INTRODUCTION

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Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and a potent angiogenic peptide secreted by a variety of cell types and tissues. Recent studies showed that VEGF is also synthesized by megakaryocytes and stored in circulating platelets, where it colocalizes with PDGF (1, 2). One speculative role for VEGF in platelets is promotion of vascular repair and wound healing in conjunction with PDGF. Platelet VEGF and PDGF are released during blood clotting or following platelet adhesion to the subendothelial basement membrane at sites of blood vessel injury (3). PDGF, as a potent mitogen for fibroblasts and smooth muscle cells, effectively promotes wound healing, whereas VEGF may initiate angiogenesis and accelerate repair of the endothelial cell lining (4). From a physiological point of view, platelets could thus be regarded as transporters of circulating VEGF that restrict its angiogenic activity to sites of vascular injury.

It is now well established that chronic vascular diseases are associated with enhanced interactions between the vessel wall and the circulating platelets. Among these diseases, pulmonary hypertension is characterized by thickening of pulmonary artery walls, narrowing of pulmonary artery lumina, and elevation of pulmonary vascular resistance (5). Associated functional abnormalities include constriction of small pulmonary arteries, focal vascular injury, and impaired endothelial function. Platelet activation has been suggested as an important contributing factor in pulmonary vascular remodeling and pulmonary hypertension (6). An imbalance between the generation of thromboxane A2 and prostacyclin (7) as well as impaired release of endothelial NO (8, 9) have been reported in the primary and secondary forms of pulmonary hypertension. These vascular abnormalities, which favor interactions between platelets and pulmonary vessels, may lead to release by platelets of vasoconstrictors or smooth muscle cell mitogens, such as PDGF or serotonin. On the other hand, VEGF released from platelets and acting specifically on endothelial cells may play a pivotal role (4). In systemic vessels, increasing VEGF bioavailability at sites of endothelial denudation has been shown to accelerate endothelial repair and to limit neointima formation (10). Moreover, VEGF overexpression within the vascular wall has been shown to restore endothelium-dependent relaxation and to protect against vasoconstriction and platelet activation (11). The overall balance between VEGF and other platelet-derived nonspecific mitogens such as PDGF may, therefore, be of importance in pulmonary vascular diseases and pulmonary hypertension.

In the present study, we investigated serum and platelet VEGF and PDGF in a large population of patients with pulmonary arterial hypertension, either primary or secondary to various diseases, as well as in patients with chronic hypoxemic lung disease. Moreover, we sought to determine whether continuous prostacyclin infusion or other drugs influence platelet VEGF and PDGF contents in patients with pulmonary arterial hypertension.

	METHODS

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Study Population

The study population comprised 68 patients and 29 normal subjects. Among the patients, 41 had pulmonary arterial hypertension, either primary or secondary to various diseases, and 27 had chronic hypoxemic lung disease (CHLD).

Patients with pulmonary arterial hypertension. Twenty-nine patients, including 11 men and 18 women, with a mean age (mean ± SD) of 44 ± 13 yr (range 18-65 yr) were studied for basal determination of biological parameters. Twenty-one had primary pulmonary hypertension (primary PH; 20 sporadic and 1 familial). Of the eight patients with secondary pulmonary hypertension (secondary PH), three had portal hypertension, two had vasculitis associated with a connective tissue disease (one case each of scleroderma and mixed connective tissue disease), one had Eisenmenger's syndrome, one had a glycogen storage disease, and one had chronic thromboembolic heart disease. All 29 PH patients underwent right-sided cardiac catheterization within the 6 mo preceding the study. Pulmonary hypertension was defined as a mean pulmonary artery pressure > 30 mm Hg with a normal pulmonary artery wedge pressure. A thermodilution cardiac output was obtained in all patients. The patients with primary PH had no evidence of primary liver disease, no clinical or serologic evidence of connective tissue disease, and no chest radiography or spirometry evidence of airway or interstitial pulmonary disease. All 29 patients were studied in a stable phase of their disease; 25 were functional class II and class III, and 4 were functional class IV. Sixteen patients were treated with continuous infusion of prostacyclin (12 patients with primary PH and 4 with secondary PH). The remaining 13 patients (9 patients with primary PH and 4 with secondary PH) were treated with nifedipine, diltiazem, or converting enzyme inhibitors.

Twelve additional patients, four men and eight women with a mean age (± SD) of 49 ± 15 yr, were investigated to assess the effects of prostacyclin infusion on biological parameters. In 6 of these patients (four with primary PH and two with secondary PH), biological parameters were determined prior to and 10 d after initiation of continuous prostacyclin therapy. The prostacyclin infusion rate was increased gradually over 7 d to a mean value of 10 ± 2 ng/kg/min. The six other patients, two with primary PH and four with secondary PH, received conventional therapy during the same period of time and were used as controls.

Patients with chronic hypoxemic lung disease. This group included 27 patients, 23 men and 4 women with a mean age of 63 yr (range 38 to 72 yr). Twenty-two of these patients had chronic obstructive lung disease (COLD) diagnosed based on a history of chronic bronchitis and on evidence of chronic airflow limitation on standard pulmonary function tests. The remaining five patients had predominantly restrictive lung disease with a reduction in total pulmonary capacity. All 27 CHLD patients were in a stable phase of their disease. Absence of electrocardiographic abnormalities suggesting ischemic left heart disease and absence of echocardiographic left ventricular dysfunction were verified in each patient prior to the study. Treatment consisted of oral and inhaled bronchodilators, but no medications were taken during the 24 h before the study. Four patients received supplemental oxygen. CHLD patients were then classified into two groups based on whether their Hb level was lower or greater than 15.5 g/dl. Physiological parameters recorded in the normocythemic (n = 19) and polycythemic (n = 8) groups are summarized in Table 3.

Control subjects. Twenty-nine normal subjects were studied. Fourteen subjects aged 31 to 49 yr (mean, 43 yr) served as controls for the PH patients and 15 subjects aged 51 to 69 yr (mean, 63 yr) as controls for the CHLD patients. All controls were healthy and active in a variety of physical recreational activities. None was known to have an acute or chronic illness, except for mild systemic hypertension treated with beta-blocking drugs or angiotensin-converting enzyme inhibitors in six of them. All had normal findings from a thorough physical examination.

The study was approved by our institutional review board, and informed consent was obtained from each subject before the study.

Derived Hemodynamic Measurements and Determination of Blood Gas Parameters

Derived hemodynamic variables were calculated according to standard formulas: cardiac index (CI) = cardiac output/body surface area (L/m²); and total pulmonary resistance index (TPR) = Pap/CI (mm Hg/L/min/m²). Blood gas tensions and pH were determined using an ABL 30, and total hemoglobin, hemoglobin oxygen saturation, and carboxyhemoglobin were measured using an OSM 3 hemoximeter (Radiometer, Copenhagen, Denmark).

Blood Sample Preparation

Blood samples were obtained from a peripheral forearm vein in the patients and normal controls. Serum was prepared by drawing 5 ml of blood on Venoject II tubes containing a clot activator (Terumo Europe N.V., Leuven, Belgium), then allowing the tubes to stand for 60 min at 22° C to ensure full clotting of the serum. Plasma was separated from blood sampled in chilled EDTA tubes. The samples were centrifuged at 800 × g for 10 min at 4° C, and the supernatants were aliquoted and frozen at 20° C pending analysis. Fresh platelet-rich plasma (PRP) was obtained from blood collected on plastic EDTA tubes, by differential centrifugation (600 × g, 10 min) at room temperature. A PRP platelet count was obtained using an automated cell counter. The fresh PRP was then centrifuged at 800 × g for 10 min at 4° C, and the supernatant was collected for subsequent analysis. Platelet lysis was achieved by suspension of the pellet in the same volume of cold saline followed by four cycles involving rapid freezing in liquid nitrogen and thawing at 20° C. VEGF and PDGF were then assayed in serum, platelet supernatant, and platelet homogenate fractions. Because nonsignificant amounts of VEGF and PDGF were detected in the platelet supernatant fraction, only the serum and platelet fractions were taken into account for further analysis.

Platelet Aggregation

In six normal volunteers, VEGF and PDGF were assayed in platelets aggregated in vitro. Blood mixed with a 3.8% citrate dextrose solution was used to obtain PRP by differential centrifugation. Aggregation tests were performed using a Labintec aggregometer (Montpellier-France) by adding calcium chloride and thrombin 0.4 U/ml (Sigma). Five minutes after maximal changes in optical transmission, a 100-µl sample was taken for VEGF and PDGF assays. Because concentrations of VEGF and PDGF in platelets aggregated by thrombin were similar to those obtained in serum, only serum concentrations were used for the remainder of the study.

ELISA for VEGF and PDGF

VEGF and PDGF concentrations were measured in duplicate in each sample using a commercially available sandwich enzyme-linked immunoabsorbent assay (ELISA) (R&D Systems Europe, Oxon, UK). According to the manufacturer, the VEGF assay has a detection threshold of 9 pg/ml (0.2 pM) and does not cross-react with PDGF or other homologous cytokines. The detection threshold of the PDGF-AB assay is 420 pg/ml. The PDGF assay was done on samples diluted 50-fold with assay buffer. Optical density at 450 nm was measured on an MRX microplate reader (Dynatech Laboratories, Guyancourt, France), and growth factor concentration was determined by linear regression from a standard curve obtained using VEGF165 or PDGF-AB as the standard.

Statistical Analysis

All data are reported as means ± SEMs. Between-group comparisons of physiological and biological parameters were performed using ANOVA. When ANOVA indicated significant between-group differences, groups were compared using a Scheffe test. To evaluate the effects of prostacyclin infusion, we used a two-way ANOVA for repeated measures and tested for group effect, treatment effect, and interaction. Because interaction was significant, a Wilcoxon test was used to compare groups before and after treatment; p values less than 0.05 were considered significant.

	RESULTS

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Platelet VEGF and PDGF in Patients with Pulmonary Arterial Hypertension

The clinical characteristics of the two PH groups are shown in Table 1. There was a trend toward lower TPR values in secondary than in primary PH patients. The only significant difference was a higher cardiac index in the secondary PH subgroup than in the primary PH subgroup.

The platelet counts and platelet VEGF and PDGF levels in the primary and secondary PH patients and in the control patients are shown in Figures 1 and 2. The platelet count was decreased in primary PH patients and unaltered in secondary PH patients, as compared with control patients (177 ± 12, 208 ± 26, and 238 ± 16 platelets × 10³ per microliter, respectively; p < 0.01) (Figure 1). The platelet lysate VEGF concentration was elevated in the primary PH patients (despite the platelet count decrease) and in secondary PH patients, as compared with control patients (838 ± 110, 1197 ± 185, and 373 ± 54 pg/ml, respectively; p < 0.01) (Figure 1). As a result, the platelet VEGF content, defined as the amount of VEGF per 10⁵ platelets, was markedly increased in patients with primary PH or secondary PH as compared with control patients (517 ± 88, 674 ± 156, and 166 ± 29 fg/10⁵ platelets, respectively; p < 0.01) (Figure 2). In contrast, the platelet lysate PDGF concentration was slightly decreased in the primary PH patients as compared with the control patients, but was not significantly altered in the secondary PH patients (Figure 1). The platelet PDGF content, defined as the amount of PDGF per 10⁵ platelets, did not differ among the primary PH, secondary PH, and control groups (31 ± 2, 36 ± 4, and 33 ± 3 pg/10⁵ platelets, respectively; NS) (Figure 2). No correlations were found between hemodynamic variables (Pap, TPR, or CI) and platelet VEGF or PDGF in patients with either form of PH.

View larger version (24K): [in this window] [in a new window]   Figure 1.   Platelet count and concentrations of VEGF and PDGF in platelet lysate from normal control patients and patients with primary or secondary pulmonary hypertension (PH). Results are expressed as means ± SEMs. *p < 0.05; **p < 0.01 versus values in control patients. There were no differences between the two pulmonary hypertension subgroups.

View larger version (20K): [in this window] [in a new window]   Figure 2.   Individual values of platelet VEGF content and platelet PDGF content in normal control patients and in patients with primary or secondary pulmonary hypertension (PH). **p < 0.01 versus values in control patients. There were no differences between the two pulmonary hypertension subgroups.

Serum VEGF concentration was also elevated in primary PH patients and secondary PH patients as compared with control patients (547 ± 95, 832 ± 172, and 169 ± 24 pg/ml, respectively; p < 0.01) whereas serum PDGF was unaltered (38 ± 3, 35 ± 6, and 50 ± 3 ng/ml, respectively; NS). The ratio of serum to platelet VEGF content was similar to that of PDGF in the overall group of patients with PH (0.65 ± 0.03 versus 0.67 ± 0.05, respectively; NS), in the group of patients with primary PH (0.64 ± 0.04 versus 0.71 ± 0.06, respectively; NS), and in the group of patients with secondary PH (0.68 ± 0.05 versus 0.63 ± 0.05, respectively; NS).

Patients on prostacyclin had a higher platelet VEGF content than did patients not on prostacyclin (689 ± 120 versus 403 ± 51 fg/10⁵ platelets, respectively; p < 0.05), whereas they had a similar platelet PDGF content (32 ± 2 versus 32 ± 2 pg/10⁵ platelets, respectively; NS) and platelet count (179 ± 15 versus 195 ± 19 platelets × 10³ per microliter, respectively; NS) (Figure 3). There was a trend toward a higher mean Pap value in prostacylin-treated than in non-prostacyclin-treated patients (65 ± 3 versus 60 ± 3 mm Hg, respectively; NS); TPR (33 ± 2 versus 32 ± 7 U/m², respectively; NS) and cardiac index (2.1 ± 0.1 versus 2.3 ± 0.3 L/min/m², respectively; NS) were similar in these two subgroups. The ratio of serum over platelet VEGF content was similar to that of PDGF in the prostacylin-treated patients (0.70 ± 0.03 versus 0.62 ± 0.05, respectively; NS). When primary PH patients with or without prostacyclin were analyzed separately, their platelet VEGF content was elevated as compared with control patients (575 ± 145 fg/10⁵ platelets, 441 ± 76 fg/10⁵ platelets, and 166 ± 29 fg/10⁵ platelets, respectively; p < 0.001).

View larger version (21K): [in this window] [in a new window]   Figure 3.   Platelet count and platelet contents of VEGF and PDGF in patients treated with oral vasodilator drugs (n = 13) or with a continuous prostacyclin infusion (n = 16). Results are expressed as means ± SEMs. *p < 0.05 for between-group comparison.

Measurements obtained before and 10 d after prostacyclin therapy initiation in six additional patients showed an increase in serum VEGF concentration (p < 0.05), platelet lysate VEGF concentration (p < 0.05), and platelet VEGF content (p < 0.05), whereas platelet count and platelet PDGF content remained unchanged (Table 2). In the control group under conventional therapy, no differences were found between values obtained before and after 10 d of treatment: serum VEGF concentrations were 817 ± 176 and 816 ± 178 pg/ml (NS), platelet lysate VEGF concentrations were 1,178 ± 318 and 1,217 ± 293 pg/ml (NS), platelet VEGF contents were 546 ± 195 and 529 ± 153 fg/10⁵ platelets (NS), platelet counts were 242 ± 20 and 242 ± 17 cells × 10³/µl (NS), and platelet PDGF contents were 29 ± 6 and 30 ± 7 pg/10⁵ platelets (NS), respectively. The prostacyclin and control groups did not differ regarding basal Pap (55 ± 3 versus 50 ± 7 mm Hg, NS), CI (1.9 ± 0.4 versus 2.0 ± 0.6 L/min/m², NS), or TPR (29 ± 2 versus 27 ± 5 UI, NS).

Platelet VEGF and PDGF in Patients with Chronic Hypoxemic Lung Disease

The 27 CHLD patients were older (63 ± 5 yr) than the PH patients, and all had a long history of smoking. Current smoking was noted in seven CHLD patients at study inclusion. Polycythemic and normocythemic patients did not differ with respect to age or pulmonary function tests (Table 3). There was a trend for polycythemic patients to have a lower arterial PO₂. A higher arterial PCO₂ in the polycythemic than in the normocythemic group was the only significant difference.

The platelet count was slightly decreased in the polycythemic patients but not in the normocythemic patients as compared with the control patients (189 ± 85, 267 ± 16, and 237 ± 17 platelets × 10³ per microliter, respectively; p < 0.001; Figure 4). No significant differences were noted between the platelet lysate VEGF concentrations in the polycythemic and normocythemic patients and those in the control patients (602 ± 79, 528 ± 83, and 420 ± 83 pg/ml, respectively). The platelet lysate PDGF concentration was increased in the polycythemic patients but not in the normocythemic patients as compared with the control patients (92 ± 18, 75 ± 5, and 55 ± 4 ng/ml, respectively; p < 0.05). Lastly, both the platelet VEGF content and the platelet PDGF content, defined as the amount of peptide per 10⁵ platelets, remained unchanged in the normocythemic patients but were increased in the polycythemic patients as compared with the control patients (VEGF: 372 ± 65 versus 185 ± 25 fg/10⁵ platelets, respectively, p < 0.01; PDGF: 49 ± 7 versus 24 ± 2 fg/10⁵ platelets, respectively; p < 0.01) (Figure 4). Positive correlations were found between the Hb concentration and the platelet PDGF and VEGF contents in the entire population of CHLD patients (Figure 5). Carboxyhemoglobin, which was available in a subgroup of nine patients, was positively correlated with platelet PDGF and VEGF contents (p < 0.05). Pulmonary artery pressure, available in 11 patients, did not correlate with platelet VEGF and PDGF contents, nor did arterial PO₂ in the overall group of CHLD patients.

View larger version (28K): [in this window] [in a new window]   Figure 4.   Platelet count and platelet contents of VEGF and PDGF in 15 age-matched normal control patients, 19 normocythemic chronic hypoxemic lung disease (CHLD) patients, and eight polycythemic CHLD patients. Results are expressed as means ± SEMs. *p < 0.05; **p < 0.01 versus values in control patients. Polycythemic differed from normocythemic patients with respect to platelet count (p < 0.05), platelet VEGF content (p < 0.01), and platelet PDGF content (p < 0.01).

View larger version (17K): [in this window] [in a new window]   Figure 5.   The relationship between Hb concentration and platelet VEGF content or platelet PDGF content in 27 patients with chronic hypoxemic lung disease.

Platelet VEGF and PDGF in Control Subjects

Platelet PDGF content was lower in patients older than 50 yr of age than in those younger than 50 yr (24 ± 2 versus 33 ± 3 pg/10⁵ platelets, respectively; p < 0.05). Although no significant age-related difference was found for platelet VEGF, there was a trend toward a higher VEGF content in patients older than 50 yr of age as compared with those younger than 50 yr of age (185 ± 40 versus 166 ± 29 fg/10⁵ platelets, respectively, NS).

	DISCUSSION

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We found that platelet VEGF content was markedly elevated in patients with primary or secondary PH as compared with normal controls, whereas platelet PDGF content was unchanged. These findings imply that sustained Pap elevation, regardless of its cause, may lead to an increase in platelet VEGF content and, potentially, to an increase in platelet VEGF release at sites of vascular injury. Interestingly, platelet VEGF content was increased by continuous prostacyclin infusion, indicating that continuous prostacyclin therapy increases circulating VEGF levels. In polycythemic patients with severe chronic hypoxemic lung disease (CHLD), only moderate increases in platelet VEGF and PDGF contents were observed, suggesting that hypoxemia was the main factor leading to the increases in platelet VEGF and PDGF contents. We suggest that platelet VEGF elevation during PH may be a protective mechanism against pulmonary vascular injury and remodeling.

PH is associated with persistent vasoconstriction and structural remodeling of pulmonary vessels. Although the mechanisms leading to these vascular abnormalities are still undefined, an increasing body of published data suggests that enhanced interactions between platelets and the pulmonary arterial wall may contribute to the functional and structural alterations of pulmonary vessels (5, 7). Upon activation, platelets release vasoactive factors such as thromboxane, serotonin, and platelet-activating factor (PAF), as well as mitogens such as PDGF and serotonin, which may contribute to pulmonary vascular remodeling. The recent observation that platelets contain VEGF provides new insight into the mechanisms by which platelets may affect the pulmonary arterial wall. VEGF, a homodimeric, 34- to 42-kD heparin-binding glycoprotein, is a peptide mitogen specific for endothelial cells that fulfills its function by binding to Flt-1 and KDR/flk-1, two highly specific tyrosine kinase receptors expressed almost exclusively on endothelial cells (12). The impact of platelet-derived VEGF in vascular diseases may depend on several factors, including the amount released locally, the number of platelets involved, and the overall balance with other platelet-derived nonspecific mitogens such as PDGF. In the present study, we simultaneously examined platelet counts, serum and platelet VEGF, and serum and platelet PDGF in patients with PH or CHLD.

Platelet VEGF was markedly increased in patients with primary or secondary PH as compared with control patients. Serum VEGF was also markedly elevated in these patients as a result of its release from platelets during the clotting process. Patients with PH had a lower platelet count that normal control patients, indicating that the increase in circulating VEGF in these patients was due to an increase in the platelet VEGF content. In contrast, platelet PDGF content was unaltered in patients with PH; circulating PDGF was slightly decreased as compared with normal control subjects, reflecting the decrease in platelet count. It should be pointed out that the platelet VEGF increase was not related to the cause of PH: platelet VEGF content was the same in patients with primary and secondary PH, who had similar levels of Pap and PVR. Our results, therefore, demonstrate a link between the presence of PH and the amount of VEGF contained in platelets, although we found no correlation in PH patients between hemodynamic variables and platelet VEGF. To explain these results, one may suggest that any form of pulmonary hypertension could lead to removal of older platelets. If there is normally a gradual loss of VEGF during the life of the platelet, this might account for the higher levels observed. This hypothesis, however, is unlikely since it has been demonstrated that VEGF resides in the alpha-granules of platelets, where it colocalizes with PDGF (3). An increase in platelet VEGF content with no change in platelet PDGF may indicate increased VEGF synthesis by platelet precursor cells. An alternative explanation may be a lower rate of release by activated platelets of VEGF than of PDGF. However, the similarity in PH patients of serum over platelet ratios for VEGF and PDGF argues against this last hypothesis. Our results therefore support the possibility that VEGF elevation in platelets from PH patients may be related to the PH process and may occur as a consequence of increased VEGF production by megakaryocytes.

In contrast to patients with PH, polycythemic patients with CHLD exhibited similar increases in platelet VEGF and PDGF. An important feature of VEGF is its sensitivity to hypoxia (12). Hypoxia is also a strong inducer of PDGF expression in vitro and in vivo (13). It follows that one possible explanation to the increased platelet VEGF and PDGF contents in patients with CHLD is a direct effect of low oxygen tension on PDGF and VEGF synthesis. The fact that platelet VEGF and PDGF contents correlated directly with hemoglobin concentration in patients with CHLD is consistent with this possibility. Polycythemic patients showed more severe gas exchange alterations than normocythemic patients, with trends toward a lower arterial oxygen tension and more severe respiratory acidosis. Moreover, polycythemia is known to be a reliable index of long-term tissue oxygenation in patients with CHLD (14). In addition, carboxyhemoglobin, which was available only in a subgroup of patients, was positively correlated with platelet VEGF.

The mechanism underlying the increased platelet VEGF content in our PH patients was unrelated to hypoxia. Patients with PH had a larger increase in platelet VEGF than patients with CHLD, although their arterial PO₂ and arterial O₂ transport were within normal ranges. In vitro studies of megakaryocytes have shown that stimulation of megakaryocyte proliferation by hematopoietic cytokines resulted in increased constitutive production of VEGF (2, 15). One hypothesis suggested by these studies is that stimulation of megakaryocytopoiesis may lead to increased production and secretion of VEGF. This possibility is consistent with our results, since both our PH patients and our polycythemic CHLD patients had low platelet counts, probably because of increased platelet consumption with a secondary increase in the rate of platelet formation. To date, serum VEGF increases have been reported mainly in cancer patients. Although the mechanism of VEGF up-regulation in cancer patients has not yet been clarified, it has been suggested that thrombopoietic cytokines, such as interleukin-6, may play an important role (16). Interleukin-6 is also known to be increased in patients with PH (17). Thus, PH, polycythemia, and cancer may all be characterized by increases in the rate of platelet formation and/or in the release of specific cytokines, which may contribute to enhance VEGF synthesis by megakaryocytes. From a physiological point of view, increased platelet VEGF production during thrombopoiesis may be a protective mechanism against excessive platelet consumption in vascular diseases associated with enhanced interactions between platelets and the endothelium (2, 16).

An additional observation made during our study is that prostacyclin-treated patients had a higher platelet VEGF content than patients not treated with prostacyclin. Moreover, data obtained in six patients at prostacyclin therapy initiation showed a significant increase in platelet VEGF content. Continuous intravenous prostacyclin is now widely used to treat severe PH and has been found in prospective randomized clinical trials to improve hemodynamics and survival in functional class III and class IV patients (18, 19). The mechanism of action of the chronic effects of prostacyclin in PH is unknown, but is probably multifactorial. Although the predominant effect of prostacyclin may be pulmonary blood vessel dilation, studies have demonstrated that absence of an acute vasodilator response to prostacyclin does not preclude a beneficial effect during chronic prostacyclin therapy (18, 19). Thus, the long-term effects of prostacyclin in primary pulmonary hypertension may be related only partially to its vasodilator properties and may be due, at least in part, to poorly defined effects on vascular growth, remodeling, or platelet function. Our results indicate that continuous prostacylin infusion leads to an increase in the amount of VEGF in platelets. One likely explanation of this effect is that prostacyclin may increase VEGF synthesis by megakaryocytes. In our study, platelet PDGF was not increased in prostacyclin-treated patients, suggesting that the effect of the drug was unrelated to inhibition of platelet aggregation and release. Moreover, prostacyclin, as well as other mediators that work via cAMP formation, have been shown to induce VEGF synthesis in various cell types (20, 21). It follows that the therapeutic effect of prostacyclin in PH patients may be related in part to increased VEGF synthesis by cells other than megakaryocytes. This observation may be of clinical relevance not only to the treatment of PH with prostacyclin but also to the treatment of peripheral arterial disease since continuous prostacyclin infusion may improve limb ischemia in this condition.

An important issue raised by our findings is whether increased platelet VEGF content and potentially, increased VEGF release at sites of vascular injury, notably in the pulmonary vasculature, have protective or deleterious effects. The exact role of VEGF in the genesis of human PH and the vascular remodeling inherent in this condition remains unknown. Recently, evidence has been provided that the plexiform lesion, a specific vascular abnormality of the pulmonary arteries during PH, results from prominent endothelial cell proliferation (22). Since increased amounts of VEGF protein and transcripts have also been found in the plexiform lesions of vessels from patients with primary PH, one current hypothesis is that VEGF may be involved in the formation of these lesions (23). On the other hand, it is now well known that VEGF protects against neointimal thickening following catheter-mediated local endothelial vascular injury. In balloon-injured rat carotid artery, local delivery of VEGF accelerates reendothelialization and attenuates neointimal thickening due to smooth muscle cell proliferation (10). Moreover, VEGF accelerates restitution of endothelial integrity and endothelium-dependent function following endothelial injury (11). Similar findings have now been obtained in the pulmonary circulation: we recently obtained evidence that lung VEGF expression was unchanged in the experimental model of hypoxic PH (24), and that adenoviral-mediated lung VEGF overexpression protected against hypoxic PH and development of pulmonary vascular remodeling (unpublished data). Our findings from the present study are consistent with these observations. Since the angiogenic activity of platelet VEGF may be restricted to sites of wound healing, it is conceivable that increased circulating amounts of VEGF during PH may contribute to improved endothelial function, prevent platelet aggregation, and protect against vascular remodeling.