Journal of Bioequivalence & Bioavailability Open Access · 2019. 11. 11. · Matsumoto, 2005;...

Journal of Bioequivalence & Bioavailability - Open Access Expert Review JBB/Vol.1 July-August 2009

J Bioequiv Availab Volume 1(2): 052-062 (2009) - 052

ISSN:0975-0851 JBB, an open access journal

The Matrix Reloaded: New Insights from Type IV Collagen Derived

Endogenous Angiogenesis Inhibitors and their Mechanism of Action

Akulapalli Sudhakar1,2,3,*

1Cell Signaling and Tumor Angiogenesis Laboratory,

Department of Genetics, Boys Town National Research Hospital, Omaha

2 Department of Biomedical Sciences, Creighton University School of Medicine, Omaha

3Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha

*Corresponding author: Akulapalli Sudhakar, MS., M.Phill & Ph.D, Assistant Professor/Staff Scientist II, Creighton

University School of Medicine and University of Nebraska Mecical Center, Director: Cell Signaling and Tumor

Angiogenesis Laboratory, Boys Town National Research Hospital, 555 N, 30th Street, Omaha NE 68131,

USA, Tel: (402) 498-6681, (402) 498-6322; Fax: (402) 498-6331; E-mail: [email protected]

Received August 19, 2009; Accepted August 21, 2009; Published August 22, 2009

Citation: Sudhakar A (2009) The Matrix Reloaded: New Insights from Type IV Collagen Derived Endogenous Angio-

genesis Inhibitors and their Mechanism of Action. J Bioequiv Availab 1: 052-062. doi:10.4172/jbb.1000009

Copyright: © 2009 Sudhakar A. This is an open-access article distributed under the terms of the Creative Commons

Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original

author and source are credited.

Abstract

Angiogenesis, the process of neovascularization from parent blood vessels, is a prerequisite for many physiologi-

cal and pathological conditions that is regulated by a balance between the levels of endogenous angiogenic stimu-

lators and matrix reloaded angiogenic regulators. Several non-collagenous carboxy terminal end domains in

chains of type IV collagen matrix reloaded molecules selectively interact with proliferating endothelial cells by

binding to distinct integrins and regulate intracellular signaling and inhibit angiogenesis. This review will focus on

the current understanding of extra cellular matrix type IV collagen reloaded endogenous angiogenesis inhibitors

and their mechanism of action.

Introduction

An emerging area of research in cell biology is the dis-

covery of extra cellular matrix (ECM) that is reloaded

with proteolytic fragments from the basement membrane,

which exert powerful anti-angiogenic or anti-tumarogenic

activities. Angiogenesis, the process by which new blood

vessels are originated from parent blood vessels, is essen-

tial in normal development and also pathological condi-

tions such as cancer (solid tumor growth), rheumatoid

arthritis and diabetes retinopathy (Marneros and Olsen,

2001; Ortega and Werb, 2002; Sottile, 2004; Sudhakar,

2007). In cancerous conditions, tumor cells need new

blood vessels for nourishment, local growth and escape

to remote organs sites through blood circulation, a pro-

cess called metastasis. Without angiogenesis, within the

tumor, tumor cells cannot stay alive for longer time. For

this reason, matrix reloaded endogenous inhibitors of an-

giogenesis, which might not produce the same sort of re-

sistance and intolerance as exogenous compounds, might

be important candidates for anticancer therapy, either by

themselves or in combination with other inhibitors that

are used routinely in the clinics.

The first discovered ECM reloaded endogenous angio-

genic inhibitory molecule is endostatin, a 20-kDa frag-

ment of heparan sulfate proteoglycan derived from type

XVIII collagen non-collagenous (NC1) domain is best

characterized among endogenous angiogenic inhibitors.

Since initial discovery of endostatin as endogenous an-

giogenesis inhibitor, there has been an outburst in anti-

angiogenic research and in the number of endogenous

molecules that are known to promote or inhibit angiogen-

esis (Iozzo and San Antonio, 2001; Marneros and Olsen,

2001; Ortega and Werb, 2002; Schenk and Quaranta, 2003;

Sottile, 2004; Sudhakar, 2007) (Figure 1). Several endog-

enous anti-angiogenic protein fragments were discovered

that were derived from ECM constituents. Although they

are structurally different they have remarkable similari-

α




ties that must be more than coincidental. More recently,

type IV collagen α1, α2, α3 and α6 chain carboxy ter-

minal NC1 domains were demonstrated to be anti-angio-

genic. These molecules that are needed to assemble vas-

cular basement membrane (VBM) to maintain the integ-

rity of blood vessels and to prevent the leakage of fluids

and loss of proteins can do the opposite actions under the

different circumstances (called matrix reloaded); that is,

they have both pro-angiogenic and anti-angiogenic ac-

tivities (Iozzo and San Antonio, 2001; Nakamura and

Matsumoto, 2005; Sudhakar and Boosani, 2008).

Therefore, matrix reloaded endogenous type IV collagen

derived angiogenic inhibitors mechanism of action are of

high significance and is updated in this review.

Structural Integrity and Biological Insights of Type IV

Collagen Matrix

The discovery of endostatin as an endogenous angio-

genesis inhibitor initiated researchers to hasten their in-

vestigation and to find other ECM derived components

that are antiangiogenic and anti-tumorogenic with the po-

tential to modulate angiogenesis. Type IV collagen is

ECM-specific and abundant collagen with various

isoforms whose network assembly is essential for the struc-

tural integrity and biological function of basement mem-

branes (Myllyharju and Kivirikko, 2004). Type IV col-

lagen has six α chains and exists in atleast three

heterotrimeric triple helical forms [α1(IV)]2α2(IV),

[α3(IV)]2α4(IV) and [α5(IV)]

2α6(IV) (Hudson et al.,

1993; Hudson et al., 2003; Kalluri, 2003). Each α(IV)

chain has a long, collagenous domain with Gly-X-Y re-

peats (X and Y represent any amino acids other than gly-

cine, but are often proline and hydroxyproline, respec-

tively) interrupted by short NC sequences, an N-terminal

cysteine-rich 7S domain and a C-terminal NC1 domain

of 230 amino acids. These superstructures self-associate

from triple helical monomers to form either dimers (via

NC1–NC1 interactions) or tetramers (via 7S-domain in-

Figure 1: The angiogenic balance between angiogenic activators and angiogenic inhibitors regulate vascular homeosta-

sis. Angiogenesis under physiological and pathological conditions is associated with up-regulation of angiogenic factors

and/or down-regulation of angiogenic inhibitors. Up-regulation of angiogenic inhibitors and/or down-regulation of an-

giogenic activators may be associated with impaired neovascularization capacity. VEGF, vascular endothelial growth

factor; bFGF, basic fibroblast growth factor; IGF-I, insulin-like growth factor-I; IL-8, interleukin-8; PDGF, platelet-

derived growth factor; PlGF, placenta growth factor; TGF-α and β, transforming growth factor-α and β.




teractions) with 56 possible combinations (Kalluri, 2003;

Sudhakar and Boosani, 2008).

Type IV collagen molecules that contain different α(IV)

chains have different cellular interactions and localiza-

tions. Furthermore, proteolysis of ECM might expose cells

to new, cryptic interaction sites within type IV collagen

(Kalluri, 2003). The α1(IV) and α2(IV) chains are ubiq-

uitous and [α1(IV)]2α2(IV) heterotrimers are predomi-

nant, whereas α3(IV) chain expression is limited. Inter-

estingly, in many patients with Alport’s syndrome (either

X-linked autosomal recessive hereditary kidney disease

that is characterized by a defect in glomerular BM) muta-

tions in α5(IV) chain led to loss of α3(IV) chain which

indicates that their expression is co-dependent (Cosgrove

et al., 1996; Hudson et al., 2003). Type IV collagen is

highly conserved, structurally and functionally in verte-

brates and in invertebrates (Blumberg et al., 1987; Netzer

et al., 1998). Normally, type IV collagen promotes cell

adhesion, differentiation, migration and growth (Sarras et

al., 1993). In C. elegans two genes encoding type IV col-

lagen have been identified, emb-9 (α1-like) and let-2 (α2-

like). Mutations in these two genes might cause either in-

hibited embryonic development or variably severe tem-

perature-sensitive mutants (Myllyharju and Kivirikko,

2004). In Drosophila, type IV collagen genes DCG1 and

viking are involved in muscle attachment and pericardin

is involved in development of heart. A mutation in α1(IV)

was identified with perinatal cerebral hemorrhage stroke,

porencephaly, endoplasmic reticulum stress and geneti-

cally modifiable ocular dysgenesis (Gould et al., 2007;

Gould et al., 2005; Gould et al., 2006). In humans or in

mice, no specific disease is yet linked to mutations in

α2(IV) chains.

Type IV Collagen Matrix Reloaded Angiogenesis In-

hibitors and their Mechanism of Action

Arresten: Arresten (α1(IV)NC1), a 26-kDa protein is

derived from C-terminal NC1 domain of α1 chain type

IV collagen by proteases (Boosani and Sudhakar, 2006;

Colorado et al., 2000; Sudhakar et al., 2005). Not much is

known about this angiogenesis inhibitor but its anti-

antiangiogenic actions are presumably mediated through

α1β1 integrins (Sudhakar et al., 2005). Integrin α1β1 is a

collagen binding receptor that also binds to other base-

ment membrane components such as laminin (Keely et

al., 1995; Zutter and Santoro, 1990). Blocking of α1β1

integrin interactions with ECM inhibits angiogenesis in-

dicating that the integrin α1β1 acts as a pro-angiogenic

receptor (Senger et al., 1997). Among the integrin recep-

tors for collagen, α1β1 integrin activates the Ras/Shc

mitogen activated protein kinase (MAPK) pathway pro-

moting cell proliferation (Senger et al., 1997; Sudhakar et

al., 2005). Arresten binds to α1β1 integrin in a collagen

type IV dependent manner and mediates its anti-angio-

genic and pro-apoptotic functions and inhibits angiogen-

esis by inhibiting endothelial cell proliferation, migration

and tube formation (Boosani et al., 2009; Nyberg et al.,

2008; Sudhakar et al., 2005). Significant halt in patho-

logical angiogenesis and tumor growth was reported in

α1 integrin knockout mice (Pozzi et al., 2000). Whereas

arresten had no effect in α1 integrin null endothelial cells,

on the contrary it significantly inhibited proliferation of

wild type mouse endothelial cells, which confirms the sig-

nificance of integrin α1β1 mediated signaling of arresten

(Sudhakar et al., 2005). Arresten inhibits phosphorylation

of FAK/Ras/Raf/MEK1/2 and p38 MAPK when endot-

helial cells are plated on collagen type IV matrix (Figure

2). Similar inhibition of MAPK signaling was not observed

with arresten treatment to α1 integrin null endothelial cells

(Sudhakar et al., 2005). Downstream to FAK, Akt/PKB

plays an important role in endothelial cell survival signal-

ing. Arresten does not inhibit Akt or phosphatidyl-3-ki-

nase (PI3 kinase) phosphorylation suggesting that arresten

regulates migration of endothelial cells in an Akt-inde-

pendent manner (Sudhakar et al., 2005).

Interestingly proteolysis of ECM reloaded arresten in-

hibits hypoxia (lack of oxygen) inducible factor alpha

(HIF-1α) and VEGF expression in endothelial cells in an

α1β1 and FAK/Ras/Raf/MEK1/2 and p38 MAPK depen-

dent manner (Sudhakar et al., 2005) (Figure. 2). HIF-1α

is an oxygen-dependent transcriptional factor which plays

prominent roles in tumor angiogenesis (Semenza, 2003).

HIF-1α regulates cellular responses to physiological and

pathological hypoxia, and studies so far demonstrate that

HIF-1α is a potential target to inhibit tumor angiogenesis

(Unruh et al., 2003). HIF-1α transcriptionally regulates

VEGF expression in hypoxic cells and promotes angio-

genesis in solid tumors (Carmeliet et al., 1998; Kung et

al., 2000; Sudhakar et al., 2005). These findings suggest

that HIF-1α is a prime target for anticancer therapies and

this hypoxic inhibitory activity might be exploited for

antiangiogenic therapy in the treatment of different solid

tumor cancers, but more pre-clinical laboratory studies

are needed.

In addition, recently we also reported that arresten in-

hibits VEGF mediated angiogenesis by promoting

apoptosis in different endothelial cells. Arresten activates

caspase-3/PARP [Poly (ADP-ribose) polymerase] activa-

tion and negatively impacts FAK/p38-MAPK phospho-

rylation via down-regulation of anti-apoptotic Bcl-2 and




Figure 2: Schematic illustration of distinct antiangiogenic and anti-tumorogenic signaling mediated by different type IV

collagen matrix reloaded molecules. Tumstatin, arresten and canstatin interact with αVβ3/α3β1, α1β1 and αVβ3/αVβ5

integrins, respectively, to inhibit the phosphorylation of focal adhesion kinase (FAK). Tumstatin: It binds to αVβ3 and

3β1 integrins and inhibits the pathway that includes phosphorylation of FAK, PI3-K, Akt, mTOR, 4E-BP1 and eIF4E to

decrease endothelial cell protein synthesis and proliferation. In addition tumstatin also inhibits NFκB mediated signaling

in hypoxic conditions leading to the inhibition of COX-2, VEGF and bFGF expressions, resulting in inhibition of hy-

poxic tumor angiogenesis. Arresten: It binds to α1β1 integrin and inhibit phosphorylation FAK, causes inhibition of Ras,

Raf, extra cellular signal related kinase 1 (ERK1) and p38 MAPK pathways that leads to inhibition of HIF-1α and VEGF

expression resulting in inhibition of endothelial cell migration, proliferation and tube formation. In addition arresten

initiates two apoptotic pathways, involving activation of caspase-9 and -8, leading to activation of caspase-3 and PARP

cleavage. (a) Arresten activates caspase-3 directly through inhibition of FAK/p38-MAPK/Bcl-2/Bcl-xL and activation of

caspase-9; (b) Integrin α1β1 cross talk with Fas-L through mitochondrial pathway and leads to activation of caspase-8

and-3 in proliferating endothelial cells. Canstatin: It binds to αVβ3/αVβ5 integrins and inhibits two apoptotic pathways,

involving activation of caspase-8 and casoase-9, leading to activation of caspase-3. Canstatin activates procaspase-9 not

only through inhibition of the FAK/PI3K/AKT pathways but also by integrins cross talking mitochondrial pathway

through Fas-L dependent caspase-8 activation leads to endothelial cell apoptosis. CM represents cell membrane.




Bcl-xL expressions with no effect on pro-apoptotic Bax

expression leading to endothelial cell death (Boosani et

al., 2009; Nyberg et al., 2008). These Bcl-family mem-

bers are considerably involved in the balance of pro and

anti-apoptotic signals at the mitochondrial level. The bal-

ance of pro and anti-apoptotic protein expression deter-

mines whether cells undergo apoptosis (Cory et al., 2003).

These studies demonstrate that activation of apoptotic sig-

naling in proliferating endothelial cells is sufficient to in-

hibit new blood vessel formation. These results are in con-

sistent with earlier reports that anti-angiogenic and anti-

tumorogenic activity of arresten is mediated via integrin

cross talk with a cell-intrinsic apoptotic pathway (Boosani

et al., 2009) (Figure 2). The endothelial specific inhibi-

tory actions of ECM reloaded arresten may be of benefit

in the treatment of a variety of diseases with a neovascular

component.

Canstatin: Proteolytic degradation of type IV collagen

liberates a 24-kDa protein fragment from C-terminal NC1

domain of α2 chain, called canstatin [α2(IV)NC1] that

was reported to inhibit tumor angiogenesis and tumor

growth (Kamphaus et al., 2000; Petitclerc et al., 2000).

Canstatin binds to endothelial and tumor cells in an αVβ3

and αVβ5 integrin dependent manner (Magnon et al.,

2005). Canstatin competes with type IV collagen of ECM

for cell surface integrin binding and reverses the

proliferatory and migratory effects induced by cell-ECM

interactions. Thus, αVβ3 and αVβ5 integrins appear to

mediate the antiangiogenic and pro-apoptotic actions of

canstatin (Magnon et al., 2005; Panka and Mier, 2003).

Canstatin inhibits the growth of many tumors in mouse

xenograft models and histological studies revealed de-

creased CD31 positive vasculature. Canstatin binds to

αVβ3 and αVβ5 integrins and inhibits endothelial migra-

tion and proliferation (Kamphaus et al., 2000; Magnon et

al., 2005; Panka and Mier, 2003; Roth et al., 2005). More-

over, these events are mediated by inhibiting phosphory-

lation of FAK, Akt, mammalian target of rapamycin

(mTOR), eukaryotic initiation factor 4E binding protein-

1 (4E-BP1) and ribosomal S6 kinase (Panka and Mier,

2003). Canstatin binds to αVβ3 and αVβ5 integrins and

initiates two apoptotic pathways that includes (a) activa-

tion of caspase-8 and -9 and leads to activation of caspase-

3 and (b) activation of caspase-8 by downregulation of

Flip levels (Magnon et al., 2005; Narazaki and Tosato,

2006). Upregulation of Fas/Fas ligand triggers not only

cell death directly through caspase-3 activation but also

indirectly through mitochondrial damage via activation of

caspase-9 within the apoptosome (Magnon et al., 2005;

Magnon et al., 2008; Wang et al., 2008). On the other

hand, phosphorylated FAK/PI3K is known to inactivate

the mitochondrial apoptotic pathway by inhibition of

caspase-9 (Figure. 2). Canstatin directly activates

procaspase-9 through inhibition of FAK/PI3K pathway

and amplifies the Fas-dependent pathway in mitochondria.

An intravitreous injection of canstatin causes selective

apoptosis in endothelial cells resulting in inhibition of

neovascularization when given prior to the onset of new

vessel sprouting (Magnon et al., 2005; Roth et al., 2005).

Importantly, when canstatin was given after

neovascularization had already developed, it caused the

new vessels to regress.

In tumor cells canstatin activates caspase-3 only in the

mitochondrial pathway (Figure. 2). Canstatin suppresses

tumor angiogenesis in different mice xenograft models

and also laser induced choroidal nonvascular (CNV) in

mice, indicating it as a strong antiangiogenic agent in the

choroid and as a therapeutic candidate for treatment of

CNV in age related macular degeneration (Lima et al.,

2006). These results demonstrate that canstatin binds to

αVβ3 and αVβ5 integrins and inactivates FAK down

stream signaling leading to inhibition of cell proliferation

and migration, and thus leading to activation of apoptosis

and inhibition of tumor angiogensis.

Tumstatin: Physiological proteolysis of α3 chain type

IV collagen by MMP-9 results in tumstatin (α3(IV)NC1)

a 28-kDa C-terminal NC1 domain (Hamano et al., 2003).

The anti-angiogenic activity of tumstatin was observed

with pharmacological doses of this protein associated with

inhibition of protein synthesis in endothelial cells

(Maeshima et al., 2002; Sudhakar et al., 2003). The ex-

pression of tumstatin is down-regulated in renal carcinoma

growth (Xu et al., 2009). Inhibition of melanoma and fib-

rosarcoma cell migration using tumstatin peptide (185-

205 amino acids) was reported with a decrease in expres-

sion of membrane-bound metalloproteinase (MT1-MMP)

and activated MMP-2 (Han et al., 1997). Tumstatin in-

hibits tube formation in mouse aortic endothelial cells

embedded in Matrigel plugs and inhibits tumor growth in

different mouse models [renal cell carcinoma (786-O),

CT26 (colon adenocarcinoma), prostate carcinoma (PC3),

lewis lung carcinoma (LLC), human lung cancer (H1299),

human prostate cancer (DU145), human fibrosarcoma

(HT1080), teratocarcinoma (SCC-PSA1) and lymphatic

metastasis in an orthotopic oral squamous cell carcinoma

model] (Boosani et al., 2007; Chung et al., 2008; Hamano

et al., 2003; Maeshima et al., 2000b; Miyoshi et al., 2006;

Pedchenko et al., 2004; Petitclerc et al., 2000; Sudhakar

and Boosani, 2008). In addition tumstatin gene therapy




enhanced anti-tumor effect of gemcitabine in murine mod-

els (Yao et al., 2005). But what integrins are engaged in

these antiangiogenic and anti-tumorogenic actions of

tumstatin? It is clear that different integrins are key tar-

gets of tumstatin to mediate its antiangiogenic and anti-

tumorogenic actions. Tumstatin interacts with endothelial

cell via αVβ3 integrin in a vitronectin and RGD indepen-

dent fashion (Maeshima et al., 2000a). Integrin αVβ3 in-

teracts with tumstatin through two separate regions. The

first region 54-132 amino acids is involved in the

antiangiogenic activity, whereas the second region 185-

203 amino acids is involved in anti-proliferative activity

in cancer cell lines (Boosani et al., 2007; Eikesdal et al.,

2008; Maeshima et al., 2000a; Pedchenko et al., 2004).

These results substantiate exact regulatory sub-domains

in tumstatin controlling adhesion and proliferation in vari-

ous cell types. The functional specificity of these two sub

domains from tumstatin in cancer or endothelial cells is

very interesting. Indeed published 3D crystal structure of

type IV collagen NC1 domain reveals N and C-homolo-

gous sub-domains (Than et al., 2002). The major differ-

ence between these sub-domains for each chain is in the

region from residues 86-95 in the N-sub-domain and 196-

209 in the C-sub-domain. These regions overlap two se-

quences that were previously identified to be having

antiangiogenic and anti-proliferative effects in cancer cells

(Borza et al., 2006). Tum-1, a tumstatin peptide, gene de-

livery into hepatocellular carcinoma cells suppressed tu-

mor growth by inhibiting angiogenesis (Goto et al., 2008).

Tumstatin or its peptides interaction with integrins seems

to be involved in the disruption of contacts between en-

dothelial or tumor cells leading to apoptosis in these cells,

but the mechanism of apoptosis activation is still not clear.

Tumstatin also binds to endothelial cells through other

integrins α6β1 and αVβ5, but the significance of these

integrins interaction is not yet clear (Boosani et al., 2007;

Maeshima et al., 2000a).

Tumstatin induces endothelial cell apoptosis by inter-

acting with integrin αVβ3 and inhibits VEGF adhesion to

matrix, and this effect was overturned by integrin αVβ3

blocking antibody. The antiangiogenic activity of tumstatin

is conferred by its interaction with integrin αVβ3 and in-

hibiting activation of focal adhesion kinase (FAK),

phosphatidylinositol 3-kinase (PI-3K), serine/threonine

kinase (Akt/protein kinase B), mammalian target of

rapamycin (mTOR) and prevents dissociation of eukary-

otic translation initiation factor 4E (eIF4E) from 4E bind-

ing protein (4E-BP1) leading to the inhibition of Cap-de-

pendent translation in proliferating endothelial cells

(Maeshima et al., 2002; Sudhakar et al., 2003) (Figure.

2). PTEN/Akt pathway dictates the direct αVβ3 integrin

dependent growth inhibitory action of T7 peptide of

tumstatin in glioma cells in-vitro and in-vivo (Kawaguchi

et al., 2006). Furthermore, these results conforms a spe-

cific function for integrins in mediating endothelial cell

specific inhibition of cap-dependent translation, signify-

ing a possible specific mechanism of tumstatin. Whereas

α3β1 integrins bind to C-terminal region 185-203 resi-

dues associated with antiangiogenic and anti-tumorogenic

activity (Borza et al., 2006), 17426256}. These results

compare earlier findings that tumstatin binds to α3β1

integrin and transdominantly inhibits expression of αVβ3

integrin (Boosani et al., 2007). Interestingly recent stud-

ies clearly show tumor suppressive action of tumstatin or

its peptide (T3; C-terminal end comprising amino acid

residues 133-244 region of the tumstatin) that directly

inhibits growth of glioma cells (Kawaguchi et al., 2006).

In addition a cyclopeptide derived from tumstatin

(YSNSG) was also shown to inhibit human melanoma

cell proliferation (Thevenard et al., 2006).

Recently we discovered that tumstatin inhibits hypoxia

induced cyclo-oxygenase-2 (COX-2) expression in endot-

helial cells via FAK/Akt/NFkB (nuclear transcription fac-

tor-kappa B) pathway leading to decreased

tumorangiogenesis and tumor growth in a α3β1 integrin

dependent manner (Figure 2). In addition to COX-2 inhi-

bition, the down stream VEGF and bFGF protein expres-

sion was also inhibited upon tumstatin treatment to en-

dothelial cells (Boosani et al., 2007). Moreover, research-

ers have confirmed that inhibition of COX-2 signaling

serves as a therapeutic benefit in different cancer models

and potential target for tumor angiogenesis (Boosani et

al., 2007; Harris, 2002). These findings indicate that there

may be several targets for the inhibitory effects of tumstatin

in tumor-angiogenesis. All these studies support that the

antiangiogenic and anti-tumorogenic activity of tumstatin

is mediated through αVβ3 and α3β1 integrins. Astonish-

ingly, aberrant tumor growth and tumor vasculature is

observed in tumstatin knockout mice, demonstrating that

tumstatin is playing a prominent role in pathological an-

giogenesis to decrease tumor progression (Hamano and

Kalluri, 2005; Hamano et al., 2003). However in a con-

trolled physiological angiogenic process such as wound

healing, tumstatin does not affect the physiological an-

giogenesis and /or neovascularization. The amino acids

residues 185-191 that contain the sequence CNYYSNS

in which the YSNS motif forms a β-turn have the same

anti-tumor activity compared to intact tumstatin

(Thevenard et al., 2006). Furthermore, replacing Cys185

with Asp, but not Met prevents the anti-tumor activity,

which indicates the importance of a sulfur atom in its struc-

ture and function (Thevenard et al., 2006; Thevenard et




al., 2009). Although tumstatin is efficient in reducing

tumorangiogenesis and /or tumor neovascularization, its

exact role needs to be deciphered.

Signaling Convergence and Complementarities Be-

tween Arresten, Canstatin and Tumstatin

The carboxy terminal localization might be necessary

for different proteases to access and liberate the

antiangiogenic domains from type IV collagen that are

embedded in the ECM. These protease-generated domains

exert their effects by binding to different integrins that

help modulate how cells interact with and are affected by

matrix components. The functional integrin receptor for

each anti-angiogenic molecule liberated from type IV col-

lagen are different and specific; α1β1 for arresten, αVβ5/

αVβ3 for canstatin and αVβ3/α3β1/αVβ5/α6β1 for

tumstatin. This indicates that the mechanism of integrin

receptors engagement and downstream signaling mecha-

nism might also be different and specific for matrix re-

loaded endogenous angiogenesis inhibitors, although some

common intracellular signaling components vary between

these angiogenic inhibitors. Arresten and canstatin seem

to exert their effects on the endothelial cytoskeleton that

is essential for the cells to migrate and form capillary like

structures. Canstatin and tumstatin function by inhibiting

protein synthesis and growth and survival of proliferating

endothelial cells. Arresten, canstatin and tumstatin can

directly affect the integrin receptor complement and have

functional consequences (Table 1). Finally, these three

antiangiogenic domains are different structurally, which

explains why they interact with different integrins and have

different effects on angiogenesis. By limiting our focus to

these three antiangiogenic domains, we hope to highlight

similarities that might also apply to other undiscovered

antiangiogenic ECM fragments and further our under-

standing of angiogenesis inhibition with potential thera-

peutic implications against cancer.

Significance of Bioavailability of ECM Reloaded En-

dogenous Angiogenesis Inhibitors

At present there are several ECM derived endogenous

angiogenesis inhibitors in pre-clinical trails including type

IV collagen derived arresten, canstatin and tumstatin

(Table 1). These endogenous matrix reloaded molecules

were found available in patients at nano molar levels.

Owing to their strong anti-angiogenic and anti-

tumorogenic actions and their availability in meager quan-

tities, these endogenous molecules have been synthesized,

expressed and purified from different heterologus systems

to increase their bioavailability and to treat patients with

neovascular diseases such as cancer, rheumatoid arthritis

and diabetic retinopathy.

Concluding Remarks

Angiogenesis is a dynamic process. In-vivo, endogenous

molecules have been identified that stimulate, inhibit and

modulate this process. A common, revolutionary theme

has gradually emerged for endogenous, ECM derived anti-

Table 1: Type IV collagen matrix reloaded angiogenesis inhibitors and their mechanisms of action.

Angiogenesis

inhibitors name Arresten Canstatin Tumstatin

Origin α1 chain Type IV collagen NC1 domain

α2 chain Type IV collagen NC1 domain

α3 chain Type IV collagen NC1 domain

Generated Matrix metalloprotensae-9 Matrix metalloprotensae-9 and-2 Matrix metalloprotensae-9,-3 and -13

Integrin

binding α1β1,? αVβ3, αVβ5,? αVβ3, α3β1, α6β1, αVβ5,?

Proliferation Inhibits endothelial

proliferation Inhibits endothelial proliferation Inhibits endothelial proliferation

Migration Inhibits endothelial

migration Inhibits endothelial migration No effect on endothelial migration

Tube formation Inhibits endothelial tube

formation

Inhibits endothelial tube

formation Inhibits endothelial tube formation

Antiangiogenic

activity

Inhibits endothelial

migration and promoted

apoptosis

Inhibits protein synthesis by

activating endothelial apoptosis

Inhibits endothelial growth and

protein synthesis

Pathways

affected

FAK/MAPK/HIF1α and FasL/Bcl-2/Bcl-xL mediated

signaling

FAK/Akt/PI3/mTOR/eIF-4E/4E-

BP1 and FasL mediated

signaling

FAK/Akt/PI3K/mTOR/eIF-4E/4E-

BP1 and NFkB/COX-2 mediated

signaling




angiogenic molecules: C-terminal end fragments of many

type IV collagen matrix exert their inhibitory effects (at

least in part) by interacting with integrins. It is feasible

that proteases that degrade ECM or VBM and enable en-

dothelial cell migration during angiogenesis also gener-

ate C-terminal anti-angiogenic peptides. In pathological

conditions such as cancer, anti-angiogenic signals might

be overwhelmed by pro-angiogenic (growth factors) sig-

nals that are produced by tumor cells.

Arresten canstatin and tumstatin are antiangiogenic and

are also potent anti-tumorogenic molecules that are de-

rived from the C-terminal end of type IV collagen seem

to have diverse complementary effects. In addition to di-

rect functional interactions among these type IV collagen

NC1 domains, the antiangiogenic activity of these NC1

domains is modulated by partially overlapping signal

transduction pathways. However, till date several ques-

tions remain to be answered and numerous issues need to

be solved like how these antiangiogenic NC1 domains

are reloaded from matrix? Are these domains regulating

different signaling mechanisms? Is it significant that these

domains are C-terminally derived? Are there any other

cellular receptors for these domains in addition to cell

surface integrins? To what extent does the overlap in sig-

naling affect the anti-angiogenic potencies of the indi-

vidual domains in-vivo? Are the physiological blood lev-

els of these domains generated from type IV collagen (all

are in nano-molar range) sufficient to exert anti-tumor

activity? Is the presence of these domains in the circulat-

ing blood a mere indicator of turnover of the ECM re-

loaded? Should their in-vivo activity be viewed as a phar-

macological effect? Most importantly, will any of these

collagen type IV derived endogenous angiogenesis inhibi-

tors be a successful clinical tool in the fight against can-

cer and other vascular diseases such as choroidal

neovascularization of age related macular degeneration?

Whatever the final results, by recognizing the common-

alities of type IV collagen ECM derived endogenous anti-

angiogenic domains, we should gain invaluable, clinically

significant insights for optimal therapeutic interventions.

Acknowledgements

This work was supported by Flight Attendant Medical

Research Institute Young Clinical Scientist Award Grant

FAMRI 062558 (AS); Dobleman Head and Neck Cancer

Institute Grant 61905 (AS); startup funds of Cell Signal-

ing and Tumor Angiogenesis Laboratory at Boys Town

National Research Hospital (AS). I thank Dr. Chandra S.

Boosani for critical reading of the review.

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TitleAuthorsAffiliationsCorresponding authorDatesCitationCopyright

AbstractIntroductionStructural Integrity and Biological Insights of Type IVCollagen MatrixType IV Collagen Matrix Reloaded Angiogenesis Inhibitorsand their Mechanism of ActionCanstatinTumstatin

Signaling Convergence and Complementarities BetweenArresten, Canstatin and TumstatinSignificance of Bioavailability of ECM Reloaded EndogenousAngiogenesis InhibitorsConcluding Remarks

AcknowledgementsFiguresFigure 1Figure 2

TablesTable 1

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