Rhinovirus upregulates transient receptor potential channels in a human neuronal cell line: Implications for respiratory virus-induced cough reflex sensitivity

ABSTRACT (250 words)
BACKGROUND: The mechanism underlying respiratory virus-induced cough hypersensitivity is unknown. Up-regulation of airway neuronal receptors responsible for sensing physical and chemical stimuli is one possibility and the transient receptor potential (TRP) channel family are potential candidates. We have used an in vitro model of sensory neurones and human rhinovirus (HRV-16) to study the effect of virus infection on TRP expression.
METHODS: IMR32 neuroblastoma cells were differentiated in culture to express three TRP channels, TRPV1, TRPA1 and TRPM8. Flow cytometry and qRT-PCR were used to measure TRP channel protein and mRNA levels following inoculation with live virus, inactivated virus, virus- induced soluble factors or pelleted virus particles. Multiplex bioassay was used to determine nerve growth factor (NGF), interleukin (IL)-1ß, IL-6 and IL-8 levels in response to infection.
RESULTS: Early up-regulation of TRPA1 and TRPV1 expression occurred 2 to4 hours post infection. This was independent of replicating virus as virus induced soluble factors alone were sufficient to increase channel expression 50 and 15 fold, respectively. NGF, IL-6 and IL-8 levels, increased in infected cell supernatants, represent possible candidates. In contrast, TRPM8 expression was maximal at 48 hours (9.6 fold) and required virus replication rather than soluble factors
CONCLUSIONS We show for the first time that rhinovirus can infect neuronal cells. Furthermore, infection causes up-regulation of TRP channels by channel specific mechanisms. Increase in TRPA1 and TRPV1 levels can be mediated by soluble factors induced by infection whereas TRPM8 requires replicating virus. TRP channels may be novel therapeutic targets for controlling virus-induced cough.

Oxidised, glycated LDL selectively influences tissue inhibitor of metalloproteinase-3 gene expression and protein production in human retinal capillary pericytes

Aims/hypothesis: Matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitor of metalloproteinases (TIMPs), regulate important biological processes including the homeostasis of the extracellular matrix, proteolysis of cell surface proteins, proteinase zymogen activation, angiogenesis and inflammation. Studies have shown that their balance is altered in retinal microvascular tissues in diabetes. Since LDLs modified by oxidation/glycation are implicated in the pathogenesis of diabetic vascular complications, we examined the effects of modified LDL on the gene expression and protein production of MMPs and TIMPs in retinal pericytes. Methods: Quiescent human retinal pericytes were exposed to native LDL (N-LDL), glycated LDL (G-LDL) and heavily oxidised and glycated LDL (HOG-LDL) for 24 h. We studied the expression of the genes encoding MMPs and TIMPs mRNAs by analysis of microarray data and quantitative PCR, and protein levels by immunoblotting and ELISA. Results: Microarray analysis showed that MMP1, MMP2, MMP11, MMP14 and MMP25 and TIMP1, TIMP2, TIMP3 and TIMP4 were expressed in pericytes. Of these, only TIMP3 mRNA showed altered regulation, being expressed at significantly lower levels in response to HOG- vs N-LDL. Quantitative PCR and immunoblotting of cell/matrix proteins confirmed the reduction in TIMP3 mRNA and protein in response to HOG-LDL. In contrast to cellular TIMP3 protein, analysis of secreted TIMP1, TIMP2, MMP1 and collagenase activity indicated no changes in their production in response to modified LDL. Combined treatment with N- and HOG-LDL restored TIMP3 mRNA expression to a level comparable with that after N-LDL alone. Conclusions/interpretation: Among the genes encoding for MMPs and TIMPs expressed in retinal pericytes, TIMP3 is uniquely regulated by HOG-LDL. Reduced TIMP3 expression might contribute to microvascular abnormalities in diabetic retinopathy. © 2007 Springer-Verlag.

Effects of modified low-density lipoproteins on human retinal pericyte survival

According to a current paradigm cardiovascular diseases can be initiated by exposure of vascular cells to qualitatively modified low-density lipoproteins (LDL). Capillary leakage, an early feature of diabetic retinopathy, results in the exposure of retinal pericytes to modified LDL, including glycated (G-LDL) and heavily oxidized glycated LDL (HOG-LDL). We demonstrate here that modified LDL inhibits the proliferation and survival of cultured human retinal pericytes. Modified LDL also induced DNA fragmentation in bovine retinal pericytes. Overall, HOG-LDL produced a significantly higher extent of cytotoxicity and apoptosis in retinal pericytes. These results indicate that exposure of pericytes to HOG-LDL could be implicated in the development of diabetic retinopathy.

Glycation does not alter LDL-induced secretion of tissue plasminogen activator and plasminogen activator inhibitor-1 from human aortic endothelial cells

Diabetes may induce both quantitative and qualitative changes in lipoproteins, especially low-density lipoprotein (LDL). Effects of LDL glycation on endothelial cell secretion of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) have not been fully elucidated. Human aortic endothelial cell (HAEC) tPA and PAI-1 production were determined after incubation with LDL (50 to 500 microg/mL protein, 24 h) from three sources: (1) nondiabetic LDL (N-LDL) modified in vitro to form six preparations: native, nonmodified (N); glycated (G); minimally oxidized (MO); minimally oxidized and glycated (MOG); heavily oxidized (HO); and heavily oxidized and glycated (HOG); (2) in vivo glycated and relatively nonglycated LDL subfractions from type 1 diabetic patients; (3) LDL from type 1 diabetic patients and matched controls, which was subfractionated using density gradient ultracentrifugation. In experiments using LDL modified in vitro, the rate of tPA release by HAECs incubated with N-LDL (83 +/- 4 ng/mg cell protein/24 h) did not differ significantly from those incubated with G-LDL (73 +/- 7), MO-LDL (74 +/- 13), or MOG-LDL (66 +/- 15) and was not influenced by LDL concentration. The rate of PAI-1 release was similar in HAECs incubated with N-LDL (5.7 +/- 0.6 mug/mg cell protein/24 h), G-LDL (5.7 +/- 0.7), MO-LDL (5.5 +/- 0.8), or MOG-LDL (5.7 +/- 0.9) and was not influenced by LDL concentration. In contrast, tPA release was significantly decreased in cells incubated with LDL (10 microg/mL) modified extensively by oxidation, and averaged 45.2 +/- 5.0 and 43.7 +/- 9.9 ng/mg/24 h for HO-LDL and HOG-LDL, respectively, and was further decreased with increasing concentrations of the heavily oxidized LDL preparations. PAI-1 release was not significantly decreased relative to N-LDL in cells incubated with low concentrations (5 to 50 microg/mL) of HO-LDL and HOG-LDL, but was decreased to 3.2 +/- 0.5 and 3.1 +/- 0.7 microg/mg/24 h for HO-LDL and HOG-LDL at 200 microg/mL, respectively. Results using in vivo glycated versus nonglycated LDL showed that tPA and PAI-1 release did not differ between subfractions. Release of tPA averaged 5.11 +/- 0.6 and 5.12 +/- 0.7 ng/mg/24 h, whereas release of PAI-1 averaged 666 +/- 27 ng/mg/24 h and 705 +/- 30 ng/mg/24 h for nonglycated and glycated LDL subfractions, respectively. Using LDL of different density subclasses, tPA and PAI-1 release in response to LDL from diabetic patients compared with control subjects did not differ when HAECs were incubated with LDLs of increasing density isolated from each subject pair. We conclude that oxidation of LDL, but not glycation, may contribute to the altered fibrinolysis observed in diabetes.

Interaction of very-low-density lipoprotein isolated from type I (insulin-dependent) diabetic subjects with human monocyte-derived macrophages

Very-low-density lipoproteins (VLDL) (density less than 1.006 g/mL) were isolated from type I (insulin-dependent) diabetic patients in good to fair glycemic control and from age-, sex-, and race-matched, nondiabetic, control subjects. VLDL were incubated with human, monocyte-derived macrophages obtained from nondiabetic donors, and the rates of cellular cholesteryl ester synthesis and cholesterol accumulation were determined. VLDL isolated from diabetic patients stimulated significantly more cholesteryl ester synthesis than did VLDL isolated from control subjects (4.04 +/- 1.01 v 1.99 +/- 0.39 nmol 14C-cholesteryl oleate synthesized/mg cell protein/20 h; mean +/- SEM, P less than .05). The stimulation of cholesteryl ester synthesis in macrophages incubated with VLDL isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation (P less than .05). The increase in cholesteryl ester synthesis and accumulation in macrophages were mediated by a significant increase in the receptor mediated, high affinity degradation (2.55 +/- 0.23 v 2.12 +/- 0.20 micrograms degraded/mg cell protein/20 h) and accumulation (283 +/- 35 v 242 +/- 33 ng/mg cell protein/20 h) of 125I-VLDL isolated from diabetic patients compared with VLDL from control subjects. To determine if changes in VLDL apoprotein composition were responsible for the observed changes in cellular rates of cholesteryl ester synthesis and accumulation, we also examined the apoprotein composition of the VLDL from both groups. There were no significant differences between the apoproteins B, E, and C content of VLDL from both groups. We also determined the chemical composition of VLDL isolated from both groups of subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of interleukin-6 polymorphisms on human longevity:a systematic review and meta-analysis

Several studies have assessed changes in frequency of -174 interleukin (IL)-6 single nucleotide polymorphism (SNP) with age. If IL-6 tracks with disability and age-related diseases, then there should be reduction, in the oldest old, of the frequency of homozygous GG subjects, who produce higher IL-6 levels. However, discordant results have been obtained. To explore the relationship between this polymorphism and longevity, we analyzed individual data on long-living subjects and controls from eight case-control studies conducted in Europeans, using meta-analysis. There was no significant difference in the IL-6 genotype between the oldest old and controls (Odds Ratio [OR]=0.96; 95% C.I.: 0.77-1.20; p=0.71), but there was significant between-study heterogeneity (I2=55.5%). In a subgroup analyses when male centenarians from the three Italian studies were included, the frequency of the IL-6 -174 GG genotype was significantly lower than the other genotypes (OR=0.49; 95% C.I.: 0.31-0.80; p=0.004), with no evidence of heterogeneity (I2=0%). Our data supports a negative association between the GG genotype of IL-6 SNP and longevity in Italian centenarians, with males who carry the genotype being two times less likely to reach extreme old age compared with subjects carrying CC or CG genotypes. These findings were not replicated in other European groups suggesting a possible interaction between genetics, sex and environment in reaching longevity.