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Pulmonary hypertension is associated with reduced nitric oxide bioavailability and early mortality in sickle cell disease (SCD). We previously demonstrated that placenta growth factor (PlGF), an angiogenic factor produced by erythroid cells, induces hypoxiaindependent expression of the pulmonary vasoconstrictor endothelin-1 in pulmonary endothelial cells. Using a lentivirus vector, we simulated erythroid expression of PlGF in normal mice up to the levels seen in sickle mice. Consequently, endothelin-1 production increased, right ventricle pressures increased, and right ventricle hypertrophy and pulmonary changes occurred in the mice within 8 weeks. These findings were corroborated in 123 patients with SCD, in whom plasma PlGF levels were significantly associated with anemia, endothelin-1, and tricuspid regurgitant velocity; the latter is reflective of peak pulmonary artery pressure. These results illuminate a novel mechanistic pathway linking hemolysis and erythroid hyperplasia to increased PlGF, endothelin-1, and pulmonary hypertension in SCD, and suggest that strategies that block PlGF signaling may be therapeutically beneficial. IntroductionPulmonary hypertension (PH) occurs in 10% to 30% of patients with sickle cell disease (SCD) [1][2][3] and is associated with a 17% 2-year mortality in adults. 1 Factors implicated in SCD PH include endothelial dysfunction, pulmonary vasoconstriction, and remodeling, all mechanistically associated with chronic hemolysis, hypoxia, hemostatic activation, and inflammation. [4][5][6] Nitric oxide (NO) and endothelin-1 are opposing pulmonary vasoactive factors that regulate pulmonary vascular tone. 7,8 Hemolysis in SCD results in quenching of NO by extracellular hemoglobin and reduces availability of NO-synthase substrate. 6,8 Endothelin-1, a potent pulmonary vasoconstrictor, is normally induced from endothelial cells by hypoxia-mediated up-regulation of hypoxia inducible factor-1␣ (HIF-1␣), and its levels are elevated in PH. 9 Indeed, endothelin-1 receptor antagonists are used for the treatment of primary PH. Endothelin-1 levels are also significantly elevated in patients with SCD, 10,11 and endothelin-1 receptor antagonists have been recently found to be beneficial in sickleAntilles-hemoglobin-D mice. 12 We recently showed the mechanism of endothelin-1 induction via a novel hypoxia-independent up-regulation of HIF-1␣ in cultured human pulmonary microvascular endothelial cells by placenta growth factor (PlGF). 13 Bone marrow erythroid cells produce PlGF, and PlGF levels are significantly increased in patients with chronic hemolytic anemias, SCD, and ␤-thalassemia, as part of the compensatory erythroid hyperplasia response. 14,15 This study was designed to test the hypothesis that chronically elevated PlGF in SCD contributes to PH. Methods Vector constructsThe mouse PGF cDNA was cloned downstream of the ␤-globin promoter to replace green fluorescent protein (GFP) in the lentiviral vector s␤-GFP, 16 to generate s␤-PlGF (Figure 1). We have shown this transcription cassette to exp...

Objective-The objectives of this study were to determine whether hypercholesterolemia promotes platelet-endothelial cell (P/E) adhesion in murine postcapillary venules and define the contributions of endothelial or platelet associated P-selection to hypercholesterolemia-induced P/E interactions. Methods and Results-Wild-type (WT) or P-selectin deficient (P-sel Ϫ/Ϫ ) platelets were isolated and labeled with the fluorochrome CFSE and administered to either WT or P-sel Ϫ/Ϫ mice placed on a normal diet (ND) or high cholesterol diet (HCD). Intravital videomicroscopy was used to quantify platelet saltation and firm adhesion. HCD-WT mice exhibited a time-dependent increase in P/E cell interactions (relative to ND-WT). Flow cytometry revealed an increased expression of P-selectin on circulating platelets of HCD-WT mice at 2 weeks compared with ND-WT mice. When WT platelets were monitored in HCD-P-sel Ϫ/Ϫ mice, P/E adhesion was dramatically reduced. However, when P-sel Ϫ/Ϫ platelets were monitored in HCD-WT recipients, P/E adhesive interactions were reduced even further, comparable to ND-WT mice. Conclusions-These results indicate that elevated cholesterol levels promote P/E adhesion in postcapillary venules and that whereas both endothelial and platelet P-selectin contribute to hypercholesterolemia-induced recruitment of platelets, platelet-associated P-selectin seems to play a more important role in producing the prothrombogenic phenotype in venules.

The influence of reductions in venular shear rate on platelet-endothelial (P/E) cell adhesion has not been previously addressed. The objectives of this study were to define the effects of reductions in venular shear rate on P/E cell adhesion and to determine the interdependence of P/E cell adhesion and leukocyte-endothelial (L/E) cell adhesion at low shear rates. Intravital videomicroscopy was used to quantify P/E and L/E cell adhesion in rat mesenteric venules exposed to shear rates ranging between 118 Ϯ 9 and 835 Ϯ 44 s Ϫ1 . Shear rate was altered in postcapillary venules by rapid, graded blood withdrawal, without retransfusion of shed blood. Reducing shear rate from Ͼ600 s Ϫ1 to Ͻ200 s Ϫ1 resulted in an eightfold increase in L/E cell adhesion, whereas P/E cell adhesion increased 18-fold. A blocking antibody directed against P-selectin blunted both the P/E and L/E cell adhesion elicited by low shear rates. Immunoneutralization of CD11/CD18 on leukocytes or rendering animals neutropenic also blocked the shear rate-dependent recruitment of both platelets and leukocytes. These findings indicate that 1) low shear rates promote P/E and L/E cell adhesion in mesenteric venules, and 2) adherent neutrophils (mediated by CD11/CD18) create a platform onto which platelets can bind to the venular wall at low shear rates. P-selectin; neutrophils; CD18; ischemia BLOOD FLOW AND THE ACCOMPANYING shear forces generated by blood moving within venules can exert a significant influence on the behavior of both platelets and neutrophils (5, 10, 30). The laminar flow profile of blood flowing in microvessels results in generation of lateral displacement forces that allow red blood cells to push leukocytes and platelets toward the vessel wall where interactions with endothelial cells can occur. Blood flow-induced shear forces also play an important role in disrupting the adhesive bonds between blood cells (leukocytes and platelets) and vascular endothelium (19).Hence, high shear rates might be expected to oppose blood cell-to-endothelial cell adhesion, whereas low shear rates should promote this cell-to-cell adhesion. This phenomenon has potentially important implications for the recruitment of circulating blood cells onto the wall of blood vessels under inflammatory conditions associated with elevated (e.g., chronic inflammatory diseases) or reduced (e.g., in postischemic tissues) blood flow (and shear rate). For example, reperfusion of ischemic tissues is generally associated with an enhanced adhesion of both leukocytes (9) and platelets (20, 21) within postcapillary venules. These adhesive interactions are generally accompanied by a reduction in venular shear rate. It appears likely, therefore, that the lower shear rates experienced by postischemic venules will contribute to the recruitment of the leukocytes and platelets during the reperfusion period.Although the influence of low venular shear rates on leukocyte-endothelial (L/E) cell adhesion has been extensively studied (3,4,26), it remains unclear if and how low shear rates modu...