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Positioning a Therapeutic Concept for Endogenous Augmentation of cEPCs — Therapeutic Indications for Macrovascular Disease: Coronary, Cerebrovascular and Peripheral

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

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

• Introduction
• Biomarker Discovery – a comprehensive Post on this topic is forthcoming
• What are our Contributions in the Domain of Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk
• Postulates of Multiple Indications for the Method Presented: Positioning of a Therapeutic Concept for Endogenous Augmentation of cEPCs — Potential Therapeutic Indications for ElectEagle
• A Three Component Method for Endogenous Augmentation of cEPCs — Macrovascular Diseases – Therapeutic Potential of cEPCs
• The Promise of the Proposed Pharmacotherapy as a Method of CVD Risk Reduction
• Emergence of Clinical Trial Results on Genous R stent — Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth – (HEALING II)
• Conclusions
• References

Key words: coronary artery disease, circulating Endothelial Progenitor Cells (cEPCs), Endothelial Progenitor Cells (EPC), genetic engineering, CVD, CAD, CHF, myocardial infarction, neovascularization, vascular repair, “multimarker biomarker”, macrovascular disease, Endogenous Augmentation of cEPCs, Primary Endpoint, Secondary Endpoint.

Abbreviations used: ED, endothelial dysfunction; CAD, coronary artery disease; CVD, cardiovascular disease; cEPCs, circulating Endothelial Progenitor Cells; EPC, Endothelial Progenitor Cells; CHF, congestive heart failure; MI, myocardial infarction; MNC, mononuclear cells; VEGF, vascular endothelial growth factor; BM–MNCs, bone marrow-derived mononuclear cells; G-CSF, granulocyte colony-stimulating factor; SDF, stromal derived factor; PB-MNCs, peripheral blood-mononuclear cells; EF, ejection fraction; PO₂, partial pressure of oxygen; BMS, bare-metal stent; CABG, coronary artery bypass graft; DES, drug-eluting stent; GP, glycoprotein; LAD, left anterior descending; LCx, left circumflex; MI, myocardial infarction; RCA, right coronary artery; S/P , status-post stent implantation; MACE, Major Adverse Cardiac Events; TLR, target lesion revascularization; TVR, target vessel revascularization; TVF, target lesion vessel failure; eNOS, endothelial Nitric Oxide Synthase 

Introduction

Cardinal to the study of reendothelialization and neovascularization is the mechanism of action (MOA) of EPCs. It requires exact biological phenotype of the true EPC and its MOA on the endothelium. Is the EPC autocrine or paracrine in its functional role? It is critical to understand this biological unknown for planning therapeutic approaches. Patients with unstable angina and no evidence of cardiac necrosis exhibited increased cEPCs. Systemic inflammation and recognized growth factors may play a role in peripheral mobilization of EPCs in patients with unstable anginal syndromes. Proportion of cEPCs in coronary ischemia, acute or chronic and its potential for restoring left ventricular dysfunction is still experimental. EC injury facilitates an accelerated development of atherosclerotic plaque which triggers cardiovascular risk factors where the magnitude of the endothelial dysfunction predicts the level of risk for a macrovascular event (George, 2004).

Diminished level of cEPCs is associated with risk factors for CVD implicating impaired endothelial repair as a contributor to a dynamic state of endothelial dysfunction. cEPCs is further reduced if multiple risk factors for CVD are present. Endothelial dysfunction is associated with cEPCs counts. It is only if cEPCs counts are low then endothelial dysfunction (ED) emerges. In the case of ED, the cells were more senescent compared with an age group without CVD and the risk factors involved with it. Impaired repair capacity due to reduced availability of cEPCs enhances the exposure to risk factors when injury occurs due to endothelial denudation, ischemic tissue, neointima build up and remodeling.

Mobilization and EPC-mediated neovascularization is critically regulated. Statins and physical exercise are stimulatory while risk factors for CAD are inhibitory in the modulation function of the level of cEPCs. Recruitment of cEPCs requires a coordinated sequence of adhesive and signaling events including adhesion and migration by integrins, chemoattraction of SDF-1/CXCR4 and differentiation of EC.

Bone-marrow derived cells in the circulating blood have an endothelial phenotype and peripheral blood can be cultured to generate ECs. cEPCs provide both diagnostic and prognostic information on CVD. EPCs are analyzed by their phenotypic markers, as discerned by fluorescence-activated cell sorting (FACS) analysis as well as by their functional capability to produce colonies in culture conditions.

Kiernan (2006) identifies the two classes of therapeutic applications of cEPCs: (a) induction of angiogenesis and (b) large vessel repair. Transplantation of autologous EPCs over-expressing eNOS in injured vessels enhances the vasculoprotective properties of the reconstituted endothelium, leading to inhibition of neointimal hyperplasia. This cell-based gene therapy strategy may be useful in treatment of vascular disease. Stents coated in CD34 antibody which binds to the CD34 antigen of cEPCs have the capability to promote re-endothelialisation in minutes to hours. This mechanism seeks to restore the normal biology of the vessel wall rather than perpetuate the wall disruption as drug eluting stents are found recently to be implicated to cause both restenosis and thrombosis (Tung et al., 2006). Thus, cEPCs are of cardinal importance in healing cardiovascular injury. Identification of augmentation methods which are endogenous in nature, are systemic rather than local, as cell-based therapy is, and therefore, it will deliver systemic protective measures against atherosclerosis delaying angioplasty and potentially avoiding cell implantation or vascular engrafting.

Biomarker Discovery – a comprehensive Post on this topic is forthcoming

A comprehensive review of “Traditional” vs. “Novel” risk markers for cardiovascular disease was recently undertaken by Folsom et al., (2006) and the Editorial to this article by Lloyd-Jones and Tian (2006). Among the “Traditional” Risk Markers, they list: Age, Race, Sex, Total/HDL levels, Smoking Status, Diabetes, Systolic BP and Use of antihypertensive  drugs. The list of “Novel” Risk Markers is impressively longer and includes: CRP, Lp-PLA2, E-Selectin, Fibrinogen, PAI-1, Vitamin B6, D-dimer, ICAM-1, Homocysteine, IL-6, HSV-1 Antibody, CMV Antibody and Folate.

Only two risk factors make the top five list following the data adjustment to Age and /or All the Traditional Risk Factors, respectively, I would conclude that only the following two are of paramount importance for clinical application and drug therapy design.

• Tissue inhibitor of Metalloproteinase1
• Intracellular adhesion molecule

Risk Factor RANKING	Risk Factor RANKING if Data Adjusted to AGE	Risk Factor RANKING if Data Adjusted to All “Traditional” Risk Factors
1	Chlamydia	Intracellular adhesion molecule
2	Lp-PLA2 lipoprotein-associatedphospholipase A2	Cytomegalovirus
3	Tisshe Plasminogen Activator	D-Dimer
4	Tissue inhibitor of Metalloproteinase1	IL-6
5	Intracellular adhesion molecule	Tissue inhibitor of Metalloproteinase1

In light of these results, chiefly edified by Folsom et al., (2006)  conclusion that: “Based on the totality of evidence, however, CRP level does not emerge as a clinically useful addition to basic risk factor assessment for identifying patients at risk of a first CHD event.” (Folsom, 2006, 1372).

What are our Contributions in the Domain of

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

(a) This is the first paper to look at cEPCs from two academic schools of thought.  One, represented by the review article of Dzau et al., Hypertension, 2005 with 122 references which treats cEPCs from two perspectives: Vascular Biology and Molecular Cardiology. The other, is the review article by Lapidot & Petit, Experimental Hematology, 2002 with 86 references which treats cEPCs as stem cells and covers the research in Immunology and in Hematology, cEPCs is circulating in our blood, it is a stem cell! The overlap between the references N=122 in Dzau and N=86 in Lapidot & Petit is zero. These two schools do not cite the findings of the other school. That happens when both schools (Vascular Biology/Molecular Cardiology) and (Immunology/Hematology), BOTH schools are researching the same biologic phenomenon, i.e., one circulating EPC. We are the first to put together in one paper the two schools in the context of cEPCs. The pathophysiology of cECs, cEPCs and Trans-Endothelium Cell Migration in one location.

(b) Table of content of Part I yielded a theoretical treatment of cEPCs not in existence anywhere.  We defined for the first time that the Clinical Frontier for cEPCs is of quadruple nature: (Vascular Biology/Molecular Cardiology) PLUS (Immunology/Hematology). We made the statement that the Clinical Frontier has 20 Future Fast Acting Therapy modality currently under research. We cited the limitation of exogenous methods for augmentation of cEPCs as a scientifically derived justification for our selection of an endogenous augmentation method.

Upon selection of the endogenous method, we specified three components:

–   inhibition of ET-1

–   induction of eNOS

–   stimulation of PPAR-gamma

The proposed combination drug therapy yielded a new multimarker biomarker for reduction of CVD risk for macrovascular events, called the ElectEagle Version I. We specified the potential indications for the ElectEagle Version I method in terms of cardiovascular disease and co-morbidity with other endothelial dysfunction derived disease.

Method name:            ElectEagle

E.L.E.C.T.

E – Efficient

L – Ligands of cEPCs

E – Elective and Individualized Diagnosis and Therapy

C – Cardiovascular diseases & secondary sequalea

T – Treatment adjustable by three agents

E.A.G.L.E.

E – Endogenous

A – Augmentation

G – Gamma-PPAReceptor

L – Ligand occupied ET_A and ET_A-ET_B – binding Nitric Oxide

E – EPCs fast generator

ElectEaglestands for an Efficient Ligands of cEPCs Elective and Individualized Diagnosis and Therapy for Cardiovascular diseases & secondary vascular sequalea, using Treatment adjustable by three agents. It is a method for Endogenous Augmentation of circulating EPCs by using Gamma-PPAR agonists, inhibitors of Ligand occupied ET_A and ET_A-ET_B and agonist for binding Nitric Oxide and induce eNOS.

A Three Component Method for Endogenous Augmentation of cEPCs — Macrovascular Diseases – Therapeutic Potential of cEPCs

Observations on Intellectual Property Development For an Unrecognized Future Fast Acting Therapy for Patients at High Risk for Macrovascular events

ElectEagle represents a discovery of a novel “multimarker biomarker” for cardiovascular disease that innovates on four counts.

First, it proposes new therapeutic indications for acceptable drugs.

Second, it defines a specific combination of therapeutic agents, thus, it put forth a proprietary drug combination.

Third, it targets receptor systems that have not been addressed in the context of cEPCs augmentation methods. Chiefly, modulation of the following three-targeted receptor systems: (a) inhibition of ET-1, ET_A and ET_A-ET_B receptors by antagonists (b) induction of eNOS, by agonists and NO stimulation and (c) upregulation of PPAReceptor-gamma by agonists (TZD). While (b) and (c) are implicated as having favorable effects of cEPCs count, each exerting its effect by a different pathway, it is suggested in this project that (a) might be identify to be the more powerful of the three markers. Our method, ElectEagleis the FIRST to postulate the following: (1) time concentration dependence on eNOS reuptake (2) dose concentration dependence on NO production (3) time and dose concentration dependence for ET-1, ET_A and ET_A-ET_B inhibition, and (4) dose concentration dependence on PPAReceptor-gamma. Points First, Second and Third are covered in Part II where a special focus is placed on ET-1, ET_A and ET_A-ET_B receptors.

Fourth, ElectEagle proposes a platform with triple modes of delivery and use of the test, as described in Part III. The triple modes are as follows: (A) an automated platform from a centralized lab with integration to Lab’s information management system. (B) a point-of-care testing device with appropriate display of test results (small benchtop analyzers in PCP office). (C) a device used for home monitoring of analytes (the hand-held device facilitates rapid read of scores and their translation to drug concentration of each of the three therapeutic agents, with computation of the three drug concentrations done by the device. Thus, it offers quicker optimization of treatment.  ElectEagle is the FIRST to propose a CVD patient kit, hand-held device, which calculates on demand an adjustable therapeutic regimen as a function of cEPCs count biomarker. In this regard, a similarity to the pump, in management of blood sugar in DM patients, exists. Since there is a high co-morbidity between DM and CVD, our methods, ElectEagle may eventually become a targeted therapy for the DM Type 2 population.

Postulates of Multiple Indications for the Method Presented: Positioning of a Therapeutic Concept for Endogenous Augmentation of cEPCs — Potential Therapeutic Indications for ElectEagle

ElectEagle can become the drug therapy of choice for the following indications:

•      CAD patients
•      Endothelial Dysfunction in DM patients with or without Erectile Dysfunction
•      Atherosclerosis patients: Arteries and or veins
•      pre-stenting treatment phase
•      post-stenting treatment phase
•      if stent is a Bare Metal stent (BMS)
•      if stent is Drug Eluting stent (DES)
•      if stent is EPC antibody coated (the ElectEagle method increase cEPCs generation in vitro) so availability of cEPCs is increased
•      post CABG patients (the ElectEagle enhances healing by endogenous augmentation of cEPCs)
•      target sub segments of CVD patients on blood thinner drugs (the ElectEagle does not require treatment with antiplatelet agents, it is suitable for all patients on Coumadin. This population have a counter indication for antiplatelet agents which is a follow up treatment after stent implantation for 30 days, with stent-eluting long term regimen of antiplatelet agents, 6 months and in some cases indefinitely (Tung, 2006).
•      ElectEagle is based on systemic therapeutics (versus the localized stent solution requiring multiple and even overlapping stents)
•      ElectEagle will be having potential in three contexts

(a) Coronary disease

(b) Periphery vascular disease

(c) Cerebrovascular

Comparative analysis of endogenous and exogenous cEPCs augmentation methods:

A. Endogenous augmentation method properties:

•         temporal – while drug therapy in use – drug action is interruptible
•         time concentration on eNOS reuptake
•         dose concentration on NO production
•         time and dose concentration manner for ETB inhibition
•         dose concentration on PPAR-gamma

B.  Cell-based and other exogenous methods

• permanent colonization till apoptosis if no repeated attempts of re-transfer,
• re-implantation as the protocol usually has several stages

The Promise of the Proposed Pharmacotherapy as a Method of CVD Risk Reduction

It is expected that ElectEagle will be resulting in potential delay of stenting implantation. Patients that are target for stenting may benefit form ElectEagle that will facilitate and accelerate healing after the stent is in place. EPC antibody coated stents will work if and only if the patient has more that just low cEPCs, most patient undergoing stenting tend to have low level of cEPC. The ElectEagle method can be coupled with that type of new stents, called Genous, now in clinical trials (HEALING II, III). These stents enhance the body ability in mobilization of cEPCs, only. However, if the initial population of cEPCs is low, an endogenous fast acting cell augmentation method is needed for pretreatment before the PCI procedure with Genous is scheduled.

Emergence of Clinical Trial Results on Genous R stent — Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth – (HEALING II)

Latest publications on HEALING II – Clinical Trial of EPC coated stent

Genous R stent
n=63
Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth – II

S Silber et al; 12 Month Outcomes of the e-HEALING (Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth) Worldwide Registry; EuroIntervention 2011;6:819-825

P Damman et al; Coronary Stenting With the Genous Bio-engineered R stent in Elderly Patients – 12-month Outcomes From the e-HEALING Registry; Circulation Journal 2011;75(11):2590-2597

P Damman et al; Twelve-month Outcomes After Coronary Stenting With the Genous Bio-Engineered R Stent in Diabetic Patients from the e-HEALING Registry; Journal of Interventional Cardiology 2011;24(4):285-94 

J Aoki et al; Endothelial progenitor cell capture by stents coated with antibody against CD34: the HEALING-FIM (Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth-First In Man) Registry.J.Am.Coll.Cardiol. 2005 May 17;45(10):1574-9

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