Murray J. Clifford. Angiogenesis Protocols - Methods in Molecular Medicine, Vol. 46

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12 Zhang and Bicknell

involved the purification of angiogenic inhibitory activity from the serum and

urine of experimental animals bearing transplantable tumors that suppressed

the growth of micrometastases in distant organs. Peptide sequencing showed

that angiostatin and endostatin are proteolytic fragments of plasminogen and

collagen type XVIII, respectively. More importantly, the discovery of

angiostatin and endostatin has revealed a novel regulatory mechanism of

angiogenesis. That is, potent negative regulators of angiogenesis are “stored”

encrypted within larger proteins that are devoid of antiangiogenic activity. Pro-

totypes of cryptic antiangiogenics, a 29-kDa fragment of fibronectin (54) and a

16-kDa fragment of prolactin (55), had been described several years earlier but

these findings were not immediately followed up. The repertoire of cryptic

fragments that are antiangiogenics is expanding (Table 4; for further discus-

sion see ref. 56).

Angiostatin has been shown to be released from plasminogen by the action

of elastases such as metalloelastase (MME) released by tumor-infiltrating mac-

rophages (57) and serine proteases from several human prostate carcinoma

lines (58). Further efforts are needed to elucidate the mechanisms by which

angiogenic inhibitory fragments are released from the parent molecules.

Screening of cancer cell lines for circulating angiogenic inhibitory activity in, for

example, a mouse corneal neovascularisation model (59), helps to show what

cell types normally serve to release cryptic inhibitors and under what circum-

stances.

Table 3

Tumor Suppressor Genes and Other Candidates

for an Angiogenic Off-Switch

Tumor suppressor genes Mechanisms

Much studied genes

p53 Induction of TSP-1 and VEGF synthesis

RB Induction of TSP-1 synthesis

VHL Inhibition of VEGF synthesis

Some other reported candidates

Locus Loh2 on mouse chromosome 16 Induction of an unknown angiogenic

inhibitory activity

A gene in baby hamster kidney Induction of TSP-1 synthesis

line BHK21/cl13

A gene on human chromosome 10q Induction of TSP-1 synthesis

Therapeutic Inhibition 13

5.2. Overview of Angiogenesis Inhibitors

Several hundred antiangiogenics have now been identified, some of which

are listed in Tables 5–7. Most antiangiogenics can be grouped into the follow-

ing broad categories: (1) physiologically occurring inhibitors (Table 5), many

of which appear to play a role in suppressing unwanted angiogenesis and may

also render healthy tissues resistant to tumor invasion, (2) natural products

(Table 6); (3) microbial derivatives (Table 6), and (4) synthetic or semisyn-

thetic molecules (Table 7). Among the wide variety of antiangiogenics, some

are in clinical trials (Table 8) while others are drugs that are already in use in

Table 4

Physiological Angiogenic Inhibitors Encrypted

Within Larger Molecules Lacking Activity

Main action(s) on ECs

Precursor proteins Cleavage products (inhibition of)

Collagen type IV MMP-2-generated fragments Adhesion

Collagen type XVIII 20-kDa C-terminal fragment Migration and

(endostatin) proliferation

EGF Synthetic fragment of amino Inhibition of EGF- and

acid 33–42 laminin- dependent EC

migration

Fibronectin 29-kDa fragment Proliferation

HGF Spliced form containing the Unknown

first two domains

High-molecular-weight His-rich fragment Adhesion

kininogen

Osteopontin Thrombin-generated fragment

containing RGD

Plasminogen 38-kDa fragment (angiostatin) Migration and

proliferation

Platelet factor-4

N-terminal processed form Proliferation

Prolactin 16-kDa N-terminal fragment Migration and

Proliferation

SPARC

Fragment(s) containing KGHK Stimulation of

proliferation

Enhancement of tube

formation

The parent protein has angiogenesis inhibitory activity but the cleavage products are more

potent.

The parent protein is a reversible G1-phase inhibitor and exhibits, if any, a negative effect on

angiogenesis while the proteolytic product stimulates angiogenesis in vivo.

14 Zhang and Bicknell

the clinic because of other activities, e.g., bleomycin, D-penicillinamine, meth-

otrexate, pacliltaxel, and (inter alia) doxorubicin.

There is some perception that endogenous angiogenic inhibitors have greater

specificity and potency than other antiangiogenics. Indeed, it has been demon-

strated that angiostatin not only retarded tumor growth in mice but also

induced dramatic shrinkage of large tumors (e.g., 400 mg, about 2% of the

body weight of the mouse) to a microscopic size (60). More dramatically,

endostatin induced complete regression and the indefinite dormancy of three

fast growing tumor types in mice (11). Similar results have not been seen with

other antiangiogenics or even cytotoxic chemotherapeutic drugs. These unprec-

edented observations may well be a hopeful prelude to the use of endogenous

antiangiogenics in the treatment of human primary tumors and metastases.

6. Delivery of Antiangiogenic Agents

Delivery is an important consideration. To this end, numerous options have

been proposed and the scattered reports in the literature can be grouped

according to the nature of the agents and vectors used.

Table 5

Examples of Physiological, Endogenous Angiogenesis Inhibitors

Inhibitors Main targets/mechanisms

Angiostatin Inhibition of EC proliferation and migration

Endostatin Inhibition of EC proliferation and migration

Glioblastoma-derived Inhibition of EC migration

angiogenesis inhibitory factor

Heparinases Blockage of cytokine binding to ECs

Interferons Reduction of release of antigenic factors,

inhibition of EC proliferation and migration

Platelet factor-4 Inhibition of EC proliferation and tube

formation, down-regulation of FGF

receptors, inhibition of VEGF activity

Proliferin-related protein Inhibition of EC proliferation and migration

Protease inhibitors Inhibition of ECM-degrading proteases,

TIMPs inhibition of EC proliferation and

PAIs migration, interference with vessel

CDI (cartilage-derived inhibitors) remodelling

Thrombospondin (TSP-1) Modulation of EC adhesion, inhibition of

collagenase, inhibition of EC proliferation

and migration

Therapeutic Inhibition 15

1. Direct administration: Low-molecular-weight drugs such as suramin analogs

(61) and AGM-1470 (62) can normally be delivered free of specially designed

vehicles or targeting partners. Other antiangiogenic agents that have been directly

administered are molecules that possess an intrinsic targeting property such as

synthetic peptides, neutralizing antibodies, growth factor/receptor antagonists,

and integrin antagonists. Anti-VEGF (63) and anti-VEGFR2 (64) antibodies and

integrin _v`3 antagonists (39) have been shown to inhibit angiogenesis and thus

suppress tumor growth and/or tumor invasion and metastasis.

2. Antibody-directed therapy: Specific antibodies can be conjugated with various

therapeutic agents such as toxins (i.e., immunotoxins), radioisotopes, or enzymes.

Genetically engineered antibody fragments such as Fab and single chain anti-

body variable domains (scFv) (65) and bispecific antibodies (12) also serve this

purpose.

3. Chimeric protein therapy: Here, a fragment of an angiogenic factor or its receptor

is engineered as the targeting partner and fused with a toxin or other therapeutic

agent. The fragment of growth factor or receptor serves at the same time as an

antagonist to interrupt ligand-receptor activating interactions. For example,

chimeric toxins (aFGF-PE) composed of aFGF fused to a mutant form of

Pseudomonas exotoxin) (66) and chimeric proteins of VEGF receptor linked to

IgG heavy chain (67) have been shown to inhibit angiogenesis in vitro and

in vivo, respectively.

4. Peptide guided therapy: As demonstrated by Arap et al. (15), anticancer drugs

can be delivered as conjugates with peptides that home to tumor vessels. Specific

antiangiogenic agents can also be delivered in this way and the pattern of pep-

tides used for targeted delivery will soon expand to those that bind to ECs in an

organ-specific manner.

5. Gene transfer-based therapy: Examples include ribozyme targeting of

pleiotrophin (PTN) (68), antisense targeting of bFGF and bFGFR-1 (42) dominant-

negative VEGF receptor (Flk-1) mutant (69) and vascular targeting of gene

therapy using EC-specific promoters (70), which have been employed to achieve

an antiangiogenic effect or vascular targeted therapy. Interested readers are

Table 6

Some Natural Product Antiangiogenics

Chemotherapeutic Extracts from natural

(Anti-)microbial agents compounds materials

Fumagillin Paclitaxel Gensenoside (ginseng)

Erbstatin Bleomycin Isoliquiritin (Licorice root)

Staurosporine Magnosalin Genistein (soy beans)

Tecogalan Squalamine (shark tissue)

CM101

16 Zhang and Bicknell

Table 7

Miscellaneous Antiangiogenics

General categories/

Factors Description Main mechanisms

Cytokine/chemokines

IL-12 75-kDa glycoprotein Induction of IP-10

heterodimer with an indirect

anti-tumor activity

gro-` Growth-related oncogene ` Inhibition of EC

protein proliferation

IP-10 Interferon-inducible protein 10 Inhibition of EC growth

and differentiation

(Anti-)microbial

products and analogs

Heparinases I Purified from Flavobacterium Depletion of heparan

and III (but heparinum sulfate receptors

not II)

FR-111142 Fumagillin analog Inhibition of collagenase

WF16775 A2 Antibiotic from soil fungus Cytostatic to ECs

C. erysiphoides

Minocycline Semisynthetic tetracycline Inhibition of collagenase

and EC proliferation

Herbimycin A Benzoquinoid ansamycin Inhibition of EC

proliferation

Peptides and

polypeptides

RGD-containing Inhibition of EC migration

peptides and tube formation

CDPGYIGSY-NH2 Synthetic laminin peptide Inhibition of EC migration

Vitreal inhibitor 5.7-kDa glycoprotein Inhibition of

metaloproteinases

and EC proliferation

RNasin 51-kDa RNase inhibitor Inhibition of angiogenin-

induced angiogenesis

Hyaluronic acid High molecular weight Inhibition of EC

glycosaminoglycan proliferation

Polysaccharides

SP-PG Sulphated polysaccharide- Inhibition of EC

peptidoglycan complex proliferation

DS-4152 As above Inhibition of bFGF

binding to ECs,

inhibition of EC

proliferation

SCM-chitin III Sulphate polysaccharide Inhibition of type IV

collagenase and of EC

migration

Therapeutic Inhibition 17

Table 7

(continued)

General categories/

Factors Description Main mechanisms

Polycationic and

polyanionic compounds

Suramin 1.4-kDa polyanionic compound Binding to various growth

factors, cell membrane,

and intracellular

enzymes

Protamine 4.3-kDa arginine-rich cationic Blockage of heparin-

protein binding and heparin-

induced EC migration,

inhibition of EC

proliferation

Distamycin A analog Sulphonic derivatives of Blocking bFGF and

diastmycin PDGF-b binding to their

receptors

Vitamin derivatives

Retinoids Natural or synthetic Induction of cell

differentiation

Vitamin D3 analog Vitamin D3 metabolites Induction of cell

differentiation

Steroids

Glucocorticoids Antiinflammatory, Inhibition of IL-1_

(cortisone) immunosuppressive production and action,

agents inhibition of collageous

protein synthesis

Methyl- Steroids without glucocorticoid Dissolution of BM,

prednisolone and activity, with a distinct inhibition of EC

tetrahydrocortisol structure on the D ring proliferation and

migration

Synthetic

Medroxyprogestrone Progestogen analog Inhibition of collagenase

facetate and plasminogen activator

D-penicillamine Antirheumatic agent Chelation of copper,

inhibition of EC

proliferation, collagenase

and collagen synthesis,

and plasminogen

activation

Gold salts Antirheumatic agents Inhibition of EC mitosis and

production of angiogenic

factors by macrophages

18 Zhang and Bicknell

Table 8

Examples of Antiangiogenic Agents in Clinical Trials

Agents Main mechanism Disease

Batimastat/BB-2516 Inhibition of metalloproteinases Women with malignant

ascites

CAI Blocking of Ca

channels Various types of cancers

CM101 Polyexotoxin produced Various solid tumors,

by Group B Streptococcus Kaposi’s sarcoma

CT-2584 Regulation of Solid tumors

tumor phospholipase-D (PLD)

enzyme activity and

phosphatidic acid production

IL-12 Induction of the antiangiogenic Renal carcinoma

protein IP-10

Marimastat Inhibition of metalloproteinases Advanced solid tumors

(pancreatic, lung,

colorectal, ovarian,

prostate, and brain

cancer)

Platelet factor-4 Inhibition of EC proliferation and Kaposi’s sarcoma, renal

tube formation, down-regulation and colon cancer,

of FGF receptors, melanoma

inhibition of VEGF activity

Suramin Binding to various growth Prostate cancer

factors, cell membrane, and

intracellular enzymes

Tecogalan sodium, Inhibition of EC growth and Kaposi’s sarcoma and

D-gluco-

D-glalactan migration replased cancer of

sulfate/DS4152 breast, lung, head-

neck and soft tissues

Thalidomide ? Brain, breast, and

prostate cancer

Razoxane ? Kaposi’s sarcoma,

psoriasis, and Crohn’s

disease

TNP470/AGM1470 Inhibition of EC growth and Breast, renal, cervical

migration cancer, Kaposi’s

sarcoma, glioblastoma

multiforme

Therapeutic Inhibition 19

referred to (refs. 6,71–74 ) for further discussion.

6. Cell transfer-based therapy: Proliferating ECs and putative EC progenitors have

been shown to incorporate into sites of active angiogenesis (75–77), suggesting

that ECs can be transduced and then seeded. This may be useful for the delivery

of anti- (or pro-) angiogenic agents to sites of pathologies. Antiangiogenesis

might also be delivered via cells of other lineages. For example, lymphokine-

activated killer (LAK) cells and, in particular, tumor-infiltrating lymphocytes

(TILs) (78) may be useful in homing antiangiogenic agents to the tumor vasculature.

More sophisticated approaches use combinations of the above methods to

achieve highly specific and efficient delivery. A good example of this is antibody-

directed enzyme prodrug therapy (ADEPT) (79), where antibody-enzyme con-

jugates are combined with direct administration of a prodrug.

7. Potential Drawbacks of Antiangiogenic Therapy

Despite the fact that antiangiogenic therapy is in advanced clinical trials,

there remain reservations concerning its utility. There is the possibility that

antiangiogenic strategies may interfere with normal physiological angiogen-

esis, impairing wound healing, stunting the growth of young children, or

inducing amenorrhea in women. However, arguments in favor of anti-

angiogenic therapies are well documented (80). Potential adverse effects due

to inhibition of physiological angiogenesis may be less prevalent than once

feared. First, toxicity of antiangiogenesis in young patients may well be negli-

gible in that normal angiogenesis appears to level off after infancy and EC

turnover is thought to be extremely low in young infants, children, and adoles-

cent males. Secondly, for women receiving therapy, pregnancy may be unde-

sirable and amenorrhea may actually be advantageous. It has even been

speculated that antiangiogenesis might offer a novel means of contraception in

healthy women. Thirdly, reservations regarding impairment of wound healing

apply equally to conventional chemotherapy and radiotherapy. In situations

where wound healing is required, antiangiogenic therapy could be interrupted

or alternatively angiogenic stimulation applied locally.

8. Future Perspectives

The therapeutic inhibition of angiogenesis is clearly an exciting area of

research with potential for improving the care of patients with numerous disor-

ders. However, several issues require further evaluation.

8.1. Optimal Targets and Delivery Systems

Unanswered concerns about clinical importance are stimulating the search

for EC-specific and organ-specific “angiogenic” markers and delivery strate-

gies to obtain more precise selectivity and efficiency. Novel natural and

recombinant polypeptides, nonpolypeptide pharmaceuticals, and gene therapy

approaches are expected to be described for this purpose. Technically, the dif-

20 Zhang and Bicknell

ficulty in the culture and transfection of primary ECs and the lack of useful EC

lines has hampered research in this area.

8.2. Monitoring the Efficacy of Antiangiogenic Therapy

Quantitative assessment of angiogenesis may be made by obtaining the

tumor microvessel density (MVD). High vascular density has been shown to

be a significant prognostic factor for many cancers. It will be possible in future

to monitor the levels in patients of both positive and negative regulators of

angiogenesis and establish an “angiogenic profile” for angiogenesis-associated

diseases. Until now, most, if not all, published studies have focused on positive

regulators such as bFGF and VEGF in the serum, plasma, or urine of patients.

Other angiogenic factors detectable in patients include angiogenin, EGF, aFGF,

HGF, TGF-_, TGF-`, TNF-_ and IL-2, -6, -7, -8, and -10. It is important in future

to also assess the level of natural, endogenous inhibitors of angiogenesis. Angio-

genic profiles will not only increase our understanding of angiogenesis-associated

diseases and permit design of more selective antiangiogenesis therapies and

tailoring of therapies to the needs of individuals, but it will also provide param-

eters for diagnosis, prognosis, monitoring response to therapies, and follow-up.

Equally important is the quantitation of endothelial specific molecules

released into the circulation as a result of EC damage after antiangiogenic treat-

ment. In this regard, soluble E-selectin has been shown to be a useful marker of

therapeutic efficiency in cancer patients treated with CM101 (Group B Strep-

tococcus polyexotoxin) (81).

Other EC products selectively expressed in different vascular environments

include endothelial-specific molecule (ESM-1) (82) and ptx3, a new member

of the pentraxin family (83). These provide hope that specific molecules may

be targeted for therapy and assessed for diagnosis and follow-up.

8.3. Synergy of Antiangiogenesis With Other Therapies

A frequently raised objection is that therapeutic antiangiogenesis might com-

promise the success of other therapeutic treatments. For example, chemothera-

peutic drugs may no longer be effectively delivered to tumors because of the

reduced number of tumor vessels, and the therapeutic efficacy of radiotherapy

could be reduced due to increased tumor hypoxia. Paradoxically, there is good

evidence that antiangiogenesis potentiates chemotherapy and radiotherapy

(reviewed in refs. 3,80,84 ). An explanation of these observations is related to

the reduction in interstitial pressure and the so-called “re-oxygenation” phe-

nomenon that follows the “first wave” of death of sensitive cancer cells

(unpacking) (85). Clearly, no one would envisage that therapeutic inhibition of

angiogenesis will make conventional therapies obsolete, rather that it will aug-

Therapeutic Inhibition 21

ment them. It will certainly extend treatment options for patients who are not

promising candidates for conventional therapies.

9. Conclusions

Clearly, antiangiogenesis now ranks prominently amongst novel and prom-

ising strategies to fight cancer as well as other pathologies. Clinical application

of antiangiogenic strategies now looks an increasingly realistic prospect, given

the plethora of agents with antiangiogenic properties. In particular, a number

of carefully designed clinical trials are underway and it is hoped that answers

to some of the questions raised in this chapter will soon be forthcoming. It is

envisaged that the benefits of antiangiogenic approaches will soon be realized

in other clinical settings aside from oncology.

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