Research Article - Biomedical Research (2017) Volume 28, Issue 12

The effect of VEGF on deep venous thrombosis in the perioperative period of elderly fracture patients

Hui-Liang Zhang¹, Li-Geng Li¹, Qing-hai Kong¹, Cheng Luo¹, Zhi-jie Feng¹, Hong Zhang² and Jun-sheng Liang^1*

¹Geriatric Department of Orthopedics, the Second Hospital of Tangshan City, Hebei province, PR China

²Department of Orthopedics, First Affiliated Hospital of PLA General Hospital, PR Chinas

*Corresponding Author:

Jun-sheng Liang
Geriatric Department of Orthopedics
The Second Hospital of Tangshan City
Tangshan City, PR China

Accepted on May 4, 2017

Abstract

Objective: We compared the correlation between serum Vascular Endothelial Growth Factor (VEGF) with D-Dimer, fibrinogen and thromboelastogram in order to investigate the effects of VEGF on deep venous thrombosis in the perioperative period of elderly fracture patients.

Methods: 68 elderly fracture patients that had undergone surgery were divided into two groups according to whether they were diagnosed with deep venous thrombosis in the perioperative period or not. We used the ELISA assay to detect the VEGF, D-Dimer and fibrinogen levels before the operation and 1 d, 5 d and 10 d post-operation. Thromboelastogram of patients before operation and after operation were recorded and further statistical analysis was conducted.

Results: The serum VEGF levels in the thrombosis group and non-thrombosis group both increased first and then decreased post-operation. After the operation at each of the different time points, the thrombosis group showed significant higher levels of serum VEGF compared to the non-thrombosis group (p<0.05). Serum VEGF levels in the thrombosis group increased significantly after operation and its peak level was higher than that of the non-thrombosis group. In addition, VEGF levels in the thrombosis group after the operation were positively correlated with the D-Dimer and fibrinogen content (R²=0.892, p<0.05; R²=0.921, p<0.05). However, for non-thrombosis group, the positive correlations between VEGF levels and the content of D-Dimer and fibrinogen were relatively weak (R²=0.634, p<0.05; R²=0.611, p<0.05).

Conclusion: VEGF levels have a certain relationship with the D-Dimer and fibrinogen content in the formation of deep venous thrombosis in elderly patients with fracture. This relationship indicates the changing of blood coagulation state and fibrinolysis process in the formation of deep venous thrombosis. The up-regulated expression of VEGF, to some extent, will guide the treatment process and prognosis of deep venous thrombosis.

Keywords

VEGF, D-dimer, Deep venous thrombosis, Fibrinogen.

Introduction

68 cases of elderly fracture patients (>65 y old), who were treated in the orthopedic clinic of our hospital from January 2013 to December 2015, were recruited for our study. 41 cases were male and 27 cases were female; their ages ranged from 65 to 84 with an average of (72.03 ± 4.36). There were 18 femoral neck fractures cases, 16 pelvic fractures cases, 12 femoral shaft fractures cases, 12 tibial plateau fracture cases and 10 ankle joint fracture cases. All patients were operated on and 29 of them experienced Deep Venous Thrombosis (DVT) during perioperative period. This study was approved by the ethics committee of our hospital, and all the patients signed informed consent.

Groups and Testing Method

68 cases were divided into the thrombosis group (29 cases) and non-thrombosis group (39 cases). We extracted 5 ml of the upper limb venous blood through the ulnar vein in the morning after having fasted all night for different times: before operation (T1), after operation for 1 day (T2), 5 days (T3) and 10 days (T4). We detected VEGF, D-Dimer and fibrinogen using the ELISA test and thromboelastogram test was performed for all patients before and post-operation.

Statistical analysis

SPSS20.0 (IBM US) statistical software was used in our research data statistical analysis. The measurement data was evaluated by using mean ± standard deviation. Mono-factor analysis of variance was used for analyzing the measurement difference between different groups. If the difference was statistically significant, a further LSD test proceeded between the two different groups. Correlations between VEGF, DDimer and fibrinogen content were analysed by Pearson correlation coefficient analysis, R² represents the strength of the correlation. p<0.05 is regarded as statistically significant.

Results

Comparison of general conditions

In the thrombosis group, there were 16 cases of males and 13 cases of females with an average age of 73.12 ± 4.62 y old, an average weight of 79.48 ± 8.76 Kg and an average BMI of 25.59 ± 2.28. In the non-thrombosis group, there were 25 cases of males and 14 cases of females with an average of age 71.65 ± 4.14 y old, an average weight of 75.32 ± 7.43 Kg and an average BMI of 24.24 ± 2.19. Comparison of the general conditions showed that there were evident differences in weight and BMI between these two groups (p<0.05). It indicated that the heavier group and higher BMI group were more likely have DVT; there were no significant differences in gender and age between these two groups (p>0.05). Results are shown in Table 1.

Table 1. Comparation of general conditions.

Serum VEGF levels

We compared the serum VEGF levels of the thrombosis group and the non-thrombosis group before operation; differences were not statistically significant (p>0.05). After the operation, serum VEGF levels in both groups showed an increase at first, followed by a decrease, which indicates that the operation can cause an increase in serum VEGF levels. The thrombosis group showed higher VEGF levels compared to the non-thrombosis group at different time points after the operation; differences were statistically significant (p<0.05). Serum VEGF levels in the thrombosis group increased significantly and its peak levels were significantly higher than the non-thrombosis group (p<0.05), which indicates that the formation of thrombus was closely related to the increase in VEGF level (Table 2).

Table 2. VEGF level (x̄ ± s, pg/ml) in peripheral venous serum.

Serum D-Dimer content

We compared the serum D-Dimer content of the thrombosis and non-thrombosis group before the operation; differences were not statistically significant (p>0.05). The serum D-Dimer content in the thrombosis and non-thrombosis group increased at the beginning, followed by a decrease which indicates that the operation can cause an increase in serum D-Dimer content. The thrombosis group showed a higher serum D-Dimer content compared to the non-thrombosis group after treating for different days; differences were statistically significant (p<0.05). The serum D-Dimer content in the thrombosis group increased significantly and its peak level was significantly higher than the non-thrombosis group (p<0.05), which indicates that the formation of thrombosis was closely related to the increase of D-Dimer content (Table 3).

Table 3. D-Dimer content in peripheral venous serum (x̄ ± s, ng/ml).

The positive correlation between VEGF levels and D-Dimer content in the thrombosis group at T2 was strong (R2=0.892, p<0.05). For the non- thrombosis group, this positive correlation was relatively weak (R²=0.634, p<0.05).

Fibrinogen content in peripheral venous serum

The comparison of fibrinogen content between the thrombosis and non-thrombosis group showed that the difference was not statistically significant before operation (p>0.05). Fibrinogen content in both groups showed an increase at the beginning, followed by a decrease post-operation, which indicates that operation can cause an increase in serum fibrinogen content. We compared the fibrinogen content between two groups at different time points after operation. Thrombosis group had higher rates than the non-thrombosis group; difference was statistically significant (p<0.05). The thrombosis group experienced a more significant increase of fibrinogen content and the peak level of fibrinogen content was significantly higher than the non-thrombosis group (p<0.05), which indicates that the formation of thrombosis was closely related to the increase of fibrinogen content (Table 4).

Table 4. Fibrinogen content in peripheral venous serum (x̄ ± s, mg/dL).

The positive correlation between VEGF levels and fibrinogen content in the thrombosis group at T2 was strong (R²=0.921, p<0.05). For the non-thrombosis group, this positive correlation was relatively weak (R²=0.611. p<0.05).

Table 5. Thromboelastogram results comparation between before and after operation.

The thromboelastogram results comparison between before and after operation

Before the operation, 7 cases in thrombosis group (24.14%) and 10 cases in non- thrombosis group (25.64%) showed low blood coagulation; the difference was not statistically significant (p>0.05). After the operation, 4 cases in thrombosis group (13.79%) and 16 cases in non-thrombosis group (41.03%) experienced a reduction of blood coagulability, and differences were statistically significant (p<0.05).

Discussion

Deep venous thrombosis is a very common complication of orthopedic patients who are on bed rest. Its clinical manifestations include unilateral or bilateral lower limb swelling, post-thrombotic syndrome and lethal pulmonary embolism which can severely affect a patients’ health [1-5]. It has been reported that the incidence of deep venous thrombosis is approximately 1-2%worldwide [6-8]. With regards to China, despite the sparsity of multicenter epidemiology investigation data available for deep venous thrombosis, it still be discerned that its incidence is high according to the analysis of literature and clinical data [7,8]. Deep venous thrombosis refers to the abnormal coagulation of blood in the lower limbs deep vein system which will block the venous duct and cause acute obstruction of lower limbs venous backflow and increase of internal pressure; it will also damage the venous valve and cause a series of symptoms and signs [9,10]. German physiologist Virchow pointed that the main causes of deep venous thrombosis are slow venous blood flow, hypercoagulable state and venous wall damage; the first two are the main reasons. Any disease which can cause the three problems mentioned above will be the risk factors for deep venous thrombosis [11].

VEGF is a specific vascular permeability factor and chemotactic factor of vascular endothelial cells [12]. During the embryonic growth and development stage, the formation of the embryonic vascular system is dependent on the correct expression of VEGF. After birth, VEGF still plays an important role in the formation of blood vessels. Endothelial progenitor cells can migrate to peripheral blood when signalled by exogenous VEGF. VEGF can improve the proliferation, migration and chemokines of endothelial progenitor cells, which can also promote angiogenesis. It has been reported that the number of endothelial progenitor cells in the recycling of peripheral blood increased significantly in a VEGF-rich microenvironment. Also a hypoxic microenvironment can further improve the proliferation and differentiation of endothelial progenitor cells and finally enhance the effects of endothelial progenitor cells on improving angiogenesis. After the formation of thrombosis, due to the blood flow stasis and hypoxic microenvironment, VEGF and BFGF content inside of the emboli increase significantly. Therefore, the thrombolysis and organization speed increases [13]. The possible antithrombotic mechanism of VEGF is as follows: 1. act as an endogenous regulator of body parts, not only participate in the maintenance of the integrity of vascular endothelial cells, but also in the maintenance of normal physiological functions to prevent the occurrence of endogenous and exogenous coagulation. 2. VEGF can increase the production of NO and PGI [14]. NO can activate Soluble Guanylate Cyclase (SGC) to increase cGMP content inside of the cell, which can finally cause platelet aggregation and block property decrease. 3. VEGF can also promote the expression and activity of serine protease, fibrinoclase, urokinase and tissue-plasminogen. These enzymes can cause the conversion of plasminogen to plasmin which will finally improve the thrombolysis [15].

It has been reported that D-Dimer, fibrinogen and inflammatory cellular factors in the peripheral blood are closely related to the formation of deep venous thrombosis [16]. After the activation of the fibrinolytic system of the body, fibrinogen will convert to a fibrin monomer to crosslink with the factor XIII. D-Dimer is the product of the fibrinolytic enzyme hydrolysis process. The accumulation of fibrinogen in plasma can enhance fibrinolytic activity and also increase DDimer content. Therefore, D-Dimer can specifically induce the hypercoagulable state of blood and hyperfunction of the fibrinolysis process at the molecular level [17,18]. D-Dimer index can increase several times during the formation of deep venous thrombosis [19,20]. The clinical way to detect D-Dimer and fibrinogen is easy and efficient, which is of great significance in the diagnosis of deep venous thrombosis and evaluation of the prognosis.

Our study has found that VEGF levels in both these two groups showed an increase at first and followed by a decrease, which indicates that the angiogenesis process after operative injury can cause an increase of serum VEGF levels. The thrombosis group showed higher VEGF levels compared to the nonthrombosis group after the operation for different days; differences were statistically significant (p<0.05). Serum VEGF levels in the thrombosis group increased significantly and its peak level was significantly higher than the nonthrombosis group which indicates that the formation of deep venous thrombosis can stimulate the expression of VEGF tos improve the thrombolysis process. Meanwhile, VEGF levels in the thrombosis group after the operation days were positively correlated with the D-Dimer and fibrinogen content (R²=0.892, p<0.05; R²=0.921, p<0.05). However, for the non-thrombosis group, the correlation between VEGF levels and the content of D-Dimer and fibrinogen was relatively weak (R²=0.634, p<0.05; R²=0.611, p<0.05). This indicated that fractures trauma and operations can stimulate the expression of VEGF to improve neovascularization and repair the trauma. In the meantime, the formation of deep venous thrombosis can further increase the expression of VEGF in order to aid the thrombolysis process.

Moreover, thromboelastogram can fully reflect the shear stress changing with time in the dynamic clotting process and precisely describe the clotting mechanism. Our study has found that the amount of patients with lower blood coagulability in the thrombosis group is significantly smaller than that in nonthrombosis group (p<0.05). The dynamic monitoring of the coagulation process demonstrated that the blood coagulability of patients in the thrombosis group was in a rising state.

In conclusion, VEGF level was positively correlated with DDimer and fibrinogen content in the formation of deep venous thrombosis of elderly fracture patients. This correlation indicates the changing of blood coagulation state and the fibrinolysis process in the formation of deep venous thrombosis. The up-regulated expression of VEGF to some extent will guide the treatment process and prognosis of the deep venous thrombosis.

References

1. Brummel-Ziedins KE, Vossen CY, Butenas S, Mann KG, Rosendaal FR. Thrombin generation profiles in deep venous thrombosis. J Thromb Haemost 2005; 3: 2497-2505.
2. Stawicki SP, Grossman MD, Cipolla J, Hoff WS, Hoey BA. Deep venous thrombosis and pulmonary embolism in trauma patients: an overstatement of the problem? Am Surg 2005; 71: 387-391.
3. Wells PS, Anderson DR, Rodger M, Forgie M, Kearon C. Evaluation of D-dimer in the diagnosis of suspected deep-vein thrombosis. N Engl J Med 2003; 349: 1227-1235.
4. Bozoglu E, Dinc A, Erdem H, Pay S, Simsek I, Kocar IH: Vascular endothelial growth factor and monocyte chemoattractant protein-1 in Behcets patients with venous thrombosis. Clin Exp Rheumatol 2005; 23: 42-48.
5. Jacobson G. Relationship between venous thromboembolism and vascular endothelial growth factor in cancer. Support Cancer Ther 2005; 3: 35.
6. Hoaglund FT. Incidence and natural history of deep-vein thrombosis after total hip arthroplasty. J Bone Joint Surg Br 2004; 86: 621.
7. Ko PS, Chan WF, Siu TH, Khoo J, Wu WC. Deep venous thrombosis after total hip or knee arthroplasty in a low-risk Chinese population. J Arthroplasty 2003; 18: 174-179.
8. Leung V, Leung V, Lui W, Chan T, Wong RS, Cheng G. Incidence of deep vein thrombosis in hospitalized Chinese medical patients is similar to that in western populations. Thromb Res 2006; 118: 763-764.
9. Sillesen H, Just S, Jorgensen M, Baekgaard N. Catheter directed thrombolysis for treatment of ilio-femoral deep venous thrombosis is durable, preserves venous valve function and may prevent chronic venous insufficiency. Eur J Vasc Endovasc Surg 2005; 30: 556-562.
10. Malone PC, Agutter PS. Deep venous thrombosis: The valve cusp hypoxia thesis and its incompatibility with modern orthodoxy. Med Hypotheses 2016; 86: 60-66.
11. Cervantes J, Rojas G. Virchows Legacy: deep vein thrombosis and pulmonary embolism. World J Surg 2005; 29: 30-34.
12. Trelinski J, Wierzbowska A, Krawczynska A, Sakowicz A, Pietrucha T, Smolewski P, Robak T, Chojnowski K. Circulating endothelial cells in essential thrombocythemia and polycythemia vera: Correlation with JAK2-V617F mutational status, angiogenic factors and coagulation activation markers. Int J Hematol 2010; 91: 792-798.
13. Cacciola RR, Di Francesco E, Giustolisi R, Cacciola E. Elevated serum vascular endothelial growth factor levels in patients with polycythemia vera and thrombotic complications. Haematologica 2002; 87: 774-775.
14. Wei W, Chen ZW, Yang Q, Jin H, Furnary A, Yao XQ, Yim AP, He GW. Vasorelaxation induced by vascular endothelial growth factor in the human internal mammary artery and radial artery. Vascul Pharmacol 2007; 46: 253-259.
15. Chun YS, Yeo EJ, Choi E, Teng CM, Bae JM, Kim MS, Park JW. Inhibitory effect of YC-1 on the hypoxic induction of erythropoietin and vascular endothelial growth factor in Hep3B cells. Biochem Pharmacol 2001; 61: 947-954.
16. Wuillemin WA, Korte W, Waser G, Lammle B. Usefulness of the D-dimer/fibrinogen ratio to predict deep venous thrombosis. J Thromb Haemost 2005; 3: 385-387.
17. Chan WS, Lee A, Spencer FA, Chunilal S, Crowther M, Wu W, Johnston M, Rodger M, Ginsberg JS. D-dimer testing in pregnant patients: Towards determining the next level in the diagnosis of deep vein thrombosis. J Thromb Haemost 2010; 8: 1004-1011.
18. Aguilar FC, Martinez BA, Martinez SA, Del RMC, Villar SV, Vazquez SM, Rodriguez RF. Diagnostic value of D-dimer in patients with a low pretest probability of deep venous thrombosis of lower extremities. Med Clin (Barc) 2002; 118: 539-542.
19. Bates SM, Jaeschke R, Stevens SM, Goodacre S, Wells PS, Stevenson MD, Kearon C, Schunemann HJ, Crowther M, Pauker SG, Makdissi R, Guyatt GH. Diagnosis of DVT: Antithrombotic therapy and prevention of thrombosis. American college of chest physicians Evidence-Based clinical practice guidelines (9th edn.). Chest 2012; 141: 351-418.
20. Schutgens RE, Ackermark P, Haas FJ, Nieuwenhuis HK, Peltenburg HG, Pijlman AH, Pruijm M, Oltmans R, Kelder JC, Biesma DH. Combination of a normal D-dimer concentration and a non-high pretest clinical probability score is a safe strategy to exclude deep venous thrombosis. Circulation 2003; 107: 593-597.