Jennyfer Bultinck

Systemic NO production during (septic) shock depends on parenchymal and not on hematopoietic cells: in vivo iNOS expression pattern in (septic) shock

Septic shock is the leading cause of death in noncoronary intensive care units and the 10th leading cause of death overall. Several lines of evidence support an important role for the vasodilator NO in hypotension, a hallmark of septic shock. However, NO may also positively or negatively regulate inflammation, apoptosis, and oxidative stress. These dual effects of NO may relate to its isoform specific production but also to differences in cellular and/or temporal expression. Via bone marrow transplantations, we examined the contribution of hematopoietic cells to the dramatically elevated NO levels seen in (septic) shock. Surprisingly, hematopoietic cells are not responsible at all for the production of circulating NO after systemic tumor necrosis factor or lipopolysaccharide challenge and contribute only marginally in a bacteremic (Salmonella) model of septic shock. Immunohistochemistry identified the nonhematopoietic sources of NO as hepatocytes, paneth cells, and intestinal and renal epithelial cells. In contrast, during granulomatous Bacillus Calmette‐Guérin inflammation, the hematopoietic cell population represents the sole source of systemic NO. These mouse data demonstrate that, in contrast to the general conjecture, the dramatically elevated levels of NO during (septic) shock are not produced by hematopoietic cells such as monocytes/macrophages but rather by parenchymal cells in liver, kidney and gut.—Bultinck, J., Sips, P., Vakaet, L., Brouckaert, P., Cauwels, A. Systemic NO production during (septic) shock depends on parenchymal and not on hematopoietic cells: in vivo iNOS expression pattern in (septic) shock FASEB J. 20, E1619 –E1627 (2006)

Bcl9/Bcl9l Are Critical for Wnt-Mediated Regulation of Stem Cell Traits in Colon Epithelium and Adenocarcinomas

Canonical Wnt signaling plays a critical role in stem cell maintenance in epithelial homeostasis and carcinogenesis. Here, we show that in the mouse this role is critically mediated by Bcl9/Bcl9l, the mammalian homologues of Legless, which in Drosophila is required for Armadillo/beta-catenin signaling. Conditional ablation of Bcl9/Bcl9l in the intestinal epithelium, where the essential role of Wnt signaling in epithelial homeostasis and stem cell maintenance is well documented, resulted in decreased expression of intestinal stem cell markers and impaired regeneration of ulcerated colon epithelium. Adenocarcinomas with aberrant Wnt signaling arose with similar incidence in wild-type and mutant mice. However, transcriptional profiles were vastly different: Whereas wild-type tumors displayed characteristics of epithelial-mesenchymal transition (EMT) and stem cell-like properties, these properties were largely abrogated in mutant tumors. These findings reveal an essential role for Bcl9/Bcl9l in regulating a subset of Wnt target genes involved in controlling EMT and stem cell-related features and suggest that targeting the Bcl9/Bcl9l arm of Wnt signaling in Wnt-activated cancers might attenuate these traits, which are associated with tumor invasion, metastasis, and resistance to therapy.

Foam cell replication and smooth muscle cell apoptosis in human saphenous vein grafts

Occlusion of saphenous vein grafts is a major problem after coronary artery bypass grafting. Segments of occluded and suboccluded implanted aortocoronary grafts were obtained during re‐intervention bypass grafting in 47 patients yielding a total of 80 vein grafts. The grafts were studied by immunohistochemistry for smooth muscle cells (ÉL‐SMC actin), macrophages (HAM56), cell replication (PCNA, Ki‐67) and transmission and scanning electronmicroscopy (TEM, SEM). In 81% of the examined grafts the (sub)occlusion was due to a myo‐intimal thickening and an associated luminal accumulation of foam cells and mural thrombi. The foam cells were constantly found at the luminal site of the myo‐intimal thickening and within the luminal part of adherent thrombi. Transmission electronmicroscopy demonstrated phagocytosis of platelets and platelet fragments by the foam cells. A significant fraction of the foam cells demonstrated nuclear immunoreactivity for Ki‐67 and PCNA. The myo‐intimal thickening of the vein grafts was composed of smooth muscle cells lying in a fibrous tissue matrix. The smooth muscle cells were surrounded by prominent basal lamina and showed ultrastructural features of apoptosis. Our results support the hypothesis that phagocytosis of lipid rich platelets by monocytes set up a mechanism for foam cell formation and replication in human saphenous vein grafts. The transformation of a smooth muscle cell rich myo‐intimal thickening towards a fibrous, cell poor intimal thickening could be induced by progressive smooth muscle cell loss through apoptosis.

Tumor cell-specific inhibition of MYC function using small molecule inhibitors of the HUWE1 ubiquitin ligase

Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARF‐BP1, MULE) associates with both MYC and the MYC‐associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high‐throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC‐dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC‐activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells.

Dual role of endogenous nitric oxide in tumor necrosis factor shock: induced NO tempers oxidative stress.

Abstract.Tumor necrosis factor (TNF) is involved in pathologies like septic shock, inflammatory bowel disease and rheumatoid arthritis. TNF and lipopolysaccharide can incite lethal shock, in which cardiovascular collapse is centrally orchestrated by the vasodilating free radical nitric oxide (NO). However, NO synthase (NOS) inhibition causes increased morbidity and/or mortality, suggesting a dual role for NO. To investigate the potential protective role of NO during TNF shock, we treated mice with TNF with or without NOS inhibition. Experiments in endothelial- NOS- and inducible NOS-deficient mice identified inducible NOS as the source of protective NO. Distinctive TNF-induced lipid peroxidation, especially in liver and kidney, was aggravated by NOS inhibition. In addition, various antioxidant treatments and a phospholipase A2 (PLA2) inhibitor prevented sensitization by NOS inhibition. Together, these in vivo results indicate that induced NO not only causes hemodynamic collapse, but is also essential for curbing TNF-induced oxidative stress, which appears to hinge on PLA2-dependent mechanisms.

Protein-protein interactions: network analysis and applications in drug discovery

Physical interactions among proteins constitute the backbone of cellular function, making them an attractive source of therapeutic targets. Although the challenges associated with targeting protein-protein interactions (PPIs) -in particular with small molecules - are considerable, a growing number of functional PPI modulators is being reported and clinically evaluated. An essential starting point for PPI inhibitor screening or design projects is the generation of a detailed map of the human interactome and the interactions between human and pathogen proteins. Different routes to produce these biological networks are being combined, including literature curation and computational methods. Experimental approaches to map PPIs mainly rely on the yeast two-hybrid (Y2H) technology, which have recently shown to produce reliable protein networks. However, other genetic and biochemical methods will be essential to increase both coverage and resolution of current protein networks in order to increase their utility towards the identification of novel disease-related proteins and PPIs, and their potential use as therapeutic targets.

TLR2 activation causes no morbidity or cardiovascular failure, despite excessive systemic nitric oxide production

AIMS Septic shock is the leading cause of death in intensive care units worldwide, resulting from a progressive systemic inflammatory reaction causing cardiovascular and organ failure. Nitric oxide (NO) is a potent vasodilator and inhibition of NO synthases (NOS) can increase blood pressure in septic shock. However, NOS inhibition does not improve outcome, on the contrary, and certain NO donors may even provide protection. In addition, NOS produce superoxide in case of substrate or cofactor deficiency or oxidation. We hypothesized that excessive systemic iNOS-derived NO production is insufficient to trigger cardiovascular failure and shock. METHODS AND RESULTS We found that the systemic injection with various synthetic Toll-like receptor-2 (TLR2), TLR3, or TLR9 agonists triggered systemic NO production identical to that of lipopolysaccharide (LPS) or tumour necrosis factor. In contrast to the latter, however, these agonists did not cause hypothermia or any other signs of discomfort or morbidity, and inflammatory cytokine production was low. TLR2 stimulation with the triacylated lipopeptide Pam3CSK4 not only caused identical NO levels in circulation, but also identical iNOS expression patterns as LPS. Nevertheless, Pam3CSK4 did not cause hypotension, bradycardia, reduced blood flow, or inadequate tissue perfusion in the kidney or the liver. CONCLUSION We demonstrate that excessive iNOS-derived NO in circulation is not necessarily linked to concomitant cardiovascular collapse, morbidity, or mortality. As such, our data indicate that the central role of iNOS-derived NO in inflammation-associated cardiovascular failure may be overestimated.

Nitric oxide production by endotoxin preparations in TLR4-deficient mice.

Sepsis and septic shock result from an exacerbated systemic inflammatory reaction to infection. Their incidence is rising, and they have recently become the main cause of death in intensive care units. Septic shock is defined as sepsis accompanied by life-threatening refractory hypotension, for which excessive nitric oxide (NO), produced by inducible NO synthase iNOS, is thought responsible. LPS, a vital outer membrane component of Gram-negative bacteria, mimics most of the septic effects and is widely used as a model for septic shock. TLR4 is the signal-transducing receptor for LPS, evidenced by the resistance of TLR4-deficient C3H/HeJ and C57BL/10ScNJ mice. As expected, we found that TLR4 deficiency precludes LPS-induced cytokine production, independent of the purity of the LPS preparation. However, various conventional LPS preparations induced NO in TLR4-deficient mice to the same level as in control animals, while ultrapure LPS did not, indicating the presence of NO-producing contaminant(s). Nevertheless, despite identical iNOS induction pattern and systemic NO levels, the contaminant does not cause hypotension, hypothermia, or any other sign of morbidity. Using mice deficient for TLR2, TRL3, TLR4, TRL2x4, TLR9, MyD88 or TRIF, we found that the contaminant signals via TLR2 and MyD88. In conclusion, conventional LPS preparations generally used in endotoxic shock research contain TLR2 agonists that induce iNOS and high levels of systemic NO as such, and synergize with LPS towards the production of pro-inflammatory cytokines, morbidity and mortality. Surprisingly, the excessive iNOS-derived systemic NO production induced by impure LPS in TLR4⁻/⁻ is not accompanied by hypotension or morbidity.