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.

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.

Inclisiran: How Widely and When Should We Use It?

PURPOSE OF REVIEW: Plasma levels of LDL cholesterol (LDL-C) are causally associated with cardiovascular risk. Reducing LDL-C results in a decreased incidence of cardiovascular events, proportionally to the absolute reduction in LDL-C. The inhibition of proprotein convertase subtilisin kexin 9 (PCSK) is a highly effective and safe approach to reducing LDL-C levels. In this review, we discuss the available data on the efficacy and safety of inclisiran, a siRNA targeting PCSK9 and propose a clinical profile for the patients who can benefit the most from this approach. RECENT FINDINGS: Inclisiran is a small interfering RNA targeting the mRNA of PCSK9 specifically in the liver, owing to the conjugation with triantennary N-acetylgalactosamine. Randomized clinical trials have shown that inclisiran provides robust and durable reductions of PCSK9 and LDL-C levels, with a dosing schedule of once every 6 months after the initial and 3-month doses. These effects are consistent in different categories of patients, including patients with atherosclerotic cardiovascular disease and/or risk equivalent or patients with heterozygous familial hypercholesterolaemia. Ultimately the administration schedule may improve patients' compliance given also the favourable safety profile of the drug. Completion of ongoing outcome clinical trials will provide information on both the expected clinical benefit and the safety of inclisiran administered for longer.

PCSK9 deficiency rewires heart metabolism and drives heart failure with preserved ejection fraction.

AIMS: PCSK9 is secreted into the circulation, mainly by the liver, and interacts with low-density lipoprotein receptor (LDLR) homologous and non-homologous receptors, including CD36, thus favouring their intracellular degradation. As PCSK9 deficiency increases the expression of lipids and lipoprotein receptors, thus contributing to cellular lipid accumulation, we investigated whether this could affect heart metabolism and function. METHODS AND RESULTS: Wild-type (WT), Pcsk9 KO, Liver conditional Pcsk9 KO and Pcsk9/Ldlr double KO male mice were fed for 20 weeks with a standard fat diet and then exercise resistance, muscle strength, and heart characteristics were evaluated. Pcsk9 KO presented reduced running resistance coupled to echocardiographic abnormalities suggestive of heart failure with preserved ejection fraction (HFpEF). Heart mitochondrial activity, following maximal coupled and uncoupled respiration, was reduced in Pcsk9 KO mice compared to WT mice and was coupled to major changes in cardiac metabolism together with increased expression of LDLR and CD36 and with lipid accumulation. A similar phenotype was observed in Pcsk9/Ldlr DKO, thus excluding a contribution for LDLR to cardiac impairment observed in Pcsk9 KO mice. Heart function profiling of the liver selective Pcsk9 KO model further excluded the involvement of circulating PCSK9 in the development of HFpEF, pointing to a possible role locally produced PCSK9. Concordantly, carriers of the R46L loss-of-function variant for PCSK9 presented increased left ventricular mass but similar ejection fraction compared to matched control subjects. CONCLUSION: PCSK9 deficiency impacts cardiac lipid metabolism in an LDLR independent manner and contributes to the development of HFpEF.

Single systemic transfer of a human gene associated with exceptional longevity halts the progression of atherosclerosis and inflammation in ApoE knockout mice through a CXCR4-mediated mechanism.

AIMS: Here, we aimed to determine the therapeutic effect of longevity-associated variant (LAV)-BPIFB4 gene therapy on atherosclerosis. METHODS AND RESULTS: ApoE knockout mice (ApoE-/-) fed a high-fat diet were randomly allocated to receive LAV-BPIFB4, wild-type (WT)-BPIFB4, or empty vector via adeno-associated viral vector injection. The primary endpoints of the study were to assess (i) vascular reactivity and (ii) atherosclerotic disease severity, by Echo-Doppler imaging, histology and ultrastructural analysis. Moreover, we assessed the capacity of the LAV-BPIFB4 protein to shift monocyte-derived macrophages of atherosclerotic mice and patients towards an anti-inflammatory phenotype. LAV-BPIFB4 gene therapy rescued endothelial function of mesenteric and femoral arteries from ApoE-/- mice; this effect was blunted by AMD3100, a CXC chemokine receptor type 4 (CXCR4) inhibitor. LAV-BPIFB4-treated mice showed a CXCR4-mediated shift in the balance between Ly6Chigh/Ly6Clow monocytes and M2/M1 macrophages, along with decreased T cell proliferation and elevated circulating levels of interleukins IL-23 and IL-27. In vitro conditioning with LAV-BPIFB4 protein of macrophages from atherosclerotic patients resulted in a CXCR4-dependent M2 polarization phenotype. Furthermore, LAV-BPIFB4 treatment of arteries explanted from atherosclerotic patients increased the release of atheroprotective IL-33, while inhibiting the release of pro-inflammatory IL-1ß, inducing endothelial nitric oxide synthase phosphorylation and restoring endothelial function. Finally, significantly lower plasma BPIFB4 was detected in patients with pathological carotid stenosis (>25%) and intima media thickness >2 mm. CONCLUSION: Transfer of the LAV of BPIFB4 reduces the atherogenic process and skews macrophages towards an M2-resolving phenotype through modulation of CXCR4, thus opening up novel therapeutic possibilities in cardiovascular disease.

Zc3h10 is a novel mitochondrial regulator.

Mitochondria are the energy-generating hubs of the cell. In spite of considerable advances, our understanding of the factors that regulate the molecular circuits that govern mitochondrial function remains incomplete. Using a genome-wide functional screen, we identify the poorly characterized protein Zinc finger CCCH-type containing 10 (Zc3h10) as regulator of mitochondrial physiology. We show that Zc3h10 is upregulated during physiological mitochondriogenesis as it occurs during the differentiation of myoblasts into myotubes. Zc3h10 overexpression boosts mitochondrial function and promotes myoblast differentiation, while the depletion of Zc3h10 results in impaired myoblast differentiation, mitochondrial dysfunction, reduced expression of electron transport chain (ETC) subunits, and blunted TCA cycle flux. Notably, we have identified a loss-of-function mutation of Zc3h10 in humans (Tyr105 to Cys105) that is associated with increased body mass index, fat mass, fasting glucose, and triglycerides. Isolated peripheral blood mononuclear cells from individuals homozygotic for Cys105 display reduced oxygen consumption rate, diminished expression of some ETC subunits, and decreased levels of some TCA cycle metabolites, which all together derive in mitochondrial dysfunction. Taken together, our study identifies Zc3h10 as a novel mitochondrial regulator.

PCSK9 deficiency reduces insulin secretion and promotes glucose intolerance: the role of the low-density lipoprotein receptor.

Aims: PCSK9 loss of function genetic variants are associated with lower low-density lipoprotein cholesterol but also with higher plasma glucose levels and increased risk of Type 2 diabetes mellitus. Here, we investigated the molecular mechanisms underlying this association. Methods and results: Pcsk9 KO, WT, Pcsk9/Ldlr double KO (DKO), Ldlr KO, albumin AlbCre+/Pcsk9LoxP/LoxP (liver-selective Pcsk9 knock-out mice), and AlbCre-/Pcsk9LoxP/LoxP mice were used. GTT, ITT, insulin and C-peptide plasma levels, pancreas morphology, and cholesterol accumulation in pancreatic islets were studied in the different animal models. Glucose clearance was significantly impaired in Pcsk9 KO mice fed with a standard or a high-fat diet for 20 weeks compared with WT animals; insulin sensitivity, however, was not affected. A detailed analysis of pancreas morphology of Pcsk9 KO mice vs. controls revealed larger islets with increased accumulation of cholesteryl esters, paralleled by increased insulin intracellular levels and decreased plasma insulin, and C-peptide levels. This phenotype was completely reverted in Pcsk9/Ldlr DKO mice implying the low-density lipoprotein receptor (LDLR) as the proprotein convertase subtilisin/kexin Type 9 (PCSK9) target responsible for the phenotype observed. Further studies in albumin AlbCre+/Pcsk9LoxP/LoxP mice, which lack detectable circulating PCSK9, also showed a complete recovery of the phenotype, thus indicating that circulating, liver-derived PCSK9, the principal target of monoclonal antibodies, does not impact beta-cell function and insulin secretion. Conclusion: PCSK9 critically controls LDLR expression in pancreas perhaps contributing to the maintenance of a proper physiological balance to limit cholesterol overload in beta cells. This effect is independent of circulating PCSK9 and is probably related to locally produced PCSK9.

Multifactorial Activation of NLRP3 Inflammasome: Relevance for a Precision Approach to Atherosclerotic Cardiovascular Risk and Disease.

Chronic low-grade inflammation, through the specific activation of the NACHT leucine-rich repeat- and PYD-containing (NLRP)3 inflammasome-interleukin (IL)-1ß pathway, is an important contributor to the development of atherosclerotic cardiovascular disease (ASCVD), being triggered by intracellular cholesterol accumulation within cells. Within this pathological context, this complex pathway is activated by a number of factors, such as unhealthy nutrition, altered gut and oral microbiota, and elevated cholesterol itself. Moreover, evidence from autoinflammatory diseases, like psoriasis and others, which are also associated with higher cardiovascular disease (CVD) risk, suggests that variants of NLRP3 pathway-related genes (like NLRP3 itself, caspase recruitment domain-containing protein (CARD)8, caspase-1 and IL-1ß) may carry gain-of-function mutations leading, in some individuals, to a constitutive pro-inflammatory pattern. Indeed, some reports have recently associated the presence of specific single nucleotide polymorphisms (SNPs) on such genes with greater ASCVD prevalence. Based on these observations, a potential effective strategy in this context may be the identification of carriers of these NLRP3-related SNPs, to generate a genomic score, potentially useful for a better CVD risk prediction, and, possibly, for personalized therapeutic approaches targeted to the NLRP3-IL-1ß pathway.

Multilevel Models to Estimate Carotid Intima-Media Thickness Curves for Individual Cardiovascular Risk Evaluation.

Background and Purpose- The value of carotid intima-media thickness (cIMT)-a marker of subclinical atherosclerosis-in defining the cardiovascular risk is still debated. The aim of this study was to estimate standard cIMT progression, adjusting values over time for the main cardiovascular risk factors, in a sample of low-to-moderate cardiovascular risk subjects, to identify normative cIMT progression values. Methods- From the progression of lesions in the intima of the carotid cohort, we selected subjects who underwent 4 planned serial clinical evaluations and ultrasound cIMT determinations, on average every 4 years. Subject taking glucose-lowering therapies in secondary cardiovascular prevention or with cardiovascular risk score >5 were excluded from the analysis. The growth of cIMT across the study period (12 years) was assessed by use of individual growth curve modeling within multilevel models. Results- A total of 1175 (36% men; mean age, 53±11 years at baseline) participants at low/intermediate cardiovascular risk have been included in this analysis. A significant and marked slope of the mean and maximum cIMT growth curves (ß=0.009 and ß=0.012, respectively) was observed, confirming that it is a function of age. A stratified analysis by decades of age highlighted a nonlinear cIMT progression over time. In addition, different patterns of cIMT development between sex were observed. Finally, different slopes in mean and maximum cIMT curves, with a significant spread since the fifth decade, were observed in the cIMT growth curve models of subjects developing multifocal carotid atherosclerosis compared with the rest of the population. Conclusions- These findings proved that the rate of change in cIMT over time is a sign of the development of atherosclerosis, which cannot be a priori assumed linear. These data, therefore, support the clinical relevance of these growth curve models for cIMT progression to be considered as useful tool to identify subjects with faster atherosclerosis progression and thus at increased cardiovascular risk.

The Interconnection Between Immuno-Metabolism, Diabetes, and CKD.

PURPOSE OF REVIEW: Metabolic reprogramming is increasingly recognized as an essential trait of functional activation of immune cells. Here, we describe the link between immuno-metabolism, diabetes, and diabetic nephropathy. RECENT FINDINGS: Crosstalk between cellular metabolic functions and immune activation occurs when plasma levels of glucose, triglycerides, and free fatty acids increase, thus promoting systemic low-grade inflammation that further boosts the development of metabolic complications. In the long run, this settles an "apparent paradox," where, despite excessive inflammation, the immune system is suppressed, further promoting progression to end-stage renal disease (ESRD) and predisposing to premature deaths from infections and cardiovascular diseases. Reviewing the effects of diabetes treatments on immuno-inflammatory responses suggests that the benefit of these drugs might extend beyond the simple control of glucose homeostasis. Hyperglycemia and dyslipidemia correlate with enhancement of the immuno-inflammatory response that can promote and worsen metabolic diseases and support the progression toward ESRD. The identification of cellular checkpoints that modulate the immuno-metabolic machinery of immune cells opens new venues for metabolic drugs.

The Interplay of Lipids, Lipoproteins, and Immunity in Atherosclerosis.

PURPOSE OF REVIEW: Atherosclerosis is an inflammatory disorder of the arterial wall, in which several players contribute to the onset and progression of the disease. Besides the well-established role of lipids, specifically cholesterol, and immune cell activation, new insights on the molecular mechanisms underlying the atherogenic process have emerged. RECENT FINDINGS: Meta-inflammation, a condition of low-grade immune response caused by metabolic dysregulation, immunological memory of innate immune cells (referred to as "trained immunity"), cholesterol homeostasis in dendritic cells, and immunometabolism, i.e., the interplay between immunological and metabolic processes, have all emerged as new actors during atherogenesis. These observations reinforced the interest in directly targeting inflammation to reduce cardiovascular disease. The novel acquisitions in pathophysiology of atherosclerosis reinforce the tight link between lipids, inflammation, and immune response, and support the benefit of targeting LDL-C as well as inflammation to decrease the CVD burden. How this will translate into the clinic will depend on the balance between costs (monoclonal antibodies either to PCSK9 or to IL-1ß), side effects (increased incidence of death due to infections for anti-IL-1ß antibody), and the benefits for patients at high CVD risk.

Translating the microRNA signature of microvesicles derived from human coronary artery smooth muscle cells in patients with familial hypercholesterolemia and coronary artery disease.

AIMS: To analyze the impact of atherogenic lipoproteins on the miRNA signature of microvesicles derived from human coronary artery smooth muscle cells (CASMC) and to translate these results to familial hypercholesterolemia (FH) and coronary artery disease (CAD) patients. METHODS: Conditioned media was collected after exposure of CASMC to atherogenic lipoproteins. Plasma samples were collected from two independent populations of diagnosed FH patients and matched normocholesterolemic controls (Study population 1, N=50; Study population 2, N=24) and a population of patients with suspected CAD (Study population 3, N=50). Extracellular vesicles were isolated and characterized using standard techniques. A panel of 30 miRNAs related to vascular smooth muscle cell (VSMC) (patho-)physiology was analyzed using RT-qPCR. RESULTS: Atherogenic lipoproteins significantly reduced levels of miR-15b-5p, -24-3p, -29b-3p, -130a-3p, -143-3p, -146a-3p, -222-3p, -663a levels (P<0.050) in microvesicles (0.1µm-1µm in diameter) released by CASMC. Two of these miRNAs, miR-24-3p and miR-130a-3p, were reduced in circulating microvesicles from FH patients compared with normocholesterolemic controls in a pilot study (Study population 1) and in different validation studies (Study populations 1 and 2) (P<0.050). Supporting these results, plasma levels of miR-24-3p and miR-130a-3p were also downregulated in FH patients compared to controls (P<0.050). In addition, plasma levels of miR-130a-3p were inversely associated with coronary atherosclerosis in a cohort of suspected CAD patients (Study population 3) (P<0.050). CONCLUSIONS: Exposure to atherogenic lipoproteins modifies the miRNA profile of CASMC-derived microvesicles and these alterations are reflected in patients with FH. Circulating miR-130a-3p emerges as a potential biomarker for coronary atherosclerosis.

Targeting PCSK9 for hypercholesterolemia.

Dyslipidemias are a predominant risk factor for cardiovascular disease. Biological and genetic research has led to the identification of several genes and proteins that may be pharmacologically targeted to improve lipoprotein profiles and possibly cardiovascular outcomes in patients with dyslipidemia. The observation that proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates the levels of circulating low-density lipoprotein C (LDL-C) by enhancing the degradation of the hepatic low-density lipoprotein receptor (LDLR) prompted the search for drugs that inhibit PCSK9 activity. Several approaches to inhibiting PCSK9 activity have been proposed; these involve inhibitory antibodies, small molecules, and gene silencing. To date, the most promising and advanced approach relates to monoclonal antibodies, which can decrease LDL cholesterol by 65-70%, even as an add-on therapy to a maximal dose of a statin. Phase III studies and large, event-driven clinical trials are ongoing and will fully address the viability and role of these drugs in clinical practice.

A simple informative intervention in primary care increases statin adherence.

PURPOSE: To assess the effectiveness of an informative intervention on general practitioners aimed at improving patients' adherence to statin therapy. METHODS: In the local health unit (LHU) of Bergamo, Lombardy (Italy), each general practitioner received a synthetic scientific document on dyslipidaemia and statins and aggregated data on adherence in 2006 for his/her patients compared to the means in the LHU and in his/her working district. Furthermore, a sample of seven districts received also a table of adherence levels for single patients. Patient's level data were retrieved from the health care utilisation databases of the LHU. Adherence parameters (proportion of patients with only one prescription, medication possession ratio [MPR] and proportion of non-persistent patients) were assessed after 1 year of follow-up. RESULTS: Overall, 5833 and 4788 new statin users were enrolled before and after the intervention, respectively. The percentage of patients with only one prescription decreased from 28.0 to 23.9 % (p < 0.001). MPR increased from 70.3 to 76.0 % (p < 0.001), and proportion of patients with MPR ≥ 80 % increased from 45.4 to 56.4 % (p < 0.001). The persistence also showed an improvement, both in terms of decreasing proportion of non-persistent (from 51.9 to 41.4 %, p < 0.001) and of increasing duration of continued therapy (from 235 to 264 mean days of persistent therapy, p < 0.001). There were not significant differences between the two types of intervention. CONCLUSIONS: This intervention resulted in an overall improvement of the short-term adherence to therapy. This tool can be replicated in other local contexts and with other chronic therapies.

HDL in infectious diseases and sepsis.

During infection significant alterations in lipid metabolism and lipoprotein composition occur. Triglyceride and VLDL cholesterol levels increase, while reduced HDL cholesterol (HDL-C) and LDL cholesterol (LDL-C) levels are observed. More importantly, endotoxemia modulates HDL composition and size: phospholipids are reduced as well as apolipoprotein (apo) A-I, while serum amyloid A (SAA) and secretory phospholipase A2 (sPLA2) dramatically increase, and, although the total HDL particle number does not change, a significant decrease in the number of small- and medium-size particles is observed. Low HDL-C levels inversely correlate with the severity of septic disease and associate with an exaggerated systemic inflammatory response. HDL, as well as other plasma lipoproteins, can bind and neutralize Gram-negative bacterial lipopolysaccharide (LPS) and Gram-positive bacterial lipoteichoic acid (LTA), thus favoring the clearance of these products. HDLs are emerging also as a relevant player during parasitic infections, and a specific component of HDL, namely, apoL-1, confers innate immunity against trypanosome by favoring lysosomal swelling which kills the parasite. During virus infections, proteins associated with the modulation of cholesterol bioavailability in the lipid rafts such as ABCA1 and SR-BI have been shown to favor virus entry into the cells. Pharmacological studies support the benefit of recombinant HDL or apoA-I mimetics during bacterial infection, while apoL-1-nanobody complexes were tested for trypanosome infection. Finally, SR-BI antagonism represents a novel and forefront approach interfering with hepatitis C virus entry which is currently tested in clinical studies. From the coming years, we have to expect new and compelling observations further linking HDL to innate immunity and infections.