Clinical immunology
Vascular endothelial growth factor (VEGF) in circulating blood of patients treated with enoxaparine after orthopaedic surgery

Introduction

The process of angiogenesis relies on a coordinated expression of pro- and anti-angiogenic factors released by various cells and tissues, which, in normal physiological conditions, are maintained in balance. However, a loss of the balance leads to generation or inhibition of neovascularisation, and this may be of utmost significance for the development and course of many diseases like psoriasis, age-related macular degeneration (AMD) and diabetic retinopathy, collagenoses, neoplasia or other illnesses connected with angiopathology. On the other hand lack of angiogenesis may be the reason of heart and other organs ischaemic diseases, or may result in disturbance of wound and bone healing [1].
VEGF, a potent and specific mitogen for endothelial cells in vessels, is one of the most important secreted glycoprotein, responsible for stimulating angiogenesis during development of embryo, as well as during tumor formation, growth and metastasis. VEGF serves as an endothelial cell survival factor, protecting endothelial cells against apoptosis, and it delays and may reverse endothelial cell senescence. There are data that confirm an important role of VEGF in wound and bone healing [2-10].
Members of VEGF family are the multifunctional cytokines that exert many effects on vascular endothelium and critically influence the angiogenic response. These include striking changes in cell morphology and cytoskeleton accompanied by stimulation of endothelial cell migration and proliferation. First and most distinctive biologic activity of VEGF is the increase of microvascular permeability. It results in leakage of plasma proteins, including fibrinogen and other clotting proteins. The clotting system is rapidly activated by means of the tissue factor pathways. VEGF is selective for vascular endothelium because its major tyrosine kinase receptors are mainly expressed on vascular endothelium. After binding to its receptors, VEGF initiates a cascade of signaling events that begins with autophosphorylation of receptor tyrosine kinases, followed by activation of numerous downstream proteins. There are 3 major transmembranous tyrosine kinase receptors for VEGF: VEGFR-1 (Flt-1), VEGFR-2 (flk-1/KDR) and VEGFR-3. Flt-1 and flk-1/KDR bind to all secreted VEGF-isoforms. This binding induces autophosphorylation and signal transduction. Endothelial cells express also neuropilin co-receptors, which bind selectively to the 165 aminoacid form of VEGF [11].
At a molecular level, VEGF reprograms endothelial cell gene expression, leading to increased expression of a number of different proteins, including the procoagulant tissue factor, proteins associated with the fibrinolytic pathway (urokinase, tissue-type plasminogen activator, type 1 plasminogen activator inhibitor, urokinase receptor), matrix metalloproteases, nitric oxide synthase, numerous mitogens, and
a number of antiapoptotic factors. Degradation of heparan sultate (HS) in the extracellular matrix (ECM) and on the cell surface by enzymes releases different polypeptides bound to HS and enables cellular invasion. Activation of mediators released from ECM and different cells like platelets, fibroblasts, macrophages, leukocytes, and others, promoting EC-receptors binding and intracellular signaling, leads to activation, proliferation and migration of endothelial cells (EC). Next step is tube and loop formation and ECM remodeling. Process is finalized by stabilization of newly-
-formed blood vessels [12, 13].
In our previous papers we have shown that enoxaparine (low-molecular weight heparin) enhanced in vivo angiogenic activity of mouse and human serum, evaluated by murine skin test, in comparison to the control. On the other hand, sera collected from nadroparine (fraxiparine) treated mice presented significantly lower neovascular reaction than the reaction observed in the skin of the recipients of control sera [14-16].
In the present study we compared angiogenic activity and VEGF content of plasma and sera of 12 patients undergoing orthopedic surgery, before and after 14 daily enoxaparine injections.

Material and methods


Patients: We collected blood samples (with or without EDTA) from 12 patients, aged 64-71 years, with coxarthrosis, gonarthrosis, avascular necrosis of the femoral head and similar others. Plasma and sera samples were separated and frozen at –70°C until testing. Blood samples were collected for the first time immediately before beginning of enoxaparine (En) treatment, and for the second time 14 days after the beginning of treatment (40 mg of En subcutaneous daily injections). We also collected, as
a control, sera from 30 healthy people, aged 40-83 years.

Angiogenic activity measurement of patients sera was done by cutaneous test performed in mice (serum-
-induced angiogenesis, SIA test) according to Barcz et al. [17]. Briefly, multiple 0.05 ml of the sera and plasma samples were injected intradermally into partly shaved, narcotized, 8 weeks old Balb/c mice. At least 3 mice for one tested material were used. In order to facilitate the localisation of injection sites later on, each serum sample was colored with 0.1% of trypan blue. After 72 hours mice were sacrificed with lethal dose of Morbital. All newly formed blood vessels were identified and counted in dissection microscope, on the inner skin surface, at
a magnification of 6 x, in 1/3 central area of the microscopic field. Identification was based on the fact that the new blood vessels, directed to the point of cells injection, are thin and differ from the background vasculature in their tortuosity and divarications. All experiments were performed in anaesthesia (3.6% chloral hydrate, 0.1 ml per 10 γ of body mass), and approved by Local Ethical Committee for Animal Experiments.

Measurement of VEGF concentration: Cytokine levels were determined in examined plasma and sera using sandwich ELISA kits (R&D Systems, USA) for human VEGF, according to the producer instructions. Optical density was measured at 450 nm using spectrophotometric reader Elx800 (Biotek Instruments, Inc., USA). Cytokines concentration was expressed as pg/ml.

Statistical analysis was performed using Student-t test.
All experiments were approved by Local Ethical Committee for human studies.

Results

Initial angiogenic activity of sera from the patients was similar to the angiogenic activity of sera collected from control people. Significant increase of neovascular response was observed when sera from enoxaparine-treated patients are injected into mice skin (figure 1).
Both serum and plasma samples collected after enoxaparine treatment presented higher in vivo angiogenic activity in mouse cutaneous test than before administration of this drug. However, the reaction induced by sera was significantly higher than that induced by plasma samples (table 1). Initial VEGF concentration in patients sera was significantly lower than that of the control group and have increased after enoxaparine treatment (figure 2).
No differences in plasma VEGF were observed. However, VEGF concentration in matched sera samples was significantly higher after surgery and enoxaparine treatment than before the beginning of the study (table 2).

Discussion

Our present study revealed that VEGF in sera of our patients was significantly lower than in the controls. However, angiogenic activity of the sera was similar to that observed in the control group. This suggests, that in vivo angiogenic activity of human serum in mouse cutaneous test (SIA) is dependent on some other factors also. In fact, we have shown important role of bFGF in this test [18]. Sera of our patients contained amounts of bFGF comparable to the concentrations present in the sera of age-matched control group [16, 19], and significant increase of bFGF amount was observed after one and fourteen enoxaparine 40 mg daily doses. Paralelly, we have shown that enoxaparine enhanced angiogenic activity of mouse and human serum [14-16]. However, enoxaparine may also behave as inhibitor of angiogenesis, what we have shown after preincubation of murine L-1 sarcoma cells with enoxaparine, or during administration of drug to L-1 sarcoma cells recipients [20, 21]. These opposite action of heparins, used in different experimental models of angiogenesis may perhaps be explained by the fact that heparin and LMWHs may exert polypharmacological actions on each level of this cascade process, and they represent complex natural mucopolysaccharide drugs that have undergone different chemical and enzymatic modifications [22-24].
We have also compared the effect of different LMWHs – enoxaparine (Clexane) and nadroparine (Fraxiparine) which were administrated for 2 or 14 days to mice, on angiogenic activity of their sera. We have found that enoxaparine stimulates angiogenic activity of sera and nadroparine inhibits such activity [14-16].
One may suppose, that enoxaparine treatment would positively influence bone healing after surgery, what was experimentally confirmed in rats [25]. However, some experimental and clinical studies revealed that longer treatment with heparin or nadroparine of rats (4 weeks) caused disorder of bone formation and intensification of bone resorption [26], and long-term exposure (1 year) to enoxaparine caused the decrease in bone mineral density in humans [27].
In the present paper we have observed higher in vivo angiogenic activity and higher VEGF levels in serum samples than in matched plasma samples. Dependence of VEGF concentration on blood collection procedures was described. Blood coagulation initiates events leading to the activation of platelets and granulocytes and to the release intracellular pool of VEGF [28- 32]. Isolated neutrophils contain considerable amounts of VEGF. It was also reported that the number of platelets is significantly correlated to VEGF concentration in serum, but not to the VEGF concentration in plasma. Some authors demonstrated that higher VEGF concentration in serum samples than in matched plasma samples was due to the presence of VEGF within platelets and its release upon their activation during coagulation [33-36].
The results of our present study suggest that enoxaparin treatment increased intracellular pool of VEGF in blood leukocytes and (or) platelets. We feel, that this increase in VEGF content and angiogenic activity of serum is mainly connected with enoxaparin treatment, and only partly dependent on the effect of surgery itself, because we have obtained similar findings in mice treated with 14 doses of enoxaparine and not subjected to surgical procedures [16].
Perhaps, the influence of heparins on the process of angiogenesis may be connected with their affinity to VEGF. The several VEGF isoforms encoded by alternative splicing have distinct physical properties. VEGF 121 is freely soluble and does not bind heparin. By contrast, isoforms 165 and 189 have increasing basic charge and bind heparin with increasing affinity; in fact, VEGF 165 was originally purified on the basis of its affinity for heparin. Heparin, heparan sulfate, and heparinase all displace the larger VEGF isoforms from proteoglycan-binding sites; plasmin cleavage also activates these isoforms by freeing them from cells or matrix. Despite these physical differences, the several VEGF isoforms apparently have identical biologic activities when free in solution. Angiogenesis is initiated only at a stage when cells express metalloproteases that liberate VEGF from matrix. Therefore, in this and likely other instances, proteases are required to push the “angiogenic switch”.

Conclusion: intracellular VEGF, released during coagulation process plays an important role in in vivo angiogenic activity of serum, and its concentration significantly increased after 2-weeks enoxaparine administration. Short-term enoxaparine treatment after surgery could improve the healing process.


Acknowledgment

This work was partly supported by Polish Scientific Committee, grant 3PO5B17223.

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