Inhibition of VEGF or TGF-{beta} signaling activates endothelium and increases leukocyte rolling.

OBJECTIVE: Motivated by the central roles that vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-beta play in the assembly and maintenance of the vasculature, we examined the impact of systemic VEGF or TGF-beta signal inhibition on endothelial activation as detected by leukocyte-endothelial interactions. METHODS AND RESULTS: VEGF or TGF-beta inhibition, accomplished using adenovirus expression of soluble Flt1 (Ad-sFlt1) or soluble endoglin (Ad-sEng), resulted in a significant increase in the number of leukocytes rolling along the mesenteric venous endothelium and a significant decrease in rolling velocity in Ad-sEng mice. Neutralization of VEGF or TGF-beta resulted in endothelial surface expression of P-selectin and impaired peripheral vasodilatation. Neither inhibition of VEGF nor TGF-beta was associated with platelet or leukocyte activation, as detected by the activation markers platelet P-selectin and the active integrin alphaIIbbetaIII, or by leukocyte expression of L-selectin. Soluble vascular cell adhesion molecule (VCAM)-1 and E-selectin were increased in sEng-expressing mice, indicating higher levels of these adhesion receptors. CONCLUSIONS: VEGF or TGF-beta neutralization leads to impaired endothelium-mediated vasodilatation and elevated expression of surface adhesion molecules, resulting in increased leukocyte adhesion. These results indicate an essential role for both VEGF and TGF-beta in maintaining the endothelium in a nonactivated state and have implications for therapeutic approaches that neutralize VEGF or TGF-beta.

Thrombocytopenia and platelet abnormalities in high-density lipoprotein receptor-deficient mice.

OBJECTIVE: High-density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediated cellular uptake of lipoprotein cholesterol controls HDL structure and plasma HDL and biliary cholesterol levels. In SR-BI knockout (KO) mice, an unusually high plasma unesterified-to-total cholesterol ratio (UC:TC) and abnormally large HDL particles apparently contribute to pathology, including female infertility, susceptibility to atherosclerosis and coronary heart disease, and anemia. Here we examined the influence of SR-BI deficiency on platelets. METHODS AND RESULTS: The high plasma UC:TC ratio in SR-BI KO mice was correlated with platelet abnormalities, including high cholesterol content, abnormal morphologies, high clearance rates, and thrombocytopenia. One day after platelets from wild-type mice were infused into SR-BI KO mice, they exhibited abnormally high cholesterol content and clearance rates similar to those of endogenous platelets. Platelets from SR-BI KO mice exhibited in vitro a blunted aggregation response to the agonist ADP but a normal response to PAR4. CONCLUSIONS: In SR-BI KO mice abnormal circulating lipoproteins, particularly their high UC:TC ratio-rather than the absence of SR-BI in platelets themselves-induce defects in platelet structure and clearance, together with a mild defect in function.

PSGL-1 regulates platelet P-selectin-mediated endothelial activation and shedding of P-selectin from activated platelets.

We have previously shown that activated platelets in circulation stimulate release of endothelial Weibel-Palade bodies thus increasing leukocyte rolling in venules. P-selectin on the activated platelets mediates adhesion to leukocytes via PSGL-1 and is rapidly shed into plasma. We were interested in studying the role of PSGL-1 in regulating expression and function of platelet P-selectin. We show here that PSGL-1 is critical for the activation of endothelial cells in venules of mice infused with activated platelets. The interaction of platelet P-selectin with PSGL-1 is also required for P-selectin shedding, as P-selectin was retained significantly longer on the surface of activated platelets infused into PSGL-1(-/-) compared to wild-type mice. The leukocyte integrin alphaMbeta2 (Mac-1) was not required for P-selectin shedding. In addition to shedding, P-selectin can be downregulated from the platelet surface through internalization and this is the predominant mechanism in the absence of PSGL-1. We demonstrate that leukocyte-neutrophil elastase, known to cleave P-selectin in vitro, is not the major sheddase for P-selectin in vivo. In conclusion, interaction of platelet P-selectin with PSGL-1 is crucial for activation of the endothelium andWeibel-Palade body secretion. The interaction with PSGL-1 also results in rapid shedding of P-selectin thus downregulating the inflammatory potential of the platelet.

Decreased plasma fibronectin leads to delayed thrombus growth in injured arterioles.

OBJECTIVE: Plasma fibronectin (FN) is decreased in several clinical conditions. We were interested to study the thrombotic and hemostatic consequences of the decrease in plasma FN (pFN), the role of FN splice variants in thrombosis, and to examine whether pFN incorporates into thrombi in vivo. METHODS AND RESULTS: We compared the thrombotic response to a vessel injury in FN heterozygous (FN+/-) mice and corresponding FN+/+ mice. Although normal thrombosis in venules was observed, a decrease to half in the pFN concentration in FN+/- mice caused a delay in the appearance of thrombi in arterioles and consequently a delay in their occlusion. We were able to rescue the thrombotic defect in the FN+/- mice by infusion of rat pFN. Additionally, we could show intense incorporation of fluorescent pFN-coated microspheres into the developing thrombi. Moreover, we found that mice expressing FN without the EIIIA or EIIIB domains specific to cellular FN including platelet FN had no thrombotic defect. CONCLUSIONS: Mice heterozygous for FN have a striking defect in thrombus initiation and growth in arterioles attributable to the decrease of pFN. Our study is an example of haploid insufficiency for FN, and it emphasizes the fundamental role of this plasma protein in thrombosis in the arterial system.

Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates GPIbalpha shedding from platelets in vitro and in vivo.

Interaction of the platelet receptor glycoprotein (GP) Ib-V-IX with von Willebrand factor exposed at a site of vascular injury is an essential step in the initiation of a hemostatic plug. Proteolytic cleavage (shedding) of the GPIbalpha subunit was first described >25 years ago, the protease mediating this event as well as its physiological function, however, have not been elucidated. We reported recently that shedding of GPIbalpha induced by platelet storage or mitochondrial injury involves a platelet-derived metalloproteinase(s). Here we show that GPIbalpha shedding in response to mitochondrial injury or physiological activation is inhibited in platelets obtained from chimeric mice, which express inactive tumor necrosis factor-alpha converting enzyme (TACE(DeltaZn/DeltaZn)) in blood cells only. Shedding was also inhibited in mouse and human platelets in the presence of 2 potent TACE inhibitors: TAP1 and TMI-1. Our data further suggest that TACE is important in the regulation of GPIbalpha expression in vivo because we observed an approximately 90% reduction in soluble GPIbalpha (glycocalicin) in plasma of TACE(DeltaZn/DeltaZn) chimeras as well as significantly increased levels of GPIbalpha on circulating platelets. In contrast, shedding of P-selectin from activated platelets was not affected by the mutation in TACE. Damaged TACE(DeltaZn/DeltaZn) platelets were further characterized by a markedly improved post-transfusion recovery and hemostatic function in mice. In conclusion, our data demonstrate that TACE is expressed in platelets and that it is the key enzyme mediating shedding of GPIbalpha.

Expression of biopterin transporter (BT1) protein in Leishmania.

The present work focuses on the growth phase regulated expression of biopterin transporter gene (BT1) from the LD1 locus on chromosome 35 of Leishmania donovani. Antiserum against recombinant BT1 detected a polypeptide of 45 kDa of equal intensity at lag, log and stationary phases of promastigote growth, both in L. donovani strain LSB-7.1 (MHOM/BL/67/ITMAP263), and strain LSB-146.1 (HOM/IR/95/X81), a natural isolate from Isfehan, Iran that caused cutaneous leishmaniasis. However, in both these strains an additional polypeptide of higher molecular mass (50 kDa) was also observed during lag phase only. In addition, polypeptides of 40, 20, 18 and 16 kDa were seen only during the lag and log phases of both strains. Analysis of L. donovani single, double and triple (null) BT1 knockout mutants confirmed that the 45-kDa polypeptide was the BT1 gene product, as it was absent in the null mutant. These results indicate that 45-kDa BT1 protein in Leishmania is consistently and constitutively expressed in all the growth stages of the parasite.

Pathogenicity and genetic variation of 3 strains of Corynebacterium bovis in immunodeficient mice.

Corynebacterium bovis has been associated with hyperkeratotic dermatitis and acanthosis in mice. We studied 3 different strains of C. bovis: one previously described to cause hyperkeratotic dermatitis (HAC), one that infected athymic nude mice without leading to the classic clinical signs, and one of bovine origin (ATCC 7715). The 3 strains showed a few biochemical and genetic differences. Immunodeficient nude mice were housed in 3 independent isolators and inoculated with pure cultures of the 3 strains. We studied the transmission of these C. bovis studies to isolator-bedding and contact sentinels housed for 5 to 12 wk in filter-top or wire-top cages in the respective isolators. Using a 16S rRNA-based qPCR assay, we did not find consistent differences in growth and transmission among the 3 C. bovis strains, and neither the incidence nor severity of hyperkeratosis or acanthosis differed between strains. Housing in filter-top compared with wire-top cages did not alter the morbidity associated with any of the strains. Our findings confirmed the variability in the gross and histologic changes associated with C. bovis infection of mice. Although bacteriology was a sensitive method for the detection of Corynebacterium spp., standard algorithms occasionally misidentified C. bovis and several related species. Our study demonstrates that PCR of skin swabs or feces is a sensitive and specific method for the detection of C. bovis infection in mice. An rpoB-based screen of samples from North American vivaria revealed that HAC is the predominant C. bovis strain in laboratory mice.

Efficacy of direct detection of pathogens in naturally infected mice by using a high-density PCR array.

We used a high-density array of real-time PCR assays for commonly reported rodent infectious agents (PRIA) to test naturally infected index mice and sentinel mice exposed by contact and soiled-bedding transfer. PRIA detected 14 pathogens--including viruses, bacteria, fur mites, pinworms, and enteric protozoa--in 97.2% of 28 pooled fecal samples, fur-perianal swabs, and oral swabs from 4 cages containing a total of 10 index mice. Among these pathogens, PRIA (like conventional health monitoring methods) failed to detect Mycoplasma pulmonis, Pasteurella pneumotropica, and Giardia spp. in all of the 9 contact and 9 soiled-bedding sentinels. PRIA demonstrated murine adenovirus and Cryptosporidium and Spironucleus spp. in contact but not soiled-bedding sentinels and detected Helicobacter and pinworms in fewer than half of the soiled-bedding sentinels. Of the 4 species of Helicobacter that species-specific PCR assays identified in index mice, only H. ganmani was found in soiled-bedding and contact sentinels. PRIA detected all of the pathogens in sentinels that were identified by conventional methods. Myobia musculi was detected by PCR in index and sentinel mice but missed by conventional parasitologic examinations. In summary, PRIA reproducibly detected diverse pathogens in heavily pooled specimens collected noninvasively from infected index mice antemortem. The inability of PRIA and conventional health monitoring methods (that is, parasitology, micro-biology, and serology) to demonstrate transmission of some pathogens to contact sentinels and the inefficient transmission of others to soiled-bedding sentinels underscores the importance of direct PCR testing to determine the pathogen status of rodents in quarantine and during routine colony surveillance.

Comparison of traditional and PCR methods during screening for and confirmation of Aspiculuris tetraptera in a mouse facility.

Pinworm detection in laboratory rodents typically is accomplished by using the tape test or various modifications of fecal flotation test to detect eggs. Direct examination of intestinal contents remains the 'gold standard' for pinworm detection, with the limitation of euthanasia of animals. Here, we compare traditional and real-time PCR methodologies during screening for and confirming the presence of Aspiculuris tetraptera. Two sets of pooled fecal samples collected from each of 521 microisolation cages in a mouse facility suspected to be pinworm-positive were tested by PCR and fecal flotation methods. The number of PCR-positive cages was 48 (9.2%) compared with 5 (0.96%) by the fecal flotation method. All of the cages determined to be positive by fecal flotation were positive by PCR. We evaluated 8 positive cages containing 26 mice from the screening group 5 wk later to confirm the initial findings; for 7 of these cages, PCR results from the initial screening were confirmed by fecal centrifugation concentration (FCC) or direct worm detection. Among the 26 mice, 4 were pinworm-positive by FCC, 5 by maceration, and 16 by PCR. All 4 mice positive by FCC were positive by PCR; PCR was positive for 7 of the 9 mice in which pinworms were detected by FCC or maceration. Our study demonstrates that real-time PCR for survival testing of mice for A. tetraptera effectively augments current detection methods for quarantine and routine health monitoring.

Assessment of rpoB and 16S rRNA genes as targets for PCR-based identification of Pasteurella pneumotropica.

Diagnosis of Pasteurella pneumotropica in laboratory animals relies on isolation of the organism, biochemical characterization, and, more recently, DNA-based diagnostic methods. 16S rRNA and rpoB gene sequences were examined for development of a real-time PCR assay. Partial sequencing of rpoB (456 bp) and 16S rRNA (1368 bp) of Pasteurella pneumotropica isolates identified by microbiologic and biochemical assays indicated that either gene sequence can be used to distinguish P. pneumotropica from other members of the Pasteurellaceae family. However, alignment of rpoB sequences from the Pasteurella pneumotropica Heyl (15 sequences) and Jawetz (16 sequences) biotypes with other Pasteurellaceae sequences from GenBank indicated that although rpoB DNA sequencing could be used for diagnosis, development of diagnostic primers and probes would be difficult, because the sequence variability between Heyl and Jawetz biotypes is not clustered in any particular region of the rpoB sequence. In contrast, alignment of 16S rRNA sequences revealed a region with unique and stable nucleotide motifs sufficient to permit development of a specific fluorogenic real-time PCR assay to confirm P. pneumotropica isolated by culture and to differentiate Heyl and Jawetz biotypes.

The adenoviral E1A oncoprotein activates the Smad7 promoter: requirement of a functional E-box.

DNA tumorviruses like adenoviruses (AdV) or human papillomaviruses (HPV) have adopted various strategies to interfere with antiproliferative transforming growth factor-beta (TGF-beta) signalling. Here we report that the AdV E1A oncoprotein is sufficient to induce Smad7 expression, an inhibitor of TGF-beta signalling. E1A but not HPV oncoproteins activated the Smad7 promoter. A promoter proximal E-box was crucial for E1A-mediated transcriptional activity. E1A but not HPV oncoproteins induced specific binding activity at this E-box, which was identified as upstream stimulatory factor. In conclusion, these results unravel a novel mechanism of how the AdV E1A oncoprotein induces a cellular inhibitor of TGF-beta signalling.

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.

Activated platelets induce Weibel-Palade-body secretion and leukocyte rolling in vivo: role of P-selectin.

The presence of activated platelets and platelet-leukocyte aggregates in the circulation accompanies major surgical procedures and occurs in several chronic diseases. Recent findings that activated platelets contribute to the inflammatory disease atherosclerosis made us address the question whether activated platelets stimulate normal healthy endothelium. Infusion of activated platelets into young mice led to the formation of transient platelet-leukocyte aggregates and resulted in a several-fold systemic increase in leukocyte rolling 2 to 4 hours after infusion. Rolling returned to baseline levels 7 hours after infusion. Infusion of activated P-selectin-/- platelets did not induce leukocyte rolling, indicating that platelet P-selectin was involved in the endothelial activation. The endothelial activation did not require platelet CD40L. Leukocyte rolling was mediated solely by the interaction of endothelial P-selectin and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1). Endothelial P-selectin is stored with von Willebrand factor (VWF) in Weibel-Palade bodies. The release of Weibel-Palade bodies on infusion of activated platelets was indicated by both elevation of plasma VWF levels and by an increase in the in vivo staining of endothelial P-selectin. We conclude that the presence of activated platelets in circulation promotes acute inflammation by stimulating secretion of Weibel-Palade bodies and P-selectin-mediated leukocyte rolling.

A direct role for C1 inhibitor in regulation of leukocyte adhesion.

Plasma C1 inhibitor (C1INH) is a natural inhibitor of complement and contact system proteases. Heterozygosity for C1INH deficiency results in hereditary angioedema, which is mediated by bradykinin. Treatment with plasma C1INH is effective not only in patients with hereditary angioedema, but also in a variety of other disease models, in which such therapy is accompanied by diminished neutrophil infiltration. The underlying mechanism has been explained primarily as a result of the inhibition of the complement and contact systems. We have shown that C1INH expresses the sialyl-Lewis(x) tetrasaccharide on its N-linked glycan, via which it binds to E- and P-selectins and interferes with leukocyte-endothelial adhesion in vitro. Here we show that both native C1INH and reactive center cleaved C1INH significantly inhibit selectin-mediated leukocyte adhesion in several in vitro and in vivo models, whereas N-deglycosylated C1INH loses such activities. The data support the hypothesis that C1INH plays a direct role in leukocyte-endothelial cell adhesion, that the activity is mediated by carbohydrate, and that it is independent of protease inhibitory activity. Direct involvement of C1INH in modulation of selectin-mediated cell adhesion may be an important mechanism in the physiologic suppression of inflammation, and may partially explain its utility in therapy of inflammatory diseases.