HDL biodistribution and brain receptors in zebrafish, using HDLs as vectors for targeting endothelial cells and neural progenitors

High density lipoproteins (HDLs) display pleiotropic functions such as anti-inflammatory, antioxidant, anti-protease, and anti-apoptotic properties. These effects are mediated by four main receptors: SCARB1 (SR-BI), ABCA1, ABCG1, and CD36. Recently, HDLs have emerged for their potential involvement in brain functions, considering their epidemiological links with cognition, depression, and brain plasticity. However, their role in the brain is not well understood. Given that the zebrafish is a well-recognized model for studying brain plasticity, metabolic disorders, and apolipoproteins, it could represent a good model for investigating the role of HDLs in brain homeostasis. By analyzing RNA sequencing data sets and performing in situ hybridization, we demonstrated the wide expression of scarb1, abca1a, abca1b, abcg1, and cd36 in the brain of adult zebrafish. Scarb1 gene expression was detected in neural stem cells (NSCs), suggesting a possible role of HDLs in NSC activity. Accordingly, intracerebroventricular injection of HDLs leads to their uptake by NSCs without modulating their proliferation. Next, we studied the biodistribution of HDLs in the zebrafish body. In homeostatic conditions, intraperitoneal injection of HDLs led to their accumulation in the liver, kidneys, and cerebral endothelial cells in zebrafish, similar to that observed in mice. After telencephalic injury, HDLs were diffused within the damaged parenchyma and were taken up by ventricular cells, including NSCs. However, they failed to modulate the recruitment of microglia cells at the injury site and the injury-induced proliferation of NSCs. In conclusion, our results clearly show a functional HDL uptake process involving several receptors that may impact brain homeostasis and suggest the use of HDLs as delivery vectors to target NSCs for drug delivery to boost their neurogenic activity

Intraplaque hemorrhage, a potential consequence of periodontal bacteria gathering in human carotid atherothrombosis

Periodontal diseases are multifactorial inflammatory diseases, caused by a bacterial biofilm involving both innate and adaptative immunity, characterized by the destruction of tooth-supporting tissues. In the context of periodontitis, the spread of weak pathogenic bacteria into the bloodstream has been described. These bacteria will preferentially localize to existing clot within the circulation. Atherothrombosis of the carotid arteries is a local pathology and a common cause of cerebral infarction. Intraplaque hemorrhages render the lesion more prone to clinical complications such as stroke. The main objective of this study is to explore the biological relationship between carotid intraplaque hemorrhage and periodontal diseases. This study included consecutive patients with symptomatic or asymptomatic carotid stenosis, admitted for endarterectomy surgical procedure (n=41). In conditioned media of the carotid samples collected, markers of neutrophil activation (myeloperoxidase or MPO, DNA-MPO complexes) and hemoglobin were quantified. To investigate the presence of DNA from periodontal bacteria in atherosclerotic plaque, PCR analysis using specific primers was performed. Our preliminary results indicate an association between neutrophil activation and intraplaque hemorrhages, reflected by the release of MPO (p<0,01) and MPO-DNA complexes (p<0,05). Presence of DNA from periodontitis-associated bacteria was found in 32/41 (78%) atheromatous plaque samples. More specifically, DNA from Pg, Tf, Pi, Aa was found in 46%, 24%, 34% and 68% of the samples, respectively. Hemoglobin levels were higher in conditioned media in carotid samples where the bacteria were found, but this was not statistically significant. Our data confirm the relationship between intraplaque hemorrhage and neutrophil activation. In addition, the presence of periodontal bacteria DNA in carotid atheromatous plaque, may contribute to this activation. Further analysis is needed to fully explore the raw data and specimens

Retention and Activation of Blood-Borne Proteases in the Arterial Wall Implications for Atherothrombosis

All forms of atheroma are characterized by a risk of arterial wall rupture leading to clinical complications. This involves medial and adventitial ruptures in abdominal aortic aneurysm (AAA) and intimal cap rupture in vulnerable atherothrombotic plaques. Extracellular proteases, including metalloproteinases, locally generated plasmin, and leukocyte elastase, are important molecular mediators of atheroma progression via their matrix degradation properties. The pathological evolution of AAA is linked to the biology of its associated mural thrombus. Indeed, in aneurysmal segments lined by a thrombus, the wall is thinner, the extracellular matrix more degraded, and the adventitial inflammatory response greater than in segments that are not. Several lines of evidence highlight the role of the thrombus, in AAA, as a reservoir of blood-borne proteases that conveys them from the lumen to the diseased wall. In stenosing atheroma, both previous and recent studies provide evidence that recurrent intraplaque hemorrhages play a dominant role in the evolution of the lesion toward vulnerability. In this review, we draw a parallel between the role of protease conveyance and activation of the mural thrombus in AAA and of intraplaque hemorrhages in stenosing atheroma. We hypothesize that intraplaque hemorrhages convey blood-borne proteases into lesions, where they are retained and activated upon thrombus/hematoma formation, thus contributing significantly to their deleterious action

Identification of novel biomarkers of abdominal aortic aneurysms by 2D-DIGE and MALDI-MS from AAA-thrombus-conditioned media

In the search for novel biomarkers, noncandidate-based proteomic strategies open up new opportunities to gain a deeper insight into disease processes regarding their molecular mechanisms, the risk factors involved, and the monitoring of disease progression. To carry out these complex analyses, the combined use of gel electrophoresis with mass spectrometry (MS) represents a powerful choice. In addition, the introduction of protein dye labeling has notably improved the reliability of differential expression studies by increasing the statistical significance of the protein candidates. Here, we describe a strategy where different layers (luminal/abluminal) from the intraluminal thrombus (ILT) of human abdominal aortic aneurysm (AAA) patients were incubated in protein-free medium. Then, the levels of the proteins released were compared by two-dimensional differential in-gel electrophoresis (2D-DIGE) and the proteins of interest identified by MS. We consider that the use of tissue-conditioned media could offer a substantial advantage in the analytical study of biological fluids, as they provide a source of proteins to be released to the bloodstream, which could serve as potential circulating biomarkers.This chapter has been supported by the EC, FAD project (FP-7, HEALTH F2-2008-200647), the Spanish MICIN (SAF2010/21852), Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Redes RECAVA (RD06/0014/0035), EUS2008-03565, and Fundacion Pro CNIC.S

Quantitative HDL Proteomics Identifies Peroxiredoxin-6 as a Biomarker of Human Abdominal Aortic Aneurysm

High-density lipoproteins (HDLs) are complex protein and lipid assemblies whose composition is known to change in diverse pathological situations. Analysis of the HDL proteome can thus provide insight into the main mechanisms underlying abdominal aortic aneurysm (AAA) and potentially detect novel systemic biomarkers. We performed a multiplexed quantitative proteomics analysis of HDLs isolated from plasma of AAA patients (N = 14) and control study participants (N = 7). Validation was performed by western-blot (HDL), immunohistochemistry (tissue), and ELISA (plasma). HDL from AAA patients showed elevated expression of peroxiredoxin-6 (PRDX6), HLA class I histocompatibility antigen (HLA-I), retinol-binding protein 4, and paraoxonase/arylesterase 1 (PON1), whereas alpha-2 macroglobulin and C4b-binding protein were decreased. The main pathways associated with HDL alterations in AAA were oxidative stress and immune-inflammatory responses. In AAA tissue, PRDX6 colocalized with neutrophils, vascular smooth muscle cells, and lipid oxidation. Moreover, plasma PRDX6 was higher in AAA (N = 47) than in controls (N = 27), reflecting increased systemic oxidative stress. Finally, a positive correlation was recorded between PRDX6 and AAA diameter. The analysis of the HDL proteome demonstrates that redox imbalance is a major mechanism in AAA, identifying the antioxidant PRDX6 as a novel systemic biomarker of AAA.We thank Simon Bartlett for language and scientific editing. This study was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2016-80843-R, BIO2012-37926 and BIO2015-67580-P), Fondo de Investigaciones Sanitarias ISCiii-FEDER (PRB2) (IPT13/0001, ProteoRed, Redes RIC RD12/0042/00038 and RD12/0042/0056, Biobancos RD09/0076/00101 and CA12/00371), Centro de Investigacion Biomedica en Red de Diabetes y Enfermedades Metabolicas Asociadas (CIBERDEM), and FRIAT. The CNIC is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505).S

Identification of soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) as a possible biomarker of subclinical atherosclerosis

OBJECTIVES: Assessment of vascular risk in asymptomatic patients and the response to medical therapy is a major challenge for prevention of cardiovascular events. Our aim was to identify proteins differentially released by healthy versus atherosclerotic arterial walls, which could be found in plasma and serve as markers of atherosclerosis. METHODS AND RESULTS: We have analyzed supernatants obtained from cultured human carotid plaques and healthy arteries by surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry ProteinChip System. Surface-enhanced laser-desorption/ionization analysis unveiled an 18.4-kDa peak released in lower amount by carotid plaques than normal endarteries. This protein was identified as soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK). To confirm that sTWEAK was the protein of interest, Western blot and enzyme-linked immunosorbent assay were performed. Both techniques confirmed that sTWEAK levels were decreased in carotid plaque supernatants. Subsequent measurement of sTWEAK in plasma showed a reduced concentration in subjects with carotid stenosis (N=30) compared with healthy subjects matched by sex and age (N=28) (P<0.001). Furthermore, in a test population of 106 asymptomatic subjects, we showed that sTWEAK concentrations negatively correlated with the carotid intima-media thickness (r=-0.4; P<0.001), an index of subclinical atherosclerosis. CONCLUSIONS: These results suggest that sTWEAK could be a potential biomarker of atherosclerosis

Porphyromonas gingivalis Participates in Pathogenesis of Human Abdominal Aortic Aneurysm by Neutrophil Activation. Proof of Concept in Rats

International audienceBACKGROUND: Abdominal Aortic Aneurysms (AAAs) represent a particular form of atherothrombosis where neutrophil proteolytic activity plays a major role. We postulated that neutrophil recruitment and activation participating in AAA growth may originate in part from repeated episodes of periodontal bacteremia. METHODS AND FINDINGS: Our results show that neutrophil activation in human AAA was associated with Neutrophil Extracellular Trap (NET) formation in the IntraLuminal Thrombus, leading to the release of cell-free DNA. Human AAA samples were shown to contain bacterial DNA with high frequency (11/16), and in particular that of Porphyromonas gingivalis (Pg), the most prevalent pathogen involved in chronic periodontitis, a common form of periodontal disease. Both DNA reflecting the presence of NETs and antibodies to Pg were found to be increased in plasma of patients with AAA. Using a rat model of AAA, we demonstrated that repeated injection of Pg fostered aneurysm development, associated with pathological characteristics similar to those observed in humans, such as the persistence of a neutrophil-rich luminal thrombus, not observed in saline-injected rats in which a healing process was observed. CONCLUSIONS: Thus, the control of periodontal disease may represent a therapeutic target to limit human AAA progression