Vitamin D Binding Protein and Renal Injury in Acute Decompensated Heart Failure.

Background: Renal function in acute decompensated heart faiulre (ADHF) is a strong predictor of disease evolution and poor outcome. Current biomarkers for early diagnostic of renal injury in the setting of ADHF are still controversial, and their association to early pathological changes needs to be established. By applying a proteomic approach, we aimed to identify early changes in the differential urine protein signature associated with development of renal injury in patients hospitalised due to ADHF. Materials and Methods: Patients (71 [64-77] years old) admitted at the emergency room with ADHF and hospitalised were investigated (N = 64). Samples (urine/serum) were collected at hospital admission (day 0) and 72 h later (day 3). Differential serum proteome was analysed by two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation-time of flight (MALDI-ToF/ToF). Validation studies were performed using ELISA. Results: Proteomic analysis depicted urinary vitamin D binding protein (uVDBP) as a two spots protein with increased intensity in ADHF and significant differences depending on the glomerular filtration rate (GFR). Urinary VDBP in patients with ADHF at hospitalisation was > threefold higher than in healthy subjects, with the highest levels in those patients with ADHF already presenting renal dysfunction. At day 3, urine VDBP levels in patients maintaining normal renal function dropped to normal values (P = 0.03 vs. day 0). In contrast, urine VDBP levels remained elevated in the group developing renal injury, with values twofold above the normal range (P < 0.05), while serum creatinine and GF levels were within the physiological range in this group. Urinary VDBP in ADHF positively correlated with markers of renal injury such as cystatin C and Kidney Injury Molecule 1 (KIM-1). By ROC analysis, urinary VDBP, when added to cystatin C and KIM-1, improved the prediction of renal injury in patients with ADHF. Conclusion: We showed increased urine VDBP in patients with ADHF at hospital admission and a differential uVDBP evolution pattern at early stage of renal dysfunction, before pathological worsening of GFR is evidenced.

Alternative C3 Complement System: Lipids and Atherosclerosis.

Familial hypercholesterolemia (FH) is increasingly associated with inflammation, a phenotype that persists despite treatment with lipid lowering therapies. The alternative C3 complement system (C3), as a key inflammatory mediator, seems to be involved in the atherosclerotic process; however, the relationship between C3 and lipids during plaque progression remains unknown. The aim of the study was to investigate by a systems biology approach the role of C3 in relation to lipoprotein levels during atherosclerosis (AT) progression and to gain a better understanding on the effects of C3 products on the phenotype and function of human lipid-loaded vascular smooth muscle cells (VSMCs). By mass spectrometry and differential proteomics, we found the extracellular matrix (ECM) of human aortas to be enriched in active components of the C3 complement system, with a significantly different proteomic signature in AT segments. Thus, C3 products were more abundant in AT-ECM than in macroscopically normal segments. Furthermore, circulating C3 levels were significantly elevated in FH patients with subclinical coronary AT, evidenced by computed tomographic angiography. However, no correlation was identified between circulating C3 levels and the increase in plaque burden, indicating a local regulation of the C3 in AT arteries. In cell culture studies of human VSMCs, we evidenced the expression of C3, C3aR (anaphylatoxin receptor) and the integrin αMß2 receptor for C3b/iC3b (RT-PCR and Western blot). C3mRNA was up-regulated in lipid-loaded human VSMCs, and C3 protein significantly increased in cell culture supernatants, indicating that the C3 products in the AT-ECM have a local vessel-wall niche. Interestingly, C3a and iC3b (C3 active fragments) have functional effects on VSMCs, significantly reversing the inhibition of VSMC migration induced by aggregated LDL and stimulating cell spreading, organization of F-actin stress fibers and attachment during the adhesion of lipid-loaded human VSMCs. This study, by using a systems biology approach, identified molecular processes involving the C3 complement system in vascular remodeling and in the progression of advanced human atherosclerotic lesions.

Aggregated low-density lipoprotein induces LRP1 stabilization through E3 ubiquitin ligase CHFR downregulation in human vascular smooth muscle cells.

OBJECTIVE: Low density lipoprotein retention and aggregation in the arterial intima are key processes in atherogenesis. Aggregated LDL (agLDL) is taken up through low-density lipoprotein receptor-related protein 1 (LRP1) by human vascular smooth muscle cells (VSMC). AgLDL increases LRP1 expression, at least in part, by downregulation of sterol regulatory element-binding proteins. It is unknown whether agLDL has some effect on the ubiquitin-proteasome system, and therefore on the LRP1 receptor turnover. The objective of this study was to analyze the effect of agLDL on the degradation of LRP1 by the ubiquitin-proteasome system in human VSMC. METHODS AND RESULTS: Human VSMC were isolated from the media of human coronary arteries. Ubiquitinylated LRP1 protein levels were significantly reduced in human VSMC exposed to agLDL (100 µg/mL) for 20 hours (agLDL: 3.70±0.44 a.u. versus control: 9.68±0.55 a.u). Studies performed with cycloheximide showed that agLDL prolongs the LRP1 protein half life. Pulse-chase analysis showed that LRP1 turnover rate is reduced in agLDL-exposed VSMC. Two-dimensional electrophoresis shows an alteration in the proteomic profile of a RING type E3 ubiquitin ligase, CHFR. Real-time PCR and Western blot analysis showed that agLDL (100 µg/mL) decreased the transcriptional and protein expression of CHFR. CHFR silencing increased VSMC, but not macrophage, LRP1 expression. However, CHFR silencing did not exert any effect on the classical low-density lipoprotein receptor protein levels. Furthermore, immunoprecipitation experiments demonstrated that the physical interaction between CHFR and LRP1 decreased in the presence of agLDL. CONCLUSIONS: Our results demonstrate that agLDL prolongs the half life of LRP1 by preventing the receptor ubiquitinylation, at least in part, through CHFR targeting. This mechanism seems to be specific for LRP1 and VSMC.

Enhanced in vivo platelet adhesion in vasodilator-stimulated phosphoprotein (VASP)-deficient mice.

Platelet adhesion and activation at the vascular wall are the initial steps leading to arterial thrombosis and vascular occlusion. Prostacyclin and nitric oxide inhibit platelet adhesion, acting via cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-dependent protein kinases. A major downstream target for both cAMP- and cGMP-dependent protein kinases is the vasodilator-stimulated phosphoprotein (VASP). To test the significance of VASP for the regulation of platelet adhesion in vivo, we studied platelet-vessel wall interactions using VASP-deficient (VASP-/-) mice. Under physiologic conditions, platelet adhesion to endothelial cells was significantly enhanced in VASP null mutants when compared with wild-type mice (P <.05). Platelet recruitment in VASP null mice involved P-selectin and the fibrinogen receptor glycoprotein IIb-IIIa (GPIIb-IIIa). Under pathophysiologic conditions, the loss of VASP increased platelet adhesion to the postischemic intestinal microvasculature, to the atherosclerotic endothelium of ApoE-deficient mice, and to the subendothelial matrix following endothelial denudation (P <.05 vs wild type). Importantly, platelet adhesion in VASP null mutants was unresponsive to nitric oxide. These data show for the first time in vivo that VASP is involved in down-regulation of platelet adhesion to the vascular wall under both physiologic and pathophysiologic conditions.