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By Athina Kyritsis, MD; Zannos G. Grekos, MD;Hector Rosario, MD; Leonel Iliano, MD

Objective;

The goal o this study isto investigate the easibility, saety,and clinical outcome o patients withIschemic Cardiomyopathy treatedwith Autologous Angiogenic andCardio-Regenerative Progenitor cells(ACP’s) in a prospective ashion.

Background;

In numerous humantrials there is evidence o improve-ment in the ejection ractions o Car-diomyopathy patients treated withACP’s. Animal experiments, not onlyshow improvement in cardiac unc-tion, but also engratment and di-erentiation o ACP’s into cardiomyo-cytes as well as neo-vascularization ininarcted myocardium. In our clinicalexperience the process has shown tobe sae as well as eective.

Methods;

We conducted a prospec-tive, non-randomized study evaluatingthe eects o ACPs (

ex vivo

expandedand dierentiated peripheral bloodstem cells) implanted in sixteen pa-tients with chronic ischemic cardio-myopathy (Ejection ractions < 45%)with congestive heart ailure symp-toms o at least NYHA class II. ACP’swhere implanted via either intra-myocardial injection or intra-coronaryinusion. Patients where optimizedmedically prior to ACP’s therapy withstandard medical therapy or CHF aswell as revascularization and upgradedto Bi-ventricular debrillators whenindicated. Ejection ractions where

recorded at baseline then at 3 and 6

months using MUGA at rest as well asat stress (dobutamine protocol). Theprimary end points were changes inrest and stress ejection ractions.

Results;

We ound treated patientsexhibiting a signicant increase incardiac ejection raction rom baseline.The increases in ejection raction were

Ischemic Cardiomyopathy Patients Treated with Autologous Angiogenicand Cardio-RegenerativeProgenitor CellsIschemic Cardiomyopathy Patients Treated with Autologous Angiogenicand Cardio-RegenerativeProgenitor Cells

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21 points (75% increase) at rest and28.5 points (80% increase) at stress.

Conclusion;

This study exempliesthat ACP’s can improve the ejec-tion raction in patients with severelyreduced cardiac unction with benetssustained to six months. These pa-tients will continue to be ollowed in asimilar ashion to determine long termoutcomes. Other secondary outcomeswill also be ollowed including cardiacevents, hospitalizations, mortality,unctional class, cardiac dimensions.

INTRODUCTION

Despite signicant advances inthe new therapeutic modalities andprevention, cardiac disorders arevery prevalent all over the world.The magnitude o the problem willincrease considerably in the uturedue to increasing lie expectancy andthe prevalence o diabetes. In spiteo considerable advances in medicaltherapy and improvements in revas-cularization procedures or coronaryartery disease, a substantial propor-tion o patients who suer rom an-gina pectoris and heart ailure are not responsive to maximal medical andsurgical treatment modalities. Impor-tantly

Cardiovascular Disease

is at thetop o the list or medical expendi-tures in the United States o America. With the majority o dollars spent onhospitalizations or congestive heart ailure. Consequently, eective al-ternative therapies or these patientswould have ar reaching benets.Regenocyte’s therapeutic strategycollects blood samples rom patients,isolates peripheral blood mononu-clear cells, and grows these cells inconditions that will cause a signicant increase o the number o progenitorcells as well as partially dierentiatethese cells into a population speci-cally targeted at cardiac regeneration

Following this culturing stage, theACPs are harvested, packaged, andtransported to the treatment centerto be injected into the coronary ves-sels and myocardium o the patients.The nal cell product is known asRegenocytes.Regenocyte therapy treats patientssuering rom angina pectoris orcardiomyopathy, not responsive tomaximal drug treatment or not will-ing or without option o undergoingcoronary artery bypass grat (CABG)surgery or PCI. The use o ACPs pro-motes the ormation o neo-vascular-ization and viable myocardial tissue.

SCIENTIFIC BACKGROUNDCELLULAR BIOLOGY

Angiogenic Cell Precursors (ACPs)or Endothelial progenitor cells (ECPs)possess the ability to dierentiateinto endothelium, the layer o cellsinvolved in both the orming o bloodvessels (neovascularization) and thelining o their lumen (endothelializa-tion). These unctions o the ACPsenable the development o new thera-pies that aim to use these cells or thetreatment o severe vascular disorders.The rst evidence indicating thepresence o ACPs in the adult circula-tion was obtained when mononuclearblood cells rom healthy humanvolunteers were shown to acquire an

endothelial cell–like phenotype

invitro

and to incorporate into capillar-ies

in vivo

(11)

. These putative ACPswere characterized via expressiono CD34 and vascular endothelial

growth factor receptor-2 (VEGFR-2),

two antigens shared by embryonic en-dothelial progenitors, and hematopoi-etic stem cells. In addition to CD34,early hematopoietic progenitor cellsexpress CD133 (AC133), which isnot expressed ater dierentiation.Currently, the widely accepted deni-tion o ACPs in circulation is, orpractical purposes, CD34

/VEGFR-2

or CD133

/VEGFR-2

cells.The act that ACPs can take part in the ormation o new blood vesselswas rst observed by Bhattacharya andcolleagues who showed the orma-tion o capillary-like structures romhematopoietic stem cells or

ex-vivo

expanded ACPs

(12,13)

. The contributiono bone marrow-derived cells, mainlyACPs, to neovascularization aterischemic injury

in vivo

, was shown inexperiments using labeled populationso stem cells to reconstitute lethallyirradiated mice. The cells or theirprogeny were shown to migrate intoischemic cardiac muscle and blood ves-sels, dierentiate to cardiomyocytes andendothelial cells, and contribute to theormation o unctional tissue

(14)

. Otherwork, involving a mouse retinopathymodel, demonstrated the important rolethat the recruitment o endothelial pre-cursors to sites o ischemic injury playsin neovascularization

(15)

.The majority o ACPs reside in thebone marrow in close association withHematopoietic Stem Cells (HSCs) andbone marrow stromal cells that providethe microenvironment or hematopoie-sis. ACPs have been shown to mobilize(i.e. migrate in increased numbers romthe bone marrow into circulation) inpatients with vascular trauma or AcuteMyocardial Inarction (AMI)

(16, 17)

, orin response to Administration o VEGFvia gene transer

(18, 19)

.The sources o Autologous Angio-genic Cell Precursors that can be usedor treatment varies and include bonemarrow, peripheral blood and dierent mesenchymal organs. The use o cellsrom peripheral blood has the advan-tage o being more uniorm easier tocharacterize and control and that theircollection is easier (without anesthe-sia). The disadvantages are the rela-tive small number o Angiogenic CellPrecursors in peripheral blood whichrequires a relatively large volume o blood and the time consuming processo augmentation.The use o Angiogenic Cell Precursorspromotes the ormation o neo-vascular-ization as well as new myocardial cellsin the ailing heart and as a consequenceattenuates congestive ailure.

CLINICAL TRIALS OF STEm CELLTHERAPY FOR CARDIAC DISEASE

Considerable work has been carriedout to elucidate the mechanisms be-hind ACP’s mobilization, localizationand unction. Progress has also beenachieved in establishing therapeuticprotocols or treating a variety o con-ditions, such as peripheral limb isch-emia, acute myocardial ischemia andinarction by using progenitor cells.The last ew years have seen signi-cant progress being achieved by clinicaltrials using therapeutic protocols ortreating a variety o vascular conditions,such as peripheral limb ischemia, acutemyocardial ischemia and inarction byusing stem and progenitor cells. Clinicaltrials have been perormed to test thesaety and potential ecacy o severaltypes o cells

(8-24)

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The trials showed considerable po-tential at alleviating these conditionswith no serious adverse eects directlyrelated to the cells administered.These studies demonstrated the po-tential saety o the administration o

other peripheral blood–derived cells in

humans suering rom myocardial andvascular diseases and the potential orenhancing myocardial unction withassociated improvement in symptomsas maniested in the patients’ physi-cal condition and in objective cardiacunction tests.Methods o cell administration wereintracoronary injection while peror-mance o angiography, intramuscularinjection at CABG operation or intra-muscular injection.The parameters o heart peror-mance were improvements in let ventricular ejection raction (LVEF),improvement in cardiac perusion andin angina score. The results o thesetrials are in general promising ater

follow-up of 4-16 months. Adverse

eects were minimal and were not related to administration o the ACPs.However, most studies have the dis-advantage o having been small series,conducted as open label trials and onlysome o them included a control group. When considering the benet o stemcell treatment there is wide agreement that these treatments are sae and carryminimal risk to patients, as supportedby “

The Consensus Of The Task ForceOf The European Society Of CardiologyConcerning The Clinical Investigation Of The Use Of Autologous Adult Stem CellsFor Repair Of The Heart”

(30)

METHODS AND PROCEDURES

Sixteen patients were selected basedon the ollowing guidelines.

INCLUSION CRITERIA:

1. Patients with ischemic cardiomyo-pathy on maximal medical therapy.

2. Ejection Fraction less than 45%.3. Age 18 to 80 years

4. Male or non-pregnant, non-lactat-ing emale5. Inormed consent obtained andconsent orm signed

EXCLUSION CRITERIA:

1. Patients who received blood trans-usions during the previous 4 weeks(to exclude the potential o non-autologous ACPs in the harvestedblood).

2. Inability to communicate (that

may interere with the clinicalevaluation o the patient)3. Ater heart transplantation4. Renal ailure5. Hepatic ailure

6. Anemia (lower than 10mg/

dl.hemoglobin or emale andlower than 11 mg/dl or male)

7. Abnormal coagulation tests [plate

lets, PT (INR), PTT]8. Malignancy

9. Concurrent chronic or acute inec-tious disease10. Severe concurrent medical disease

(e.g., septicemia, HIV-1,2/HBV/

HCV inections, systemic lupuserythematosus)11. Chronic immunomodulating orcytotoxic drug treatment

12. Patients who have rectal tempera

ture above 38.40C for 2 consecu

-tive days13. Patient unlikely to be available orollow-up

Evaluation Parameters:

The ollowing tests were perormed

at baseline and at 3 and 6 month fol

-low-up visits to measure subjective andobjective parameters o the treatment:1. Physical exam

2. Blood pressure, heart rate and ECG

3. Blood tests4. Hematology: RBC; Hemoglobin;Hematocrit; WBC; Neutrophils;Lymphocytes; Monocytes; Plate-let count.5. Blood Chemistry: Glucose; Bloodurea nitrogen (BUN); Serumcreatinine; Serum chloride; Serumpotassium; Serum sodium; HgA, C,C-Peptide, CRP, P-BNP

6. CCS (Canadian Cardiovascular

Society) grading or Angina7. NYHA (New York Heart As-sociation) grading or congestiveheart ailure

8. Assessment of cardiovascular drug

types and doses9. Echocardiography10. Dobutamine Stress MUGA11. Bruce exercise nuclear perusion test

12. Number of hospitalizations

13. Mortality14. Cardiovascular events

CELL PRODUCT

The Final Cell Product (Regenocytes)

consisted o Autologous Angiogenic CellsPrecursors isolated rom the patient’sblood and then expanded and par-tially dierentiated

ex vivo

under

sterileconditions. The cells were divided into 3syringes suspended in 15 ml sterile cellculture medium. The product was sterileand pyrogen-ree.

BIOLOGICAL ACTIVITY ANALYSES

Acceptable biological parameters as as-sessed by microscopy and fow cytometrythat were in accordance with the ollow-ing specications:1. Cell viability o greater than 75%

2. Appropriate Morphology – spin

-dle-shaped, large cells orminglong thread-like structures.3. Minimum subpopulations o cellsstaining positive or the CD34and CD 31 markers (assessed byfow cytometry).The nal cell product was also testedor saety based on the ollowing:1. Sterility

2. Gram stain

3. Bacterial Endotoxin4. Mycoplasma contamination5. Bacterial culture

TREATMENT ADMINISTRATION OFREGENOCYTES

Patients were transerred to

the car-diac catheterization laboratory approxi-mately one hour beore the anticipated

arrival o the cells. Coronary angiogra-phy was perormed to dene the arteryor arteries planned to be used or thecell injection. The administration wasperormed intracoronary utilizing anover-the-wire balloon catheter andollowing a specic delivery protocol orby intra-myocardial injection.

SAFETY

There were no adverse events as-sociated with the ACP’s. No cardiacevents occurred. There was one severeadverse event. One patient suered aCVA during one o the cardiac cathe-terizations and was thereore excludedrom the group.