Angiogenesis and Direct Myocardial Revascularization

3. Preclinical Models and Experience to Date

부산의학전문대학원한성필

neuroscience@pnumdphd.org

I. ANIMAL MODEL SELECTION CRITERIA - Anatomy of Coronary Circulation - Pre-Existing Interconnecting Arterioles and Collateral Circulation

II. MODELS OF MYOCARDIAL ISCHEMIA - Myocardial Ischemia or Infarction - Ameroid Constrictor Model - Intermittent Occlusion Model - Variation of Vineberg Direct Arterial Implantation Procedure - Minimally Invasive Coronary Stenosis Model for Imaging - Cytothermia-Induced Myocardial Infartion Model - Microembolization Model - Myocardial Infarction (Ligation) Model

III. HINDLIMB ISCHEMIA MODEL

IV. EXPERIENCE IN PROTEIN THERAPY

V. UPDATE IN GENE THERAPY - Intramyocardial Injection Via Thoracotomy - Catheter-Based Transendocardial Injection - Intramyocardial/Intramuscular Injection - Intrapericardial Delivery - Intracoronary Delivery

VI. CELL THERAPY STUDIES

VII. NONINVASIVE IMAGING TECHNIQUES

VIII. COLLATERAL-SENSITIVE MRI IN SWINE MODEL OF CHRONIC ISCHEMIA

- Catheter-Based Endomyocardial Injections with Real-Time MRI in swine

IX. ELECTROMECHANICAL LV MAPPING/INJECTION SYSTEM - Structure and Mechanism of Function

X. ANIMAL MODELS FOR ANGIOGENESIS:DEPENDABLE BASIS FOR HUMAN STUDIES?

XI. SUMMARY AND CONCLUSIONS

General Outline

• Growth factor 주입으로 손쉽게 허혈심장에 혈관네트워크를 만들어낼 수 있다면 ? (especially for No-option patients)

• 단순한 GF 직접 주입 , Adenovirus 와 plasmid 를 이용한 유전자 전달 -> 초기에 많은 실패

• 그러나 최근 급속도로 발전하고 있는 학문 분야

• Angiogenesis / Antiangiogenesis 두 분야 모두 심혈관질환 /종양 연구에 필수적임

Preclinical efforts

어떻게 Treat 할 것인가 ?

How do we treat

어떤 동물 , 어느 경로 , 어떤 농도

Treat 한 뒤 효과를 어떻게 포착 , 응용할 것인가 ?

How do we capture the treatment effect

CT, USG, MRI, 혈청학 , 조직학 , 형태학

I. ANIMAL MODEL SELECTION CRITERIA

Ideal animal model1. Small, yet easily implemented and

maintained2. Coronary anatomy and innate collateral

circulation similar to humans3. Collateral development in the virtual

absence of infarction4. Consistent, regional abnormalities in LV

perfusion and function5. Easily assessed in response to simple

interventions

Ideal animal model1. Small, yet easily implemented and

maintained2. Coronary anatomy and innate collateral

circulation similar to humans3. Collateral development in the virtual

absence of infarction4. Consistent, regional abnormalities in LV

perfusion and function5. Easily assessed in response to simple

interventions

• This difference has resulted in a preference for the pig model for angiogenesis studies.

II. MODELS OF MYOCARDIAL ISCHEMIA

1. Myocardial Ischemia or Infarction2. Ameroid Constrictor Model3. Intermittent Occlusion Model4. Variation of Vineberg Direct Arterial Implantation

Procedure5. Minimally Invasive Coronary Stenosis Model for

Imaging6. Cryothermia-Induced Myocardial Infartion Model7. Microembolization Model8. Myocardial Infarction (Ligation) Model

Myocardial Ischemia or Infarction

• Reversible myocardial ischemia and myocardial infarction are fundamentally different pathophysiological events.

• Myocardial survival (hours) vs vascular development (days)

• Video 1• Video 2

Ameroid Constrictor Model

Ameroid Constrictor Model

Ameroid Constrictor Model

• 주로 LCX 에 위치 (Left thoracotomy, through 4th intercostal space, under general anesthesia)

• C shaped device• Swelling 이 안쪽으로 심화되면서 occlusion• Mechanical endothelial damage, platelet

aggregation, thrombus formation, foreign body reaction with local scar formation

Ameroid Constrictor Model

• Advantages① Provides slow and progressive coronary artery

occlusion, mimicking chronic ischemia② Simple development of arterial occlusion after

implantation around a coronary artery• Disadvantages

① The occlusion may be influenced by vascluar tone, platelet aggregability, thrombogenicity, inflammation, and fibrosis

② Collateral 생성에서 나온 bFGF(NO-dependent vasodilatior) 등에 의해 occlusion 이 생성 안될 가능성도 있다 .

③ 다양한 경색이 존재함으로 통계학적 오차 발생 가능

Minimally Invasive Coronary Stenosis Model for Imaging

• MICS model in swine to investigate perfusion in MRI, SPECT Kritchman et al. 2000

• The aim– Open chest 의 단점을 극복 (Normal mechanics,

autoregulation 손상 등 )

• Flow-reducing fittings were advanced to a wedge position in the prox. LAD with an angioplasty cath. via carotid artery approach.

Variation of Vineberg Direct Arterial Implantation Procedure

• Internal mammary artery(IMA) and its intercostal branches were transected and tunneled directly into the myocardium (before CABG)–Vineberg 1966

• Ameroid + Vineberg = a systemic to coronary anastomoses model

Microembolization Model

• Injecting microspheres into a discrete vascular bed

• The method of choice = “Repetitive microembolization” 작은 microsphere( 주로 15um) 를 반복적으로 주입

• Artery 에서 capillary 까지 다양한 크기의 입자를 주입 가능

• 유리 , 폴리스티렌 , 플라스틱 등• Dogs and pigs• 인간의 죽상동맥 경화증과 유사한 chronic LV

dysfunction 유도 가능

Myocardial Infarction (Ligation) Model

• Simplistic model that guaratees the onset of ischemia

• Transmural infarction in pigs and smaller animals (pigs will not survive)

• Nontransmural infarctions in dogs• Can be used in rats and dogs• Complicated by cell death, myocyte

hypertrophy, and fibroblastic growth

III. HINDLIMB ISCHEMIA MODEL

• Dissection, ligation, complete excision of a portion of the femoral artery

• Advantages1. Relatively simple surgical technique2. Easy to measure indices of collateral

development - BP ratios between ischemic and normal limbs, pressure gradients

3. Readily defined zones of collateral development and easily harvested samples

Ariana G. Ojalvo, “Therapeutic angiogenesis following intramuscular gene transfer of vascular endothelialgrowth factor 121 in a dog model of hindlimb ischemia,” Electronic Journal of Biotechnology I 6, no. 3 (December 15, 2003)

세가지 치료적 접근법

1 2

Gene Therapy

3

Cell Therapy

ProteinTherapy

IV. EXPERIENCE IN PROTEIN THERAPY

• FGF, VEGF, HGF 등이 폭넓게 사용됨 (Table 2,3)

• 대다수의 연구에서 angiogenic effect, ventricular function enhancing 이 관찰되었으나 그 중 가장 폭넓게 입증된 것은 FGF-2 (Laham’s group), VEGF-A (Unger’s group, Isner’s group)

• 효능을 위한 peptide 농도 , colateralization되는 동안 투약의 타이밍 등에 대한 많은 의문이 아직 남아있음 .

V. UPDATE IN GENE THERAPY

1. Intramyocardial Injection Via Thoracotomy

2. Catheter-Based Transendocardial Injection

3. Intramyocardial/Intramuscular Injection4. Intrapericardial Delivery5. Intracoronary Delivery

Intramyocardial Injection Via Thoracotomy

• 낮은 양의 adenovirus 에 의한 Gene transfer = ventricular function 과 perfusion 의 개선 (+) – Mack et al. 1998– LCX ameroid– Ad.VEGF-121 x 10 injections– direct intramyocardial injection, pig– ECG, Radionuclide imaging

Catheter-Based Transendocardial Injection

• Kornowski et al– Ameroid– Delivery of Ad.VEGF-121 (1x1010 vp)using an

electromagnetic-based cath guidance system through a retractable needle.

–수술에 의한 transepicardial injection 과 흡사한 gene transfer

Intracoronary Delivery

• Giordano et al. Nat Med 1996• Porcrine ameroid• Ad.FGF-5• Transthoracic echocardiography(TTE) 로 확인• Improved function and perfusion were

associated with evidence of angiogenesis• Capillary angiogenesis (+), Larger caliber

angiogenesis (-)

VI. CELL THERAPY STUDIES

• 유전자와 단백질 치료 뿐만 아니라 보다 자연적인 혈관신생인자인 세포를 이용하는 시도가 일어나고 최근 다양하게 시도되고 있음 (Table 4)

• Different cell types– Allogeneic– Autologous

• Different cell sourses– Skeletal muscle– Bone marrow– Peripheral blood

VII. NONINVASIVE IMAGING TECHNIQUES

• Therapeutic angiogenic strategies 의 평가를 위한 이미지테크닉의 진보가 계속되고 있음

• Noninvasive and high tech approaches such as MRI

• 과거 , collateral circulation 을 확인하기란 매우 제한적이었음 (Conventional angiography- 180um 이상만 detection 가능 )

VIII. COLLATERAL-SENSITIVE MRI IN SWINE MODEL OF CHRONIC ISCHEMIA

• Neovascularization 을 측정하는 새로운 방법 Pearlman JS et al. Radiology 2000

• Advantages– Depicts small areas of neovascularization– Provides quantitative assessment of extent of

neovascularization– Enables serial noninvasive studies of collateral

development• Disadvantages– Is not useful in determining the time of arrival of

the contrast agent in the LV– 작은 신생혈관을 과다측정하는 경향이 있다 .

IX. ELECTROMECHANICAL LV MAPPING/INJECTION SYSTEM

• 전자기계적 좌심실 매핑– EMM 후 돼지의 myocardium 에 catheter 로

혈관신생 transgene(Ad.VEGF-121, 1x1010 viral particles X 6 sites) 을 주입하는 최신 기술 (Vale PR et al., J Am Coll Cardiol 1999)

–장점 : precisely localizes gene transfer and directs it towards the most ischemic myocardial regions.

–수술에 의한 방법과 비교해 동등한 gene transfection efficacy

X. ANIMAL MODELS FOR ANGIOGENESIS:DEPENDABLE BASIS

FOR HUMAN STUDIES?

• Points to be considered in translating the results of the preclinical studies into the clinical arena① 동물은 죽상동맥경화증을 갖고 있지 않다 .② Angiogenesis 를 필요로 하는 환자는 전형적으로

늙은데 반하여 동물 모델은 젊거나 성장 중이다 . 나이듦에 따라 angiogenic potential, resposiveness 가 감소한다는 보고가 있다

③ 약제에 의한 angiogenic 반응의 감소④ No option patients vs unselected population of

amimal

XI. CONCLUSIONS

• No option patients 에게 혈관신생은 꼭 필요한 과제이며 더 많은 연구와 지원이 요구됨

• 가장 효과적인 타이밍을 잡아 어떤 방식의 치료를 시작할 것인지를 결정하는 연구가 요구됨

• 인간과 동물과의 차이점을 극복하며 translational research 에 대한 요구가 이루어져야함 .

감사합니다 .

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