Functional genetic screen of human diversity reveals that a methionine salvage enzyme regulates inflammatory cell death.

Genome-wide association studies can identify common differences that contribute to human phenotypic diversity and disease. When genome-wide association studies are combined with approaches that test how variants alter physiology, biological insights can emerge. Here, we used such an approach to reveal regulation of cell death by the methionine salvage pathway. A common SNP associated with reduced expression of a putative methionine salvage pathway dehydratase, apoptotic protease activating factor 1 (APAF1)-interacting protein (APIP), was associated with increased caspase-1-mediated cell death in response to Salmonella. The role of APIP in methionine salvage was confirmed by growth assays with methionine-deficient media and quantitation of the methionine salvage substrate, 5'-methylthioadenosine. Reducing expression of APIP or exogenous addition of 5'-methylthioadenosine increased Salmonellae-induced cell death. Consistent with APIP originally being identified as an inhibitor of caspase-9-dependent apoptosis, the same allele was also associated with increased sensitivity to the chemotherapeutic agent carboplatin. Our results show that common human variation affecting expression of a single gene can alter susceptibility to two distinct cell death programs. Furthermore, the same allele that promotes cell death is associated with improved survival of individuals with systemic inflammatory response syndrome, suggesting a possible evolutionary pressure that may explain the geographic pattern observed for the frequency of this SNP. Our study shows that in vitro association screens of disease-related traits can not only reveal human genetic differences that contribute to disease but also provide unexpected insights into cell biology.

A genome-wide in vitro bacterial-infection screen reveals human variation in the host response associated with inflammatory disease.

Recent progress in cataloguing common genetic variation has made possible genome-wide studies that are beginning to elucidate the causes and consequences of our genetic differences. Approaches that provide a mechanistic understanding of how genetic variants function to alter disease susceptibility and why they were substrates of natural selection would complement other approaches to human-genome analysis. Here we use a novel cell-based screen of bacterial infection to identify human variation in Salmonella-induced cell death. A loss-of-function allele of CARD8, a reported inhibitor of the proinflammatory protease caspase-1, was associated with increased cell death in vitro (p = 0.013). The validity of this association was demonstrated through overexpression of alternative alleles and RNA interference in cells of varying genotype. Comparison of mammalian CARD8 orthologs and examination of variation among different human populations suggest that the increase in infectious-disease burden associated with larger animal groups (i.e., herds and colonies), and possibly human population expansion, may have naturally selected for loss of CARD8. We also find that the loss-of-function CARD8 allele shows a modest association with an increased risk of systemic inflammatory response syndrome in a small study (p = 0.05). Therefore, a by-product of the selected benefit of loss of CARD8 could be increased inflammatory diseases. These results demonstrate the utility of genome-wide cell-based association screens with microbes in the identification of naturally selected variants that can impact human health.

Cellular and molecular responses of smooth muscle cells to surface nanotopography.

Recent studies have demonstrated that surface nano-topography affect cell responses and activities. However, the molecular mechanism of the nano-structures on cellular behavior is yet to be determined. To bridge this gap, the present study was aimed to investigate the cellular and molecular responses of smooth muscle cells (SMCs) to surface nano-topography in vitro using nano-porous alumina membranes with different sizes (200 nm- and 20 nm-pores). Cellular responses such as cell adhesion, morphology, and proliferation were assessed using scanning electron microscopy (SEM), hematoxylin and eosin (HE) staining, and cell counting. The molecular cell responses were also investigated using cDNA microarrays. Results from these studies showed an unchanged response in cell adhesion, an alteration in cell morphology, and an increase in cell proliferation for cells grown on 200 nm-pore surfaces than on 20 nm-pore surfaces. In addition, exposure of SMCs to larger nano-pores induced the expression of various genes involved in cell cycle, DNA replication, cell proliferation, and signaling transduction pathways. These findings demonstrated that cellular responses of SMCs are dependent on the underlying nano-topography, and thereby suggesting nano-dimensional surface is one of the most important considerations to design of the next generation of medical devices and tissue engineering scaffolds.

Molecular responses of vascular smooth muscle cells and phagocytes to curcumin-eluting bioresorbable stent materials.

A major complication of coronary stenting is restenosis, often accompanied by inflammatory reactions and smooth muscle cell proliferation. Curcumin has been shown to possess anti-inflammatory and anti-proliferative properties, thus we hypothesize that locally released curcumin by coronary stent would diminish in-stent restenosis. As a first test of this hypothesis, curcumin-eluting PLLA films (C-PLLA) were produced and the anti-inflammatory and anti-proliferative properties were then tested using peritoneal phagocytes and human coronary artery smooth muscle cell (hCASMCs) culture systems. We find that the addition of curcumin reduced phagocyte accumulation and activation on C-PLLA films. On the other hand, C-PLLA significantly reduced the proliferation, but not the adhesion, of hCASMCs. The molecular responses of hCASMCs to C-PLLA were further assessed by cDNA microarray analysis. Curcumin up-regulated genes related to apoptosis and enhanced the expression of anti-proliferative and anti-inflammatory factors, and of antioxidants. Equally important, C-PLLA inhibited the cell cycle progression of adherent hCASMCs. The results suggest that curcumin regulates gene expression and cell function through the protein kinase (PK) and mitogen-activated protein kinase (MAPK) pathways. These results support the use of curcumin to inhibit in-stent restenosis.

A cellular genome-wide association study reveals human variation in microtubule stability and a role in inflammatory cell death.

Pyroptosis is proinflammatory cell death that occurs in response to certain microbes. Activation of the protease caspase-1 by molecular platforms called inflammasomes is required for pyroptosis. We performed a cellular genome-wide association study (GWAS) using Salmonella typhimurium infection of human lymphoblastoid cell lines as a means of dissecting the genetic architecture of susceptibility to pyroptosis and identifying unknown regulatory mechanisms. Cellular GWAS revealed that a common human genetic difference that regulates pyroptosis also alters microtubule stability. An intergenic single-nucleotide polymorphism on chromosome 18 is associated with decreased pyroptosis and increased expression of TUBB6 (tubulin, ß 6 class V). TUBB6 is unique among tubulin isoforms in that its overexpression can completely disrupt the microtubule network. Cells from individuals with higher levels of TUBB6 expression have lower microtubule stability and less pyroptosis. Reducing TUBB6 expression or stabilizing microtubules pharmacologically with paclitaxel (Taxol) increases pyroptosis without affecting the other major readout of caspase-1 activation, interleukin-1ß secretion. The results reveal a new role for microtubules and possibly specific tubulin isoforms in the execution of pyroptosis. Furthermore, the finding that there is common diversity in TUBB6 expression and microtubule stability could have broad consequences for other microtubule-dependent phenotypes, diseases, and pharmacological responses.

Studies of the cellular uptake of hydrogel nanospheres and microspheres by phagocytes, vascular endothelial cells, and smooth muscle cells.

Intensive research efforts have been placed on the development of nanospheres for targeted drug delivery for treating a variety of diseases, including coronary restenosis, cancer, and inflammatory reactions. Although most of these drug-bearing spheres are delivered via intravenous administration, little is known about the effect of sphere physical characteristics on the responses of vascular and blood cells. To find the answer, this work was aimed to investigate the cellular uptake of nanosized (100 nm) and microsized hydrogel spheres (1 microm) made of poly(N-isopropylacrylamide) by vascular cells and phagocytes under various flow conditions in vitro. We found that the cellular uptake of nanospheres depended on incubation times and sphere concentrations as well as on the introduced shear stress levels of the medium. Measurements of the intracellular-released fluorescence and confocal fluorescence microscopy revealed that nanospheres were internalized by endothelial cells and smooth muscle cells more than microspheres, whereas microspheres were rapidly taken up by phagocytes, especially at high concentration. Our results strongly suggest that hydrogel nanospheres are more effective as an intravascular delivery system compared to microspheres in the terms of vascular cellular uptake and biocompatibility.