Selective pharmacological inhibition of the sodium-dependent phosphate cotransporter NPT2a promotes phosphate excretion.

The sodium-phosphate cotransporter NPT2a plays a key role in the reabsorption of filtered phosphate in proximal renal tubules, thereby critically contributing to phosphate homeostasis. Inadequate urinary phosphate excretion can lead to severe hyperphosphatemia as in tumoral calcinosis and chronic kidney disease (CKD). Pharmacological inhibition of NPT2a may therefore represent an attractive approach for treating hyperphosphatemic conditions. The NPT2a-selective small-molecule inhibitor PF-06869206 was previously shown to reduce phosphate uptake in human proximal tubular cells in vitro. Here, we investigated the acute and chronic effects of the inhibitor in rodents and report that administration of PF-06869206 was well tolerated and elicited a dose-dependent increase in fractional phosphate excretion. This phosphaturic effect lowered plasma phosphate levels in WT mice and in rats with CKD due to subtotal nephrectomy. PF-06869206 had no effect on Npt2a-null mice, but promoted phosphate excretion and reduced phosphate levels in normophophatemic mice lacking Npt2c and in hyperphosphatemic mice lacking Fgf23 or Galnt3. In CKD rats, once-daily administration of PF-06869206 for 8 weeks induced an unabated acute phosphaturic and hypophosphatemic effect, but had no statistically significant effect on FGF23 or PTH levels. Selective pharmacological inhibition of NPT2a thus holds promise as a therapeutic option for genetic and acquired hyperphosphatemic disorders.

Extrahepatic high-density lipoprotein receptor SR-BI and apoA-I protect against deep vein thrombosis in mice.

OBJECTIVE: Deep vein thrombosis (DVT) and pulmonary embolism are frequent causes of morbidity and mortality. The goal of our study was to determine whether plasma high-density lipoprotein (HDL), which inversely correlates with the risk of cardiovascular events, affects DVT. METHODS AND RESULTS: Using a murine DVT model of inferior vena cava stenosis, we demonstrated that deficiency of the HDL receptor, scavenger receptor class B type I (SR-BI), promotes venous thrombosis. As SR-BI(-/-) mice have increased plasma cholesterol levels and abnormal HDL particles, we tested SR-BI(-/-) mice with an SR-BI liver transgene that normalizes both parameters. These mice also exhibited increased susceptibility to DVT, indicating a protective role of extrahepatic SR-BI. Mice lacking the major HDL apolipoprotein apoA-I or endothelial nitric oxide synthase (eNOS) (a downstream target of endothelial SR-BI signaling) also had a prothrombotic phenotype. Intravenous infusion of human apoA-I, an HDL component and SR-BI ligend, prevented DVT in wild-type but not SR-BI(-/-) or eNOS(-/-) mice, suggesting that its effect is mediated by SR-BI and eNOS. Intravenous apoA-I infusion abolished histamine-induced platelet-endothelial interactions, which are important for DVT initiation. CONCLUSIONS: An apoA-I (HDL)-SR-BI-eNOS axis is highly protective in DVT and may provide new targets for prophylaxis and treatment of venous thrombosis.

Elevated levels of soluble P-selectin in mice alter blood-brain barrier function, exacerbate stroke, and promote atherosclerosis.

Cerebrovascular and cardiovascular diseases are a major cause of morbidity and mortality. Soluble P-selectin (sP-selectin) is a biomarker for platelet/endothelial activation and is considered a risk factor for vascular disease. sP-selectin enhances procoagulant activity by inducing leukocyte-derived microparticle production and promotes activation of leukocyte integrins. However, it is not known whether it directly contributes to vascular complications. We investigated the effect of increased levels of sP-selectin on blood-brain barrier (BBB) function, stroke outcome, and atherosclerosis by comparing wild-type mice with P-sel(DeltaCT/DeltaCT) mice in which the endogenous P-selectin gene was replaced with a mutant that produces abnormally high plasma levels of sP-selectin. P-sel(DeltaCT/DeltaCT) mice presented several abnormalities, including (1) higher BBB permeability, with 25% of the animals showing differential permeability between the right and left hemispheres; (2) altered social behavior with increased aggression; (3) larger infarcts in the middle cerebral artery occlusion ischemic stroke model; and (4) increased susceptibility to atherosclerotic, macrophage-rich lesion development in both male and female mice on the apoE(-/-) genetic background. Thus, elevated sP-selectin is not only a biomarker for vascular disease, but also may contribute directly to atherosclerosis and cerebrovascular complications.

ADAMTS13: a new link between thrombosis and inflammation.

von Willebrand factor (VWF) levels are elevated and a disintegrin-like and metalloprotease with thrombospondin type I repeats-13 (ADAMTS13) activity is decreased in both acute and chronic inflammation. We hypothesized that by cleaving hyperactive ultralarge VWF (ULVWF) multimers, ADAMTS13 down-regulates both thrombosis and inflammation. Using intravital microscopy, we show that ADAMTS13 deficiency results in increased leukocyte rolling on unstimulated veins and increased leukocyte adhesion in inflamed veins. Both processes were dependent on the presence of VWF. Depletion of platelets in Adamts13(-/-) mice reduced leukocyte rolling, suggesting that platelet interaction with ULVWF contributes to this process. Increased levels of endothelial P-selectin and plasma VWF in Adamts13(-/-) compared with wild-type (WT) mice indicated an elevated release of Weibel-Palade bodies. ULVWF multimers released upon stimulation with histamine, a secretagogue of Weibel-Palade bodies, slowed down leukocyte rolling in Adamts13(-/-) but not in WT mice. Furthermore, in inflammatory models, ADAMTS13 deficiency resulted in enhanced extravasation of neutrophils, and this process was also dependent on VWF. Our findings reveal an important role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade body secretion, and in the regulation of leukocyte adhesion and extravasation during inflammation.

Peroxiredoxin1 prevents excessive endothelial activation and early atherosclerosis.

The peroxiredoxin (Prdx) family of antioxidant enzymes uses redox-active cysteines to reduce peroxides, lipid hydroperoxides, and peroxynitrites. Prdx1 is known to be important to protect red blood cells against reactive oxygen species and in tumor prevention. In this study, the role of Prdx1 in inflammation, thrombosis, and atherosclerosis was investigated. Using intravital microscopy, we showed that the number of leukocytes rolling per minute in unstimulated veins was increased by 2.5-fold in Prdx1(-/-) compared to Prdx1(+/+) mice. In Prdx1(-/-) mice, 50% of leukocytes rolled at a velocity <10 mum/sec compared with 10% in Prdx1(+/+) mice, suggesting that adhesion molecule density on the endothelium may have been increased by Prdx1 deficiency. Indeed, endothelial P-selectin, soluble P-selectin, and von Willebrand factor in plasma were increased in Prdx1(-/-) mice compared to Prdx1(+/+) mice, indicating elevated Weibel-Palade body release. In contrast to this excessive endothelial activation, Prdx1(-/-) platelets showed no sign of hyperreactivity, and their aggregation both in vitro and in vivo was normal. We also examined the role of Prdx1 in the apoE(-/-) murine spontaneous model of atherosclerosis. Prdx1(-/-)/apoE(-/-) mice fed normal chow developed larger, more macrophage-rich aortic sinus lesions than Prdx1(+/+)/apoE(-/-) mice, despite similar amounts and size distributions of cholesterol in their plasma lipoproteins. Thus, Prdx1 protects against excessive endothelial activation and atherosclerosis, and the Prdx1(-/-) mice could serve as an animal model susceptible to chronic inflammation.

Prothrombotic effects of fibronectin isoforms containing the EDA domain.

OBJECTIVE: Fibronectin (FN) plays an important role in the formation of stable arterial thrombi at the site of vascular injury. FN containing Extra Domain A (EDA+ FN) is absent from normal plasma, but elevated plasma levels of EDA+ FN are found in several pathological conditions. We hypothesized that EDA+ FN plays a special role in thrombosis. METHODS AND RESULTS: We used mouse strains constitutively including (EDA+/+) or excluding (EDA-/-) the EDA domain in all tissues and plasma. Using a flow chamber and the ferric-chloride injury model we found that EDA+ FN accelerates thrombosis both in vitro and in vivo at arterial shear rates. In EDA+/+ mice thrombi (>30 microm) grew faster when compared with EDA(WT/WT) (6.6+/-0.2 minutes versus 8.3+/-0.6 minutes, P<0.05) and the mean vessel occlusion time was shorter (9.9+/-0.4 minutes versus 14.6+/-1.7 minutes, P<0.05). However, the presence of EDA+ FN affected neither single platelet adhesion to subendothelium nor thrombosis in veins. In addition, the mortality rate of EDA+/+ mice after collagen/epinephrine infusion was twice that of EDA(WT/WT) or EDA-/- mice. CONCLUSIONS: Our findings reveal that EDA+ FN has prothrombotic activity, and its presence in plasma may worsen pathological conditions in which this form is elevated.

Platelet adhesion receptors do not modulate infarct volume after a photochemically induced stroke in mice.

Photochemically induced cerebral infarction has been considered a clinically relevant model for ischemic stroke. We evaluated various transgenic mice to study the role of platelet adhesion molecules in this model. Infarction to the sensorimotoric cortex was induced by erythrosin B and laser light. Infarct volumes were calculated from triphenyltetrazolium chloride stained brain slices. Thrombus formation and vessel leakage were observed in vivo by multiphoton microscopy. Mice mutant in VWF, GPIbalpha, beta3 integrin, and P-selectin did not show any significant differences in infarct volume compared to wild type (WT). This is in contrast to the intraluminal middle cerebral artery occlusion model in which alphaIIbbeta3 integrin, GPIbalpha, and P-selectin are known to modulate infarct size. Multiphoton microscopy showed that small, non-occlusive embolizing platelet thrombi formed in the photochemically injured brains. Massive vessel leakage was observed within 25 min of laser injury. Interestingly, we observed a significant increase in infarct size with aging, accordant with heightened fragility of the blood brain barrier (BBB) in older mice. This model of photochemically induced stroke is closer to a BBB injury model than a thrombotic stroke model in which platelets and their adhesion molecules are crucial. This model will be useful to study mechanisms regulating BBB permeability.

von Willebrand factor and factor VIII are independently required to form stable occlusive thrombi in injured veins.

von Willebrand factor (VWF) protects factor VIII (FVIII) from proteolysis and mediates the initial contact of platelets with the injured vessel wall, thus playing an important role in hemostasis and thrombosis. VWF is crucial for the formation of occlusive thrombi at arterial shear rates. However, with only a few conflicting studies published, the role of VWF in venous thrombosis is still unclear. Using gene-targeted mice, we show that in ferric chloride-injured veins platelet adhesion to subendothelium is decreased and thrombus growth is impaired in VWF(-/-) mice when compared with wild type (WT). We also observed increased embolization in the VWF(-/-) mice, which was due to lower FVIII levels in these mice as recombinant factor VIII (r-FVIII) restored thrombus stability. Despite normalization of blood clotting time and thrombus stability after r-FVIII infusion, the VWF(-/-) venules did not occlude. Transgenic platelets lacking the VWF receptor GPIbalpha extracellular domain showed decreased adhesion to injured veins. But, after a delay, all the injured venules occluded in these transgenic mice. Thus, VWF likely uses other adhesion receptors besides GPIbalpha in thrombus growth under venous shear conditions. Our studies document crucial roles for VWF and FVIII in experimental thrombosis under venous flow conditions in vivo.

Platelets and platelet adhesion support angiogenesis while preventing excessive hemorrhage.

Platelets contain both pro- and antiangiogenic factors, but their regulatory role in angiogenesis is poorly understood. Although previous studies showed that platelets stimulate angiogenesis in vitro, the role of platelets in angiogenesis in vivo is largely uncharacterized. To address this topic, we used two in vivo approaches, the cornea micropocket assay and the Matrigel model, in four animal models: thrombocytopenic, Lyst(bg) (platelet storage pool deficiency), glycoprotein (GP) Ibalpha/IL4R transgenic (lacking extracellular GPIbalpha, the receptor for von Willebrand factor as well as other adhesive and procoagulant proteins), and FcgammaR(-/-) (lacking functional GPVI, the collagen receptor) mice. Adult mice were rendered thrombocytopenic by i.p. administration of an antiplatelet antibody. The number of growing vessels in the thrombocytopenic mice was lower in the cornea assay, and they showed significantly increased appearance of hemorrhage compared with mice treated with control IgG. The thrombocytopenic mice also showed more protein leakage and developed hematomas in the Matrigel model. GPIbalpha/IL4R transgenic mice presented increased hemorrhage in both assays, but it was less severe than in the platelet-depleted mice. FcgammaR(-/-) and Lyst(bg) mice showed no defect in experimental angiogenesis. Intravital microscopy revealed a >3-fold increase in platelet adhesion to angiogenic vessels of Matrigel compared with mature quiescent skin vessels. Our results suggest that the presence of platelets not only stimulates angiogenic vessel growth but also plays a critical role in preventing hemorrhage from the angiogenic vessels. The adhesion function of platelets, as mediated by GPIbalpha, significantly contributes to the process.

Elevated levels of homocysteine compromise blood-brain barrier integrity in mice.

Elevated levels of plasma homocysteine (Hcy) correlate with increased risk of cardiovascular and Alzheimer diseases. We studied the effect of elevated Hcy on the blood-brain barrier (BBB) to explore the possibility of a vascular link between the 2 diseases. On a hyperhomocysteinemic diet, cystathionine beta-synthase (Cbs)-heterozygous mice develop hyperhomocysteinemia. Intravital microscopy analysis of the mesenteric venules showed that leukocyte rolling velocity was markedly decreased and numbers of adherent cells were increased in the mutant mice. This was due at least in part to increased expression of P-selectin. BBB permeability was measured by Evans blue dye permeation and was found to be 25% greater in the Cbs(+/-) cortex compared with wild-type controls. Our study suggests an important toxic effect of elevated Hcy on brain microvessels and implicates Hcy in the disruption of the BBB.