Cathepsin K gene disruption does not affect murine aneurysm formation.

Cathepsin K (catK), a lysosomal cysteine protease, exerts strong elastinolytic and collagenolytic activity and is implicated in a range of pathological disorders including cardiovascular disease. CatK expression was found to be elevated in human aortic aneurysm pointing to a role in this vasculopathy. In the angiotensin II (Ang II)-induced mouse model for aneurysm formation, catK, S and C expression was strongly upregulated. Therefore, we investigated the effect of catK deficiency on Ang II-induced aneurysm formation in the abdominal aorta of apoE-/- mice. Contrary to our expectations, catK deficiency did not protect against aneurysm formation, nor did it affect medial elastin breaks. Proteolytic activity in abdominal aortic lysates were comparable between apoE-/- and catK-//-apoE-/- mice. Adventitial presence of catS- and catC-expressing cells was significantly increased in catK-/-//apoE-/- versus apoE-/- mice, which might have compensated for the deficiency of catK-derived proteolysis in the aneurysm tissue of catK deficient apoE-/- mice. Circulating granulocytes and activated T cell numbers were significantly increased in Ang II-infused catK-/-//apoE-/- mice, which is consistent with the borderline significant increase in adventitial leukocyte content in catK-/-//apoE-/- compared to apoE-/- mice. Strikingly, despite unchanged proteolytic activity in AAA lesions, collagen content in the aneurysm was significantly increased in catK-//-apoE-/- mice. In conclusion, while catK deficiency has major impact on various vasculopathies, it did not affect murine aneurysm formation.

Molecular MRI of early thrombus formation using a bimodal alpha2-antiplasmin-based contrast agent.

OBJECTIVES: We aimed to investigate whether early thrombus formation can be visualized with in vivo magnetic resonance imaging (MRI) by the use of a novel bimodal alpha(2)-antiplasmin-based contrast agent (CA). BACKGROUND: Thrombus formation plays a central role in several vascular diseases. During the early phases of thrombus formation, activated factor XIII (FXIIIa) covalently cross-links alpha(2)-antiplasmin to fibrin, indicating the potential of alpha(2)-antiplasmin-based CAs in the detection of early thrombus formation. METHODS: A bimodal CA was synthesized by coupling gadolinium-diethylene triamine pentaacetic acid and rhodamine to an alpha(2)-antiplasmin-based peptide. For the control CA, a glutamine residue essential for cross-linking was replaced by alanine. In vitro-generated thrombi were exposed to both CAs and imaged by MRI and 2-photon laser-scanning microscopy. Immunohistochemistry was performed on human pulmonary thromboemboli sections to determine the presence of alpha(2)-antiplasmin and FXIII in different thrombus remodeling phases. In vivo feasibility of the CA in detecting early thrombus formation specifically was investigated with MRI. RESULTS: In vitro-generated thrombi exposed to the alpha(2)-antiplasmin-based CA showed hyperintense magnetic resonance signal intensities at the thrombus edge. No hyperintense signal was observed when we used the alpha(2)-antiplasmin-based CA in the presence of FXIII inhibitor dansylcadaverine nor when we used the control CA. Two-photon laser-scanning microscopy demonstrated that the alpha(2)-antiplasmin-based CA bound to fibrin. Immunohistochemistry demonstrated substantial alpha(2)-antiplasmin staining in fresh compared with lytic and organized thrombi. The administration of CA in vivo within seconds after inducing thrombus formation increased contrast-to-noise ratios (CNRs 2.28 +/- 0.39, n=6) at the site of thrombus formation compared with the control CA (CNRs -0.14 +/- 0.55, p = 0.003, n = 6) and alpha(2)-antiplasmin-based CA administration 24 to 48 h after thrombus formation (CNRs 0.11 +/- 0.23, p = 0.006, n = 6). CONCLUSIONS: A bimodal CA was developed, characterized, and validated. Our results showed that this bimodal CA enabled noninvasive in vivo magnetic resonance visualization of early thrombus formation.

Macrophage-specific expression of mannose-binding lectin controls atherosclerosis in low-density lipoprotein receptor-deficient mice.

BACKGROUND: With consideration of the central role of the innate immune system in atherogenesis and mannose-binding lectin (MBL) as an innate regulator of immunity, the role of MBL in experimental and human atherosclerosis was assessed. METHODS AND RESULTS: With the use of immunohistochemistry and polymerase chain reaction, deposition and gene expression of MBL-A and -C were assessed in murine atherosclerosis from mice deficient for the low-density lipoprotein receptor (LDLR(-/-)) after 10 or 18 weeks of high-fat feeding. MBL was present and was produced in 10-week-old lesions, whereas deposition and gene expression were minimal after 18 weeks of high-fat feeding and absent in healthy vasculature. Interestingly, deposition of MBL-A and -C differed: MBL-A predominantly localized in upper medial layers, whereas MBL-C was found in and around intimal macrophages. To further study the role of local MBL production by monocytic cells in atherosclerosis, LDLR(-/-) mice with MBL-A and -C(-/-) monocytic cells were construed by bone marrow transplantation. Mice carrying MBL-A and -C double deficient macrophages had increased (30%) atherosclerotic lesions compared with wild-type controls (P=0.015) after 10 weeks of high-fat diet. Subsequently, analysis of MBL deposition and gene expression in advanced human atherosclerotic lesions revealed the presence of MBL protein in ruptured but not stable atherosclerotic lesions. Putatively in agreement with murine data, no MBL gene expression could be detected in advanced human atherosclerotic lesions. CONCLUSIONS: These results are the first to show that MBL is abundantly present and locally produced during early atherogenesis. Local MBL expression, by myeloid cells, is shown to critically control development of atherosclerotic lesions.

Effects of surfactant protein D on growth, adhesion and epithelial invasion of intestinal Gram-negative bacteria.

Surfactant protein D (SP-D) interacts with various different microorganisms and plays an important role in pulmonary innate immunity. SP-D expression has also been detected in extrapulmonary tissues, including the gastro-intestinal tract. However, its function in the intestine is unknown and may differ considerably from SP-D functions in the lung. Therefore, the effects of porcine SP-D (pSP-D) on several strains of intestinal bacteria were studied by means of bacterial growth assays, colony-count assays, radial diffusion assays and differential fluorescent staining. Furthermore, the effect of pSP-D on the adhesion- and invasion-characteristics was investigated. All bacterial strains tested in this study were aggregated by pSP-D, but only Escherichia coli K12 was susceptible to pSP-D-mediated growth inhibition. Bacterial membrane integrity of E. coli K12 was affected by pSP-D, but this did not lead to a reduced bacterial viability. Therefore, it is unlikely that pSP-D has a direct antimicrobial effect, and the observed effects are most likely due to pSP-D-mediated bacterial aggregation. The effects of pSP-D on bacterial adhesion and invasion were studied with the porcine intestinal epithelial cell line IPI-2I. Preincubation with pSP-D results in a several-fold increase in adhesion (E. coli and Salmonella) and invasion (Salmonella), but did not affect the IL-8 production induced by the bacteria. Results obtained in this study suggest that pSP-D promotes uptake of pathogenic bacteria by epithelial cells. This may reflect a scavenger function for pSP-D in the intestine, which enables the host to generate a more rapid response to infectious bacteria.

Expression sites of the collectin SP-D suggest its importance in first line host defence: power of combining in situ hybridisation, RT-PCR and immunohistochemistry.

Surfactant proteins A and D are pattern recognition molecules that play a role in pulmonary host defence. In this paper, we describe for the first time the expression and localisation of both collectins in various porcine tissues using a combination of in situ hybridisation (ISH), RT-PCR and immunohistochemistry (IHC). SP-D was expressed in several tissues including lung, tongue, intestinal tract, thymus, skin, gall bladder and lacrimal gland. Focal SP-D expression was detected in oesophagus, stomach, kidney, liver, prostate and spleen with both histological techniques. These tissues tested negative with RT-PCR. In contrast, SP-A expression was limited to the lung as measured by ISH and IHC. Interestingly, analysis by RT-PCR showed that thymus, trachea, jejunum and duodenum are positive for the presence of SP-A mRNA. We conclude that the combination of different methods can be advantageous if tissue-specific expression is studied. The importance of SP-D in innate immune defence of the pig is underlined by its expression at the potential ports of entry of pathogens.