

English: Typical chemokine receptor structure showing seven transmembrane domains and a chanracteristic “DRY” motif in the second intracelluar domain. (Photo credit: Wikipedia)

Structure of Chemokines

Structure of Chemokines (Photo credit: Wikipedia)

Chemokine receptor

Chemokine receptor (Photo credit: Wikipedia)

 

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Nitric Oxide and it’s impact on Cardiothoracic Surgery

Author, curator: Tilda Barliya PhD

 

In the past few weeks we’ve had extensive in-depth series about nitric oxide (NO) and it’s role in renal function and donors in renal disorders, coagulation, endothelium and hemostasis. This inspired this new post regarding the impact of NO on cardiothoratic surgery.  You can read and follow up on these posts here: https://pharmaceuticalintelligence.com/category/nitric-oxide-in-health-and-disease/

Atherosclerosis in the form of peripheral arterial disease (PAD) affects approximately eight million Americans, which includes 12 to 20% of individuals over the age of 65.  Approximately 20% of patients with PAD have typical symptoms of lower extremity claudication, rest pain, ulceration, or gangrene, and one-third have atypical exertional symptoms. Persons with PAD have impaired function and quality of life even if they do not report symptoms and experience a decline in lower extremity function over time. Cardiovascular disease is the major cause of death in patients with intermittent claudication; the annual rate of cardiovascular events (myocardial infarction, stroke, or death from cardiovascular causes) is 5 to 7%.  Thus, PAD represents a significant source of morbidity and mortality. (1) (http://www.medscape.com/viewarticle/569812).

Several options exist for treating atherosclerotic lesions, including:

  • percutaneous transluminal angioplasty with and without stenting,
  • endarterectomy
  • bypass grafting

Unfortunately, patency rates for each of these procedures continue to be suboptimal secondary to the development of neointimal hyperplasia. A universal feature of all vascular surgical procedures is the removal of or damage to the endothelial cell monolayer that occurs whether the procedure performed is endovascular or open. This endothelial damage leads to a decreased or absent production of nitric oxide (NO) at the site of injury.

noendoschematic

he relationship between NO and the cardiovascular system has proven to be a landmark discovery, and the scientists credited for its discovery were awarded the Nobel Prize in Medicine in 1998. Since its discovery, NO has proven to be one of the most important molecules in vascular homeostasis. In fact, the term endothelial dysfunction has now become synonymous with the reduced biologic activity of NO.

NO produced by endothelial cells has been shown to have many beneficial effects on the vasculature.

As described above,

  • NO stimulates vascular smooth muscle cells (VSMC) relaxation, which leads to vessel vasodilatation.  
  • NO has opposite beneficial affects on endothelial cells compared with VSMCs.
  • Whereas NO stimulates endothelial cell proliferation and prevents endothelial cell apoptosis,  it inhibits VSMC growth and migration  and stimulates VSMC apoptosis.  
  • NO also has many thromboresistant properties, such as inhibition of platelet aggregation, adhesion, and activation;  inhibition of leukocyte adhesion and migration;  and inhibition of matrix formation

 As stated before, the endothelial cell monolayer is often removed or damaged during the time of vascular procedures, which leads to a local decrease in the production of NO. It is now understood that this loss of local NO synthesis by endothelial cells at the site of vascular injury is one of the inciting events that allows platelet aggregation, inflammatory cell infiltration, and VSMC proliferation and migration to occur in excess, which, taken together, leads to neointimal hyperplasia.

Reendothelialization of the injured artery can restore proper function to the artery and potentially halt the restenotic process. Many studies have attempted to improve the patency of bypass grafts and stents by coating them with endothelial cells in the hope that this would restore the thromboresistant nature of native blood vessels.

Unfortunately, although it has been possible to coat these devices with endothelial cells, these cells do not behave like normal endothelial cells and their NO production is often diminished or absent. Because the vasoprotective properties of endothelial cells are largely carried out by NO alone, investigators are engaged in research to improve the bioavailability of NO at the site of vascular injury in an attempt to reduce the risk of thrombosis and restenosis after successful revascularization. The overall goal of using a NO-based approach is to reproduce the same thromboresistive moiety observed with normal NO production.

Why of delivering NO to the injured site:

  • Systemic delivery
  • Local delivery

Systemic Delivery

One simple mechanism by which to deliver NO to the body is via inhalational therapy. Inhaled NO has been used clinically in the past to selectively reduce pulmonary vascular resistance in patients with pulmonary hypertension, as well as a potential therapy for patients with acute respiratory distress syndrome. Because the gas is delivered only to the pulmonary system and has a very short half-life, it was thought that there would be no systemic effects of the drug. Subsequently, studies in the mid- to late 1990s suggested that inhaled NO had beneficial antiplatelet and antileukocyte properties without adverse systemic side effects (2,3)

To test if inhaled NO had any beneficial systemic properties specifically on the vasculature, Lee and colleagues evaluated the effect of inhaled NO on neointimal hyperplasia in rats undergoing carotid balloon injury, Unfortunately, the treatment was required for the full 2 weeks to see any difference between the treatment and the control group, thereby limiting its clinical utility.

Despite some of the early animal studies, investigations with healthy human volunteers failed to reproduce these findings.I t was speculated that despite the obvious effects of inhaled NO on the pulmonary vasculature, systemic bioavailability could not be reliably achieved because of the immediate binding and depletion of NO by hemoglobin as soon as it entered the systemic circulation.

Hamon and colleagues tested the ability of orally supplementing l-arginine (2.25%), the precursor to NO, in the drinking water of rabbits to reduce the formation of neointimal hyperplasia after injuring the iliac arteries with a balloon.  This amount of l-arginine is approximately sixfold higher than normal daily intake. When the arteries were studied 4 weeks after injury, the l-arginine-fed group exhibited less neointimal hyperplasia and greater acetylcholine-induced relaxation compared with the control animals. The authors speculated that the improved outcomes were due to increased bioavailability of NO secondary to the l-arginine-supplemented diets. To test the ability of this supplemented diet to reduce neointimal hyperplasia in a vein bypass graft model, Davies and colleagues fed rabbits l-arginine (2.25%) 7 days prior to and 28 days after common carotid vein bypass grafts. A 51% decrease in the formation of neointimal hyperplasia was demonstrated in the l-arginine-fed groups, and their vein grafts exhibited preserved NO-mediated relaxation.

Despite some of the positive findings in animals, similar studies in humans have failed to show any benefit with l-arginine supplementation. Shiraki and colleagues studied the effects of short-term high-dose l-arginine on restenosis after PTCA.  Thirty-four patients undergoing cardiac catheterization and PTCA for angina pectoris received 500 mg of l-arginine administered through the cardiac catheter immediately prior to PTCA and 30 g per day of l-arginine administered via the peripheral vein for 5 days after PTCA. No significant statistical differences in restenosis were observed between the two groups (34% vs 44%). The authors speculated that the lack of effect was secondary to the fact that although the levels of l-arginine in the plasma increased significantly, NO and cyclic guanosine monophosphate (cGMP) did not. (4)

Table 1.  Comparison of Different Nitric Oxide Donor Drugs Currently Used for Clinical or Research Purposes
Drug Mechanism of NO Release Unique Properties
Diazeniumdiolates Spontaneous when in contact with physiologic fluidsNO release follows first-order kinetics Stable as solidsVarious reliable half-lives depending on the structure of the nucleophile it is attached to
Nitrosamines can form as by-products
S-Nitrosothiols Copper ion-mediated decomposition Stable as a solid
Direct reaction with ascorbate Must be protected from light
Homeolytic cleavage by light Present in circulating blood
Potential for unlimited NO release
Sydnonimines Requires enzymatic cleavage by liver esterases to form active metabolite Stable as a solidMust be protected from light
Requires molecular oxygen as an electron acceptor Requires alkaline pHReleases superoxide as a by-product, which may have negative effects
l-Arginine Substrate for NOS genes Stable as a solid
Ease of administration
Dependent on presence of NOS for NO production
Sodium nitroprusside Requires a one-electron reduction to release NO Stable as a solid
Must be protected from light
Light can induce NO release Must be given intravenously
Releases cyanide as a by-product
Organic nitrates Either by enzymatic cleavage or nonenzymatic bioactivation with sulfhydryl or thiol groups Stable as a solid
Must be protected from light
Ease of administration
Development of tolerance limits efficacy
NO-releasing aspirin Require enzymatic cleavage to break the covalent bond between the aspirin and the NO moiety Stable as a solid
Ease of administration
Inherent benefits of aspirin also
Does not affect systemic blood pressure

Despite the ease of administration, the reliability of drug delivery, and the relative safety of these NO-donating drugs, there are limitations associated with systemic administration. One such limitation is that NO is rapidly inactivated by hemoglobin in the circulating blood, resulting in limited bioavailability. Furthermore, in attempts to increase the amount of drug delivered to obtain the desired clinical effect, unwanted systemic circulatory effects (eg, vasodilation) and unwanted hemostatic effects (eg, bleeding) often preclude administration of biologically effective doses of NO.

Because NO produces systemic side effects, lower doses of NO have been used in many of the human studies. One of the reasons for the differences observed between the animal studies and the human studies was the 10- to 50-fold lower doses of drugs used in the human studies compared with the animal studies. Thus, local delivery of NO may achieve improved results.

Local Delivery

The local delivery of drugs allows for the administration of the maximally effective dose of a drug without the unwanted systemic side effects. Because the target vessels are easily accessible during most vascular procedures, a local pharmacologic approach to administer a drug during the intervention can be easily performed.

Suzuki and colleagues performed a prospective, randomized, single-center clinical trial. (7)

The study population consisted of patients with symptomatic ischemic heart disease who were undergoing coronary artery stent placement. After stent deployment, l-arginine (600 mg/6 mL) or saline (6 mL) was locally delivered via a catheter over 15 minutes. The patients were followed with serial angiography and intravascular ultrasonography to assess for neointimal thickness for up to 6 months. The authors found that in the l-arginine-treated groups, there was slightly less neointimal volume, but this was not statistically significant.

Because it was not known if the addition of l-arginine actually translated to increased NO production, several studies have focused on the addition of NO donors directly to the site of injury.However, Critics of some of the highlighted animal studies point out that the evaluation of neointimal hyperplasia was performed radiographically, which could be subjectively biased. Furthermore, infusing the drug through a catheter for an extended period of time during the procedure to achieve an effect is not clinically feasible. Because of this, other studies have aimed to develop a clinically applicable approach to deliver NO locally to the site of injury.

  • Hydrogels
  • Vascular grafts
  • Gene therapy

represents another method by which to locally increase the level of NO at the site of vascular injury, tested in different multiple creative animal models. Thought, most of this studies shown great preliminary results, only the gene therapy moved forward into randomized clinical trial in humans using gene therapy to reduce neointimal hyperplasia.

In December 2000, the Recombinant DNA Advisory Committee at the National Institutes of Health voted unanimously to proceed with the first phase of clinical evaluation of iNOS lipoplex-mediated gene transfer, called REGENT-1: Restenosis Gene Therapy Trial. (8). The primary objective of this multicenter, prospective, single-blind, dose escalation study was to obtain safety and tolerability information of iNOS-lipoplex gene therapy for reducing restenosis following coronary angioplasty. As of 2002, 27 patients had been enrolled overseas and the process had been determined to be safe. To date, no results have been published as it appears that this trial lost its funding and closed. On April 5, 2002, a notification was issued that the trial had been closed without enrolling any individuals in the United States.

Unfortunately, despite the promising findings shown with NOS therapy, the field of gene therapy has been mottled by two widely known complications. One case occurred as the result of administering a large viral load that led to the death of a patient. In addition, in France, there were at least two cases of malignancy following retroviral gene therapy.  (9)

Summary

Atherosclerosis in the form of coronary artery disease and peripheral vascular disease continues to be a major source of morbidity and mortality. Unfortunately, the procedures and materials that are currently used to alleviate these disease states are temporary at best because of the inevitable injury to the native endothelium and the subsequent impairment of NO release. Since the discovery of NO and its role in vascular biology, a main focus in vascular research has been to create novel mechanisms to use NO to combat neointimal hyperplasia. To date, numerous animal studies have restored NO production to the vasculature and have shown that this inhibits neointimal hyperplasia, improves patency rates, and is safe to the animal. Clinical studies using these novel NO-releasing compounds in humans are on the horizon.

Ref:

1. Daniel A. Popowich, Vinit Varu, Melina R. Kibbe. Nitric Oxide: What a Vascular Surgeon Needs to Know. Vascular. 2007;15(6):324-335. (http://www.medscape.com/viewarticle/569812).

2.  Gries A, Bode C, Peter K, et al. Inhaled nitric oxide inhibits human platelet aggregation, P-selectin expression, and fibrinogen binding in vitro and in vivo Circulation 1998;97:1481-7.

3.  Lee JS, Adrie C, Jacob HJ, et al. Chronic inhalation of nitric oxide inhibits neointimal formation after balloon-induced arterial injury Circ Res 1996;78:337-42.

4.  Shiraki T, Takamura T, Kajiyama A, et al. Effect of short-term administration of high dose l-arginine on restenosis after percutaneous transluminal coronary angioplasty J Cardiol 2004;44:13-20.

5. David A. Fullerton, MD, Robert C. McIntyre, Jr, MD. Inhaled Nitric Oxide: Therapeutic Applications in Cardiothoracic Surgery. Ann Thorac Surg 1996;61:1856-1864. http://ats.ctsnetjournals.org/cgi/content/abstract/61/6/1856

6. Owen I.Miller,Swee Fong Tang, Anthony Keech,Nicholas B.Pigott, Elaine Beller and David S. Celemajer.  Inhaled nitric oxide and prevention of pulmonary hypertension after congenital heart surgery: a randomised double-blind study. The Lancet,2000:356; 9240 Pages 1464 – 1469,  http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(00)02869-5/abstract

7. Suzuki T, Hayase M, Hibi K, et al. Effect of local delivery of l-arginine on in-stent restenosis in humans Am J Cardiol 2002;89:363-7.

8. von der Leyen HE, Chew N. Nitric oxide synthase gene transfer and treatment of restenosis: from bench to bedside Eur J Clin Pharmacol 2006;62:83-89

9.  Barbato JE, Tzeng E. iNOS gene transfer for graft disease Trends Cardiovasc Med 2004;14:267-72.

10. E. Matevossian, A. Novotny, C. Knebel, T. Brill, M. Werner, I. Sinicina, M. Kriner, M. Stangl, S. Thorban, and N. Hüser. The Effect of Selective Inhibition of Inducible Nitric Oxide Synthase on Cytochrome P450 After Liver Transplantation in a Rat Model. Transplantation Proceedings 2008, 40, 983–985. http://211.144.68.84:9998/91keshi/Public/File/29/40-4/pdf/1-s2.0-S0041134508004181-main.pdf

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Cardiovascular Risk Inflammatory Marker: Risk Assessment for Coronary Heart Disease and Ischemic Stroke – Atherosclerosis

Reporter: Aviva Lev-Ari, PhD, RN

 

Updated on 10/3/2018

Treatment concentration of high-sensitivity C-reactive protein

Published:November 13, 2017DOI:https://doi.org/10.1016/S0140-6736(17)32865-9

Interleukin 1β has multiple potential mechanisms that contribute to the pathogenesis of atherothrombotic cardiovascular disease.

Induction of interleukin 6 leads to the release of acute phase reactants including hsCRP. Thus, hsCRP serves as a surrogate marker of the overall inflammatory milieu,

often in situations where patients have multiple co-morbidities,

with a cumulative dose-response indicating a higher risk.

References

  • Ridker PM
  • Everett BM
  • Thuren T
  • et al.
Antiinflammatory therapy with canakinumab for atherosclerotic disease.

N Engl J Med. 2017; 3771119-1131

  • Libby P
Interleukin-1 beta as a target for atherosclerosis therapy: biological basis of CANTOS and beyond.

J Am Coll Cardiol. 2017; 702278-2289

  • Pokharel Y
  • Sharma PP
  • Qintar M
  • et al.
High-sensitivity C-reactive protein levels and health status outcomes after myocardial infarction.

Atherosclerosis. 2017; 26616-23

  • Wang A
  • Liu J
  • Li C
  • et al.
Cumulative exposure to high-sensitivity C-reactive protein predicts the risk of cardiovascular disease.

J Am Heart Assoc. 2017; 6e005610

    • Ridker PM
    • MacFadyen JG
    • Everett BM
    • et al.

on behalf of the CANTOS Trial Group

Relationship of C-reactive protein reduction to cardiovascular event reduction following treatment with canakinumab: a secondary analysis from the CANTOS randomised controlled trial.

Lancet. 2017; (published online Nov 13.)

SOURCE

 

 

 

 

Cardiovascular Risk Inflammatory Marker: Risk Assessment for Coronary Heart Disease and Ischemic StrokeAtherosclerosis.

 

Watch VIDEO

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Lp-PLA2 Overview Webinar

Source: http://www.plactest.com/healthcare/webinar

Watch VIDEO

 american-heart-association-2007-lppla2-highlights

American Heart Association 2007 Lp-PLA2 Presentation

Source: http://www.plactest.com/healthcare/american-heart-association-2007-lppla2-highlights

diaDexus’s PLAC, the test measuting Lp-PLA2 as a novel and valuable cardiovascular risk inflammatory marker a vascular-specific inflammatory marker implicated in the formation of rupture-prone plaque, and is the only blood test cleared by the FDA to assess risk for coronary heart disease and ischemic stroke associated with atherosclerosis. (2003 and in 2005 received additional clearance as an aid in the assessment of risk for ischemic stroke associated with atherosclerosis.)

 

In 2007 the PLAC Test was granted a Category I CPT Code (83698) by the American Medical Association and is reimbursed by the Centers for Medicare and Medicaid Services (CMS) with a National Limitation Amount (NLA) of $47.77 in the 2011 CMS Clinical Laboratory Fee Schedule.

In July 2010, diaDexus completed a reverse merger with VaxGen. diaDexus currently trades on the OTC Bulletin Board (DDXS.OB).

 

PLAC Test is an alternative to C- Reactive Protein Test

 

The PLAC® Test is a simple blood test to detect Lp-PLA2 in the bloodstream. It is used to help predict risk for coronary heart disease and ischemic stroke associated with atherosclerosis.

 

  • The PLAC Test measures Lp-PLA2
    (lipoprotein-associated phospholipase A2), a vascular-specific inflammatory enzyme implicated in the formation of rupture-prone plaque. It is plaque rupture and thrombosis, not stenosis, that causes the majority of cardiac events.
  • A substantial body of evidence, including over 100 studies and abstracts in peer-reviewed journals and conferences, support Lp-PLA2 as a cardiovascular risk marker that provides new information, over and above traditional risk factors.
  • Consistent with ATP III and European guidelines, the PLAC Test should be used as an adjunct to traditional risk factor assessment to identify which moderate or high risk patients, as initially assessed by traditional risk factors, may actually be at higher risk.
  • An elevated PLAC Test may indicate a need for more aggressive patient management.
    • 50% of cardiovascular events strike in patients with unremarkable lipid levels, highlighting the prevalence of hidden cardiovascular risk.
    • LDL-C and total cholesterol have proven not to be reliable predictors of stroke; the PLAC Test addresses this unmet clinical need.
  • Lipid lowering therapies, including statins, are proven to reduce cardiovascular events regardless of baseline LDL-C levels.

 

Basic Science of Lp-PLA2

The PLAC® Test measures Lp-PLA2 (lipoprotein-associated phospholipase A2) a vascular-specific inflammatory enzyme implicated in the formation of rupture-prone plaque. It is plaque rupture and thrombosis that cause the majority of cardiac events, not stenosis.

 

 

 

 

Lp-PLA2 is a calcium-independent serine lipase that is associated with both low-density lipoprotein (LDL) and, to a lesser extent, high-density lipoprotein (HDL) in human plasma and serum and is distinct from other phospholipases such as cPLA2 and sPLA2. Lp-PLA2 is produced by macrophages and other inflammatory cells and is expressed in greater concentrations in advanced atherosclerotic lesions than early-stage lesions.

 

Lp-PLA2 has demonstrated modest intra- and inter-individual variation, commensurate with other cardiovascular lipid markers and substantially less than C-reactive protein (CRP). In addition, Lp-PLA2 is not elevated in systemic inflammatory conditions, and may be a more specific marker of vascular inflammation. The relatively small biological variation of Lp-PLA2 and its specificity are of value in the detection and monitoring of cardiovascular risk.

SOURCE:

http://www.plactest.com/healthcare/basic-science.html

 

 

Clinical Utility of the PLAC Test

 

The PLAC® Test Measures Lp-PLA2, a Unique Marker  
The PLAC Test for Lp-PLA2 is the only blood test cleared by the FDA to aid in assessing risk for both coronary heart disease and ischemic stroke associated with atherosclerosis. The PLAC Test measures lipoprotein-associated phospholipase A2 (Lp-PLA2), a vascular-specific biomarker implicated in the formation of rupture-prone plaque. The majority of all heart attacks and strokes are caused by plaque rupture and thrombosis (clots) – not stenosis (narrowing of arteries).

Lp-PLA2 is a unique marker for vascular-specific inflammation and is produced by macrophages in inflamed plaque. Lp-PLA2 provides additive risk information when combined with other markers such as hs-CRP to help you personalize your treatment options, beyond the limitations of the traditional cardiovascular (CV) risk factors.

The PLAC Test Helps Identify Hidden Risk
Lp-PLA2 is an independent risk marker for stroke. At every level of blood pressure, an Lp-PLA2 value above the median almost doubles the risk for stroke.  Current stroke guidelines include consideration of Lp-PLA2 measurement in asymptomatic patients to identify those who may be at increased risk of stroke.

The PLAC Test Helps Improve Patient Management 
Periodic measurement of the amount of Lp-PLA2 in the blood for patients with 2 or more CVD risk factors can aid clinical decisions for at-risk patients, allowing you to assess or reassess the effect of lipid lowering therapies on vascular inflammation, intensify therapeutic lifestyle changes, and reinforces doctors’ recommendations for patient management.

 

 

 

 

Essential Information to Guide Treatment

In accordance with ATP III Guidelines, patients with 2 or more CV risk factors may be candidates for advanced lipid testing.

Measure the amount of Lp-PLA2 in your patient’s blood stream with the PLAC Test to determine whether they may be at increased risk for heart attack or stroke.

If the PLAC Test results are 200 ng/mL or greater, cardiovascular disease may be present. Review your patient’s advanced lipid panel results to determine where more aggressive patient management may be needed.

 

* additional reduction of Lp-PLA2 seen when added to statin therapy.

Based on:

Shalwitz R, et al. ATVB Annual Mtg. 2007.

Kuvin J, et al. Am J Cardiol. 2006.

Albert M, et al. Atherosclerosis 2005.

Schaefer EJ, et al. Am J Cardiol. 2005.

Saougos VG, et al. ATVB 2007.

Muhlestein JB, et al. JACC 2006.

      Early detection and more aggressive treatment can help prevent cardiovascular events.


 

SOURCE:

http://www.plactest.com/Default.aspx?PageID=4620488&A=PrinterView

 

 

REFERENCES

 

Pathophysiology and Genetics Studies

 

A Twin Study of Heritability of Plasma Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Mass and ActivityLenzini L, Antezza K, Caroccia B, Wolfert RL, Szczech R, Cesari M, Narkiewicz K, Williams CJ, Rossi GP. A Twin Study of Heritability of Plasma Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Mass and Activity. Atherosclerosis. 2009; 205(1): 181-5.

Enhanced Expression of Lp-PLA2 and Lysophosphatidylcholine in Symptomatic Carotid Atherosclerotic PlaqueMannheim D, Herrmann J, Versari D, Gössl M, Meyer FB, McConnell JP, Lerman LO, Lerman A. Enhanced Expression of Lp-PLA2 and Lysophosphatidylcholine in Symptomatic Carotid Atherosclerotic Plaque. Stroke. 2008; 39: 1448-55.

Expression of Lipoprotein-Associated Phospholipase A2 in Carotid Artery Plaques Predicts Long-term Cardiac OutcHerrmann J, Mannheim D, Wohlert C, Versari D, Meyer FB, McConnell JP, Gössl M, Lerman LO, Lerman A. Expression of Lipoprotein-Associated Phospholipase A2 in Carotid Artery Plaques Predicts Long-term Cardiac Outcome. Eur. Heart J. 2009 Dec; 30(23): 2930-8.

Lipoprotein-Associated Phospholipase A2 is an Independent Marker for Coronary Endothelial Dysfunction in HumansYang EH, McConnell JP, Lennon RJ, Barsness GW, Pumper G, Hartman SJ, Rihal CS, Lerman LO, Lerman A. Lipoprotein-Associated Phospholipase A2 is an Independent Marker for Coronary Endothelial Dysfunction in Humans. Arterioscler Thromb Vasc Biol. 2006; 26(1): 106-11.

Lipoprotein-Associated Phospholipase A2 Protein Expression in the Natural Progression of Human Coronary AtherosclerosisKolodgie FD, Burke AP, Skorija KS, Ladich E, Kutys R, Makuria AT, Virmani R. Lipoprotein-Associated Phospholipase A2 Protein Expression in the Natural Progression of Human Coronary Atherosclerosis. Arterioscler Thromb Vasc Biol. 2006; 26: 2523-9.

 

Therapeutic Modulation Studies

 

Cardiovascular Events With Increased Lipoprotein-Associated Phospholipase A2 and Low High-Density Lipoprotein-Cholesterol. The Veterans Affairs HDL Intervention Trial.Robins SJ, Collins D, JJ, Bloomfield HE, Asztalos BF. Cardiovascular Events With Increased Lipoprotein-Associated Phospholipase A2 and Low High-Density Lipoprotein-Cholesterol. The Veterans Affairs HDL Intervention Trial. Arterioscler Thromb Vasc Biol. 2008; 28(6): 1172-8.

Changes in Lp-PLA2 activity in secondary prevention predict coronary events and treatment effect by pravastatin in long term intervention with pravastatin in ischemic disease (LIPID) TrialWhite HD, Simes J, Barnes, E et al. Changes in Lp-PLA2 activity in secondary prevention predict coronary events and treatment effect by pravastatin in long term intervention with pravastatin in ischemic disease (LIPID) Trial. Circulation, abstract 14857, AHA 2011

Differential Effect of Hypolipidemic Drugs on Lipoprotein-Associated Phospholipase A2Saougos VG, Tambaki AP, Kalogirou M, Kostapanos M, Gazi IF, Wolfert RL, Elisaf M, Tselepis AD. Differential Effect of Hypolipidemic Drugs on Lipoprotein-Associated Phospholipase A2. Arterioscler Thromb Vasc Biol. 2007; 27: 2236-43.

Effects of Atorvastatin Versus Other Statins on Fasting and Postprandial C-Reactive Protein and Lipoprotein-Associated Phospholipase A2 in Patients With Coronary Heart Disease Versus Control SubjectsSchaefer EJ, McNamara JR, Asztalos BF, Tayler T, Daly JA, Gleason JL, Seman LJ, Ferrari A, Rubenstein JJ. Effects of Atorvastatin Versus Other Statins on Fasting and Postprandial C-Reactive Protein and Lipoprotein-Associated Phospholipase A2 in Patients With Coronary Heart Disease Versus Control Subjects. Am J Cardiol. 2005; 95: 1025-32.

Effects of Extended-Release Niacin on Lipoprotein Particle Size, Distribution, and Inflammatory Markers in Patients With Coronary Artery DiseaseKuvin JT, Dave DM, Sliney KA, Mooney P, Patel AR, Kimmelstiel CD, Karas RH. Effects of Extended-Release Niacin on Lipoprotein Particle Size, Distribution, and Inflammatory Markers in Patients With Coronary Artery Disease. Am J Cardiol. 2006; 98: 743-5.

Cardiovascular Events With Increased Lipoprotein-Associated Phospholipase A2 and Low High-Density Lipoprotein-Cholesterol. The Veterans Affairs HDL Intervention Trial.Robins SJ, Collins D, JJ, Bloomfield HE, Asztalos BF. Cardiovascular Events With Increased Lipoprotein-Associated Phospholipase A2 and Low High-Density Lipoprotein-Cholesterol. The Veterans Affairs HDL Intervention Trial. Arterioscler Thromb Vasc Biol. 2008; 28(6): 1172-8.

Changes in Lp-PLA2 activity in secondary prevention predict coronary events and treatment effect by pravastatin in long term intervention with pravastatin in ischemic disease (LIPID) TrialWhite HD, Simes J, Barnes, E et al. Changes in Lp-PLA2 activity in secondary prevention predict coronary events and treatment effect by pravastatin in long term intervention with pravastatin in ischemic disease (LIPID) Trial. Circulation, abstract 14857, AHA 2011

Differential Effect of Hypolipidemic Drugs on Lipoprotein-Associated Phospholipase A2Saougos VG, Tambaki AP, Kalogirou M, Kostapanos M, Gazi IF, Wolfert RL, Elisaf M, Tselepis AD. Differential Effect of Hypolipidemic Drugs on Lipoprotein-Associated Phospholipase A2. Arterioscler Thromb Vasc Biol. 2007; 27: 2236-43.

Effects of Atorvastatin Versus Other Statins on Fasting and Postprandial C-Reactive Protein and Lipoprotein-Associated Phospholipase A2 in Patients With Coronary Heart Disease Versus Control SubjectsSchaefer EJ, McNamara JR, Asztalos BF, Tayler T, Daly JA, Gleason JL, Seman LJ, Ferrari A, Rubenstein JJ. Effects of Atorvastatin Versus Other Statins on Fasting and Postprandial C-Reactive Protein and Lipoprotein-Associated Phospholipase A2 in Patients With Coronary Heart Disease Versus Control Subjects. Am J Cardiol. 2005; 95: 1025-32.

Effects of Extended-Release Niacin on Lipoprotein Particle Size, Distribution, and Inflammatory Markers in Patients With Coronary Artery DiseaseKuvin JT, Dave DM, Sliney KA, Mooney P, Patel AR, Kimmelstiel CD, Karas RH. Effects of Extended-Release Niacin on Lipoprotein Particle Size, Distribution, and Inflammatory Markers in Patients With Coronary Artery Disease. Am J Cardiol. 2006; 98: 743-5.

Cardiovascular Events With Increased Lipoprotein-Associated Phospholipase A2 and Low High-Density Lipoprotein-Cholesterol. The Veterans Affairs HDL Intervention Trial.Robins SJ, Collins D, JJ, Bloomfield HE, Asztalos BF. Cardiovascular Events With Increased Lipoprotein-Associated Phospholipase A2 and Low High-Density Lipoprotein-Cholesterol. The Veterans Affairs HDL Intervention Trial. Arterioscler Thromb Vasc Biol. 2008; 28(6): 1172-8.

Changes in Lp-PLA2 activity in secondary prevention predict coronary events and treatment effect by pravastatin in long term intervention with pravastatin in ischemic disease (LIPID) TrialWhite HD, Simes J, Barnes, E et al. Changes in Lp-PLA2 activity in secondary prevention predict coronary events and treatment effect by pravastatin in long term intervention with pravastatin in ischemic disease (LIPID) Trial. Circulation, abstract 14857, AHA 2011

Differential Effect of Hypolipidemic Drugs on Lipoprotein-Associated Phospholipase A2Saougos VG, Tambaki AP, Kalogirou M, Kostapanos M, Gazi IF, Wolfert RL, Elisaf M, Tselepis AD. Differential Effect of Hypolipidemic Drugs on Lipoprotein-Associated Phospholipase A2. Arterioscler Thromb Vasc Biol. 2007; 27: 2236-43.

Effects of Atorvastatin Versus Other Statins on Fasting and Postprandial C-Reactive Protein and Lipoprotein-Associated Phospholipase A2 in Patients With Coronary Heart Disease Versus Control SubjectsSchaefer EJ, McNamara JR, Asztalos BF, Tayler T, Daly JA, Gleason JL, Seman LJ, Ferrari A, Rubenstein JJ. Effects of Atorvastatin Versus Other Statins on Fasting and Postprandial C-Reactive Protein and Lipoprotein-Associated Phospholipase A2 in Patients With Coronary Heart Disease Versus Control Subjects. Am J Cardiol. 2005; 95: 1025-32.

Effects of Extended-Release Niacin on Lipoprotein Particle Size, Distribution, and Inflammatory Markers in Patients With Coronary Artery DiseaseKuvin JT, Dave DM, Sliney KA, Mooney P, Patel AR, Kimmelstiel CD, Karas RH. Effects of Extended-Release Niacin on Lipoprotein Particle Size, Distribution, and Inflammatory Markers in Patients With Coronary Artery Disease. Am J Cardiol. 2006; 98: 743-5.

 

 

 

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Chemical structure of etidronic acid (INN). Cr...

Chemical structure of etidronic acid (INN). Created using ACD/ChemSketch and Inkscape. (Photo credit: Wikipedia)

Diagram showing the origins of the main branch...

Diagram showing the origins of the main branches of the carotid arteries. (Photo credit: Wikipedia)

Larry H Bernstein, MD, FCAP, Reporter

Vascular Effects of Bisphosphonates—A Systematic Review

  • Leyna L. Santos, Taciana B. Cavalcanti and Francisco A. Bandeira

Division of Endocrinology and Diabetes, Agamenon Magalhães Hospital, Ministry of Health, University of Pernambuco
Medical School, Recife, Brazil. Corresponding author email: leynaleite@yahoo.com.br

Abstract
Background: Osteoporosis and cardiovascular disease are interconnected entities with pathophysiological similarities. Bisphosphonates are therapeutic options available for resorptive bone diseases; however, experimental evidence has demonstrated a role for bisphosphonates in the inhibition of atherogenesis.
Methods: A systematic review of the vascular effects of bisphosphonates on atherosclerosis was performed. Vascular effects were evaluated by the thickening of the intima-media of carotid arteries and calcification of the coronary and aorta arteries. Electronic databases PubMed, The Cochrane Library, and Embase from January 1980 to May 2011 were searched.
Results: Of 169 potentially relevant articles, 9 clinical trials were selected. Two articles showed the benefit of the use of etidronate (−0.038 mm, P < 0.005) and alendronate (−0.025 mm, P < 0.05) on carotid artery intima-media thickening (CIMT) after one year.
One article found no changes associated with the use of alendronate. The use of risedronate was associated with a reduction of plaque score on the carotid arteries (decrease of 1% at 1 year, P = 0.015). Of those studies that evaluated the effect on coronary artery calcification (CAC), the results are conflicting: one study showed no changes with use of etidronate and in another, etidronate resulted in inhibition of the process of CAC after 1 year of follow-up (−372 mm3 in CAC score, P , 0.01). Three studies showed positive effects of etidronate on the aortic calcificaton (AC) score, showing no effect with use of ibandronate, and another showed a inhibition in the progression of the abdominal AC score with use of risendronate (P = 0.043).
Conclusion: Bisphosphonates seem to have an inhibitory effect on the atherosclerotic process; however, larger placebo-controlled
studies are needed to better clarify this issue.
Keywords: Bisphosphonates, osteoporosis, atherosclerosis, carotid IMT, aortic calcification

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Endothelial Function and Cardiovascular Disease

Pathologist and AuthorLarry H Bernstein, MD, FCAP 

 

This discussion is a continuation of a series on Nitric Oxide, vascular relaxation, vascular integrity, and systemic organ dysfunctions related to inflammatory and circulatory disorders. In some of these, the relationships are more clear than others, and in other cases the vascular disorders are aligned with serious metabolic disturbances. This article, in particular centers on the regulation of NO production, NO synthase, and elaborates more on the assymetrical dimethylarginine (ADMA) inhibition brought up in a previous comment, and cardiovascular disease, including:

Recall, though, that in SIRS leading to septic shock, that there is a difference between the pulmonary circulation, the systemic circulation and the portal circulation in these events. The comment calls attention to:
Böger RH. Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, explains the ‘L-arginine paradox’ and acts as a novel cardiovascular risk factor. J Nutr 2004; 134: 2842S–7S.

This observer points out that ADMA inhibits vascular NO production at concentrations found in pathophysiological conditions (i.e., 3–15 μmol/l); ADMA also causes local vasoconstriction when it is infused intra-arterially. ADMA is increased in the plasma of humans with hypercholesterolemia, atherosclerosis, hypertension, chronic renal failure, and chronic heart failure.

Increased ADMA levels are associated with reduced NO synthesis as assessed by impaired endothelium-dependent vasodilation. We’ll go into that more with respect to therapeutic targets – including exercise, sauna, and possibly diet, as well as medical drugs.

It is remarkable how far we have come since the epic discovery of 17th century physician, William Harvey, by observing the action of the heart in small animals and fishes, proved that heart receives and expels blood during each cycle, and argued for the circulation in man. This was a huge lead into renaissance medicine. What would he think now?

Key Words: eNOS, NO, endothelin, ROS, oxidative stress, blood flow, vascular resistance, cardiovascular disease, chronic renal disease, hypertension, diabetes, atherosclerosis, MI, exercise, nutrition, traditional chinese medicine, statistical modeling for targeted therapy.

Endothelial Function
The endothelium plays a crucial role in the maintenance of vascular tone and structure by means of eNOS, producing the endothelium-derived vasoactive mediator nitric oxide (NO), an endogenous messenger molecule formed in healthy vascular endothelium from the amino acid precursor L-arginine. Nitric oxide synthases (NOS) are the enzymes responsible for nitric oxide (NO) generation. The generation and actions of NO under physiological and pathophysiological conditions are exquisitely regulated and extend to almost every cell type and function within the circulation. While the molecule mediates many physiological functions, an excessive presence of NO is toxic to cells.

The enzyme NOS, constitutively or inductively, catalyses the production of NO in several biological systems. NO is derived not only from NOS isoforms but also from NOS-independent sources. In mammals, to date, three distinct NOS isoforms have been identified:

  1. neuronal NOS (nNOS),
  2. inducible NOS (iNOS), and
  3. endothelial NOS (eNOS).

The molecular structure, enzymology and pharmacology of these enzymes have been well defined, and reveal critical roles for the NOS system in a variety of important physiological processes. The role of NO and NOS in regulating vascular physiology, through neuro-hormonal, renal and other non-vascular pathways, as well as direct effects on arterial smooth muscle, appear to be more intricate than was originally thought.

Vallance et al. described the presence of asymmetric dimethylarginine (ADMA) as an endogenous inhibitor of eNOS in 1992. Since then, the role of this molecule in the regulation of eNOS has attracted increasing attention.
Endothelins are 21-amino acid peptides, which are active in almost all tissues in the body. They are potent vasoconstrictors, mediators of cardiac, renal, endocrine and immune functions and play a role in bronchoconstriction, neurotransmitter regulation, activation of inflammatory cells, cell proliferation and differentiation.

Endothelins were first characterised by Yanagisawa et al. (1988). The three known endothelins ET-1, -2 and -3 are structurally similar to sarafotoxins from snake venoms. ET-1 is the major isoform generated in blood vessels and appears to be the isoform of most importance in the cardiovascular system with a major role in the maintenance of vascular tone.

The systemic vascular response to hypoxia is vasodilation. However, reports suggest that the potent vasoconstrictor endothelin-1 (ET-1) is released from the vasculature during hypoxia. ET-1 is reported to augment superoxide anion generation and may counteract nitric oxide (NO) vasodilation. Moreover, ET-1 was proposed to contribute to increased vascular resistance in heart failure by increasing the production of asymmetric dimethylarginine (ADMA).

A study investigated the role of ET-1, the NO pathway, the potassium channels and radical oxygen species in hypoxia-induced vasodilation of large coronary arteries and found NO contributes to hypoxic vasodilation, probably through K channel opening, which is reversed by addition of ET-1 and enhanced by endothelin receptor antagonism. These latter findings suggest that endothelin receptor activation counteracts hypoxic vasodilation.

Endothelial dysfunction
Patients with Raynaud’s Phemonenon had abnormal vasoconstrictor responses to cold pressor tests (CPT) that were similar in primary and secondary RP. There were no differences in median flow-mediated and nitroglycerin mediated dilation or CPT of the brachial artery in the 2 populations. Patients with secondary RP were characterized by abnormalities in microvascular responses to reactive hyperemia, with a reduction in area under the curve adjusted for baseline perfusion, but not in time to peak response or peak perfusion ratio.

Plasma ET-1, ADMA, VCAM-1, and MCP-1 levels were significantly elevated in secondary RP compared with primary RP. There was a significant negative correlation between ET-1 and ADMA values and measures of microvascular perfusion but not macrovascular endothelial function. Secondary RP is characterized by elevations in plasma ET-1 and ADMA levels that may contribute to alterations in cutaneous microvascular function.

ADMA inhibits vascular NO production within the concentration range found in patients with vascular disease. ADMA also causes local vasoconstriction when infused intra-arterially, and increases systemic vascular resistance and impairs renal function when infused systemically. Several recent studies have supplied evidence to support a pathophysiological role of ADMA in the pathogenesis of vascular dysfunction and cardiovascular disease. High ADMA levels were found to be associated with carotid artery intima-media-thickness in a study with 116 clinically healthy human subjects. Taking this observation further, another study performed with hemodialysis patients reported that ADMA prospectively predicted the progression of intimal thickening during one year of follow-up.

In a nested, case-control study involving 150 middle-aged, non-smoking men, high ADMA levels were associated with a 3.9-fold elevated risk for acute coronary events. Clinical and experimental evidence suggests elevation of ADMA can cause a relative L-arginine deficiency, even in the presence of “normal” L-arginine levels. As ADMA is a competitive inhibitor of eNOS, its inhibitory action can be overcome by increasing the concentration of the substrate, L-arginine. Elevated ADMA concentration is one possible explanation for endothelial dysfunction and decreased NO production in these diseases.
Metabolic Regulation of L-arginine and NO Synthesis 
Methylation of arginine residues within proteins or polypeptides occurs through N-methyltransferases, which utilize S-adenosylmethionine as a methyl donor. After proteolysis of these proteins or polypeptides, free ADMA is present in the cytoplasm. ADMA can also be detected in circulating blood plasma. ADMA acts as an inhibitor of eNOS by competing with the substrate of this enzyme, L-arginine. The ensuing reduction in nitric oxide synthesis causes vascular endothelial dysfunction and, subsequently, atherosclerosis. ADMA is eliminated from the body via urinary excretion and via metabolism by the enzyme DDAH to citrulline and dimethylamine.
Supplementation with L-arginine in animals with experimentally-induced vascular dysfunction atherosclerosis improves endothelium-dependent vasodilation. Moreover, L-arginine supplementation results in enhanced endothelium-dependent inhibition of platelet aggregation, inhibition of monocyte adhesion, and reduced vascular smooth muscle proliferation. One mechanism that explains the occurrence of endothelial dysfunction is the presence of elevated blood levels of asymmetric dimethylarginine (ADMA) – an L-arginine analogue that inhibits NO formation and thereby can impair vascular function. Supplementation with L-arginine has been shown to restore vascular function and to improve the clinical symptoms of various diseases associated with vascular dysfunction.

Beneficial Effects of L-Arginine

  • Angina
  • Congestive Heart Failure
  • Hypertension
  • Erectile dysfunction
  • Sickle Cell Disease and Pulmonary Hypertension

The ratio of L-arginine to ADMA is considered to be the most accurate measure of eNOS substrate availability. This ratio will increase during L-arginine supplementation, regardless of initial ADMA concentration. Due to the pharmacokinetics of oral L-arginine and the positive results from preliminary studies, it appears supplementation with a sustained-release L-arginine preparation will achieve positive therapeutic results at lower dosing levels.

Many prospective clinical trials have shown that the association between elevated ADMA levels and major cardiovascular events and total mortality is robust and extends to diverse patient populations. However, we need to define more clearly in the future who will profit from ADMA determination, in order to use this novel risk marker as a more specific diagnostic tool.
Elimination of ADMA by way of DDAH
Asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA) are endogenously produced amino acids that inhibit all three isoforms of nitric oxide synthase (NOS). ADMA accumulates in various disease states, including renal failure, diabetes and pulmonary hypertension, and its concentration in plasma is strongly predictive of premature cardiovascular disease and death. Both LNMMA and ADMA are eliminated largely through active metabolism by dimethylarginine dimethylaminohydrolase (DDAH) and thus DDAH dysfunction may be a crucial unifying feature of increased cardiovascular risk. These investigators ask whether ADMA is the underlying issue related to the pathogenesis of the vascular disorder.
They identified the structure of human DDAH-1 and probed the function of DDAH-1 both by deleting the Ddah1 gene in mice and by using DDAH-specific inhibitors that is shown by crystallography, bind to the active site of human DDAH-1. The loss of DDAH-1 activity leads to accumulation of ADMA and reduction in NO signaling. This in turn causes vascular pathophysiology, including endothelial dysfunction, increased systemic vascular resistance and elevated systemic and pulmonary blood pressure. The results suggest that DDAH inhibition could be harnessed therapeutically to reduce the vascular collapse associated with sepsis.
Methylarginines are formed when arginine residues in proteins are methylated by the action of protein arginine methyltransferases (PRMTs), and free methylarginines are liberated following proteolysis. Clear demonstration of an effect of endogenous ADMA and L-NMMA on cardiovascular physiology would be of importance, not only because of the implications for disease, but also because it would expose a link between post-translational modification of proteins and signaling through a proteolytic product of these modified proteins.
Which is it? ADMA or DDHA: Intrusion of a Genetic alteration.
The study showed that loss of DDAH expression or activity causes endothelial dysfunction, we believe that DDAH inhibition could potentially be used therapeutically to limit excessive NO production, which can have pathological effects. They then showed treated cultured isolated blood vessels with lipopolysaccharide (LPS) induced expression of the inducible isoform of NO synthase (iNOS) and generated high levels of NO, which were blocked by the iNOS-selective inhibitor 1400W and by DDAH inhibitors. Treatment of isolated blood vessels with DDAH inhibitors significantly increased ADMA accumulation in the culture medium. Treatment of isolated blood vessels with bacterial LPS led to the expected hyporeactivity to the contractile effects of phenylephrine, which was reversed by treatment with a DDAH inhibitor. The effect of the DDAH inhibitor was large and stereospecific, and was reversed by the addition of L-arginine.
In conclusion, genetic and chemical-biology approaches provide compelling evidence that loss of DDAH-1 function results in increased ADMA concentrations and thereby disrupts vascular NO signaling. A broader implication of this study is that post-translational methylation of arginine residues in proteins may have downstream effects by affecting NO signaling upon hydrolysis and release of the free methylated amino acid. This signaling pathway seems to have been highly conserved through evolution.

The crucial role of nitric oxide (NO) for normal endothelial function is well known. In many conditions associated with increased risk of cardiovascular diseases such as hypercholesterolemia, hypertension, abdominal obesity, diabetes and smoking, NO biosynthesis is dysregulated, leading to endothelial dysfunction. The growing evidence from animal and human studies indicates that endogenous inhibitors of endothelial NO synthase such as asymmetric dimethylarginine (ADMA) and NG-monomethyl-L-arginine (L-NMMA) are associated with the endothelial dysfunction and potentially regulate NO synthase.

Nitric Oxide Synthase

Asymmetric dimethylarginine (ADMA) is one of three known endogenously produced circulating methylarginines (i.e. ADMA, NG-monomethyl-L-arginine (L-NMMA) and symmetrically methylated NG, NG-dimethyl-L-arginine). ADMA is formed by the action of protein arginine methyltransferases that methylate arginine residues in proteins and after which free ADMA is released. ADMA and L-NMMA can competitively inhibit NO elaboration by displacing L-arginine from NO synthase (NOS). The amount of methylarginines is related to overall metabolic activity and the protein turnover rate of cells. Although methylarginines are excreted partly by the kidneys, the major route of elimination of ADMA in humans is metabolism by the dimethylarginine dimethylaminohydrolase enzymes[ dimethylarginine dimethylaminohydrolase-1 and -2 (DDAH)] enzymes. Inhibition of DDAH leads to the accumulation of ADMA and consequently to inhibition of NO-mediated endothelium dependent relaxation of blood vessels.
The potential role of ADMA in angina pectoris has been evaluated by Piatti and co-workers, who reported ADMA levels to be higher in patients with cardiac syndrome X (angina pectoris with normal coronary arteriograms) than in controls. According to preliminary results from the CARDIAC (Coronary Artery Risk Determination investigating the influence of ADMA Concentration) study, patients with coronary heart disease (n 816) had a higher median ADMA plasma concentration than age and sex matched controls (median 0.91 vs. 0.70 mol/l; p 0.0001). Further, in a prospective Chinese study, a high plasma ADMA level independently predicted subsequent cardiovascular adverse events (cardiovascular death, myocardial infarction, and repeated revascularization of a target vessel).

Protein detoxification pathway.

Protein detoxification pathway. (Photo credit: Wikipedia)

There are only few published findings concerning variations in human DDAH. However, polymorphisms in other genes potentially related to risk factors for endothelial dysfunction and cardiovascular events have been studied. Reduced NO synthesis has been implicated in the development of atherosclerosis. For example, there are some functionally important variants of the NOS that could affect individual vulnerability to atherosclerosis by changing the amount of NO generated by the endothelium.
There are probably several functional variations in genes coding DDAH enzymes in different populations. Some of them could confer protection against the harmful effects of elevated ADMA and others impair enzyme function causing accumulation of ADMA in cytosol and/or blood.
In a study of 16 men with either low or high plasma ADMA concentrations were screened to identify DDAH polymorphisms that could potentially be associated with increased susceptibility to cardiovascular diseases. In that study a novel functional mutation of DDAH-1 was identified; the mutation carriers had a significantly elevated risk for cardiovascular disease and a tendency to develop hypertension. These results confirmed the clinical role of DDAH enzymes in ADMA metabolism. Furthermore, it is possible that more common variants of DDAH genes contribute more widely to increased cardiovascular risk.
We found a rare variation in the DDAH-1 gene, which is associated with elevated plasma concentrations of ADMA in heterozygous mutation carriers. There was also an increased prevalence of CHD and a tendency to hypertension among individuals with this DDAH-1 mutation. These observations highlight the importance of ADMA as a possible risk factor and emphasize the essential role of DDAH in regulating ADMA levels.

ADMA Elevation and Coronary Artery Disease
Endothelial dysfunction may be considered as a systemic disorder and involves different vascular beds. Coronary endothelial dysfunction (CED) precedes the development of coronary. Endothelial dysfunction is characterized by a reduction in endogenous nitric oxide (NO) activity, which may be accompanied by elevated plasma asymmetric dimethylarginine (ADMA) levels. ADMA is a novel endogenous competitive inhibitor of NO synthase (NOS), an independent marker for cardiovascular risk.

English: Structure of asymmetric dimethylargin...

English: Structure of asymmetric dimethylarginine; ADMA; N,N-Dimethylarginine Deutsch: Asymmetrisches Dimethylarginin; N,N-Dimethyl-L-arginin; Guanidin-N,N-dimethylarginin (Photo credit: Wikipedia)

In a small study fifty-six men without obstructive coronary artery disease (CAD) who underwent coronary endothelial function testing were studied. Men with CED had significant impairment of erectile function (P ¼ 0.008) and significantly higher ADMA levels (0.50+0.06 vs. 0.45+0.07 ng/mL, P ¼ 0.017) compared with men with normal endothelial function. Erectile function positively correlated with coronary endothelial function. This correlation was independent of age, body mass index, high-density lipoprotein, C-reactive protein, homeostasis model assessment of insulin resistance index, and smoking status, suggesting that CED is independently associated with ED and plasma ADMA concentration in men with early coronary atherosclerosis.

ADMA and Chronic Renal Failure in Hepatorenal Syndrome
The concentration of SDMA was significantly higher in the patients with HRS compared to the patients without HRS and it was also higher than the values obtained from the healthy participants (1.76 ± 0.3 μmol/L; 1.01 ± 0.32 and 0.520 ± 0.18 μmol/L, respectively; p < 0.01). The concentrations of ADMA were higher in the cirrhotic patients with HRS than in those without this serious complication of cirrhosis. The concentration of ADMA in all the examined cirrhotic patients was higher than those obtained from healthy volunteers (1.35 ± 0.27 μmol/L, 1.05 ± 0.35 μmol/L and 0.76 ± 0.21 μmol/L, respectively). In the patients with terminal alcoholic liver cirrhosis, the concentrations
of ADMA and SDMA correlated with the progress of cirrhosis as well as with the development of cirrhosis complications. In the patients with HRS there was a positive correlation between creatinine and SDMA in plasma (r2 = 0.0756, p < 0.001) which was not found between creatinine and ADMA. The results demonstrate that the increase in SDMA concentration is proportionate to the progression of chronic damage of the liver and kidneys. Increased ADMA concentration can be a causative agent of renal insufficiency in patients with cirrhosis.

In patients with cirrhosis, ADMA, as well as SDMA could be markers for kidney insufficiency development. Accumulation of ADMA in plasma causes kidney
vasoconstriction and thereby retention of SDMA. Considering that ADMA has several damaging effects, it can be concluded that modulation of the activity of enzyme which participates in ADMA catabolism may represent a new therapeutic goal which is intended to reduce the progress of liver and kidney damage and thus the development of HRS.

ADMA Therapeutic Targets
Elevated plasma concentrations of the endogenous nitric oxide synthase
inhibitor asymmetric dimethylarginine (ADMA) are found in various clinical settings, including

  • renal failure,
  • coronary heart disease,
  • hypertension,
  • diabetes and
  • preeclampsia.

In healthy people acute infusion of ADMA promotes vascular dysfunction,
and in mice chronic infusion of ADMA promotes progression of atherosclerosis.
Thus, ADMA may not only be a marker but also an active player in cardiovascular disease, which makes it a potential target for therapeutic interventions.

This review provides a summary and critical discussion of the presently available data concerning the effects on plasma ADMA levels of cardiovascular drugs, hypoglycemic agents, hormone replacement therapy, antioxidants, and vitamin supplementation.
We assess the evidence that the beneficial effects of drug therapies on vascular function can be attributed to modification of ADMA levels. To develop more specific ADMA-lowering therapies, mechanisms leading to elevation of plasma ADMA concentrations in cardiovascular disease need to be better understood.

ADMA is formed endogenously by degradation of proteins containing arginine residues that have been methylated by S-adenosylmethionine-dependent methyltransferases (PRMTs). There are two major routes of elimination: renal excretion and enzymatic degradation by the dimethylarginine dimethylaminohydrolases (DDAH-1 and -2).

Oxidative stress causing upregulation of PRMT expression and/or attenuation of DDAH activity has been suggested as a mechanism and possible drug target in clinical conditions associated with elevation of ADMA. As impairment of DDAH activity or capacity is associated with substantial increases in plasma ADMA concentrations, DDAH is likely to emerge as a prime target for specific therapeutic interventions.

Cardiovascular diseases (CVD) in diabetic patients have endothelial dysfunction as a key pathogenetic event. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), plays a pivotal role in endothelial dysfunction. Different natural polyphenols have been shown to preserve endothelial function and prevent CVD. Another study assessed the effect of silibinin, a widely used flavonolignan from milk thistle, on ADMA levels and endothelial dysfunction in db/db mice.

Plasma and aorta ADMA levels were higher in db/db than in control lean mice. Silibinin administration markedly decreased plasma ADMA; consistently, aorta ADMA was reduced in silibinin-treated animals. Plasma and aorta ADMA levels exhibited a positive correlation, whereas liver ADMA was inversely correlated with both plasma and aorta ADMA concentrations. Endothelium-(NO)-dependent vasodilatation to ACh was impaired in db/db mice and was restored in the silibinin group, in accordance with the observed reduction of plasma and vascular levels of ADMA. Endothelium-independent vasodilatation to SNP was not modified by silibinin administration.

Endothelin Inhibitors
Endothelins are potent vasoconstrictors and pressor peptides and are important mediators of cardiac, renal andendocrine functions. Increased ET-1 levels in disease states such as congestive heart failure, pulmonary hypertension, acute myocardial infarction, and renal failure suggest the endothelin system as an attractive target for pharmacotherapy. A non-peptidic, selective, competitive endothelin receptor antagonist with an affinity for the ETA receptor in the subnanomolar range was administered by continuous intravenous infusion to beagle dogs, rats, and Goettingen minipigs. It caused mild arteriopathy characterised by segmental degeneration in the media of mid- to large-size coronary arteries in the heart of dog, but not rat or minipig.

The lesions only occurred in the atrium and ventricle. Frequency and severity of the vascular lesions was not sex or dose related. No effects were noted in blood vessels in other organs or tissue. Plasma concentrations at steady state, and overall exposure in terms of AUC(0–24h) were higher in minipig and rat than the dog but did not cause cardiac arteriopathy. These findings concur with those caused by other endothelin anatagonists, vasodilators and positive inotropic: vasodilating drugs such as potassium channel openers, phosphodiesterase inhibitors and peripheral vasodilators.

Results by echocardiography indicate treatment-related local vasodilatation in the coronary arteries. These data suggest that the coronary arteriopathy may be the result of exaggerated pharmacology. Sustained vasodilatation in the coronary vascular bed may alter flow dynamics and lead to increased shear stress and tension on the coronary wall with subsequent microscopic trauma. In our experience with a number of endothelin receptor antagonists, the cardiac arteriopathy was only noted in studies with multiple daily or continuous intravenous infusion inviting speculation that sustained high plasma levels are needed for development of the lesions.

Up-regulation of vascular endothelin type B (ETB) receptors is implicated in the
pathogenesis of cardiovascular disease. Culture of intact arteries has been shown to induce similar receptor alterations and has therefore been suggested as a suitable method for, ex vivo, in detail delineation of the regulation of endothelin receptors. We hypothesize that mitogen-activated kinases (MAPK) and protein kinase C (PKC) are involved in the regulation of endothelin ETB receptors in human internal mammary arteries.

The endothelin-1-induced contraction (after endothelin ETB receptor desensitization) and the endothelin ETA receptor mRNA expression levels were not altered by culture. The sarafotoxin 6c contraction, endothelin ETB receptor protein and mRNA expression levels were increased. This increase was antagonized by;

PKC inhibitors (10 μM bisindolylmaleimide I and 10 μM Ro-32-0432), and
inhibitors of the p38, extracellular signal related kinases 1 and 2 (ERK1/2) and C-jun terminal kinase (JNK) MAPK pathways
Endothelin Receptor Antagonist Tezosentan
The effects of changes in the mean (Sm) and pulsatile (Sp) components of arterial wall shear stress on arterial dilatation of the iliac artery of the anaesthetized dog were examined in the absence and presence of the endothelin receptor antagonist tezosentan (10 mg kg_1 I.V.; Ro 61-0612; [5-isopropylpyridine-2-sulphonic acid 6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2-(2-1H-tetrazol-5-ylpyridin-4-yl)-pyrimidin-4-ylamide]).

Changes in shear stress were brought about by varying local peripheral resistance and stroke volume using a distal infusion of acetylcholine and stimulation of the left ansa subclavia. An increase in Sm from 1.81 ± 0.3 to 7.29 ± 0.7 N m_2 (means ± S.E.M.) before tezosentan caused an endothelium-dependent arterial dilatation which was unaffected by administration of tezosentan for a similar increase in Sm from 1.34 ± 0.6 to 5.76 ± 1.4 N m_2 (means ± S.E.M.).

In contrast, increasing the Sp from 7.1 ± 0.8 to a maximum of 11.5 ± 1.1 N m_2 (means ± S.E.M.) before tezosentan reduced arterial diameter significantly. Importantly, after administration of tezosentan subsequent increases in Sp caused arterial dilatation for the same increase in Sp achieved prior to tezosentan, increasing from a baseline of 4.23 ± 0.4 to a maximum of 9.03 ± 0.9 N m_2 (means ± S.E.M.; P < 0.001). The results of this study provide the first in vivo evidence that pulsatile shear stress is a stimulus for the release of endothelin from the vascular endothelium.

Exercise and Diet
Vascular endotheliumis affected by plasma asymmetric dimethylarginine (ADMA), and it is induced by inflammatory cytokines of tumour necrosis factor (TNF)-a in vitro. Would a tight glycemic control restore endothelial function in patients with type-2 diabetes mellitus (DM) with modulation of TNF-a and/or reduction of ADMA level? In 24 patients with type-2 DM, the flow-mediated, endothelium-dependent dilation (FMD: %) of brachial arteries during reactive hyperaemia was determined by a high-resolution ultrasound method. Blood samples for glucose, cholesterol, TNF-a, and ADMA analyses were also collected from these patients after fasting. No significant glycemic or FMD changes were observed in 10 patients receiving the conventional therapy.

In 14 patients who were hospitalized and intensively treated, there was a significant decrease in glucose level after the treatment [from 190+55 to 117+21 (mean+SD) mg/dL, P , 0.01]. After the intensive control of glucose level, FMD increased significantly (from 2.5+0.9 to 7.2+3.0%), accompanied by a significant (P , 0.01) decrease in TNF-a (from 29+16 to 11+9 pg/dL) and ADMA (from 4.8+1.5 to 3.5+1.1 mM/L) levels. The changes in FMD after treatment correlated inversely with those in TNF-a (R ¼ 20.711, P , 0.01) and ADMA (R ¼ 20.717, P , 0.01) levels.
The exaggerated blood pressure response to exercise (EBPR) is an independent predictor of hypertension. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide inhibitor and higher plasma levels of ADMA are related to increased cardiovascular risk. The aim of this study is to identify the relationship between ADMA and EBPR.

A total of 66 patients (36 with EBPR and 30 as controls) were enrolled in the study. EBPR is defined as blood pressure (BP) measurements ≥200/100 mmHg during the treadmill test. All the subjects underwent 24-h ambulatory BP monitoring. L-arginine and ADMA levels were measured using a high performance lipid chromatography technique.

The serum ADMA levels were increased in the EBPR group compared to the healthy controls (4.0±1.4 vs 2.6±1.1 μmol/L respectively, P=0.001), but L-arginine levels were similar in the 2 groups (P=0.19). The serum ADMA levels were detected as an independent predictor of EBPR (odds ratio 2.28; 95% confidence interval 1.22–4.24; P=0.002). Serum ADMA levels might play a role in EBPR to exercise.

Endothelial dysfunction occurs early in atherosclerosis in response to cardiovascular risk factors. The occurrence of endothelial dysfunction is primarily the result of reduced nitric oxide (NO) bioavailabilty. It represents an independent predictor of cardiovascular events and predicts the prognosis of the patient. Therefore, endothelial function has been identified as a target for therapeutic intervention. Regular exercise training is a nonpharmacological option to improve endothelial dysfunction in patients with cardiovascular disease by increasing NO bioavailability.

Peripheral Arterial Disease (PAD) is a cause of significant morbidity and mortality in the Western world. risk factor modification and endovascular and surgical revascularisation are the main treatment options at present. However, a significant number of patients still require major amputation. There is evidence that nitric oxide (NO) and its endogenous inhibitor asymmetric dimethylarginine (ADMA) play significant roles in the pathophysiology of PAD.

This paper reviews experimental work implicating the ADMA-DDAH-NO pathway in PAD, focusing on both the vascular dysfunction and both the vascular dysfunction and effects within the ischaemic muscle, and examines the potential of manipulating this pathway as a novel adjunct therapy in PAD.

In patients with CHF, the peripheral vascular resistance is increased via activation of the neurohormonal system, namely by autonomous sympathetic nervous system, rennin -angiotensin- aldosterone system (RAAS), and endothelin system. The vascular endothelial function in patients with CHF, mainly represented by the endothelium-dependent vasodilation, is altered.

Such alteration leads to increased vascular tone and remodeling of the blood vessels, reducing the peripheral blood flow. Hence, the amount of oxygen for the skeletal muscles is compromised, with progressive exercise intolerance. The vascular endothelial dysfunction in the CHF is mainly due to the decrease of the nitric oxide production induced by the reduced gene expression of eNOS and increased oxidative stress.

The endothelium-dependent vasodilation alteration has been virtually reported in all cardiovascular diseases. Using sauna bath as therapeutic option for CHF is not very recent, since in the 1950’s the first studies with CHF patients were conducted and the potential beneficial effect of sauna was suggested. However, some time later the studies emphasized especially its risks and recommended caution in its use for cardiac patients.

Frequently, sports medicine physicians are invited to evaluate the impact of the sauna on diseases and on health in general. Sauna can be beneficial or dangerous depending on its use. In the past few years the sauna is considered beneficial for the cardiovascular diseases’ patients, as the heart failure and lifestyle-related diseases, mainly by improving the peripheral endothelial function through the increase in cardiac output and peripheral vasodilation.

It is widely known that the vasodilators, such as angiotensin converting enzyme inhibitors, improve the CHF and increase the peripheral perfusion. Since the endothelial function is altered in CHF, the endothelium is considered as a new therapeutic target in heart failure. Hence, the angiotensin converting enzyme inhibitors and physical training improve the endothelial function in CHF patients. One of the proposed mechanisms for the alteration of the endothelium-dependent vasodilation would be through the decrease of the NO production in the peripheral vessels in CHF patients. The decrease of peripheral perfusion would decrease the shear stress. The shear stress is an important stimulus for NO production and eNOS expression. On the other hand, the heat increases the cardiac output and improves the peripheral perfusion in CHF patients. Consequently, with the cardiac output improvement in CHF patients, an increase of the shear stress, NO production and eNOS expression are expected.

Sauna bath
The sauna bath represents a heat load of 300-600 W/m2 of body surface area. The skin temperature rapidly increases to ± 40o-41oC and the thermoregulatory mechanisms are triggered. Evaporative heat transfer by sweating is the only effective body heat loss channel in dry sauna. The sweating begins rapidly and reaches its maximum level in ± 15 min. The total sweat secretion represents a heat loss of about 200 W/m2 of the body surface area. The body cannot compensate for the heat load and causing elevation of internal temperature. The skin circulation increases substantially. The skin blood flow, in the thermo-neutral condition (± 20oC) and in rest corresponding to ± 5-10% of the cardiac output, can reach ± 50-70% of the cardiac output.

Thermal therapy in 60oC produced systemic arterial, pulmonary arterial and venous vasodilation, reduced the preload and afterload and improved the cardiac output and the peripheral perfusion, clinical symptoms, life quality, and cardiac arrhythmias in CHF patients. In infants with severe CHF secondary to ventricular septal defect, the sauna therapy decreased the systemic vascular resistance and increased the cardiac output. The sauna benefits in CHF patients are possibly caused by the improvement of the vascular endothelial function and normalization of the neurohormonal system .

Ikeda et al. discovered that the observed improvements in the sauna therapy are due to the eNOS expression increase in the arterial endothelium. They later showed that the thermal therapy with sauna improves the survival of the TO-2 cardiomyopathic hamsters with CHF and, more recently, showed that the repetitive therapy with sauna increases the eNOS expression and the nitric oxide production in artery endothelium of TO-2 cardiomyopathic hamsters with CHF.
Whether n-3 polyunsaturated fatty acid (PUFA) supplementation and/or diet intervention might have beneficial influence on endothelial function was assessed using plasma levels of ADMA and L-arginine. A male population (n = 563, age 70 ± 6 yrs) with long-standing hyperlipidemia, characterized as high risk individuals in 1970–72, was included, randomly allocated to receive placebo n-3 PUFA capsules (corn oil) and no dietary advice (control group), dietary advice (Mediterranean type), n-3 PUFA capsules, or dietary advice and n-3 PUFA combined and followed for 3 years. Fasting blood samples were drawn at baseline and the end of the study.

Compliance with both intervention regimens were demonstrated by changes in serum fatty acids and by recordings from a food frequency questionnaire. No influence of either regimens on ADMA levels were obtained. However, n-3 PUFA supplementation was accompanied by a significant increase in L-arginine levels, different from the decrease observed in the placebo group (p < 0.05). In individuals with low body mass index (<26 kg/m2), the decrease in L-arginine on placebo was strengthened (p = 0.01), and the L-arginine/ADMA ratio was also significantly reduced (p = 0.04). In this rather large randomized intervention study, ADMA levels were not influenced by n-3 PUFA supplementation or dietary counselling. n-3 PUFA did, however, counteract the age related reduction in L-arginine seen on placebo, especially in lean individuals, which might be considered as an improvement of endothelial function.

Traditional Chinese Medicine

Traditional Chinese Medicine (TCM) involves a broad range of empirical testing and refinement and plays an important role in the health maintenance for people all over the world. However, due to the complexity of Chinese herbs, a full understanding of TCM’s action mechanisms is still unavailable despite plenty of successful applications of TCM in the treatment of various diseases, including especially cardiovascular diseases (CVD), one of the leading causes of death.

An integrated system of TCM has been constructed to uncover the underlying action mechanisms of TCM by incorporating the chemical predictors, target predictors and network construction approaches from three representative Chinese herbs, i.e., Ligusticum chuanxiong Hort., Dalbergia odorifera T. Chen and Corydalis yanhusuo WT Wang widely used in CVD treatment, by combined use of drug absorption, distribution, metabolism and excretion (ADME) screening and network pharmacology techniques. These studies have generated 64 bioactive ingredients and identified 54 protein targets closely associated with CVD, to clarify some of the common conceptions in TCM, and provide clues to modernize such specific herbal medicines.

Ligusticum chuanxiong Hort., Dalbergia odorifera T. Chen and Corydalis yanhusuo WT Wang
Twenty-two of 194 ingredients in Ligusticum chuanxiong demonstrate good bioavailability (60%) after oral administration. Interestingly, as the most abundant bioactive compound of Chuanxiong, Ligustilide (M120) only has an adequate OB of 50.10%, although it significantly inhibits the vasoconstrictions induced by norepinephrine bitartrate (NE) and calcium chloride (CaCl2). Indeed, this compound can be metabolized to butylidenephthalide, senkyunolide I (M156), and senkyunolide H (M155) in vivo.

The three natural ingredients produce various pharmacological activities in cerebral blood vessels, the general circulatory system and immune system including spasmolysis contraction effects, inhibitory effects of platelet aggregation and anti-proliferative activity, and thus improve the therapeutic effect on patients. Cnidilide (M93, OB = 77.55%) and spathulenol (M169, OB = 82.37%) also closely correlate with the smooth muscle relaxant action, and thereby have the strongest spasmolytic activity. Carotol (M8) and Ferulic acid (M105) with an OB of 149.03% and 86.56%, respectively, demonstrate better bioavailability compared with cnidilide and spathulenol, which show strong antifungal, antioxidant and anti-inflammatory activity.

The pharmacological activity of ferulic acid results in the improvement of blood fluidity and the inhibition of platelet aggregation, which may offer beneficial effects against cancer, CVD, diabetes and Alzheimer’s disease. As for 3-n-butylphthalide (M85, OB = 71.28%), this compound is not only able to inhibit platelet aggregation, but also decreases the brain infarct volume and enhances microcirculation, thus benefiting patients with ischemic stroke. Platelet aggregation represents a multistep adhesion process involving distinct receptors and adhesive ligands, with the contribution of individual receptor-ligand interactions to the aggregation process depending on the prevailing blood flow conditions, implying that the rheological (blood flow) conditions are an important impact factor for platelet aggregation. Moreover, thrombosis, the pathological formation of platelet aggregates and one of the biggest risk factors for CVD, occludes blood flow causing stroke and heart attack. This explains why the traditional Chinese herb Ligusticum chuanxiong that inhibits platelet aggregates forming and promotes blood circulation can be used in treatment of CVD.

Twenty-six percent (24 of 93) of the ingredients in Dalbergia odorifera meet the OB > 60% criterion irrespective of the pharmacological activity. Relatively high bioavailability values were predicted for the mainly basic compounds odoriflavene (M275, OB = 84.49%), dalbergin (M247, OB = 78.57%), sativanone (M281, OB = 73.01%), liquiritigenin (M262, OB = 67.19%), isoliquiritigenin (M259, OB = 61.38%) and butein (M241, OB = 78.38%). Interestingly, all of the six ingredients show obvious anti-inflammatory property. Butein, liquiritigenin and isoliquiritigenin inhibit cell inflammatory responses by suppressing the NF-κB activation induced by various inflammatory agents and carcinogens, and by decreasing the NF-κB reporter activity. Inflammation occurs with CVD, and Dalbergia odorifera, one of the most potent anti-cardiovascular and anti-cerebrovascular agents, exerts great anti-inflammatory activity.

Corydalis yanhusuo has gained ever-increasing popularity in today’s world because of its therapeutic effects for the treatment of cardiac arrhythmia disease, gastric and duodenal ulcer and menorrhalgia. In our work, 21% (15 of 73) of chemicals in this Chinese herb display good OB (60% or even high), and the four main effective ingredients are natural alkaloid agents.

Dehydrocorydaline blocks the release of noradrenaline from the adrenergic nerve terminals in both the Taenia caecum and pulmonary artery, and thereby inhibits the relaxation or contraction of adrenergic neurons. As for dehydrocavidine with an OB of 47.59%, this alkaloid exhibits a significant spasmolytic effect, which acts via relaxing smooth muscle.

In recent years, CVD has been at the top list of the most serious health problems. Many different types of therapeutic targets have already been identified for the management and prevention of CVD, such as endothelin and others. The key question asked is

  • what the interactions of the active ingredients of the Chinese herbs are with their protein targets in a systematic manner and
  • how do the corresponding targets change under differential perturbation of the chemicals?

The study used an unbiased approach to probe the proteins that bind to the small molecules of interest in CVD on the basis of the Random Forest (RF) and Support Vector Machine (SVM) methods combining the chemical, genomic and pharmacological information for drug targeting and discovery on a large scale. Applied to 64 ingredients derived from the three traditional Chinese medicines Dalbergia odorifera, Ligusticum chuanxiong and Corydalis yanhusuo, which show good OB, 261 ligand-target interactions have been constructed, 221 of which are enzymes, receptors, and ion channels. This indicates that chemicals with multiple relative targets are responsible for the high interconnectedness of the ligand-target interactions. The promiscuity of drugs has restrained the advance in recent TCM, because they were thought to be undesirable in favor of more target-specific drugs.

Target Identification and Validation
To validate the reliability of these target proteins, the researchers performed a docking analysis to select the ligand-protein interactions with a binding free energies of ≤−5.0 kcal/mol, which leads to the sharp reduction of the interaction number from 5982 to 760. These drug target candidates were subsequently subject to PharmGkb (available online: http://www.pharmgkb.org; accessed on 1 December 2011), a comprehensive disease-target database, to investigate whether they were related to CVD or not, and finally, 54 proteins were collected and retained.

Fourty-two proteins (76%) were identified as the targets of Ligusticum chuanxiong, such as dihydrofolate reductase (P150), an androgen receptor (P210) and angiotensin-converting enzyme (P209) that were involved in the development of CVD. Of the proteins, seven and two were recognized as those of Dalbergia odorifera and Corydalis yanhusuo, respectively. For Dalbergia odorifera, this Chinese herb has 48 potential protein targets, 13 of which have at least one link to other drugs.

The three herbs share 29 common targets, accounting for 52.7% of the total number. Indeed, as one of the most important doctrines of TCM
abstracted from direct experience and perception, “multiple herbal drugs for one disease” has played an undeniable role. These studies explored the targets of the three Chinese herbs, indicating that these drugs target the same targets simultaneously and exhibit similar pharmacological effects on CVD. This is consistent with the theory of “multiple herbal drugs for one disease”.

The three Chinese herbs possess specific targets. The therapeutic efficacy of a TCM depends on multiple components, targets and pathways. The complexity becomes a huge obstacle for the development and innovation of TCM. For example, the Chinese herb Ligusticum chuanxiong identifies the protein caspase-3 (P184), a cysteinyl aspartate-specific protease, as one of its specific targets, and exhibits inhibitory effects on the activity of this protease. In fact, connective tissue growth factor enables the activation of caspase-3 to induce apoptosis in human aortic vascular smooth muscle cells.

Thus, modulation of the activity of caspase-3 with Ligusticum chuanxiong suggests an efficient therapeutic approach to CVD. The Chinese herb Dalbergia odorifera has the α-2A adrenergic receptor (P216) as its specific target and probably blocks the release of this receptor, and thus influences its action. As for Corydalisyanhusuo, the protein tyrosine-protein kinase JAK2 (P9) is the only specific target of this Chinese herb. The results indicate different specific targets possessed by the three Chinese herbs.

Ligand-Candidate Target and Ligand-Potential Target Networks
Previous studies have already reported the relationships of the small molecules with CVD, which indicates the reliability of our results [45,46]. Regarding the candidate targets, we have found that prostaglandin G/H synthase 2 (P46) and prostaglandin G/H synthase 1 (P47) possess the largest number of connected ingredients. Following are nitric-oxide synthase, endothelial (P66) and tyrosine-protein phosphatase non-receptor type 1 (P8), which have 62 and 61 linked chemicals, respectively.
The 29 targets shared by the three traditional Chinese herbs exhibit a high degree of correlations with CVD, which further verifies their effectiveness for the treatment of CVD. These results provide a clear view of the relationships of the target proteins with CVD and other related diseases, which actually link the Chinese herbs and the diseases via the protein targets. This result further explains the theory of “multiple herbal drugs for one disease” based on molecular pharmacology.

Target-Pathway Network
Cells communicate with each other using a “language” of chemical signals. The cell grows, divides,or dies according to the signals it receives. Signals are generally transferred from the outside of the cell. Specialized proteins are used to pass the signal—a process known as signal transduction. Cells have a number of overlapping pathways to transmit signals to multiple targets. Ligand binding in many of the signaling proteins in the pathway can change the cellular communication and finally affect cell growth and proliferation. The authors extracted nine signal pathways closely associated with CVD in PharmGkb (available online: http://www.pharmgkb.org; accessed on 1 December 2011).

As the main components in the VEGF system, proto-oncogene tyrosine-protein kinase Src, eNOS, and hsp90-α is also recognized as common targets of Dalbergia odorifera, Ligusticum chuanxiong and Corydalis yanhusuo, which are efficient for the treatment of CVD. This implies that the candidate drugs can target different target proteins involved in the same or different signal pathways, and thereby have potential effects on the whole signal system.

Target Prediction
In search of the candidate targets, the model that efficiently integrates the chemical, genomic and pharmacological information for drug targeting and discovery on a large scale is based on the two powerful methods Random Forest (RF) and Support Vector Machine (SVM). The model is supported by a large pharmacological database of 6511 drugs and 3999 targets extracted from the DrugBank database (available online: http://drugbank.ca/; accessed on 1 June 2011), and shows an impressive performance of prediction for drug-target interaction, with a concordance of 85.83%, a sensitivity of 79.62% and a specificity of 92.76%. the candidate targets were selected according to the criteria that the possibility of interacting with potential candidate targets was higher than 0.6 for the RF model and 0.7 for the SVM model. The obtained candidate targets were finally reserved and were further predicted for their targets.

Target Validation
Molecular docking analysis was carried out using the AutoDock software (available online: http://autodock.scripps.edu/; accessed on 1 February 2012). This approach performs the docking of the small, flexible ligand to a set of grids describing the target protein. During the docking process, the protein was considered as rigid and the molecules as flexible. The crystal structures of the candidate targets were downloaded from the RCSB Protein Data Bank (available online: http://www.pdb.org/; accessed on 1 December 2011), and the proteins without crystal structures were performed based on homology modeling using the Swiss-Model Automated Protein Modelling Server (available online: http://swissmodel.expasy.org/; accessed on 1 February 2012).

TCM is a heritage that is thousands of years old and is still used by millions of people all over the world—even after the development of modern scientific medicine. Chinese herbal combinations generally include one or more plants and even animal products.

The study identified 54 protein targets, which are closely associated with CVD for the three Chinese herbs, of which 29 are common targets (52.7%), which clarifies the mechanism of efficiency of the herbs for the treatment of CVD.

Activation of NFkB

Extracellular stimuli for NFkB activation and NFkB regulated genes
Extracellular stimuli                       Regulated genes
TNFa                                         Growth factors (G/M-CSF)
Interleukin 1                            G/M CSF, M CSF, G CSF
ROS                                              Cell adhesion molecules
UV light                            ICAM-1, VCAM, E-Selectin, P-selectin
Ischaemia                                   Cytokines
Lipopolysaccharide               TNFa, IL-1, IL-2, IL-6, interferon
Bacteria                                        Transcription regulators
Viruses                                         P53, IkB, c-rel, c-myc
Amyloid                                      Antiapoptotic proteins
Glutamate                              TRAF-1, TRAF-2, c-IAP1, c-IAP2
Pathophysiology
Reactive oxygen species (ROS) are toxic and in conditions of a dysbalance between their overproduction and the diminished activity of various antioxidant enzymes and other molecules induce cellular injury termed oxidative stress. ROS are often related to a number of diseases like atherosclerosis. However, the mechanism is not clear at all. Latest years of research have brought the idea of connection between ROS and NFkB. And indeed, in vitro studies showed a rapid activation of NFkB after exposure of certain cell types to ROS. Today, no specific receptor for ROS has been found, thus, the details of the ROS induced activation of NFkB are missing.

Natural occurring agents which actions are still a matter of debate in the theory and nouvelle small molecular derivates activate or inhibit the transcriptional factor. Synthetic oligo and polypeptide inhibitors of NFkB can penetrate the cell membrane and directly act on the Rel proteins. The most sophisticated approaches towards inhibiting the activation and translocation of NFkB into the nucleus represent gene deliveries, using plasmids or adenoviruses containing genes for various super repressors—modified IkB proteins, or so called NFkB decoys, which interact with activated NFkB and thus, inhibit the interaction between the transcription factor and nuclear DNA enhancers.

A simplified scheme of the activation of NFkB by the degradation of IkB. IkB is phosphorylated by IKK and ubiquinatated by the ubiquitine ligase system (ULS). IkB is further degradated by the 26S proteasome (26S).Activated NFkB can pass the nuclear membrane and interact with kB binding sequences in enhancers of NFkB regulated genes. LPS, lipopolysaccharide; ROS, reactive oxygen species; FasL, Fas ligand; TRAF, TNFa receptor associated factor; NIK, NFkB inducing kinase; MEKK, mitogen activated protein kinase/extracellular signal regulated kinases kinases.

The medicine of this century is a medicine of molecules, the diagnostic procedure and the therapy moves further from the “clinical picture” to the use of achievements in molecular biology and genetics. However, sober scepticism and awareness are indicated. Especially the role of NFkB in multiple signal transducing pathways and the tissue dependent variability of responses to alternations in NFkB pathway may be the reasons for unwanted side effects of the therapy that are after in vitro or in vivo experiments hardly to expect in the clinical use.

Therapeutic Targets
Modern drug discovery is primarily based on the search and subsequent testing of drug candidates acting on a preselected therapeutic target. Progress in genomics, protein structure, proteomics, and disease mechanisms has led to a growing interest in an effort for finding new targets and more effective exploration of existing targets. The number of reported targets of marketed and investigational drugs has significantly increased in the past 8 years. There are 1535 targets collected in the therapeutic target database.
Knowledge of these targets is helpful for molecular dissection of the mechanism of action of drugs and for predicting features that guide new drug design and the
search for new targets. This article summarizes the progress of target exploration and investigates the characteristics of the currently explored targets to analyze their sequence, structure, family representation, pathway association, tissue distribution, and genome location features for finding clues useful for searching for new targets. Possible “rules” to guide the search for druggable proteins and the feasibility of using a statistical learning method for predicting druggable proteins directly from their sequences are discussed.

Current Trends in Exploration of Therapeutic Targets
There are 395 identifiable targets described in 1606 patents. Of these targets, 264 have been found in more than one patent and 50 appear in more than 10 patents. The number of patents associated with a target can be considered to partly correlate with the level of effort and intensity of interest currently being directed to it. Approximately one third of the patents with an identifiable target were approved in the past year. This suggests that the effort for the exploration of these targets is ongoing, and there has been steady progress in the discovery of new investigational agents directed to these targets.

Various degrees of progress have been made toward discovery and testing of agents directed at these targets. However, for some of these targets, many difficulties remain to be resolved before viable drugs can be derived. The appearance of a high number of patents associated with these targets partly reflects the intensity of efforts for finding effective drug candidates against these targets.

There are 62 targets being explored for the design of subtype-specific drugs, which represents 15.7% of the 395 identifiable targets in U.S. patents approved in 2000 through 2004. Compared with the 11 targets of FDA approved subtype-specific drugs during the same period, a significantly larger number of targets are being explored for the design of subtype-specific drugs.

What Constitutes a Therapeutic Target?
The majority of clinical drugs achieve their effect by binding to a cavity and regulating the activity, of its protein target. Specific structural and physicochemical properties, such as the “rule of five” (Lipinski et al., 2001), are required for these drugs to have sufficient levels of efficacy, bioavailability, and safety, which define target sites to which drug-like molecules can bind. In most cases, these sites exist out of functional necessity, and their structural architectures accommodate target-specific drugs that minimally interact with other functionally important but structurally similar sites.
These constraints limit the types of proteins that can be bound by drug-like molecules, leading to the introduction of the concept of druggable proteins (Hopkins and Groom, 2002; Hardy and Peet, 2004). Druggable proteins do not necessarily become therapeutic targets (Hopkins and Groom, 2002); only those that play key roles in diseases can be explored as potential targets.

 Prediction of Druggable Proteins by a Statistical Learning Method

Currently, the support vector machine (SVM) method seems to be the most accurate statistical learning method for protein predictions. SVM is based on the structural risk minimization principle from statistical learning theory. Known proteins are divided into druggable and nondruggable classes; each of these proteins is represented by their sequence-derived physicochemical features.

These features are then used by the SVM to construct a hyperplane in a higher dimensional hyperspace that maximally separates druggable proteins and nondruggable ones. By projecting the sequence of a new protein onto this hyperspace, it can be determined whether this protein is druggable from its location with respect to the hyperplane. It is a druggable protein if it is located on the side of druggable class.
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