Heritability of chronic venous disease

Varicose veins without skin changes have a prevalence of approximately 20% in Northern and Western Europe whereas advanced chronic venous insufficiency affects about 3% of the population. Genetic risk factors are thought to play an important role in the aetiology of both these chronic venous diseases (CVD). We evaluated the relative genetic and environmental impact upon CVD risk by estimating the heritability of the disease in 4,033 nuclear families, comprising 16,434 individuals from all over Germany. Upon clinical examination, patients were classified according to the CEAP guidelines as either C2 (simple varicose veins), C3 (oedema), C4 (skin changes without ulceration), C5 (healed ulceration), or C6 (active ulcers). The narrow-sense heritability (h ²) of CVD equals 17.3% (standard error 2.5%, likelihood ratio test P = 1.4 × 10⁻¹³). The proportion of disease risk attributable to age (at ascertainment) and sex, the two main risk factors for CVD, was estimated as 10.7% (Kullback–Leibler deviance R ²). The heritability of CVD is high, thereby suggesting a notable genetic component in the aetiology of the disease. Systematic population-based searches for CVD susceptibility genes are therefore warranted.     

Cell type-specific neuroprotective activity of untranslocated prion protein

Background

A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP). However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions.

Principal Findings

Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells.

Significance

These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.     

The inflammasome-mediated caspase-1 activation controls adipocyte differentiation and insulin sensitivity

Obesity-induced inflammation originating from expanding adipose tissue interferes with insulin sensitivity. Important metabolic effects have been recently attributed to IL-1β and IL-18, two members of the IL-1 family of cytokines. Processing of IL-1β and IL-18 requires cleavage by caspase-1, a cysteine protease regulated by a protein complex called the inflammasome. We demonstrate that the inflammasome/caspase-1 governs adipocyte differentiation and insulin sensitivity. Caspase-1 is upregulated during adipocyte differentiation and directs adipocytes toward a more insulin-resistant phenotype. Treatment of differentiating adipocytes with recombinant IL-1β and IL-18, or blocking their effects by inhibitors, reveals that the effects of caspase-1 on adipocyte differentiation are largely conveyed by IL-1β. Caspase-1 and IL-1β activity in adipose tissue is increased both in diet-induced and genetically induced obese animal models. Conversely, mice deficient in caspase-1 are more insulin sensitive as compared to wild-type animals. In addition, differentiation of preadipocytes isolated from caspase-1(-/-) or NLRP3(-/-) mice resulted in more metabolically active fat cells. In?vivo, treatment of obese mice with a caspase-1 inhibitor significantly increases their insulin sensitivity. Indirect calorimetry analysis revealed higher fat oxidation rates in caspase-1(-/-) animals. In conclusion, the inflammasome is an important regulator of adipocyte function and insulin sensitivity, and caspase-1 inhibition may represent a novel therapeutic target in clinical conditions associated with obesity and insulin resistance.     

Important Role for the Murid Herpesvirus 4 Ribonucleotide Reductase Large Subunit in Host Colonization via the Respiratory Tract

Viral enzymes that process small molecules provide potential chemotherapeutic targets. A key constraint—the replicative potential of spontaneous enzyme mutants—has been hard to define with human gammaherpesviruses because of their narrow species tropisms. Here, we disrupted the murid herpesvirus 4 (MuHV-4) ORF61, which encodes its ribonucleotide reductase (RNR) large subunit. Mutant viruses showed delayed in vitro lytic replication, failed to establish infection via the upper respiratory tract, and replicated to only a very limited extent in the lower respiratory tract without reaching lymphoid tissue. RNR could therefore provide a good target for gammaherpesvirus chemotherapy.Cellular deoxyribonucleotide synthesis is strongly cell cycle dependent. DNA viruses replicating in noncycling cells must therefore either induce cellular enzymes or supply their own. Most herpesviruses encode multiple homologs of nucleotide metabolism enzymes, including both subunits of the cellular ribonucleotide reductase (RNR) (4). While most in vivo cells are resting, most in vitro cell lines divide continuously (29). The importance of viral RNRs may therefore only be apparent in vivo (14). In contrast to alpha- and betaherpesviruses, gammaherpesviruses cause disease mainly through latency-associated cell proliferation. However, gamma-2 herpesviruses show lytic gene expression in sites of latency (9, 17), and lytic reactivation could potentially alleviate some gammaherpesvirus-infected cancers (7, 8). Therefore, it is important also to understand the pathogenetic roles of gammaherpesvirus lytic cycle enzymes, such as RNR.The known human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV) have narrow species tropisms that preclude most pathogenesis studies. In contrast, murid herpesvirus 4 (MuHV-4) (21, 26) allows gammaherpesvirus host colonization to be studied in vivo. After intranasal (i.n.) inoculation, MuHV-4 replicates lytically in lung epithelial cells before seeding to lymphoid tissue (27). Long-term virus loads are independent of extensive primary lytic spread (25). However, whether persistence requires some lytic gene expression remains unclear. Replication-deficient viral DNA reached the spleen after intraperitoneal (i.p.) but not i.n. virus inoculation (15, 20, 28), suggesting that virus dissemination from the lung to lymphoid tissue requires lytic replication. In addition, less invasive inoculations may increase further the viral functions required to establish a persistent infection. Thymidine kinase (TK)-deficient MuHV-4 given i.n. without general anesthesia, in which method the wild-type virus infects the upper respiratory tract and reaches lymphoid tissue without infecting the lungs (18), fails to colonize in mice at all (12). The implication is that virions using a likely physiological route of host entry must replicate in terminally differentiated cells to establish a significant infection. However, some unusual features of gammaherpesvirus TKs (11) suggest that they have functions besides thymidine phosphorylation. We therefore targeted here another enzyme linked to viral DNA replication, the MuHV-4 RNR. We aimed to define the in vivo importance of a potential therapeutic target and to advance generally our understanding of gammaherpesvirus pathogenesis.Transposon insertions in the MuHV-4 RNR small (ORF60) and large (ORF61) RNR subunit genes have been described as either attenuating or not for lytic replication in vitro (19, 23). We disrupted ORF61 (RNR⁻) by inserting stop codons close to its 5′ end (Fig. ​(Fig.11 a). An EcoRI-L genomic clone (coordinates 80644 to 84996) in pUC19 (6) was digested with AleI to remove nucleotides 82320 to 82534 of ORF61 (82865 to 80514). An oligonucleotide encoding multiple stop codons and an EcoRI restriction site (5′-CTAGCATGCTAGAATTCTAGCATGCATG-3′) was ligated in place. Nucleotides 81365 to 83883 were then PCR amplified, including a BamHI site in the 81365 primer, cloned as a BglII/BamHI fragment into the BamHI site of pST76K-SR, and recombined into a MuHV-4 bacterial artificial chromosome (BAC) (1). A revertant virus was made by reconstituting the corresponding, unmutated genomic fragment. Southern blots (5) of viral DNA (Fig. ​(Fig.1b)1b) confirmed the expected genomic structures, and immunoblots (5) of infected cell lysates (Fig. ​(Fig.1c)1c) established that mutant viruses no longer expressed the RNR large subunit.Open in a separate windowFIG. 1.Disruption of the MuHV-4 ORF61. (a) Schematic diagram of the ORF61 (RNR large) locus, showing the mutation introduced and relevant restriction sites. (b) Viral DNA was digested with EcoRI and probed for ORF61. Oligonucleotide insertion into ORF61 changes a 4,352-bp wild-type band to 2,462 bp plus 1,676 bp. The 2,462-bp fragment is not visible because it overlaps the probe by only 331 nucleotides (nt) and comigrates with a background band of unknown origin. WT, wild type; REV, revertant; RNR⁻, mutant; RNR⁻ ind, independent mutant. WT luc⁺ is MuHV-4 expressing luciferase from an ORF57/ORF58 intergenic cassette. RNR⁻ luc⁺ and RNR⁻ luc⁺ind have ORF61 disrupted on this background. (c) Infected cell lysates were immunoblotted for gp150 (virion envelope glycoprotein, monoclonal antibody [MAb] T1A1), ORF17 (capsid component, MAb 150-7D1), TK (tegument component, MAb CS-4A5), and ORF61 (MAb PS-8A7). (d) BHK-21 cells were infected with RNR⁺ or RNR⁻ viruses (0.01 eGFP units/cell, 2 h, 37°C), washed two times with phosphate-buffered saline (PBS) to remove unbound virions, and cultured at 37°C to allow virus spread. Infectivity (in eGFP units) at each time point was determined on fresh BHK-21 cells in the presence of phosphonoacetic acid to prevent further viral spread, with the number of eGFP-postive cells counted 18 h later by flow cytometry. (e) BHK-21 cells were infected with RNR⁺ or RNR⁻ viruses (2 eGFP units/cell, 2 h, 37°C), washed in medium (pH 3) to inactivate nonendocytosed virions, and cultured at 37°C to allow virus replication. The infectivity of replicate cultures was then assayed as described in the legend of panel d. (f) BHK-21 cells were incubated with RNR⁺ or RNR⁻ viruses (0.3 eGFP units/cell, 37°C) for the times indicated, and the numbers of eGFP-positive cells in the cultures were then determined by flow cytometry.RNR⁻ viruses were noticeably slower than RNR⁺ viruses when spreading through BHK-21 cell monolayers after BAC DNA transfection. Normalizing by immunoblot signal, RNR⁻ virus stocks had titers similar to that of the wild type by viral enhanced green fluorescent protein (eGFP) expression but 10- to 100-fold lower plaque titers. Using eGFP expression as a readout, RNR⁻ virion production after a low multiplicity of infection lagged 1 day behind that of the wild type (Fig. ​(Fig.1d).1d). Maximum infectivity yields were also reduced, but once BHK-21 cells become confluent, they support MuHV-4 lytic infection poorly, so this was probably a consequence of the slower lytic spread. After a high multiplicity of infection (Fig. ​(Fig.1e),1e), RNR⁻ mutants showed a 10-h lag in virion production and no difference in the final yield. They showed no defect in single-cycle eGFP expression (Fig. ​(Fig.1f),1f), implying normal virion entry. Therefore, the main RNR⁻ defect lay in infectious virion production.For in vivo experiments, the loxP-flanked viral BAC-eGFP cassette must be removed (1). Therefore, to monitor infection in vivo without having to rely on new virion production as a readout, we transferred the RNR mutation onto a luciferase-positive (luc⁺) background (18). Viral luciferase expression (from an early lytic promoter) by in vitro luminometry (18) was independent of either viral DNA replication or RNR expression (Fig. ​(Fig.22 a). After i.n. inoculation of anesthetized mice, RNR⁻ luciferase signals measured in vivo by i.p. luciferin injection and IVIS Lumina charge-coupled-device (CCD) camera scanning (18) were visible in lungs (Fig. ​(Fig.2b)2b) but were 100-fold lower than those of the RNR⁺ controls (Fig. ​(Fig.2c).2c). A severe impairment of RNR⁻ lytic replication was confirmed by plaque assay (18) (Fig. ​(Fig.2d);2d); the difference between RNR⁻ and RNR⁺ plaque titers greatly exceeded any difference in plaquing efficiency.Open in a separate windowFIG. 2.Host colonization by RNR⁻ MuHV-4 mutants. (a) BHK-21 cells were left uninfected or infected overnight with RNR⁺ or RNR⁻ luc⁺ MuHV-4 and then assayed for luciferase expression by luminometry. Phosphonoacetic acid (PAA; 100 μg/ml) was either added or not to cultures to block viral late gene expression. Each point shows the mean ± standard deviation from triplicate cultures. (b) BALB/c mice were infected i.n. under general anesthesia with RNR⁻ or RNR⁺ luc⁺ MuHV-4 (5 × 10³ PFU) and then assayed for luciferase expression by luciferin injection and CCD camera scanning. The images are from 5 days postinfection. Note that the RNR⁺ and RNR⁻ images have different sensitivity scales. (c) For quantitation, dorsal and ventral luciferase signals were summed. Each point shows 1 mouse. The dashed lines show detection thresholds. The RNR⁺ signal was significantly greater than the RNR⁻ signal for all sites and time points (P < 0.001 by Student''s t test). (d) C57BL/6 mice were infected i.n. under anesthesia with RNR⁻ or RNR⁺ MuHV-4 (5 × 10³ PFU). Five days later, infectious virus loads in noses and lungs were measured by plaque assay. Each point shows 1 mouse. RNR⁻ infections yielded no plaques and therefore are shown at the sensitivity limits of each assay. (e) BALB/c mice were infected i.n. with RNR⁻ or RNR⁺ MuHV-4 without anesthesia and then monitored by luciferin injection and CCD camera scanning. Each point shows the summed ventral and dorsal signals of the relevant region for 1 mouse. Neck signals correspond to the superficial cervical lymph nodes (SCLN). The dashed lines show detection thresholds. RNR⁻ luciferase signals were undetectable at all time points.No RNR⁻ luciferase signals were visible in noses, nor did RNR⁻ MuHV-4 give signals in the superficial cervical lymph nodes (SCLN), which drain the nose (Fig. ​(Fig.2c).2c). This lack of live imaging signals from the upper respiratory tract was confirmed by ex vivo imaging of SCLN at day 14 postinfection. We examined upper respiratory tract infection further with an independently derived luc⁺ RNR⁻ mutant, inoculating i.n. without anesthesia so as to avoid virus aspiration into the lungs. No RNR⁻ luciferase signals were detected, while wild-type signals were readily observed in the nose and superficial cervical lymph nodes (Fig. ​(Fig.2e2e).Like RNR⁻ MuHV-4, TK⁻ mutants are severely attenuated for lytic replication in the lower respiratory tract. However, they eventually establish a reactivatable latent infection and induce virus-specific antibody (3). Latent virus titers in spleens peak at 1 month postinoculation. Infectious center assays showed no RNR⁻ infection of spleens at that time (Fig. ​(Fig.33 a). We also looked for viral DNA in spleens by quantitative PCR (Fig. ​(Fig.3b).3b). Genomic coordinates 4166 to 4252 were amplified and hybridized to a probe with coordinates 4218 to 4189. Viral genome copies, relative to the cellular adenosine phosphoribosyl transferase copy number, were calculated from standard curves of cloned plasmid DNA (10). No RNR⁻ viral DNA was detected. ELISA for MuHV-4-specific serum IgG (24) detected an antibody response after lung infection but not upper respiratory tract infection of BALB/c mice with RNR⁻ MuHV-4 (Fig. ​(Fig.3c).3c). There was a similar lack of antibody 1 month after upper respiratory tract infection of C57BL/6 mice with independently derived RNR⁻ mutants (Fig. ​(Fig.3d)3d) and 3 months after exposure of 6 BALB/c mice to RNR⁻ luc⁺ MuHV-4. In contrast, i.p. RNR⁻ luc⁺ MuHV-4 gave lower luciferase signals than RNR⁺ luc⁺ MuHV-4 (Fig. ​(Fig.44 a), but RNR⁻ infectious centers (Fig. ​(Fig.4b)4b) and viral genomes (Fig. ​(Fig.4c)4c) were detected in spleens, and enzyme-linked immunosorbent assays (ELISAs) (Fig. ​(Fig.4d)4d) showed MuHV-4-specific serum IgG.Open in a separate windowFIG. 3.Spleen colonization by RNR⁻ MuHV-4. (a) BALB/c or C57BL/6 mice were infected i.n. either with general anesthesia (lung infection) or without (nose infection). One month later, spleens were assayed for recoverable latent virus by infectious center assay. Lower detection limit, 10 infectious centers per spleen. (b) The spleens described in the legend of panel a were further analyzed for viral DNA by quantitative PCR. Copy numbers are expressed relative to the cellular adenosine phosphoribosyl transferase copy number in each sample. The dashed lines show lower detection limits (1 viral copy/10,000 cellular copies). (c) Sera from BALB/c mice after i.n. infection either with (lung infection) or without (nose infection) general anesthesia were assayed for MuHV-4-specific IgG by ELISA. Each line shows the absorbance curve for 1 mouse. The dashed lines show naive serum. (d) Sera from C57BL/6 mice 1 month after infection with independent RNR mutants were analyzed for MuHV-4-specific IgG, as described in the legend to panel c.Open in a separate windowFIG. 4.Intraperitoneal infection with RNR⁺ and RNR⁻ MuHV-4. (a) Mice were infected i.p. with RNR⁻ luc⁺ or RNR⁺ luc⁺ MuHV-4 and then monitored for luciferase expression. Each point shows the total abdominal signal of 1 mouse. The x axis is at the lower limit of signal detection above the background. (b) Spleens were assayed for recoverable virus by infectious center assay 10 days after i.p. infection with RNR⁻ luc⁺ or RNR⁺ luc⁺ MuHV-4. Each point shows the titer of 1 mouse. One log₁₀ infectious center per mouse corresponds to the lower limit of detection. (c) Spleen DNA was analyzed for viral genome content by quantitative PCR. Each point shows viral copy/cellular copy for the mean of triplicate reactions for 1 mouse. (d) Sera taken 10 days after i.p. infection with RNR⁻ luc⁺ or RNR⁺ luc⁺ MuHV-4 were assayed for MuHV-4-specific IgG by ELISA. Each line shows the absorbance values for the serum of 1 mouse. “Naive” represents age-matched, uninfected controls.The failure of both the RNR large subunit (ORF61) and TK⁻ MuHV-4 mutants to infect via the upper respiratory tract argues that this requires viral replication in a nucleotide-poor cell. The additional lack of lymphoid RNR⁻ infection after inoculation into the lungs seemed likely to reflect a defect in virus transport, as RNR⁻ MuHV-4 did colonize the spleen after i.p. inoculation. It is also possible that the first cells infected simply produced no infectious virions, although this seemed a more likely explanation for upper respiratory tract infection being undetectable; lung infection progressed sufficiently to give detectable luciferase expression and to induce an antiviral antibody response. How transport from lung to lymphoid tissue occurs is unknown, but likely scenarios include latently infected dendritic cells (22) carrying MuHV-4 along afferent lymphatics to germinal centers and cell-free virions being captured in lymph nodes by subcapsular sinus macrophages (13). Therefore, RNR may be important for MuHV-4 to spread from myeloid cells to B cells.The difference between RNR⁻ and TK⁻ mutants in host colonization via the lung—TK⁻ mutants reached lymphoid tissue whereas RNR⁻ mutants did not—could reflect additional ORF61 functions, as precedent exists for functional drift (2, 16). Alternatively, RNR may be needed more than TK for MuHV-4 replication in some cell types. Formidable hurdles to RNR-based therapies remain: human gammaherpesvirus infections rarely present until latency is well established, so blocking virus spread to lymphoid tissue may have a limited impact, and no drugs sufficiently selective to target viral RNRs in a clinical setting have yet emerged. Nevertheless, the severe in vivo attenuation of RNR⁻ MuHV-4 suggested that RNR may be a viable target for limiting gammaherpesvirus lytic spread.     

Metabolic rate of Arctic charr eggs depends on their parentage

The metabolic rate (specific heat output) of individual eyed-stage eggs of Arctic charr Salvelinus alpinus (Linnaeus, 1758) originating from different families was measured with direct microcalorimetry. Metabolic rates varied between 2.3–7.9 μW ind⁻¹ and 0.06–0.22 μW mg⁻¹. Absolute heat output was unrelated to egg size, but size-scaled or specific heat output was negatively correlated with egg size, measured as diameter, dry mass or fresh mass. Metabolic rates varied significantly between families, suggesting that genetic and/or maternal effects affect embryonic metabolism in Arctic charr. Heat output increased almost linearly from 3.4 to 16.7 μW ind⁻¹ (0.09–0.67 μW mg⁻¹) during the embryonic development. Although the metabolic rate varied between the families and egg metabolic rate increased during development, there was an unexpected disconnect between metabolic rate and hatching time.     

Interaction between TRPC channel subunits in endothelial cells

Transient Receptor Potential Canonical (TRPC) proteins have been identified in mammals as a family of plasma membrane calcium-permeable channels activated by different kinds of stimuli in several cell types. We have studied TRPC subunit expression in bovine aortic endothelial (BAE-1) cells, where stimulation with basic fibroblast growth factor (bFGF), a potent angiogenetic factor, induces calcium entry carried at least partially by TRPC1 channels. By means of a RT-PCR approach, we have found that, in addition to TRPC1, only TRPC4 is expressed, both at the mRNA and protein level, as confirmed by immunoblotting and immunocytochemical analysis. Because functional TRPC channels are formed by assembly of four subunits in either homo- or heterotetrameric structures, we have carried out immunoprecipitation experiments and showed that TRPC1 and TRPC4 interact to form heteromers in these cells, independently from culture conditions (high or low percent of fetal calf serum, stimulation with bFGF). Moreover, the data show that TRPC subunits are not tyrosine-phosphorylated after bFGF stimulation and they do not co-immunoprecipitate with the type 1 FGF receptor. These results suggest that BAE-1 cells are a suitable model to study function and regulation of endogenous TRPC1/TRPC4 heteromers.     

Enzymatic processing of collagen IV by MMP-2 (gelatinase A) affects neutrophil migration and it is modulated by extracatalytic domains

Proteolytic degradation of basement membrane influences the cell behavior during important processes, such as inflammations, tumorigenesis, angiogenesis, and allergic diseases. In this study, we have investigated the action of gelatinase A (MMP-2) on collagen IV, the major constituent of the basement membrane. We have compared quantitatively its action on the soluble forms of collagen IV extracted with or without pepsin (from human placenta and from Engelbreth-Holm-Swarm [EHS] murine sarcoma, respectively). The catalytic efficiency of MMP-2 is dramatically reduced in the case of the EHS murine sarcoma with respect to the human placenta, probably due to the much tighter packing of the network which renders very slow the speed of the rate-limiting step. We have also enquired on the role of MMP-2 domains in processing collagen IV. Addition of the isolated collagen binding domain, corresponding to the fibronectin-like domain of whole MMP-2, greatly in hibits the cleavage process, demonstrating that MMP-2 interacts with collagen type IV preferentially through its fibronectin-like domain. Conversely, the removal of the hemopexin-like domain, using only the catalytic domain of MMP-2, has only a limited effect on the catalytic efficiency toward collagen IV, indicating that the missing domain does not have great relevance for the overall mechanism. Finally, we have investigated the effect of MMP-2 proteolytic activity ex vivo. MMP-2 action negatively affects the neutrophils' migration across type IV coated membranes and this is likely related to the production of lower molecular weight fragments that impair the cellular migration.     

Subcellular localization and regulation of type-1C and type-5 phosphodiesterases

We investigated the subcellular localization of PDE5 in in vitro human myometrial cells. We demonstrated for the first time that PDE5 is localized in discrete cytoplasmic foci and vesicular compartments corresponding to centrosomes. We also found that PDE5 intracellular localization is not cell- or species-specific, as it is conserved in different animal and human cells. PDE5 protein levels are strongly regulated by the mitotic activity of the smooth muscle cells (SMCs), as they were increased in quiescent, contractile myometrial cultures, and conditions in which proliferation was inhibited. In contrast, PDE1C levels decreased in all conditions that inhibited proliferation. This mirrored the enzymatic activity of both PDE5 and PDE1C. Increasing cGMP intracellular levels by dbcGMP or sildenafil treatments did not block proliferation, while dbcAMP inhibited myometrial cell proliferation. Together, these results suggest that PDE5 regulation of cGMP intracellular levels is not involved in the control of SMC cycle progression, but may represent one of the markers of the contractile phenotype.