Elevated plasma Galectin-3 is associated with major adverse kidney events and death after ICU admission.

BACKGROUND: Galectin-3 (Gal-3) is a proinflammatory and profibrotic protein especially overexpressed after Acute Kidney Injury (AKI). The early renal prognostic value of Gal-3 after AKI in critically ill patients remains unexplored. The objective was to evaluate the prognostic value of plasma level of Gal-3 for Major Adverse Kidney Events (MAKE) and mortality 30 days after ICU admission across AKI stages. METHODS: This is an ancillary study of a prospective, observational, multicenter cohort (FROG-ICU). AKI was defined using KDIGO definition. RESULTS: Two thousand and seventy-six patients had a Gal-3 plasma level measurement at ICU admission. Seven hundred and twenty-three (34.8%) were females and the median age was 63 [51, 74] years. Eight hundred and seven (38.9%) patients developed MAKE, 774 (37.3%) had AKI and mortality rate at 30 days was 22.4% (N = 465). Patients who developed MAKE had higher Gal-3 level at admission compared to patients without (30.2 [20.8, 49.2] ng/ml versus 16.9 [12.7, 24.3] ng/ml, p < 0.001, respectively. The area under the receiver operating characteristic curve of Gal-3 to predict MAKE was 0.76 CI95% [0.74-0.78], p < 0.001. Gal-3 was associated with MAKE (OR 1.80 CI95% [1.68-1.93], p < 0.001, non-adjusted and OR 1.37 CI95% [1.27-1.49], p < 0.001, adjusted). The use of Gal-3 improved prediction performance of prediction model including SAPSII, Screatadm, pNGAL with a NRI of 0.27 CI95%(0.16-0.38), p < 0.001. Median Gal-3 was higher in non-survivors than in survivors at 30 days (29.2 [20.2, 49.2] ng/ml versus 18.8 [13.3, 29.2] ng/ml, p < 0.001, respectively). CONCLUSION: Plasma levels of Gal-3 were strongly associated with renal function, with an increased risk of MAKE and death after ICU admission. Trial registration ClinicalTrials.gov NCT01367093. Registered on 6 June 2011.

Endothelial-specific deletion of connexin40 promotes atherosclerosis by increasing CD73-dependent leukocyte adhesion.

BACKGROUND: Endothelial dysfunction is the initiating event of atherosclerosis. The expression of connexin40 (Cx40), an endothelial gap junction protein, is decreased during atherogenesis. In the present report, we sought to determine whether Cx40 contributes to the development of the disease. METHODS AND RESULTS: Mice with ubiquitous deletion of Cx40 are hypertensive, a risk factor for atherosclerosis. Consequently, we generated atherosclerosis-susceptible mice with endothelial-specific deletion of Cx40 (Cx40del mice). Cx40del mice were indeed not hypertensive. The progression of atherosclerosis was increased in Cx40del mice after 5 and 10 weeks of a high-cholesterol diet, and spontaneous lesions were observed in the aortic sinuses of young mice without such a diet. These lesions showed monocyte infiltration into the intima, increased expression of vascular cell adhesion molecule-1, and decreased expression of the ecto-enzyme CD73 in the endothelium. The proinflammatory phenotype of Cx40del mice was confirmed in another model of induced leukocyte recruitment from the lung microcirculation. Endothelial CD73 is known to induce antiadhesion signaling via the production of adenosine. We found that reducing Cx40 expression in vitro with small interfering RNA or antisense decreased CD73 expression and activity and increased leukocyte adhesion to mouse endothelial cells. These effects were reversed by an adenosine receptor agonist. CONCLUSIONS: Cx40-mediated gap junctional communication contributes to a quiescent nonactivated endothelium by propagating adenosine-evoked antiinflammatory signals between endothelial cells. Alteration in this mechanism by targeting Cx40 promotes leukocyte adhesion to the endothelium, thus accelerating atherosclerosis.

A silanized hydroxypropyl methylcellulose hydrogel for the three-dimensional culture of chondrocytes.

Articular cartilage has limited intrinsic repair capacity. In order to promote cartilage repair, the amplification and transfer of autologous chondrocytes using three-dimensional scaffolds have been proposed. We have developed an injectable and self-setting hydrogel consisting of hydroxypropyl methylcellulose grafted with silanol groups (Si-HPMC). The aim of the present work is to assess both the in vitro cytocompatibility of this hydrogel and its ability to maintain a chondrocyte-specific phenotype. Primary chondrocytes isolated from rabbit articular cartilage (RAC) and two human chondrocytic cell lines (SW1353 and C28/I2) were cultured into the hydrogel. Methyl tetrazolium salt (MTS) assay and cell counting indicated that Si-HPMC hydrogel did not affect respectively chondrocyte viability and proliferation. Fluorescent microscopic observations of RAC and C28/I2 chondrocytes double-labeled with cell tracker green and ethidium homodimer-1 revealed that chondrocytes proliferated within Si-HPMC. Phenotypic analysis (RT-PCR and Alcian blue staining) indicates that chondrocytes, when three-dimensionnally cultured within Si-HPMC, expressed transcripts encoding type II collagen and aggrecan and produced sulfated glycosaminoglycans. These results show that Si-HPMC allows the growth of differentiated chondrocytes. Si-HPMC therefore appears as a potential scaffold for three-dimensional amplification and transfer of chondrocytes in cartilage tissue engineering.

Regulation of human COL2A1 gene expression in chondrocytes. Identification of C-Krox-responsive elements and modulation by phenotype alteration.

To identify control motifs involved in human type II collagen gene transcription in both differentiated and dedifferentiated rabbit articular chondrocytes, transient transfection experiments were performed. A 715-base pair (bp) region of the first intron (+2127/+2842), including a 153-bp sequence so far uncharacterized (+2689/+2842), was found to mediate enhancer activity. In dedifferentiated chondrocytes, this enhancer activity was shown to be less effective than in primary cultures but still present. We then demonstrated that a zinc finger protein, C-Krox, activates COL2A1 gene transcription in differentiated chondrocytes through the enhancer region, whereas in subcultured cells, it inhibited the gene activity via a 266-bp promoter. Multicopies of the C-Krox binding site were found to mediate transactivation in both primary cultures and passaged cells, whereas C-Krox overexpression inhibited transcription in dedifferentiated chondrocytes. Additionally, we showed that C-Krox binds to several cis sequences that mediate its transcriptional effects. During chondrocyte dedifferentiation, the protein levels and binding activity of C-Krox were reduced, whereas those of NF-kappaB were increased. This was not associated with variations of mRNA levels, suggesting that post-transcriptional regulatory mechanisms could be involved in C-Krox expression. These results suggest that C-Krox plays a major role in type II collagen expression and the chondrocyte phenotype modulation.

Sp3 represses the Sp1-mediated transactivation of the human COL2A1 gene in primary and de-differentiated chondrocytes.

Sp1 and Sp3 effects on the transcription of the human alpha1(II) procollagen gene (COL2A1) were investigated in both differentiated and de-differentiated rabbit articular chondrocytes. Transient transfection with constructs of deleted COL2A1 promoter sequences driving the luciferase reporter gene revealed that the region spanning -266 to +121 base pairs showed Sp1-enhancing effects, whatever the differentiation state. In contrast, Sp3 did not influence COL2A1 gene transcription. Concomitant overexpression of the two Sp proteins demonstrated that Sp3 blocked the Sp1 induction of COL2A1 promoter activity. Moreover, inhibition of Sp1/Sp3 binding to their target DNA sequence decreased both COL2A1 gene transcription and Sp1-enhancing effects. DNase I footprinting and gel retardation assays revealed that Sp1 and Sp3 bind specifically to cis-sequences of the COL2A1 gene promoter whereby they exert their transcriptional effects. Sp1 and Sp3 levels were found to be reduced in de-differentiated chondrocytes, as revealed by DNA-binding and immunochemical study. Sp1 specifically activated collagen neosynthesis whatever the differentiation state of chondrocytes, suggesting that this factor exerts a major role in the expression of collagen type II. However, our data indicate that type II collagen-specific expression in chondrocytes depend on both the Sp1/Sp3 ratio and cooperation of Sp1 with other transcription factors, the amounts of which are also modulated by phenotype alteration.