Hemolysis and hypoxia have been identified as risk factors for elevated tricuspid regurgitation velocity in children and adolescents with sickle cell disease 
. Here we investigated whether markers of other biologic pathways implicated in pulmonary hypertension in conditions other than sickle cell disease may be associated with elevated tricuspid regurgitation velocity in sickle cell disease as well. We also examined the relationship of these markers to mitral valve E/Etdi, an indicator of left ventricular diastolic function. In this process, we found that, in addition to a composite marker of hemolysis, circulating angiogenic and inflammatory markers were associated with elevated tricuspid regurgitation velocity but not with elevated mitral valve E/Etdi ratio in children and adolescents with sickle cell disease. In multivariate analyses, higher levels of PDGF and lower levels of VEGF were associated with elevated regurgitation velocity.
Pulmonary hypertension is characterized by abnormal proliferation of pericytes and smooth muscle cells in the pulmonary microvasculature 
. PDGF activates vascular smooth muscle cells/pericytes in the process of angiogenesis 
and promotes the development of experimental and hypoxia-related pulmonary hypertension by inducing the proliferation and migration of smooth muscle cells and fibroblasts 
. In the present study, we found that greater plasma concentrations of PDGF correlated significantly with higher values for tricuspid regurgitation velocity in both bivariate and multivariate analyses among children and adolescents with sickle cell disease. Other clinical and translational studies also support a role for PDGF in promoting 
the development of pulmonary hypertension in settings other than sickle cell disease. The pharmacologic agent, imatinib, serves as a PDGF receptor antagonist and reverses vascular remodeling and cor pulmonale in experimental pulmonary hypertension 
. Recent studies indicate that PDGF receptor antagonists such as imatinib and sunatinib are effective therapy for experimental pulmonary hypertension 
, and case reports suggest that imatinib has efficacy in human patients as well 
. In addition, activated platelets might be a source of increased plasma PDGF levels in sickle cell disease 
In contrast to the stimulation of vascular smooth muscle cells by PDGF, VEGF stimulates proliferation and migration of endothelial cells in the process of angiogenesis 
and has not consistently been found to contribute to experimental pulmonary hypertension 
. VEGF expression is controlled by a number of complex regulatory mechanisms 
. Specifically during hypoxia VEGF transcription is induced from the rapid stabilization of hypoxia inducible factor-1 
. During hemolysis and subsequent hypoxia in sickle cell disease, one would expect VEGF to be induced, and the present data shows a strong inverse correlation of circulating VEGF concentration with hemoglobin concentration. Consistent with a recent study in adults 
, we did not observe a significant relationship of plasma concentrations of VEGF with tricuspid regurgitation velocity in bivariate analysis. However, we found that greater VEGF concentrations were associated with lower tricuspid regurgitation velocity in multivariate analysis. This observation is noteworthy in the light of recent research by other investigators indicating that PDGF and VEGF have dichotomous roles in the regulation of vascular smooth muscle cells/pericytes: PDGF mediates the growth of pericytes by a receptor-mediated mechanism in a model of angiogenesis, and increased VEGF prevents this effect of PDGF 
. Another potential mechanism whereby VEGF may associate with a lower regurgitation velocity is through induction of nitric oxide: VEGF induces nitric oxide synthesis by endothelial nitric oxide synthesis via an Akt-mediated pathway 
. Other clinical and translational studies also support a role for VEGF in inhibiting 
the development of pulmonary hypertension in settings other than sickle cell disease. Thus, higher VEGF expression conceivably could protect from elevation of pulmonary artery pressure in sickle cell disease by two pathways, namely inhibition of pulmonary vascular smooth muscle proliferation and increased production of nitric oxide by endothelial cells.
Although we previously observed significant, independent associations of both higher hemolytic index and lower hemoglobin oxygen saturation with elevated tricuspid regurgitation velocity in this cohort of children and adolescents with sickle cell disease 
, the present study indicated that the hemoglobin oxygen saturation was not significantly associated after adjustment for VEGF, PDGF-BB and interleukin-6. Our findings are therefore consistent with the possibility that effects of hypoxia on systolic pulmonary arterial pressure are at least in part mediated by changes in angiogenic and inflammatory responses.
Sickle cell disease is characterized by chronic low-grade inflammation and endothelial activation 
as manifested by leukocytosis and monocytosis 
and increased soluble vascular cell adhesion molecules 
. There are differing reports regarding the involvement of cytokines as mediators of inflammation in sickle cell disease 
. In the present study, we observed higher plasma levels of interleukin-8 and interleukin-10 in sickle cell disease patients compared to control subjects. Among the patients with sickle cell disease, we observed bivariate associations of higher interleukin-6, interleukin-8, interferon-γ, tumor necrosis factor-α and RANTES concentrations with higher tricuspid regurgitation velocity, and an independent association of increased interleukin-6 concentration with elevated tricuspid regurgitation velocity. These observations are compatible with the possibility that pro-inflammatory processes contribute to pulmonary hypertension in sickle cell disease as proposed by other investigators 
and as observed in other settings 
. Our observation that the relationship of VEGF with tricuspid regurgitation velocity is modified by the degree of elevation of the inflammatory marker, interleukin-6, is paralleled by a report from the literature that pulmonary hypertension in tumor necrosis factor-α-over-expressing mice is associated with decreased VEGF expression 
There are a number of limitations to our study. Thirty-eight percent of the patients were being treated with hydroxyurea and 13% were on a chronic transfusion program. Although plasma concentration of PDGF was lower in the patients receiving hydroxyurea and higher in those on a chronic transfusion program, these forms of treatment were not significant covariates in examining the relationships of the biologic markers with tricuspid regurgitation velocity or mitral valve E/Etdi. The reliability of single echocardiographic measurements of tricuspid regurgitation velocity and E/Etdi has not been established in children with sickle cell disease. Other than a greater decline in oxygen saturation during the six minute walk test in patients with elevated tricuspid regurgitation velocity, we have not observed functional impairment in patients with elevated tricuspid regurgitation velocity or mitral valve E/Etdi in children and adolescents with sickle cell disease 
. Other limitations to our study are that circulating concentrations of biologic markers may not reflect the levels to which cells of the pulmonary microvasculature are exposed, that this was a cross-sectional rather than a longitudinal study, and that multiple comparisons dilute the statistical significance of individual observations. Furthermore, the negative association of VEGF with tricuspid regurgitation velocity appears only after adjustment for PDGF concentration. However, the pathway analysis lends support to the finding of contrasting associations of VEGF and PDGF with tricuspid regurgitation velocity.
Although the clinical importance of elevated tricuspid regurgitation velocity in children and adolescents with sickle cell disease has not been clarified by long-term follow-up studies, it seems possible that elevated velocity in this age group may identify individuals at risk for developing pulmonary hypertension later in life. From this standpoint, the PUSH cohort could have unique research importance for studying the development of pulmonary hypertension in sickle cell disease. Our observation of opposing VEGF and PDGF profiles in normal versus elevated tricuspid regurgitation velocity groups supports the idea that elevated velocity associates with known factors involved in pulmonary hypertension, and that this clinical measure may be useful for dissecting the pathogenesis of the early stages of cardiopulmonary complications of sickle cell disease. In general, our findings support the idea that the etiology of pulmonary hypertension in sickle cell disease is multi-factorial, and that pro-inflammatory and angiogenic pathways may interact with the degree of hemolysis in contributing to the development of pulmonary hypertension.