High levels of placenta growth factor in sickle cell disease promote pulmonary hypertension.

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 hypoxia-independent 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.

Apolipoprotein A-I and serum amyloid A plasma levels are biomarkers of acute painful episodes in patients with sickle cell disease.

BACKGROUND: Acute painful episodes are the clinical hallmark of sickle cell disease and have been linked to morbidity and mortality in the sickle cell population. DESIGN AND METHODS: We undertook exploratory proteomic studies on paired plasma samples collected from a cohort of 26 adult sickle cell patients during steady state and on the first day of an acute painful episode. We screened for changes in abundance of specific protein peaks via surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS), and confirmed the identify of candidate protein peaks by specific immunoassays. RESULTS: The levels of hemoglobin, hematocrit, total protein, and albumin were lower and the levels of lactate dehydrogenase and absolute reticulocytes higher during acute painful episodes than during the steady state. Surface-enhanced laser desorption/ionization time of flight mass spectrometry spectral analysis consistently showed a mass-to-charge peak at 11.7 kDa with elevated intensities during acute painful episodes, which correlated significantly with the serum amyloid A immunoassay. Serum amyloid A levels were significantly elevated during acute painful episodes, especially in four patients with marked end-organ complications of such episodes. A second, recurring peak, less abundant during acute painful episodes, was present at 28.1 kDa; this peak was correlated significantly with immunoassay measurements of apolipoprotein A1. CONCLUSIONS: On the average, plasma serum amyloid A rises and apolipoprotein AI falls during acute painful episodes. The serum amyloid A/apolipoprotein AI ratio increased in 81% of the patients during acute painful episodes, potentially making it a useful objective marker of such episodes. We propose that these protein alterations, known to contribute to endothelial dysfunction in other settings, might do likewise acutely in acute painful episodes and present a new target for therapeutic intervention in sickle cell disease. (ClincalTrials.gov Identifier: NCT00081523).

Hypercholesterolemia promotes P-selectin-dependent platelet-endothelial cell adhesion in postcapillary venules.

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-/-) platelets were isolated and labeled with the fluorochrome CFSE and administered to either WT or P-sel-/- 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-/- mice, P/E adhesion was dramatically reduced. However, when P-sel-/- 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.

Hypercholesterolemia promotes inflammation and microvascular dysfunction: role of nitric oxide and superoxide.

Relatively brief periods (days) of hypercholesterolemia can exert profound effects on endothelium-dependent functions of the microcirculation, including dilation of arterioles, fluid filtration across capillaries, and regulation of leukocyte recruitment in postcapillary venules. Hypercholesterolemia appears to convert the normal anti-inflammatory phenotype of the microcirculation to a proinflammatory phenotype. This phenotypic change appears to result from a decline in nitric oxide (NO) bioavailability that results from a reduction in NO biosynthesis, inactivation of NO by superoxide (O(2)(*)(-)), or both. A consequence of the hypercholesterolemia-induced microvascular responses is an enhanced vulnerability of the microcirculation to the deleterious effects of ischemia and other inflammatory conditions. Hence, therapeutic strategies that are directed towards preventing the early microcirculatory dysfunction and inflammation caused by hypercholesterolemia may prove effective in reducing the high mortality associated with ischemic tissue diseases. Agents that act to maintain the normal balance between NO and reactive oxygen species (ROS) in vascular endothelial cells may prove particularly useful in this regard.

Roles of platelet and endothelial cell COX-1 in hypercholesterolemia-induced microvascular dysfunction.

Aspirin is a common preventative therapy in patients at risk for cardiovascular diseases, yet little is known about how aspirin protects the vasculature in hypercholesterolemia. The present study determines whether aspirin, nitric oxide-releasing aspirin (NCX-4016), a selective cyclooxygenase (COX)-1 inhibitor (SC560), or genetic deficiency of COX-1 prevents the inflammatory and prothrombogenic phenotype assumed by hypercholesterolemic (HC) venules. Aspirin or NCX-4016 (60 mg/kg) was administered orally for the last week of a 2-wk HC diet. COX-1-deficient (COX-1(-/-)) and wild-type (WT) mice were transplanted with WT (WT/COX-1(-/-)) or COX-1(-/-) (COX-1(-/-)/WT) bone marrow, respectively. HC-induced adhesion of platelets and leukocytes in murine intestinal venules, observed with intravital fluorescence microscopy, was greatly attenuated in aspirin-treated mice. Adhesion of aspirin-treated platelets in HC venules was comparable to untreated platelets, whereas adhesion of SC560-treated platelets was significantly attenuated. HC-induced leukocyte and platelet adhesion in COX-1(-/-)/WT chimeras was comparable to that in SC560-treated mice, whereas the largest reductions in blood cell adhesion were in WT/COX-1(-/-) chimeras. NCX-4016 treatment of platelet recipients or donors attenuated leukocyte and platelet adhesion independent of platelet COX-1 inhibition. Platelet- and endothelial cell-associated COX-1 promote microvascular inflammation and thrombogenesis during hypercholesterolemia, yet nitric oxide-releasing aspirin directly inhibits platelets independent of COX-1.

Mechanisms of platelet and leukocyte recruitment in experimental colitis.

Both leukocytes and platelets accumulate in the colonic microvasculature during experimental colitis, leading to microvascular dysfunction and tissue injury. The objective of this study was to determine whether the recruitment of leukocytes and platelets in inflamed colonic venules are codependent processes. The rolling and adherence of leukocytes and platelets in colonic venules of mice with dextran sodium sulfate (DSS)-induced colitis were monitored by intravital videomicroscopy. DSS elicited an increased recruitment of both rolling and adherent leukocytes and platelets. DSS-colitic mice rendered thrombocytopenic with anti-platelet serum exhibited profound reductions in leukocyte adhesion. Neutropenia, induced with anti-neutrophil serum, significantly reduced the adhesion of leukocytes and the accumulation of platelet-leukocyte aggregates while greatly enhancing the number of platelets that roll and adhere directly to venular endothelial cells. The enhanced platelet adhesion associated with neutropenia was mediated by platelet P-selectin interactions with endothelial cell P-selectin glycoprotein ligand (PSGL-1). DSS colitis was also associated with an increased expression of PSGL-1 in the colonic vasculature. These findings indicate that the recruitment of leukocytes and platelets in inflamed colonic venules are co-dependent processes.

TLR2 and TLR4 as Potential Biomarkers of Environmental Particulate Matter Exposed Human Myeloid Dendritic Cells.

In many subjects who are genetically susceptible to asthma, exposure to environmental stimuli may exacerbate their condition. However, it is unknown how the expression and function of a family of pattern-recognition receptors called toll-like receptors (TLR) are affected by exposure to particulate pollution. TLRs serve a critical function in alerting the immune system of tissue damage or infection-the so-called "danger signals". We are interested in the role that TLRs play in directing appropriate responses by innate immunity, particularly dendritic cells (DC), after exposing them to particulate pollution. Dendritic cells serve a pivotal role in directing host immunity. Thus, we hypothesized that alterations in TLR expression could be further explored as potential biomarkers of effect related to DC exposure to particulate pollution. We show some preliminary data that indicates that inhaled particulate pollution acts directly on DC by down-regulating TLR expression and altering the activation state of DC. While further studies are warranted, we suggest that alterations in TLR2 and TLR4 expression should be explored as potential biomarkers of DC exposure to environmental particulate pollution.

Platelet-vessel wall interactions in the microcirculation.

Platelet adhesion in the microcirculation is being reported and studied in a growing number of animal models of human disease. The adhesion molecules utilized by platelets to attach to the walls of microscopic blood vessels have been defined in most model systems, with P-selectin-PSGL-1, GPIIb/IIIa-fibrinogen-ICAM-1, and vWF-GPIba interactions serving as major adhesion pathways. The binding of platelets to adherent leukocytes appears to contribute significantly to the platelet-vessel wall interactions elicited in many models of disease. Shear forces generated by the movement of blood through the microcirculation exert an important influence on platelet adhesion. Physiologic regulation of platelet adhesion is mediated by both nitric oxide and superoxide, with the former inhibiting and the latter promoting platelet adhesion. The interactions between platelets and the walls of microscopic blood vessels appear to have important implications in the initiation and/or progression of tissue injury associated with different experimental models of human disease.

Hypercholesterolemia promotes leukocyte-dependent platelet adhesion in murine postcapillary venules.

OBJECTIVE: Leukocyte-platelet aggregates form in blood during the development of cardiovascular diseases, including atherosclerosis. The study determined whether leukocytes contribute to the platelet adhesion induced by hypercholesterolemia in postcapillary venules. METHODS: Wild-type (WT) C57Bl/6 or CD18-deficient (CD18-/-) mice were placed on a normal (ND) or high-cholesterol (HC) diet for 2 weeks. Platelets isolated from ND, HC, or CD18-/- mice were fluorescently labeled with CFSE, and administered to either ND, HC, HC-CD18-/- or anti-neutrophil serum (HC-ANS)-treated mice. Rhodamine 6G was administered to label and visualize leukocytes. Intravital fluorescence microscopy was used to quantify leukocyte and platelet adhesion in intestinal postcapillary venules. RESULTS: HC increased both leukocyte and platelet adhesion (relative to ND). Greater than 50% of adherent platelets in HC mice were bound to adherent leukocytes. When HC platelets were examined in HC-ANS-treated or HC-CD18-/- mice, leukocyte-dependent platelet adhesion was significantly attenuated. Conversely, when HC-CD18-/- platelets were observed in HC recipients both leukocyte-dependent and endothelium-dependent platelet adhesion was comparable to HC mice receiving WT platelets. CONCLUSIONS: The findings demonstrate that the pro-thrombogenic phenotype assumed in the microvasculature during hypercholesterolemia is largely attributed to leukocyte-dependent platelet adhesion.

HMG-CoA reductase inhibitor attenuates platelet adhesion in intestinal venules of hypercholesterolemic mice.

Whereas the anti-inflammatory properties of statins have been extensively studied, less attention has been devoted to the antithrombogenic effects of these drugs. We evaluated the effect of short-term (18 h) treatment with pravastatin (1 mg/kg) on hypercholesterolemia-induced platelet-endothelial (P/E) cell adhesion in intestinal venules. Mice were placed on either a normal diet (ND) or cholesterol-enriched diet (HCD) for 2 wk. Wild-type mice fed a HCD exhibited significantly elevated blood serum cholesterol levels, which were unaltered by pravastatin treatment. ND or HCD platelets were isolated, fluorescently labeled, and administered to either ND or HCD recipients. Intravital videomicroscopy was used to quantify transient (saltation) and firm adhesion of platelets. HCD mice receiving platelets from either ND or HCD mice exhibited increased P/E cell interactions compared with ND mice receiving platelets from ND or HCD mice. P/E adhesion was dramatically reduced when platelets from donor mice, recipient mice, or both were treated with pravastatin. The protective effect of pravastatin in hypercholesterolemia-induced P/E cell adhesion was abolished in N(G)-nitro-l-arginine methyl ester-treated mice. These results indicate that 1). hypercholesterolemia-induced P/E cell adhesion is mediated by changes in the vascular wall rather than circulating platelets; 2). pravastatin treatment inhibits the prothrombogenic effects of hypercholesterolemia via an action on both endothelial cells and platelets; and 3). the protective effect of pravastatin is nitric oxide dependent.

Low venular shear rates promote leukocyte-dependent recruitment of adherent platelets.

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 +/- 9 and 835 +/- 44 s(-1). Shear rate was altered in postcapillary venules by rapid, graded blood withdrawal, without retransfusion of shed blood. Reducing shear rate from >600 s(-1) to <200 s(-1) 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.