Angiogenesis

Mariano B. Mikulic, M.D.

 

Angiogenesis

The development of new blood vessels defines angiogenesis.1 Vasculogenesis is the formation of new vessels in an embryonic environment. In the early 1970'2 Dr. Folkman studied the involvement of angiogenic growth factors in tumor growth.2 The inhibition of this angiogenic factors adds a new treatment to the armamentarium against cancer.3 In patients with coronary artery disease with no options in terms of revascularization the possiblity of angiogenesis opens a new promise. The process of increasing blood flow to areas that are ischemic have been attempted through the following mechanisms:

  1. transmyocardial laser revascularization, and
  2. factors that increase blood vessel growth.

Transmyocardial Laser Revascularization

Transmyocardial laser revascularization (TMR) is a new therapy that improves symptoms in patients with stenotic coronary lesions not amendable to percutaneus or surgical treatments.4 By percutaneus or open heart surgery the prior detected ischemic areas of the heart are drilled with laser holes. This channels however do not remain open in the long term. but laser is able to induce the formation of new capillaries (angiogenesis) and ameliorate the ischemic symptoms by improving the perfusion to the affected myocardium. Investigators have noted an increase in the number of capillaries and the levels of angiogenic growth factors in patients who underwent TMR. Two patients who died had increased capillary density in the areas treated by laser at autopsy. 5

Angiogenic Growth Factors

Muliple growth factors have been detected, however most of the research has been done with vascular endothelial growth factor and fibroblast growth factor.

How does angiogenesis work?

The serial events that leads to angiogenesis begins with the endothelial cell being stimulated by an insulting process like hypoxia. 6 This generates an activation of the endotelial cell with prodution of vascular growing factors. A cascade of events follows: Fisrt there is migration of the endothelial cells with disruption of the basement membrane. The next steps include proliferation and the formation of tubes with periendothelial support. A new blood vessel is then formed. This was shown in experimental models of rat corneas were endothelial cells signaled the beginning of the formation new capillaries.7 The promotion of migration of endothelial cells though is a complex process were proteases like plasmin have a critical role in stimulating the migration of endothelial cells across the matrix of the cells.8 Multiple studies have demonstrated the importance of smooth muscle cells together with endothelial cells in the process of developing new blood vessels.9 Research have demonstrated the notion that proliferation of smooth muscle cells is an intrinsic element in angiogenesis.

Cytokines

Many of the mechanisms delineated above are modulated by factors called cytokines. Cytokines regulate these activities by binding to a tyrosinase kinase membrane receptors. Example of cytokines include vascular endothelial growth factor (VEGF) and fibroblast growth factor.

Vascular Endothelial Growth Factor (VEGF)

This cytokine is produced by the endothelial cells when they are under hypoxic conditions and exerts its efect by binding to two types of tyrosinase kinase receptors on the endothelial cells. These are VGEF receptor 1: which is responsible of the organization of endothelial cells in tubes, and VGEF recptor 2: that induces endothelial cell migration and proliferation. Both receptors are upregulated in an ischemic environment.10 VGEF is a glycoprotein that increases the mitosis of endothelial cells as well as their permeability. The most important form is VEGF 165.

VGEF can be delivered in two ways. The first one as a gene that encodes this peptide and the second one as a recombinant protein.

  1. Recombinant VEGF (Rh VGEF)

Rh VGEF was administered in a rabbit model of hind limb ischemia by the St. Elizabeth Hospital Group in Boston. It produced an increase of angiographic collaterals with improvement in hemodynamics in the ischemicgroup 5. Later experiments in pigs and dogs, Rh VGEF increased myocarial flow in ishemic induced myocardium.5,11 The VIVA study (vascular endothelial growth factor for in ischemia for vascular angiogenesis) was a double blind placebo contrlled trial of patients who were not candidates for percutaneus or surgical revascularization with viable myocardium who received different doses of recombinant VEGF. The results of this clinical study demonstrated an improvement in anginal class in the VGEF group as compared with placebo but no improvement in exercise time or SPECT-sesta Mibi perfusion defects. The doses of VGEF were limited by hypotension. The angiography results are pending.12

  1. Gene Transfer of VGEF

Different animal and human studies showed that naked DNA that encodes VGEF could be administrated with important effects. The delivery of DNA could be done in three ways, which are: arterial, intramuscular and myocardial gene therapy.

  • Arterial delivery of naked plasmid DNA

Dr. Isner and collaborators enrolled eight patients with critical limb ischemia (pain at rest or nonhealing ulcers) not candidates for percutaneus or surgical management in a Phase I study that delivered the VEGF 165 gene intra-arterially in escalating doses. Magnetic resonance angiography and contrast angiography demonstrated an increase in distal flow. Pig and dog studies revealed that this flow is the result of the development of new collaterals less than 180um in diameter, which is below the sensitivity of angiography. In this Phase I study three patients become free of rest pain after three months and one patient showed angiographic evdence of collaterals.

  • Intramuscular Gene Therapy

The St. Elizabeth group in Boston studies nine patients with critical limb ischemia. Each patient was injected at four sites two weeks apart. Enzyme-linked immunosorbent assay (ELISA) documented an increase in the VGEF serum levels. Mra and angiography documented newly deneloped collaterals.At twelve weeks there was an increase in mean ankle-brachial index from 0.33 to 0.48. Three patients have resolution of the rest pain, while four have complete healing of the ischemic ulcers. Since, over seventy- five patients with critical limb ischemia have been treated with fvorable clinical results. Interestingly younger patients have better results and outcomes. The main adverse side efeect is edema due to the increase permeability of VGEF.13

  • Myocardial Naked DNA Transfer

The Boston group of Dr. Isner initiated Phase I study to assess this therapy in symptomatic patients with coronary artery disease (CAD) with no option for surgical or percutaneus revascularization. Thirty-nine patients, ages 53 to 71 years with class III or IV angina, refractory to medical therapy and which ischemic defects on SPECT-sesta Mibi or by electro-mechanical mapping of the myocardium have been enrolled. (Electro-mechanical mapping is a new modality for the detection of myocardial viability mainly used during percutaneus myocardial revascularization by a specially designed catheter placed in the left ventrcle). All patients have multivessel CAD. After a mini thoracotomy or by a specially designed electro-mechanical catheter placed percutaneusly in the left ventricle, naked plasmid DNA for VGEF 165 was directly administrated to the ischemic myocardium. Thirty patients were studied. ELIZA was used to monitor serum VGEF levels. The main adverse effect were premature ventricular contractions. SPECT and electro-mechanical mapping showed a reduction of ischemia in twenty-nine patients. Angina was also reduced after 21 days of treatment with a significant decrease in nitroglycerin intake.The favorable results of this clinical study are highlighted by the fact that that VGEF gene was the only modality of treatment in contrast with other angiogenesis trials were a combination treatment of revascularization (CABG: coronary artery bypass) and VGEF administration was used. The limitation of this study was the lack of a placebo control.14,15

Fibroblast Growth Factor

This cytokine belongs to a family of nine factors. The most important one in the myocardium is basic fibroblast growth factor (bFGF). It has a mitogenic (increase mitosis) and angiogenic properties. Research in animals showed that FGF increases myocardial perfusion and function in acute and chronic ischemia. This was demonstrated by an increase in capillary networks. Several studies have been done with bFGF.16 In a randomized trial of delivery of bFGF in the subepicardial fat along the ischemic but not graftable territory in patients with CAD undergoing CABG, the initial results show an improvement in the group assigned to 100ug of bFGF in terms of symptoms, myocardial ischemic defects and wall motion abnormalities.17 The problem in evaluation the results of this study is that bFGF was not the only modality of treatment, because it was combined with CABG, so the outcomes are difficult to analyze.

Combination Therapy

Vascular endotheium growth factor is specific for the endothelial cells. Basic FGF acts on other cells including smooth muscle cells. The association of these effects may synergistically improve angiogenesis. The therapeutic implications of this combination will be determined with on going research.

Adverse Effects of Angiogenic Factors

The main concern with these factors is tumor growth. However there is no evidence that this can happen. In the VIVA trial the incidence of tumors was higher in the placebo arm. Other side effects include interstitial edema. This is seen with VGEF because of the increase in permeability. However, this is easily treated with diuretics.

Hypotension can be seen when high systemic doses of the peptide are administrated. The mechanism is likely the release of nitric oxide and vasodilation. This is not seen with the naked DNA injection. Other side effects are less common and include anemia, neointimal proliferation and renal toxicity (only seen in monkeys treated with bFGF).

Conclusions

Angiogenesis provides a new treatment for patients with no other revascularization options. Peripheral and myocardial ischemia has been the target of this novel medical management. Present studies have documented an increase in collaterals with minimal side effects. However many issues need to be resolved including the extent of improvement of symptoms. Future clinical trials help to answer these questions.

References

  1. Dorland's Medical Dictionary. W.B. Saunders Company. Philadelphia. 1989
  2. Folkman J: Tumor angiogenesis: therapeutic implications. N Engl J Med 1971, 285:1185-1186
  3. O'Reilly ms, Boehm T, Shing Y, et al. :Endostatin an endogeous inhibitor of angiogenesis and tumor growth. Cell 1997, 88: 277-285
  4. Narins R, Topol E: Percutaneus myocardial revascularization and angiogenesis in Topol E. Textbook of Interventional Cardiology. Third Edition. W.B. Saunders Company. Philadelphia. 1999
  5. Bashir R, Vale P, Isner J et al: Angiogenesis, mechanisms of effacy:safety concerns and early results. Current Interventional Cardiology Reports 2000, 2:196-203
  6. D' Amore PA, Thompson RW: Mechanism of angiogenesis. Ann Rev Physiol 1987, 49:453-464
  7. Sholley MM, Ferguson GP, Sebel HR, et al: Mechanisms of neovascularization. Lab invest 1984, 51:624-634
  8. Pepper MS, Montesano R: Proteolytic balance and capillary morphogenesis. Cell Differ Devel 190, 32: 319-328
  9. Schaper W, Schaper J, Xhonneux R et al: The morphology of intercoronary anastomosis in chronic coronary artery occlusions. Cardiovasc Res 1969, 3:315-323
  10. Brogi E, Schatteman G, Wu T et al: Hypoxia induced paracrine regulation of VGEF receptor. J Clin Invest 1996, 97:468-476
  11. Banai S: Angiogenic-induced enhancement of collateral blood flow to ischemic myocardiumby VGEF in dogs. Circulation 1998, 31:65A
  12. Henry TD, Rocha-Sing K, Isner J, et al: Results of intracoronary recombinant human VGEF administartion trial (abstract). Circulation 1998, 31:65A
  13. Baumgartner I, Pieczek A, Manor O, et al: Constitutive expression of phVGEF165 following intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation 1998, 97: 1114-1123
  14. Symes JF, Losordo DW, Vale PR, et al: Gene therapy with VGEF for inoperable CAD. Ann Thorac Surg 1999, 68: 830-836
  15. Losordo DW, Vale P, Symes J et al: Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of pH VEGF165 as sole therapy for myocardial ischemia. Circulation 1998, 98:2800-2804
  16. Schumasher B, Pecher P, von Specht BU et al: Induction of neoangiogenesis in ischemic myocardium by human growth factor: first clinical results of a new treatment for CAD. Circulation 1998, 97:645-650
  17. Ibukiyama C: Angiogenesis. Angiogenic therapy using FG
December, 2000/ Jacksonville Medicine

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