Leukocytic-Vascular Endothelial Growth Factor and Integrin ...

THE EGYPTIAN JOURNAL OF IMMUNOLOGY Vol. 15 (2), 2008 Page: 81-91

Leukocytic-Vascular Endothelial Growth Factor and Integrin αvß3 in Acute Myeloid Leukemia: Relation to Clinical Outcome

1Lobna A. Abou-Shamaa and 2Gihan N. Mahmoud

Departments of 1Immunology, 2Hematology, Medical Research Institute, Alexandria University, Alexandria, Egypt.

Different signaling routes seem to be simultaneously triggered in leukemia, with distinct and overlapping activities. Different reports emphasize the interaction between vascular endothelial growth factor (VEGF) and integrin αvß3 as a key control system of angiogenesis, oncogenesis and metatasis. The current study was undertaken to investigate leukocytic-VEGF and integrin αvß3 as correlated with clinical outcome in patients with acute myeloid leukemia (AML). The study groups included 10 newly diagnosed AML patients before the start of any chemotherapeutic medication and 10 normal healthy control subjects. The level of VEGF was estimated in culture supernatant of peripheral blood mononuclear cells (PBMN) of both groups using commercially available ELISA kit. The degree of integrin αvß3 expression on PBMN was estimated by indirect immunoflourescence. Obtained results showed that the level of VEGF and degree of expression of integrin αvß3 were significantly higher in AML patients than in normal healthy subjects. However, no significant correlation was observed between the levels of VEGF and the degree of expression of integrin αvß3.When clinical findings were concerned, there was a significant positive correlation between VEGF and the percentage of blasts, both in peripheral blood & bone marrow. On the other hand, such correlations were not observed in case of integrin αvß3. In addition no significant correlation was observed between either VEGF or integrin αvß3 and clinical staging, age, and sex. In conclusion, our results proved the importance of VEGF and integrin αvß3 in the pathogenesis of AML. However, the per se increased production or/and secretion of VEGF and integrin αvß3 by leukemic PBMN cells, respectively can not be used as independent predictor (s) for clinical outcome in AML patients. It is more comprehensive to study changes of intracellular signaling pathways when such critically interacting factors are concerned in the leukemic process.

cute myelocytic leukemia (AML) is a

malignant neoplasm of hematopoietic

cells characterized by an abnormal

proliferation of myeloid precursor cells,

decreased rate of self-destruction and an arrest

in cellular differentiation. The leukemic cells

have an abnormal survival advantage. Thus,

the bone marrow and peripheral blood are

characterized by leukocytosis with a

predominance of immature cells, primarily

blasts. As the immature cells accumulate in

the bone marrow, they replace the normal

myelocytic cells, megakaryocytes, and

erythrocytic cells. This leads to a loss of

normal bone marrow function and associated

complications of bleeding, anemia, and

infection (Löwenberg et al., 1999).

The endothelium forms a network of

vasculature spanning the whole cavity of the

bone marrow, thus providing a blood supply

like in solid organs (Mazo et al., 1999).

Accordingly, increased bone marrow

angiogenesis, the process of new blood vessel

formation from endothelial precursors, is

crucial for the pathogenesis and progression

of AML and other hematological

malignancies similar to solid invasive tumors

(Aguayo et al., 2000). A critical event in the

regulation of angiogenesis is the signaling

cascade involving vascular endothelium

growth factor (VEGF-A, VEGF), which is a

multifunctional, secreted cytokine that

stimulates endothelial cells to proliferate, to

migrate, and to increase their permeability to

plasma proteins, (Karamysheva, 2008). The

ability of this factor to enhance vascular

permeability defines its important role in

tumor cell penetration into vascular networks

A

Leukocytic-VEGF and Integrin αvß3 in AM 82

and metastasis (Sierra, 2005). VEGF exists as

five different isoforms of 121, 145, 165, 189

and 206 amino acids (Neufeld et al., 1996).

On adult endothelial cells it exhibits high-

affinity binding sites corresponding to two

distinct tyrosine kinase receptors, the VEGF

receptor (VEGFR)-1 encoded by Flt-1 and

VEGFR-2 encoded by KDR/ Flk-1 with much

greater binding affinity to VEGFR1.

(Robinson et al., 2001). It now appears that

VEGF also has autocrine functions acting as a

survival factor for tumour cells protecting

them from stresses such as hypoxia,

chemotherapy and radiotherapy. The

mechanisms of action of VEGF are still being

investigated with emerging insights into

overlapping pathways and cross-talk with

other receptors (Byrne et al., 2005).

Adhesion molecules, including integrins,

mediate critical cytosolic signaling events that

regulate both physiologic and pathologic

events, including complex processes such as

leucocyte migration, angiogenesis, tumor

growth, and metastasis (Moschos et al.,

2007). Several examples of crosstalk between

integrins and growth factor receptors indicate

that integrin ligation is required for growth

factor–induced biological processes.

Furthermore, integrins can directly associate

with growth factor receptors, thereby

regulating the capacity of integrin/growth

factor receptor complexes to propagate

downstream signaling (reviewed in Eliceiri,

2001). Integrin αvß3 (leukocyte response

integrin) is the cell surface receptor for

vitronectin, fibronectin, fibrinogen, and

denatured collagen and is widely distributed

on monocytes, vascular endothelial cells and

tumor cells (Capo et al., 1999; Kerr et al.,

2002; Kumar, 2003). It plays an important

role in regulation of cell growth,

differentiation, migration, and angiogenesis.

The adhesion of integrin αvß3 to the ECM

proteins induces focal adhesion contacts

which generate the cascade of

phosphorylation of many signal transduction

molecules, including tyrosine kinases (FAK,

pp60src), cytoskeletal proteins (vinculin,

paxillin), Grb-2, and MAP kinase (reviewed

in Hodivala-Dilke K. 2008). Previous reports

have provided evidences for the interaction

between VEGF and integrin αvß3 as regards

tumor progression (De et al., 2005; Soldi et

al., 1999). So, in the current study we

investigated leukocytic-VEGF and integrin

αvß3 as related to clinical outcome in patients

with AML.

Subjects and Methods

The present study was conducted on ten newly

diagnosed AML patients before the start of any

chemotherapeutic medication. Ten normal healthy

subjects were included as a normal control group.

Patients were obtained from Hematology Department,

Medical Research Institute, University of Alexandria.

Patients were subjected to detailed history taking and

thorough clinical examination with special emphasis on

lymph nodes, liver and spleen.

Diagnosis of AML based on standard morphology

and cytochemistry of peripheral blood and bone

marrow films according to the French-American-

British (FAB) criteria (Bennett et al., 1985). Diagnosis

was confirmed by immunophenotyping using a

comprehensive panel of monoclonal antibodies against

myeloid associated antigens as proposed by the EGIL

group (Bene et al., 1995).

Routine Hematological Investigations

Complete blood picture including hemoglobin

concentration, platelet count as well as total and

differential leukocytic counts was done using

automated blood cell counter (Hema Star II). Films

from fresh blood samples and bone marrow aspirate

were stained by Leishmann stain and cytochemical

staining to confirm the differential blood leukocytic

count and detect abnormal leukocyte morphology and

characteristic abnormalities in the bone marrow (Bain

et al., 2001).

Cell Culture

Peripheral blood mononuclear cells (PBMN) and

leukemic blast cells were isolated from normal control

subjects and AML patients, respectively. Simply,

heparinized venous blood samples were centrifuged

over ficoll-hypaque density gradient (Sigma) at 1800

rpm for 45 min., washed three times with saline (NaCl

0.9%), and resuspended in RPMI-1640 medium

(Gibco, UK) supplemented with 10% fetal calf serum,

THE EGYPTIAN JOURNAL OF IMMUNOLOGY 83

100 IU/mL penicillin, 100 mg/mL streptomycin and 2

mM glutamine. Viability of the isolated cells was tested

using dye exclusion technique which is based on the

impermeability of viable cells to trypan blue.

Resuspended cells were cultured in 96 wells flat bottom

polystyrene culture plates (2x105

cells/well) and

stimulated with 20µg/ml phytohaemagglutinin (PHA;

Sigma). After incubation for 72 hrs in a humidified 5%

CO2 incubator at 37°C, culture supernatant was

collected and stored at -70°C till time of assay for

VEGF (Fiedler et al., 1997).

Measurement of Leukocytic-VEGF-A (VEGF)

The level of VEGF was measured in culture

supernatant using commercially available enzyme

linked immunosorbent assay [ELISA] kit [Human

VEGF-A ELISA Kit, Bender MedSystems, Austria]

and following manufacturer recommendations. The

sensitivity of the applied ELISA kit is 20 pg/ml.

Concentrations were deduced from a manually

constructed standard curve using supplied standard

concentrations and corresponding optical densities.

Expression of Leukocytic-Integrin αvß3

The degree of expression of integrin αvß3 on PBMN

was assessed by indirect immunoflourescent technique

as previously described (Ylänne et al., 1990) with mild

modification. Briefly, PBMN cells were attached to

glass slides by cytospin centrifugation and fixed in

100% ethanol. PBMN monolayers were washed in PBS

(pH 7.4) containing 0.2% bovine serum albumin and

then incubated with the first monoclonal antibody

[1/100, mouse anti-human integrin alphavbeta3,

Chemicon International, Austria] for 45 min at room

temperature. After washing with PBS, pH 7.4, samples

were incubated with goat anti-mouse IgG coupled to

fluorescein isothiocyanate [1/50, Goat-anti-mouse IgG

(Fc specific), FITC conjugate, Sigma] for 45 min at

room temperature in the dark. Slides were then washed

in PBS (pH 7.4) and mounted in glycerol-PBS (9–1).

Expression was scored blindly [(+), (++), and (+++)]

by fluorescent microscopic examination considering

intensity and density of fluorescence on the periphery

of the cells.

Statistical Analysis

Statistical analysis was done using Statistical Package

for Social Sciences (SPSS/version 15) software. Chi-

square test and student t-test were used for comparing

qualitative and quantitative data of each two groups,

respectively. Person correlation coefficient was used to

detect the correlation between different variables. The

level of significant was 0.05. Numerical values are

presented as mean ± SD.

Results

Subjects under study included ten newly

diagnosed AML patients (2 males and 8

females, mean age 43.5±13.52 years) before

the start of any chemotherapeutic medication

and ten normal healthy control subjects (4

males and 6 females, mean age 41.6±10.33

years). No significant difference was observed

between the two groups as regards age and

sex.

Clinical data

All control subjects were completely healthy

with normal complete blood picture. Total

leukocytic count (TLC) and percentage of

leukemic blasts in peripheral blood (PB) and

bone marrow (BM) in AML patients at

presentation are presented in Table (1).

According to FAB classification, AML

patients were either M1, M2, or M4 (number=

4, 2, and 4, respectively).

Table 1. TLC and leukemic blasts in AML patients.

Min. Max. Mean S.D.

TLC(x109 / L) 8600.0 75000.0 34551.0 22502.96

Blasts in PB (%) 12.0 80.0 51.30 22.80

Blasts in BM (%) 22.0 95.0 60.8 31.70


Leucocytic-VEGF

The level of VEGF in culture supernatant of

PBMN from AML patients was significantly

higher than corresponding level in normal

control subjects, (253.8±121.49 vs

143.5±20.94 pg/ml, respectively, P= 0.0093).

(Table 2, Figure 1). In AML patients,

estimated level of VEGF had a significant

positive correlation with the percentage of

blasts, both in PB & BM. (P= .033 & .011,

respectively). However, no significant

correlation was observed when clinical

staging, age, and sex were concerned in

relation to VEGF.

Leucocytic- integrin αvß3

The intensity of expression of integrin αvß3 on

PBMN from AML patients was significantly

higher than that in normal control subjects, P=

0.0006. (Table 2, Figure 2, 3). However, there

was no significant correlation between

leukocytic- VEGF and integrin αvß3.

Similarly, no significant correlation was

observed when percentage of blasts, clinical

staging, age, and sex were concerned in

relation to integrin αvß3 in AML patients.

1010N =

Normal controlAML patients

VE

GF

600

500

400

300

200

100

0

2

5

Figure 1. Leukocytic-VEGF in AML patients and normal control subjects.

Le

uko

cytic-V

EG

F p

g/m

l


Table 2. Leukocytic-VEGF and integrin αvß3 in AML patients and normal control subjects.

AML patients

n=10

Normal control

n=10

VEGF(pg/ml)

Range

Mean ± S.D.

68 – 480

253.8±121.49

120 – 184

143.5±20.94

P 0.0093*

Integrin αvß3

+

++

+++

0 (0.0%)

2 (20.0%)

8 (80.0%)

6 (60.0%)

4 (40.0%)

0 (0.0%)

P 0.0006*

P<0.05 is significant.

+60.0%

++40.0%

++20.0%

+++80.0%

Normal controlAML patients

Figure 2. Leukocytic- integrin αvß3 in AML patients and normal control subjects.


Figure 3. Different intensities of Leukocytic- integrin αvß3

Discussion

AML is a rapid progressive disease

characterized by high proliferation of

leukemic blasts with consequent repression of

normal hematopoiesis in the bone marrow.

AML shows the characteristic malignant

behavior through the ability of immature cells

to circulate in blood and to invade peripheral

tissues. In fact, the leukemic process is very

complicated where it is "fed" by intensively

B (+ve, 1+) A (-ve control)

B (+ve, 2+) B (+ve, 3+)


interacting leukemic cells, endothelial cells

and extracellular matrix. Among the

important candidates of this interplay are

agiogenic growth factors and adhesion

molecules (McKenzie, 2005; Wood, 2007). In

the current study we are concerned with two

important candidates; VEGF and integrin

αvβ3.

Our results revealed that leukemic PBMN

produce significantly higher levels of VEGF

than normal PBMN. Such data support

pervious evidences for the importance of

leukocyte-associated VEGF in the

pathogenesis of AML. It was previously

observed that leukemic blasts from AML

patients express and produce significantly

increased levels of VEGF (Aguayo et al.,

2000; Dias et al., 2000; Fiedler et al., 1997;

Hussong et al, 2000; Loges et al., 2006).

It is now quite evidenced that acute

leukemia cells use angiogenic growth factor

signaling pathways, namely those activated by

VEGF in autocrine and paracrine fashions.

The presence of VEGFR-1 and -2 on many of

leukemic blasts implies that expansion of the

leukemic population may be facilitated by an

autocrine loop (Bellamy et al., 2001). Such

autocrine action of VEGF was supported by

other more recent studies (Fragoso et al.,

2007, Vales et al., 2007). Moreover, the

leukemia-derived VEGF can also stimulate

the production of growth factors, including

interleukin-6 [IL-6] and granulocyte-

macrophage colony stimulating factor [GM-

CSF], by human endothelial cells, which in

turn further promotes the growth of leukemia

cells (the paracrine loop). In a system where

leukemia cells are co-cultured with

endothelial cells, IMC-2C6, a fully human

anti-VEGFR2 antibody, inhibits both the

production of IL-6 and GM-CSF by

endothelial cells and the growth of leukemia

cells. In addition, IMC-2C6 effectively blocks

VEGF-induced migration of KDR+ human

leukemia cells, and when administered in

vivo, significantly prolonged survival of mice

inoculated with KDR+ human leukemia cells

(Zhang et al., 2004). Autocrine and paracrine

actions of VEGF results in leukemic cell

proliferation, increased survival and

migration. Paracrine growth stimulation may

not only be restricted to the bone marrow

microenvironment, but may also take place at

extramedullary sites. Circulating AML blasts

may profit from paracrine provision of growth

factors in various capillary beds. This may

result in their expansion in peripheral blood

(Fragoso et al., 2007).

On the contrary, Litwin et al., (2002);

showed a discrepancy between bone marrow

vascularity and VEGF expression in vivo and

VEGF expression and angiogenesis from 2-

day conditioned medium ex vivo. They

suggested that angiogenesis in AML likely

represents a response to microenvironmental

factors in vivo, rather than being an intrinsic

property of leukemic cells. Actually, the

control of angiogenesis involves not only

changes in the profiles of environmental

angiogenic cues, such as the increase in

VEGFR2 in response to VEGF, but also

changes in the adhesive capacity of the

endothelial cells. In particular, the changes in

one family of adhesion molecules, integrins,

are thought to regulate several of these

angiogenic steps. In this respect the

interaction of co-expressed endothelial

VEGFR2 and integrin αvβ3 is suggested to

play an important role (Hodivala-Dilke,

2008). Recent studies have identified a

significant increase in activated αvβ3 on

angiogenic vessels, and these cells also appear

to express high levels of VEGFR2

(Mahabeleshwar et al., 2008). It was

previously shown that VEGF increases the

expression of αvβ3 by human microvascular

endothelial cells and also enhances cell

adhesion and migration mediated by αvβ3

(Byzova et al., 2000; Senger et al., 1996).

Current results also showed that the

expression of leucocytic- integrin αvß3 was

significantly higher in AML patients than in


normal control subjects. Up to our knowledge,

current study is the first report looking for the

expression of integrin αvß3 by peripheral

blood blast cells in AML patients. It was

previously observed that integrin αvß3 is

expressed by leukemic cell lines (Aswald et

al., 2004, Atkins et al., 1998, Benedetto et al.,

2006) and other metastatic malignant cells

(Felding-Habermann et al., 2001, 2002).

In cancer growth, the modification of

integrin structure is often associated with both

quantitative and qualitative alterations in

integrin cell surface patterns (Mizejewski,

1999). A characteristic feature of integrins,

including αvß3, is the capacity to transmit

signals bidirectionally, both inside-out and

outside-in. (Byzova et al., 2000). Integrin

activation, or inside-out signaling, is a tightly

governed process involving conformational

changes within the highly conserved

cytoplasmic tail of integrin receptor ß

subunits (Hughes et al., 1996) and provides a

mechanism of integrin regulation. This

process occurs when an agonist binds to a

traditional receptor that ultimately changes the

activation state of an integrin (Banno et al.,

2008). Previous studies emphasize the role of

integrin αvß3 as a ‘‘gatekeeper’’ of VEGF-

mediated processes (Borges et al., 2000,

Reynolds et al., 2004, Soldi et al., 1999).

More recently, De S. et al., (2005), have

defined a mechanism where by integrin αvß3,

through activation, clustering, and signaling,

regulates the production of VEGF in tumor

cells expressing this integrin. Tumors with

‘‘activatable’’ but not ‘‘inactive’’ ß3 integrin

secrete high levels of VEGF, which in turn

promotes extensive neovascularization and

augments tumor growth in vivo. This

stimulation of VEGF expression depends

upon the ability of αvß3 integrin to cluster and

promote phosphorylation of p66 Shc (Src

homology 2 domain containing).

We could not find a significant correlation

between released VEGF and expressed

integrin αvß3.When clinical and hematological

findings were concerned, our data revealed a

significant positive correlation between

VEGF and the percentage of blasts, both in

PB & BM. On the other hand, such

correlations were not observed in case of

integrin αvß3. In addition, no significant

correlation was observed between either

VEGF or integrin αvß3 and clinical staging,

age, and sex. Several reports pointed to the

correlation between increased VEGF and

worse prognosis (Aguayo et al, 1999, 2002;

de Bont et al., 2002). In addition, increased

expression of integrin αvß3 was associated

with poor prognosis and increased metastasis

in solid tumors (Gasparini et al., 1998;

Kageshita et al., 2000). However, our results

suggested that it is not just the quantitative

analysis which reflects the importance of

VEGF and integrin αvß3 in the leukemic

process. Looking for qualitative changes as

well as intracellular signaling pathways is

more indicative for the ultimate interaction

between these two importantly cooperating

factors.

In conclusion, obtained data confirm the

importance of VEGF and integrin αvß3 VEGF

in the pathogenesis of AML. The above

mentioned discussion reflects the complex

nature of interaction between VEGF and

intgrin αvß3 in controlling the leukemic

process. So, it is better to rely on related

qualitative rather than just quantitative

changes. The intimate link between intgrin

αvß3 and VEGF in tumor growth and

angiogenesis may influence anti-integrin as

well as anti-VEGF therapeutic strategies.

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