Serum levels of VEGF and bFGF in hypoxic patients with exacerbated COPD

ARTICLE

Auteur(s) : Gordana Pavlisa¹, Goran Pavlisa², Vesna Kusec³, Slobodanka Ostojic Kolonic⁴, Asja Stipic Markovic⁵, Branimir Jaksic⁴

¹Special Hospital for Pulmonary Diseases, Zagreb, Croatia
²Clinical Institute of Diagnostic and Interventional Radiology, University Hospital Center Zagreb, Croatia
³Clinical Institute of Laboratory Diagnosis, University Hospital Center Zagreb, Zagreb, Croatia
⁴Department of Internal Medicine, “Merkur” University Hospital, Zagreb, Croatia
⁵Department of Internal Medicine, University Hospital “Sveti Duh”, Zagreb, Croatia

accepté le 25 F�vrier 2010

Chronic obstructive pulmonary disease (COPD) is frequently complicated by hypoxia. The response to hypoxia occurs through several, well described mechanisms, but none of them alone, or in combination, can fully explain the adaptation to hypoxia. The regulation of gene expression has recently been recognized as an important adaptive response to hypoxia. The cell response to hypoxia is mediated by hypoxia-inducible factor-1 (HIF-1), whose expression rises exponentially in hypoxic conditions. HIF-1 participates in the initiation of transcription of erythropoietin (EPO), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) genes, and in the regulation of metabolic processes by activation of the transcription of several glycolytic enzymes and glucose-carrier-1 [1]. The activation of EPO production and subsequent erythropoesis is the most frequently mentioned model of adaptation to systemic hypoxia. However, only a minor subset of hypoxic COPD patients develop secondary polycythemia and elevated levels of EPO [2]. These data indicate other important mechanisms, such as angiogenesis, that may be involved in the adaptation to hypoxia. VEGF and bFGF are the most potent proangiogenic factors [3]. VEGF is a 45 kDa, dimeric glycoprotein. The biological potency of VEGF depends on its reaction with specific receptors: VEGF R1 and VEGF R2. VEGF R1 and VEGF R2 are expressed on endothelial cells and tumor cells which produce VEGF [4]. Basic fibroblast growth factor is a single-chain, non-glycolysed cation polypeptide with a molecular mass of approximately 18 kDa [5]. VEGF and bFGF mediate a number of processes necessary for angiogenesis [4, 5]. VEGF and bFGF play a significant role during normal angiogenesis, such as wound healing, fetal and neonatal life [4, 6]. Several investigational models have shown that VEGF and bFGF are a part of the adaptive reaction to hypoxia. Their production is enhanced in conditions of local and systemic hypoxia [7]. HIF-1, VEGF and bFGF have been found in the serum and atherosclerotic lesions of patients with coronary artery disease, and play a role in collateral vessel formation [8, 9]. In patients with sleep apnea syndrome and hypoxia, the level of VEGF is elevated [10].

The regulation of these proangiogenic factors in COPD associated with hypoxia is unclear. The aim study were to investigate whether these factors are up-regulated in COPD patients with respiratory failure, and to assess which factors may influence their expression in this complex clinical model.

DONORS AND METHODS

Selection of the patients

The quantitative characteristics of the groups are summarized in table 1.

Group 1: Patients with exacerbated COPD

All patients had the clinical characteristics of respiratory inflammation such as an increase in sputum production, purulent sputum and dyspnea progression. According to the GOLD criteria, 67% of the patients were GOLD IV stage.
Table 1 Characteristics of groups and differences between groups

Group 2: Patients with stable COPD

The maximum value for the forced expiratory volume in the first second/forced expiratory vital capacity (FEV1/FVC) was 60% of the predicted value. Based on the GOLD criteria, 67% of the patients had moderate, and 33% had severe COPD. All the patients had irreversible airflow limitation.

Group 3: Healthy blood donors

Laboratory tests

Pulmonary function studies were performed in patients with stable COPD on the day of the blood sample collection. In the exacerbated COPD group, pulmonary function studies were done before the discharge from the hospital.

Blood samples for the arterial blood gas analyses were drawn anaerobically from the radial artery into heparinized syringes and were promptly analyzed. Routine hematological analyses were performed using standard biochemical methods.

The serum samples for the determination of VEGF, bFGF and IL-6 were collected in vacutainer tubes without additive. Within 30 minutes of blood collection, the samples were centrifuged at 1,000 x g for 15 minutes. The serum was carefully transferred to new tubes. Serum samples were frozen within one hour of collection at a temperature of - 70^oC. All samples were analyzed after the study period, at the same time. The samples were analyzed using commercially available ELISA kits for VEGF (DVE00), bFGF (HSFB75) and IL-6 (D6050) (R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer`s instructions. Sensitivity for bFGF was 3 pg/mL, VEGF 9 pg/mL, and for IL-6 0.7 pg/mL.

Statistical analysis

Results

VEGF and bFGF findings in the three study groups

We did not find any statistically significant difference in the VEGF levels between the stable COPD group and blood donor group (p = 0.1071). In the stable COPD group, the mean VEGF value was 197.68 ± 178.06 pg/mL (ranging from undetectable levels to 784 pg/mL). In theblood donor group, the mean VEGF value was 257.69 ±170.4 pg/mL (ranging from 12.48 to 605.73 pg/mL). Basic FGF was undetectable in the stable COPD and blood donor groups (figures 1 and 2).

Correlation between parameters analyzed in the COPD patients

We found no statistically significant correlation with lung function parameters or GOLD stages of disease when analyzing the overall COPD population or groups 1 and 2 separately.

Also, bFGF significantly correlated with almost all parameters studied (table 3). The strongest correlation of bFGF was with PaO₂ (r = - 0.816, p < 0.0001), VFGF (r = 0.455, p < 0.0001) and RBC (r = 0.437, p < 0.0001).
Table 2 Correlation between serum VEGF and selected continuous data

Multivariate analysis

Step-wise regression was performed with VEGF and bFGF as dependent variables. Thirteen independent variables were included in the model. These were: PaO₂, arterial carbon dioxide tension (PaCO₂), pH, red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), WBC, platelets, IL-6, age, VEGF and/or bFGF.

Table 4 shows the final step in a step-wise regression analysis of VEGF, and describes variables that remain in the model, while all other variables ended the analysis out of the model. VEGF is independently determined by PaO₂ (F-to-remove = 14.9), WBC (F-to-remove = 23.2) and IL-6 (F-to-remove = 9.3). Table 5 shows that bFGFis independently determined by PaO₂ (F-to-remove = 151.35) and pH (F-to-remove = 11.26), while other parameters analyzed ended the analysis out of model. A strongly significant, simple correlation between VEGF and bFGF was entirely lost during a step-wise regression analysis. This suggests that the significant, simple correlation between the two angiogenic factors is not independent, but is essentially due to their individual association with hypoxia.
Table 4 Stepwise regression analysis of VEGF versus 13 independent variables (final step)

Discussion

Hypoxia is a strong inducer of VEGF release in physiological and numerous pathological situations with consequent neoangiogenesis. Physical strain increases the expression of VEGF in muscles, and this increase correlates with the lactate concentration in venous plasma of the involved muscles [12]. VEGF is up-regulated in ischemic myocardial tissue [8], ischemic brain tissue [13], and ischemic limbs [14]. In patients suffering from sleep apnea syndrome and severe hypoxia, levels of VEGF are elevated [10]. Several investigations have studied serum levels of VEGF in COPD patients. Valipour et al. found that patients with exacerbated COPD have higher circulating VEGF concentrations compared to stable COPD patients and healthy controls [15]. The mean serum VEGF levels in stable COPD patients and healthy controls in their study are comparable to our results. Conversely, the mean serum VEGF levels in patients with exacerbated COPD were higher in our study. Our patients with exacerbated COPD had more severe hypoxia, which may have been the cause of the higher VEGF levels. Alydonyte et al. did not find increased expression of serum VEGF in stable COPD patients, and the mean values were comparable to our results [16]. In the study by Kierszniewska-Stepie et al., the serum VEGF concentration was elevated in patients with stable COPD, and even in patients with mild disease [17]. In this study, patients with mild COPD had lower mean PaO₂ compared to our group with stable COPD. The studies mentioned did not address the role of hypoxia as a potential trigger of VEGF synthesis in COPD patients. A statistically significant negative correlation of VEGF withPaO₂ found in our study confirms the hypothesis that hypoxia is strongly associated with VEGF synthesis in this clinical model.

Studies of the concentration dynamics of VEGF are scarce. It has been shown that the concentration of VEGF reaches amaximum 24 hours after an acute myocardial infarction [18]. In our patients, the progression of the disease, as well as the hypoxia lasted longer than 24 hours. Significantly higher parameters of the red blood count in the exacerbated compared to stable COPD group indicate that those patients were possibly hypoxemic prior to the exacerbation with consequently activated erythropoeisis. Also, since this group of patients had more significantly impaired lung function, it is to be expected that the level of hypoxia was lower, even in a stable phase of the disease.

Since the COPD is a complex clinical syndrome, hypoxia may not be the only factor involved with the increased expression of VEGF. Systemic inflammation can influence the VEGF level [19, 20]. Neutrophils are recognized as asignificant source of VEGF [21]. Also, inflammatory cytokines, such as IL-6, may induce VEGF expression [22]. COPD is characterized by the chronic inflammation of the tracheobronchial tree, further activated in the case ofexacerbation [23]. It is well known that COPD patients exhibit elevated markers of systemic inflammation [24, 25].

In our study, WBC and IL-6 values were, not surprisingly, significantly higher in a group of exacerbated COPD patients compared to stable COPD patients or the blood donor group. A significantly positive correlation, as well as an independent relationship of VEGF with the white blood cell count and IL-6, may suggest that both acute and chronic inflammation contribute to high VEGF level in the model studied. The positive correlation of WBC and VEGF cannot be explained by direct VEGF release from leukocytes under the influence of hypoxia, since white blood cells do not produce significant quantities of VEGF in response to isolated hypoxia [26].

Recent investigations have indicated that VEGF may have a role in the pathogenesis of different manifestations and different stages of stable COPD [27, 28]. In our study, there was no significant correlation between the VEGF levels and lung function parameters. It is possible that in the exacerbated COPD group, systemic hypoxia and inflammation have a much stronger influence on serum VEGF level, than local lung tissue production. Also, in our group of patients with exacerbation, the lung function testing was not performed at the same time as the VEGF measurements.

The most important and consistent finding of our study wasthat serum bFGF levels are significantly elevated in exacerbated COPD patients, which has been scarcely addressed in the literature. Serum bFGF levels were undetectable in patients with stable disease and in healthy blood donors. Basic FGF synthesis was activated only in patients with significant hypoxia. Therefore, it is probably not a linear phenomenon, but occurs only after a certain level of hypoxia is reached. A strong negative correlation in this model indicates that hypoxia is the most important stimulus of bFGF production. Also, step-wise regression analysis disclosed that the independent correlation is due to hypoxia and acid-base status. Based on these results, we believe that hypoxia is one of the factors leading to its increased expression.

It is well known that the synthesis of bFGF is strongly activated in hypoxic conditions by the regulation of HIF[5]. The increased expression of this factor has been found in states of local and systemic hypoxia. The expression of the bFGF gene is enhanced in skeletal muscles after physical exercise [29]. It has been shown in the model ofischemic myocardium, that monocytes in hypoxia produce bFGF [30]. In patients with solid tumors and concomitant anemia there is a significant correlation between systemic hypoxia and serum b-FGF [31]. In the condition of chronic hypoxia, bFGF is increasingly expressed in the brain tissue [32].

Larsson et al. measured bFGF in healthy blood donors [33]. In this study, the levels of bFGF were higher than in our group of healthy blood donors, and levels in women were higher than in men. The mean age of female patients in that study was 43 years, with the expected reproductive cycle in a significant percentage of patients. One of rare situations in which angiogenesis exist in adults is during the female reproductive cycle [6]. The mean age of female patients in our study was 54 years and they were all experiencing the menopause, which may be a reason for the lower levels of bFGF.

Several clinical models have also indicated the association between chronic inflammation and bFGF expression. It hasbeen shown that systemic inflammation in patients on peritoneal dialysis is linked to increased plasma levels of bFGF [34]. Synovial fibroblasts of patients suffering from rheumatoid arthritis produce bFGF [35]. Some studies have suggested that IL-6 could enhance expression of bFGF [36].

However, in contrast to VEGF, in our study, we did not confirm any independent correlation with inflammatory markers.

The highly significant, simple correlation of VEGF and bFGF is completely lost in multivariate analysis, suggesting that their correlation is not independent, but is due to the high correlation with common, causative factor(s) thatremain in the model after step-wise regression. Most important of these are essentially linked to the level of hypoxia.

In this study, the patients with exacerbated COPD had significantly higher serum VEGF and bFGF levels compared to the COPD patients with stable disease or the healthy blood donor group. Multivariate analysis disclosed interesting interactions. Step-wise regression analysis showed that hypoxia was the independent factor that contributed to the model, for both VEGF and bFGF. The only two additional factors influencing VEGF were WBC and IL-6, both essentially linked to inflammation. The only additional factor influencing bFGF was alkalosis, the mechanism of which is presently unclear. All other parameters regarding VEGF and bFGF ended the analysis outof the model. A strong, significant correlation between VEGF and bFGF was entirely lost in multivariate step-wise regression analysis. This suggests that their correlation was not independent, but was due to the high correlation with hypoxia, the common factor that remained in the model after step-wise regression. This finding illustrates the predominant role of hypoxia.

The elevated values of VEGF and bFGF in a group of exacerbated COPD patients may indicate that the process of neoangiogenesis has been activated in this group of patients, which may have lead to increased perfusion and an improvement in tissue oxygenation.

Financial support

Disclosure. None of the authors has any conflict of interest to disclose.

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