Longevity-associated BPIFB4 gene counteracts the inflammatory signaling.

BACKGROUND: Increased levels of pro-inflammatory proteins in plasma can be detected in older individuals and associate with the so called chronic low-grade inflammation, which contributes to a faster progression of aged-related cardiovascular (CV) diseases, including frailty, neurodegeneration, gastro-intestinal diseases and disorders reflected by alterations in the composition of gut microbiota. However, successful genetic programme of long-living individuals alters the trajectory of the ageing process, by promoting an efficient immune response that can counterbalance deleterious effects of inflammation and the CV complications. This is the case of BPIFB4 gene in which, homozygosity for a four single-nucleotide polymorphism (SNP) haplotype, the Longevity-Associated Variant (LAV) correlates with prolonged health span and reduced risk of CV complications and inflammation. The relation between LAV-BPIFB4 and inflammation has been proven in different experimental models, here we hypothesized that also human homozygous carriers of LAV-BPIFB4 gene may experience a lower inflammatory burden as detected by plasma proteomics that could explain their favourable CV risk trajectory over time. Moreover, we explored the therapeutic effects of LAV-BPIFB4 in inflammatory disease and monolayer model of intestinal barrier. RESULTS: We used high-throughput proteomic approach to explore the profiles of circulating proteins from 591 baseline participants selected from the PLIC cohort according to the BPIFB4 genotype to identify the signatures and differences of BPIFB4 genotypes useful for health and disease management. The observational analysis identified a panel of differentially expressed circulating proteins between the homozygous LAV-BPIFB4 carriers and the other alternative BPIFB4 genotypes highlighting in the latter ones a higher grade of immune-inflammatory markers. Moreover, in vitro studies performed on intestinal epithelial organs from inflammatory bowel disease (IBD) patients and monolayer model of intestinal barrier demonstrated the benefit of LAV-BPIFB4 treatment. CONCLUSIONS: Homozygosity for LAV-BPIFB4 results in the attenuation of inflammation in PLIC cohort and IBD patients providing preliminary evidences for its therapeutic use in inflammatory disorders that need to be further characterized and confirmed by independent studies.

High dietary inflammatory index associates with inflammatory proteins in plasma.

BACKGROUND AND AIM: Unhealthy dietary habits and highly caloric foods induce metabolic alterations and promote the development of the inflammatory consequences of obesity, insulin resistance, diabetes and cardiovascular diseases. Describing an inflammatory effect of diet is difficult to pursue, owing lacks of standardized quali-quantitative dietary assessments. The Dietary Inflammatory Index (DII) has been proposed as an estimator of the pro- or anti-inflammatory effect of nutrients and higher DII values, which indicate an increased intake of nutrients with pro-inflammatory effects, relate to an increased risk of metabolic and cardiovascular diseases and we here assessed whether they reflect biologically relevant plasmatic variations of inflammatory proteins. METHODS: In this cross-sectional study, seven days dietary records from 663 subjects in primary prevention for cardiovascular diseases were analyzed to derive the intake of nutrients, foods and to calculate DII. To associate DII with the Normalized Protein eXpression (NPX), an index of abundance, of a targeted panel of 368 inflammatory biomarkers (Olink™) measured in the plasma, we divided the population by the median value of DII (1.60 (0.83-2.30)). RESULTS: 332 subjects with estimated DII over the median value reported a higher intake of saturated fats but lower intakes of poly-unsaturated fats, including omega-3 and omega-6 fats, versus subjects with estimated dietary DII below the median value (N = 331). The NPX of 61 proteins was increased in the plasma of subjects with DII > median vs. subjects with DII < median. By contrast, in the latter group, we underscored only 3 proteins with increased NPX. Only 23, out of these 64 proteins, accurately identified subjects with DII > median (Area Under the Curve = 0.601 (0.519-0.668), p = 0.035). CONCLUSION: This large-scale proteomic study supports that higher DII reflects changes in the plasmatic abundance of inflammatory proteins. Larger studies are warranted to validate.

ASGR1 deficiency diverts lipids toward adipose tissue but results in liver damage during obesity.

BACKGROUND: Asialoglycoprotein receptor 1 (ASGR1), primarily expressed on hepatocytes, promotes the clearance and the degradation of glycoproteins, including lipoproteins, from the circulation. In humans, loss-of-function variants of ASGR1 are associated with a favorable metabolic profile and reduced incidence of cardiovascular diseases. The molecular mechanisms by which ASGR1 could affect the onset of metabolic syndrome and obesity are unclear. Therefore, here we investigated the contribution of ASGR1 in the development of metabolic syndrome and obesity. METHODS: ASGR1 deficient mice (ASGR1-/-) were subjected to a high-fat diet (45% Kcal from fat) for 20 weeks. The systemic metabolic profile, hepatic and visceral adipose tissue were characterized for metabolic and structural alterations, as well as for immune cells infiltration. RESULTS: ASGR1-/- mice present a hypertrophic adipose tissue with 41% increase in fat accumulation in visceral adipose tissue (VAT), alongside with alteration in lipid metabolic pathways. Intriguingly, ASGR1-/- mice exhibit a comparable response to an acute glucose and insulin challenge in circulation, coupled with notably decreased in circulating cholesterol levels. Although the liver of ASGR1-/- have similar lipid accumulation to the WT mice, they present elevated levels of liver inflammation and a decrease in mitochondrial function. CONCLUSION: ASGR1 deficiency impacts energetic homeostasis during obesity leading to improved plasma lipid levels but increased VAT lipid accumulation and liver damage.

The inhibition of inner mitochondrial fusion in hepatocytes reduces non-alcoholic fatty liver and improves metabolic profile during obesity by modulating bile acid conjugation.

AIMS: Mitochondria are plastic organelles that continuously undergo biogenesis, fusion, fission, and mitophagy to control cellular energy metabolism, calcium homeostasis, hormones, sterols, and bile acids (BAs) synthesis. Here, we evaluated how the impairment of mitochondrial fusion in hepatocytes affects diet-induced liver steatosis and obesity. METHODS AND RESULTS: Male mice selectively lacking the key protein involved in inner mitochondrial fusion, optic atrophy 1 (OPA1) (OPA1ΔHep) were fed a high fat diet (HFD) for 20 weeks. OPA1ΔHep mice were protected from the development of hepatic steatosis and obesity because of reduced lipid absorption; a profile which was accompanied by increased respiratory exchange ratio in vivo, suggesting a preference for carbohydrates in OPA1ΔHep compared to controls. At the molecular level, this phenotype emerged as a consequence of poor mitochondria-peroxisome- endoplasmic reticulum (ER) tethering in OPA1 deficient hepatocytes, which impaired BAs conjugation and release in the bile, thus impacting lipid absorption from the diet. Concordantly, the liver of subjects with non-alcoholic fatty liver disease (NAFLD) presented an increased expression of OPA1 and of the network of proteins involved in mitochondrial function when compared with controls. CONCLUSION: Patients with NAFLD present increased expression of proteins involved in mitochondrial fusion in the liver. The selective deficency of OPA1 in hepatocytes protects mice from HFD-induced metabolic dysfunction by reducing BAs secretion and dietary lipids absorption as a consequence of reduced liver mitochondria-peroxisome-ER tethering.

Apolipoprotein E isoforms differentially affect LCAT-dependent cholesterol esterification.

BACKGROUND AND AIMS: LCAT esterifies cholesterol in both HDL (α-activity) and apoB-containing lipoproteins (ß-activity). The main activator of LCAT ß-activity is apoE, which in humans exists in 3 main different isoforms (E2, E3 and E4). Here, to gather insights into the potential role of LCAT in apoB-containing lipoprotein metabolism, we investigated the ability of apoE isoforms to promote LCAT-mediated cholesterol esterification. METHODS: We evaluated the plasma cholesterol esterification rate (CER) in 311 individuals who express functional LCAT and either apoE2, apoE3, or apoE4 and in 28 individuals who also carried LCAT mutations causing selective loss of LCAT α-activity (Fish-Eye Disease (FED)-causing mutations). The association of carrier status with CER was determined using an adjusted linear regression model. The kinetic of LCAT activity towards reconstituted HDLs (rHDLs) containing each apoE isoform was determined using the Michaelis-Menten model. RESULTS: Plasma CER was ∼20% higher in apoE2 carriers compared to apoE3 carriers, and ∼30% higher in apoE2 carriers compared to apoE4 carriers. After adjusting for age, sex, total cholesterol, HDL-C, apoA-I, apoB, chronic kidney disease diagnosis, zygosity, and LCAT concentration, CER remained significantly different among carriers of the three apoE isoforms. The same trend was observed in carriers of FED-causing mutations. rHDLs containing apoE2 were associated with a lower affinity but higher maximal esterification rate, compared to particles containing apoE3 or apoE4. CONCLUSION: The present results suggest that the apoE2 isoform is associated with a higher LCAT-mediated cholesterol esterification. This observation may contribute to the characterization of the peculiar functional properties of apoE2.

Consensus document on Lipoprotein(a) from the Italian Society for the Study of Atherosclerosis (SISA).

AIMS: In view of the consolidating evidence on the causal role of Lp(a) in cardiovascular disease, the Italian Society for the Study of Atherosclerosis (SISA) has assembled a consensus on Lp(a) genetics and epidemiology, together with recommendations for its measurement and current and emerging therapeutic approaches to reduce its plasma levels. Data on the Italian population are also provided. DATA SYNTHESIS: Lp(a) is constituted by one apo(a) molecule and a lipoprotein closely resembling to a low-density lipoprotein (LDL). Its similarity with an LDL, together with its ability to carry oxidized phospholipids are considered the two main features making Lp(a) harmful for cardiovascular health. Plasma Lp(a) concentrations vary over about 1000 folds in humans and are genetically determined, thus they are quite stable in any individual. Mendelian Randomization studies have suggested a causal role of Lp(a) in atherosclerotic cardiovascular disease (ASCVD) and aortic valve stenosis and observational studies indicate a linear direct correlation between cardiovascular disease and Lp(a) plasma levels. Lp(a) measurement is strongly recommended once in a patient's lifetime, particularly in FH subjects, but also as part of the initial lipid screening to assess cardiovascular risk. The apo(a) size polymorphism represents a challenge for Lp(a) measurement in plasma, but new strategies are overcoming these difficulties. A reduction of Lp(a) levels can be currently attained only by plasma apheresis and, moderately, with PCSK9 inhibitor treatment. CONCLUSIONS: Awaiting the approval of selective Lp(a)-lowering drugs, an intensive management of the other risk factors for individuals with elevated Lp(a) levels is strongly recommended.

Neutrophil aging exacerbates high fat diet induced metabolic alterations.

BACKGROUND: High fat diet (HFD) chronically hyper-activates the myeloid cell precursors, but whether it affects the neutrophil aging is unknown. PURPOSE: We characterized how HFD impacts neutrophil aging, infiltration in metabolic tissues and if this aging, in turn, modulates the development of metabolic alterations. We immunophenotyped neutrophils and characterized the metabolic responses in physiology (wild-type mice, WT) and in mice with constitutively aged neutrophils (MRP8 driven conditional deletion of CXCR4; herein CXCR4fl/flCre+) or with constitutively fresh neutrophils (MRP8 driven conditional deletion of CXCR2; CXCR2fl/flCre+), following 20 weeks of HFD feeding (45 % kcal from fat). FINDINGS: After 20 weeks HFD, the gluco-metabolic profile of CXCR4fl/flCre+ mice was comparable to that of WT mice, while CXCR2fl/flCre+ mice were protected from metabolic alterations. CXCR4fl/flCre+ infiltrated more, but CXCR2fl/flCre+ neutrophils infiltrated less, in liver and visceral adipose tissue (VAT). As consequence, while CXCR4fl/flCre+ resulted into hepatic "suicidal" neutrophils extracellular traps (NETs) and altered immune cell architecture in VAT, CXCR2fl/flCre+ promoted proresolutive hepatic NETs and reduced accumulation of pro-inflammatory macrophages in VAT. In humans, higher plasma levels of Cxcl12 (CXCR4 ligand) correlated with visceral adiposity while higher levels of Cxcl1 (the ligand of CXCR2) correlated with indexes of hepatic steatosis, adiposity and metabolic syndrome. CONCLUSIONS: Neutrophil aging might contribute to the development of HFD induced metabolic disorders.

Adherence to Mediterranean Diet: A Population-Based Longitudinal Cohort Study.

Adherence to the Mediterranean diet (MedDiet) is recommended for cardiovascular disease prevention. However, recent epidemiological studies report a shift toward lower adherence to MedDiet. We have conducted a prospective cohort study to evaluate changes in individual determinants of MedDiet adherence over time. Clinical information and MedDiet adherence score (MEDAS) were collected in 711 subjects (mean age 68 ± 10 years; 42% males), enrolled in the PLIC study (Progression of Intimal Atherosclerotic Lesions in Carotid arteries), during two visits conducted, on average, 4.5 years apart. MEDAS score worsening and improvements (absolute change, ΔMEDAS) and the variation in the proportion of subjects reporting to meet each MEDAS criteria were assessed. Overall, 34% of the subjects improved their MedDiet adherence (ΔMEDAS: +1.87 ± 1.13), by consuming more olive oil, legumes and fish and use of dishes seasoned with sofrito and 48% subjects worsened their MedDiet adherence (ΔMEDAS: -2.02 ± 1.14) by consuming less fruit, legumes, fish and nuts, with higher rates of worsening in women and subjects aged 50-65 years. Subjects who improved the score were more obese, had higher plasma glucose levels, and metabolic syndrome at the basal visit. In summary, we report an overall decrease in MedDiet adherence, evaluated during a timeframe heavily affected by the COVID-19 pandemic, underlining the need for better dietary interventions.

The High Fat Diet Impacts the Plasticity between Fresh and Aged Neutrophils.

Metabolic alterations induced by unhealthy lifestyles, including obesity and insulin resistance are often associated with increased innate immune response and chronic inflammation. Cholesterol has been identified as a key metabolite driving the activation of the inflammasome and the "epigenetic memory" in long-term living hematopoietic stem cells. In addition to these mechanisms, the physiological aging of short-living neutrophils is a relevant modifier of their immune competency, as while they egress from medullary niches as "fresh", fully competent, cells, they turn into "aged", disarmed cells, when they extravasate into peripheral tissues to fight against pathogens or they reach the spleen for disposal. We recently observed that cardio-metabolic alterations induced by a lipid enriched unhealthy diet critically accelerate this process. Indeed, the chronic feeding with a high fat diet (HFD) results in the increase of aged neutrophils in the circulation and their accumulation in liver. This profile is associated with a deteriorated insulin response and obesity. The HFD primes aged, but not fresh neutrophils, to infiltrate in the liver and promotes inflammation coupled to altered cell immune architecture in visceral adipose tissue. Preventing the aging of neutrophils via selective ablation of CXCR2, reduces the development of obesity and improves the sensitivity to insulin. In humans, plasma levels of CXCL1, one of the cytokines binding CXCR2 and promoting neutrophil aging, are directly associated with abdominal adiposity and fatty liver independently of other risk factors. Together these findings point to a direct role of aged neutrophils in the development of metabolic disorders.

Special Issue: Nutraceutical Approaches to Cardiovascular and Metabolic Diseases: Evidence and Opportunities.

The effective prevention and treatment of cardiovascular and metabolic diseases is a major task for health systems since these pathological conditions are still major causes of mortality, morbidity, and disability worldwide [...].

Targeted Plasma Proteomics to Predict the Development of Carotid Plaques.

BACKGROUND: Cardiovascular risk stratification in primary prevention is a clinical challenge. We recently identified a large set of circulating proteins improving the risk prediction for cardiovascular events. We now evaluate which of these proteins predicts the development of subclinical carotid atherosclerosis (SCA) in primary cardiovascular prevention. METHODS: Three hundred sixty-eight proteins were quantified, by proximity extension assay, from the plasma collected at basal visit from 586 subjects without previous cardiovascular events and without preclinical atherosclerosis. These subjects were reevaluated 11 years after median follow-up (10-12) in a longitudinal observational analysis, to assess the development of SCA, defined as the formation of focal lesion in any carotid tract and detected by carotid ultrasound at basal visit and after follow-up. Common carotid (intima-media thickness [IMT]) was also measured by ultrasound during the same follow-up to identify subjects with faster common carotid intima-media thickness (IMT) progression (increase IMT)>1.3 mm in the common carotid tract). RESULTS: The variation of 68 proteins predicted SCA development and, among them, higher levels of PIgR2 (polymeric immunoglobulin receptor), chemokine (C-C motif) ligand 18, CA1 (carbonic anhydrase 1), Fc gamma receptor IIa and reduced MMP10 (matrix metallopeptidase 10), GT (gastrotropin), IL7R (interleukin 7 receptor) were the most predictive for SCA development. These 7 proteins improved the sensitivity and the specificity for SCA development versus risk factors (age, sex, overweight, hypertension, low HDL-cholesterol, high triglyceride); area under the curve: 0.747 ([0.707-0.784] versus 0.620 [0.577-0.663]; P<0.001). Vice versa, 25 proteins (not in common with the previous 68) predicted faster common carotid IMT progression. Among them, increased IL7D (interleukin 7), chemokine (C-X-C motif) ligand 1, and reduced TNFS13B (TNF superfamily member 13b) significantly increased the sensitivity and the specificity to predict faster common carotid IMT progression as compared with same risk factors (area under the curve: 0.719 [0.680-0.756] versus 0.569 [0.527-0.610]; P<0.001). CONCLUSIONS: A new set of circulating proteins have been identified that may be considered as markers of preclinical atherosclerosis development. The difference of the protein identified to predict SCA versus IMT progression may reflect different etiological factors.

Adherence to the Mediterranean Diet: Impact of Geographical Location of the Observations.

The Mediterranean diet has emerged as a comprehensive lifestyle, including specific foods and meal composition and a set of behavioural and social features. Adherence to the Mediterranean diet has been shown to promote health and reduce the prevalence of chronic diseases. The actual implementation of the Mediterranean diet is affected by several sociocultural factors as well as geographical components. Indeed, the geographical location, such as a specific country or different areas in a country and specific latitude and climate, appears to be an important factor that may strongly affect the implementation of the Mediterranean diet or some of its principles as well as the adherence to it. Another dynamic component affecting personal nutritional choices, also regarding adherence to the Mediterranean diet and its principles, is the individual life-long trajectory of food preference and nutrition habits and awareness. In this review, we discuss the current evidence on the impact of geographical location on adherence to the Mediterranean diet.

PCSK9 promotes arterial medial calcification.

BACKGROUND AND AIMS: A complex interplay among chronic kidney disease (CKD), lipid metabolism and aortic calcification has been recognized. Here we investigated the influence of kidney function on PCSK9 levels and its potential direct action on smooth muscle cells (SMCs) calcification. METHODS AND RESULTS: In a cohort of 594 subjects, a negative association between glomerular filtration rate and plasma PCSK9 was found. Atherosclerotic cardiovascular disease, as co-morbidity, further increased PCSK9 plasma levels. Diet-induced uremic condition in rats led to aortic calcification and increased total cholesterol and Pcsk9 levels in plasma, livers, and kidneys. Both human and rat SMCs overexpressing human PCSK9 (SMCsPCSK9), cultured in a pro-calcific environment (2.0 mM or 2.4 mM inorganic phosphate, Pi) showed a significantly higher extracellular calcium (Ca2+) deposition compared to control SMCs. The addition of recombinant human PCSK9 did not increase the extracellular calcification of SMCs, suggesting the involvement of intracellular PCSK9. Accordingly, the further challenge with evolocumab did not affect calcium deposition in hSMCsPCSK9. Under pro-calcific conditions, SMCsPCSK9 released a higher number of extracellular vesicles (EVs) positive for three tetraspanin molecules, such as CD63, CD9, and CD81. EVs derived from SMCsPCSK9 tended to be more enriched in calcium and alkaline phosphatase (ALP), compared to EVs from control SMCs. In addition, PCSK9 has been detected in SMCsPCSK9-derived EVs. Finally, SMCsPCSK9 showed an increase in pro-calcific markers, namely bone morphogenetic protein 2 and ALP, and a decrease in anti-calcific mediator matrix GLA protein and osteopontin. CONCLUSIONS: Our study reveals a direct role of PCSK9 on vascular calcification induced by higher inorganic phosphate levels associated with renal impairment. This effect appears to be mediated by a switch towards a pro-calcific phenotype of SMCs associated with the release of EVs containing Ca2+ and ALP.

The zebrafish model system for dyslipidemia and atherosclerosis research: Focus on environmental/exposome factors and genetic mechanisms.

Dyslipidemias and atherosclerosis play a pivotal role in cardiovascular risk and disease. Although some pathophysiological mechanisms underlying these conditions have been unveiled, several knowledge gaps still remain. Experimental models, both in vitro and in vivo, have been instrumental to our better understanding of such complex processes. The latter have often been based on rodent species, either wild-type or, in several instances, genetically modified. In this context, the zebrafish may represent an additional very useful in vivo experimental model for dyslipidemia and atherosclerosis. Interestingly, the lipid metabolism of zebrafish shares several features with that present in humans, recapitulating some molecular features and pathophysiological aspects in a better way than that of rodents. The zebrafish model may be of help to address questions related to exposome factors as well as to genetic features, aiming to dissect selected aspects of the more complex scenario observed in humans. Indeed, exposome-related dyslipidemia/atherosclerosis research in zebrafish may target different scientific questions, related to nutrition, microbiota, temperature, light exposure at the larval stage, exposure to chemicals and epigenetic consequences of such external factors. Addressing genetic features related to dyslipidemia/atherosclerosis using the zebrafish model is already a reality and active research is now ongoing in this promising area. Novel technologies (gene and genome editing) may help to identify new candidate genes involved in dyslipidemia and dyslipidemia-related diseases. Based on these considerations, the zebrafish experimental model appears highly suitable for the study of exposome factors, genes and molecules involved in the development of atherosclerosis-related disease as well as for the validation of novel potential treatment options.

Genetically determined hypercholesterolaemia results into premature leucocyte telomere length shortening and reduced haematopoietic precursors.

AIMS: Leucocyte telomere length (LTL) shortening is a marker of cellular senescence and associates with increased risk of cardiovascular disease (CVD). A number of cardiovascular risk factors affect LTL, but the correlation between elevated LDL cholesterol (LDL-C) and shorter LTL is debated: in small cohorts including subjects with a clinical diagnosis of familial hypercholesterolaemia (FH). We assessed the relationship between LDL-C and LTL in subjects with genetic familial hypercholesterolaemia (HeFH) compared to those with clinically diagnosed, but not genetically confirmed FH (CD-FH), and normocholesterolaemic subjects. METHODS AND RESULTS: LTL was measured in mononuclear cells-derived genomic DNA from 206 hypercholesterolaemic subjects (135 HeFH and 71 CD-FH) and 272 controls. HeFH presented shorter LTL vs. controls (1.27 ± 0.07 vs. 1.59 ± 0.04, P = 0.045). In particular, we found shorter LTL in young HeFH as compared to young controls (<35 y) (1.34 ± 0.08 vs. 1.64 ± 0.08, P = 0.019); moreover, LTL was shorter in statin-naïve HeFH subjects as compared to controls (1.23 ± 0.08 vs. 1.58 ± 0.04, P = 0.001). HeFH subjects presented shorter LTL compared to LDL-C matched CD-FH (1.33 ± 0.05 vs. 1.55 ± 0.08, P = 0.029). Shorter LTL was confirmed in leucocytes of LDLR-KO vs. wild-type mice and associated with lower abundance of long-term haematopoietic stem and progenitor cells (LT-HSPCs) in the bone marrow. Accordingly, HeFH subjects presented lower circulating haematopoietic precursors (CD34 + CD45dim cells) vs. CD-FH and controls. CONCLUSIONS: We found (i) shorter LTL in genetically determined hypercholesterolaemia, (ii) lower circulating haematopoietic precursors in HeFH subjects, and reduced bone marrow resident LT-HSPCs in LDLR-KO mice. We support early cellular senescence and haematopoietic alterations in subjects with FH.

Impact of metabolic disorders on the structural, functional, and immunological integrity of the blood-brain barrier: Therapeutic avenues.

Mounting evidence has linked the metabolic disease to neurovascular disorders and cognitive decline. Using a murine model of a high-fat high-sugar diet mimicking obesity-induced type 2 diabetes mellitus (T2DM) in humans, we show that pro-inflammatory mediators and altered immune responses damage the blood-brain barrier (BBB) structure, triggering a proinflammatory metabolic phenotype. We find that disruption to tight junctions and basal lamina due to loss of control in the production of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) causes BBB impairment. Together the disruption to the structural and functional integrity of the BBB results in enhanced transmigration of leukocytes across the BBB that could contribute to an initiation of a neuroinflammatory response through activation of microglia. Using a humanized in vitro model of the BBB and T2DM patient post-mortem brains, we show the translatable applicability of our results. We find a leaky BBB phenotype in T2DM patients can be attributed to a loss of junctional proteins through changes in inflammatory mediators and MMP/TIMP levels, resulting in increased leukocyte extravasation into the brain parenchyma. We further investigated therapeutic avenues to reduce and restore the BBB damage caused by HFHS-feeding. Pharmacological treatment with recombinant annexin A1 (hrANXA1) or reversion from a high-fat high-sugar diet to a control chow diet (dietary intervention), attenuated T2DM development, reduced inflammation, and restored BBB integrity in the animals. Given the rising incidence of diabetes worldwide, understanding metabolic-disease-associated brain microvessel damage is vital and the proposed therapeutic avenues could help alleviate the burden of these diseases.

The Association of Proprotein Convertase Subtilisin/Kexin Type 9 to Plasma Low-Density Lipoproteins: An Evaluation of Different Methods.

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is key regulator of low-density lipoprotein (LDL) metabolism. A significant proportion of PCSK9 is believed to be associated with LDL in plasma as it circulates, although this finding is still a matter of debate. The purpose of this study was to establish an experimental method to investigate the presence of such an interaction in the bloodstream. We compared a number of well-established methods for lipoprotein (LP) isolation to clarify whether PCSK9 associates differently to circulating lipoproteins, such as KBr gradient ultracentrifugation, physical precipitation of ApoB-LPs, fast protein liquid chromatography (FPLC) and iodixanol gradient ultracentrifugation. Our data show heterogeneity in PCSK9 association to lipoproteins according to the method used. Two methods, iodixanol ultracentrifugation and column chromatography, which did not involve precipitation or high salt concentration, consistently showed an interaction of PCSK9 with a subfraction of LDL that appeared to be more buoyant and have a lower size than average LDL. The percent of PCSK9 association ranged from 2 to 30% and did not appear to correlate to plasma or LDL cholesterol levels. The association of PCSK9 to LDL appeared to be sensitive to high salt concentrations. FPLC and iodixanol gradient ultracentrifugation appeared to be the most suitable methods for the study of this association.