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In atherogenesis, free oxygen radicals cause a lipid peroxidation of apoB100-containing lipoproteins in the blood, at the blood–endothelium-interface and in the subendothelial space. These lipoproteins easily aggregate, bind to their receptor heparan sulfate proteoglycan and calcify to arteriosclerotic nanoplaques (ternary complexes). Nanoplaque formation was measured by ellipsometry, both in vitro on an HS-PG coated hydrophobic silica surface and also in vivo on living human coronary endothelial cells, which had overgrown the silica surface. Reversely, we show with the same techniques that, in dependence on the degree of peroxidation and epitope in concern, oxLDL attacks its molecular receptor and thus can induce degradation of arteriosclerotic plaques and, in a combined action with inflammatory processes, even a plaque rupture. In order to delay or stop this process, cytokines circulating in the blood such as TNFα and TGFβ upregulate PML-NB especially in the vulnerable shoulder region of the plaque. PML-NB influences via two transcription factors, CIITA and NFκB, the collagen and proteoglycan synthesis both negatively and positively. We could prove that the purely negative effect of CIITA does not play any role, while the altogether positive effect of NFκB predominates. NFκB is inhibited with the help of the transcription mediator SMAD4 within this molecular feedback control circuit; simultaneously, NFκB inhibits the collagen and proteoglycan synthesis in the fibrous cap of the plaque. This double check (disinhibition) causes a stabilization of the fibrous cap through a specially strong collagen and proteoglycan production, which in addition is supported by circulating TGFβ. TGFβ furthers also calcification, so that fibrous cap tensile strength and resistance to shear stress are imparted. This way, a plaque rupture can possibly be averted.

This study tested the hypothesis that neuropeptide Y (NPY) and NPY receptors 1 (Y1) and 2 (Y2) participate in lung allograft rejection. Inflammation in grafts may include interaction between blood leukocytes and graft endothelial cells and marked accumulation of intravascular blood leukocytes. Fewer leukocytes accumulate in lung than in kidney allografts. Lung transplantion was performed in the Dark Agouti to Lewis rat strain combination. Intravascular and intraalveolar leukocytes were isolated from the grafts, and we evaluated the mRNA expression of NPY, Y1, and Y2 by real-time RT-PCR as well as the peptide expression of NPY by radioimmunoassay and immunohistochemistry. NPY and Y1 were expressed by pulmonary intravascular and intraalveolar leukocytes. Y1 was up-regulated by pulmonary intravascular and intraalveolar leukocytes during allograft rejection while Y2 could not be detected. Higher NPY expression levels in intravascular leukocytes were observed in lung compared to kidney allografts, which were investigated previously. Our findings suggest that an increased leukocytic expression of NPY in lung compared to kidney allografts results in a reduced accumulation of leukocytes in allograft vessels.

Heart surgery involving cardiopulmonary bypass induces systemic inflammation that is, at least in part, caused by extracellular ATP originating from damaged cells and by proteases secreted by activated neutrophils. The anti-protease α1-antitrypsin (AAT) forms complexes with several proteases including neutrophil elastase, resulting in a mutual loss of activity. We demonstrated recently that AAT inhibits the ATP-induced release of the pro-inflammatory cytokine interleukin-1β by human monocytes by a mechanism involving activation of metabotropic functions at nicotinic acetylcholine receptors. Interleukin-1β importantly contributes to the pathogenesis of sterile inflammatory response syndrome. Thus, AAT might function as an endogenous safeguard against life-threatening systemic inflammation. In this preliminary study, we test the hypothesis that during cardiopulmonary bypass, AAT is inactivated as an anti- protease and as an inhibitor of ATP-induced interleukin-1β release. AAT was affinity-purified from the blood plasma of patients before, during and after surgery. Lipopolysaccharide-primed human monocytic U937 cells were stimulated with ATP in the presence or absence of patient AAT to test for its inhibitory effect on interleukin-1β release. Anti-protease activity was investigated via complex formation with neutrophil elastase. The capacity of patient AAT to inhibit the ATP-induced release of interleukin-1β might be slightly reduced in response to heart surgery and complex formation of patient AAT with neutrophil elastase was unimpaired. We conclude that surgery involving cardiopulmonary bypass does not markedly reduce the anti-inflammatory and the anti-protease activity of AAT. The question if AAT augmentation therapy during heart surgery is suited to attenuate postoperative inflammation warrants further studies in vivo.

Objective Significant sex differences exist in cardiovascular diseases. Although an impact of gonadal hormones is presumed, it is largely unknown whether sexually dimorphic gene expression also plays a role and whether cells themselves show intrinsic sex differences. Methods We performed whole genome expression analyses in human umbilical vein endothelial cells (HUVEC) from 20 male and 20 female donors and compared levels of gene transcription between the sexes. To further assess whether there is a sex-specific response to stress, we subjected male and female HUVEC to shear for 24 h and analysed changes in gene expression. Results Genes indicative for greater immune responsiveness were stronger expressed in female compared to male HUVEC. There was a significant enrichment of 77 immune-related genes in female HUVEC. These increased transcriptional levels in female cells were verified for 20 genes by real-time RT-PCR. 6.7% of all mRNAs were regulated by shear stress. Female HUVEC showed a more pronounced transcriptional response to shear than did their male counterparts. In addition to quantitative differences, a number of genes were regulated in the opposite direction between the two sexes by shear stress. Functionally, female HUVEC showed a higher cell viability after serum starvation and an increased tube formation capacity compared to male cells. Conclusion These findings underscore the importance for differentiation between male and female cells in cell culture experiments. This may apply not only to endothelial cells but might be generalized to other cell types as well. The observed sexual dimorphisms in gene expression in endothelial cells may contribute to sex differences between males and females in endothelial function.

Transient receptor potential canonical (TRPC) channels type 6 play an important role in the function of human podocytes. Diabetic nephropathy is characterized by altered TRPC6 expression and functions of podocytes. Thus, we hypothesized that high glucose modifies TRPC6 channels via increased oxidative stress and syndecan-4 (SDC-4) in human podocytes. Human podocytes were exposed to control conditions (5.6 mmol/L d-glucose), high glucose (30 mmol/L d-glucose or l-glucose), 100 μmol/L peroxynitrite, or high glucose and the superoxide dismutase mimetic tempol (100 μmol/L). TRPC6 and SDC-4 transcripts and protein expression were measured using RT-PCR and in-cell Western assay. Intracellular reactive oxygen species (ROS) and cytosolic calcium were measured using fluorescent dye techniques. High d-glucose increased TRPC6 transcripts to 8.66 ± 4.08 (p < 0.05) and TRPC6 protein expression to 1.44 ± 0.07 (p < 0.05) without altering SDC-4 transcripts or protein expression. The d-glucose induced increase of TRPC6 expression was blocked by tempol. Increased oxidative stress using peroxynitrite significantly increased TRPC6 transcripts to 4.29 ± 1.26 (p < 0.05) and TRPC6 protein expression to 1.28 ± 0.05 (p < 0.05) without altering SDC-4 transcripts or protein expression. In human podocytes transfected with scrambled siRNA, high d-glucose increased ROS after 90 min to 3.55 ± 0.08 arbitrary units while 5.6 mmol/L d-glucose increased ROS to 2.49 ± 0.09 (p < 0.001) only. The increase in ROS was inhibited by tempol and by SDC-4 knockdown. High glucose modifies TRPC6 channels and ROS production via SDC-4 in human podocytes.

Mast cell (MC) accumulation has been demonstrated in the lungs of idiopathic pulmonary fibrosis (IPF) patients. Mediators released from MCs may regulate tissue remodeling processes, thereby contributing to IPF pathogenesis. We investigated the role of MC–fibroblast interaction in the progression of lung fibrosis. Increased numbers of activated MCs, in close proximity to fibroblast foci and alveolar type II cells, were observed in IPF lungs. Correspondingly elevated tryptase levels were detected in IPF lung tissue samples. Coculture of human lung MCs with human lung fibroblasts (HLFs) induced MC activation, as evinced by tryptase release, and stimulated HLF proliferation; IPF HLFs exhibited a significantly higher growth rate, compared with control. Tryptase stimulated HLF growth in a PAR-2/PKC-α/Raf-1/p44/42–dependent manner and potentiated extracellular matrix production, but independent of PKC-α, Raf-1, and p44/42 activities. Proproliferative properties of tryptase were attenuated by knockdown or pharmacological inhibition of PAR-2, PKC-α, Raf-1, or p44/42. Expression of transmembrane SCF, but not soluble SCF, was elevated in IPF lung tissue and in fibroblasts isolated from IPF lungs. Coculture of IPF HLFs with MCs enhanced MC survival and proliferation. These effects were cell-contact dependent and could be inhibited by application of anti-SCF antibody or CD117 inhibitor. Thus, fibroblasts and MCs appear to work in concert to perpetuate fibrotic processes and so contribute to lung fibrosis progression.

During self-limiting acute rejection preceding chronic vasculopathy, large amounts of leukocytes, predominantly monocytes, interact with the endothelium of renal allografts. We aim to characterize them and to identify targets for functional and interventional studies. Leukocytes were harvested by vascular perfusion from Fischer 344 to Lewis renal allografts or Lewis isografts, followed by flow cytometry, quantitative RT-PCR and genome-wide transcriptional profiling. Leukocyte accumulation peaked in allografts on day 9. The percentage of monocytes expressing MHC class II and CD161 was increased whereas CD4, CD11a, CD43, and CD71 expression remained unchanged. IFN-γ, IL-1β, IL-2, IL-10, TNF-α, and iNOS mRNA increased in allograft leukocytes but IL-4, IL-6, IL-12, TGF-β, and tissue factor did not. During acute rejection, 1783 genes were differentially expressed. In conclusion, graft blood leukocytes display a unique state of partial activation during self-limiting rejection. Numerous differentially expressed genes deserve further investigation as potential factors in deciding the fate of the allograft.

La cicatrisation et le bourgeonnement des lambeaux libres de tissu dependent principalement du developpement de vaisseaux sanguins, autrement dit de l'invasion angiogenique par les cellules endotheliales, qui est surtout reduite chez les fumeurs, les patients souffrant de microangiopathies (par exemple diabetique), ou ceux traites par immunosuppresseurs. Bien que plusieurs facteurs angiogeniques aient ete evalues pour accelerer la cicatrisation chez ces patients en particuliers, leurs combinaisons n'ont pas encore ete etudiees de facon systematique. Cette etude a ete realisee pour indiquer quelle combinaison de proangiogeniques et de facteurs promaturants est la plus efficace dans un modele de cicatrisation endotheliale. Des cellules endotheliales humaines de veine ombilicale ont ete isolees, cultivees a confluence, et soumises a un modele de plaie par eraflure, avec l'addition de facteur de croissance endothelial vasculaire (VEGF-A165), de facteur de croissance de derives plaquettaires (PDGF-AB), d'angiopoietine-1 (ANG1), ou ANG2, ainsi que l'ensemble de leurs 16 combinaisons possibles. VEGF-A165 et ANG1 etait la combinaison la plus efficace pour accelerer la cicatrisation endotheliale de la plaie. De plus, VEGF-A165 a stimule la cicatrisation dans toutes les combinaisons examinees, alors qu'elle a ete attenuee par PDGF-AB. Ainsi, en ce qui concerne leurs effets sur les cellules endotheliales, la combinaison de VEGF-A avec ANG1 est la plus prometteuse et est superieure aux combinaisons avec PDGF-AB.

The number of patterns discovered by data mining can become tremendous, in some cases exceeding the size of the original database. Therefore, there is a requirement for querying previously generated mining results or for querying the database against discovered patters. In this paper, we focus on developing methods for the storage and querying of large collections of sequential patterns. We describe a family of algorithms, which address the problem of considering the ordering among elements, that is crucial when dealing with sequential patterns. Moreover, we take into account the fact that the distribution of elements within sequential patterns is highly skewed, to propose a novel approach for the effective encoding of patterns. Experimental results, which examine a variety of factors, illustrate the efficiency of the proposed method.