Arteriosclerosis, Thrombosis, and Vascular Biology最新文献

Background: We aimed to compare the molecular and clinical characteristics of patients identified in Italy as affected by either familial chylomicronemia syndrome (FCS) or multifactorial chylomicronemia syndrome (MCS) and to assess the overall benefit of novel triglyceride-lowering therapies prescribed to these patients within the routine clinical care.

Methods: From the national LIPIGEN-sHTG (Lipid Transport Disorders Italian Genetic Network-Severe Hypertriglyceridemia) registry, 169 patients (57 FCS, 51 MCS, 61 variant-negative, variant-negative MCS) were retrospectively analyzed. Data on clinical and genetic characteristics, medical history, and medications were collected. Peak triglyceride levels were used to define untreated lipid phenotypes.

Results: In FCS, 72% exhibited biallelic LPL and 28% non-LPL variants; in MCS, 38% (n=19) carried LPL variants, and 38% (n=19) carried APOA5 variants, whereas the remaining individuals were carriers of LMF1 (n=3), GPIHBP1 (n=2), and CREB3L3 or GPD1 variants (n=8), respectively. Peak TGs were highest in FCS (3000 mg/dL [interquartile range, 2116.0-4265.0]), followed by MCS (1817 mg/dL [interquartile range, 1370.0-3062.0]) and variant-negative MCS (1340.0 mg/dL [interquartile range, 946.5-2508.5]; P<0.001). FCS showed a 3.4-fold higher risk of acute pancreatitis than others, whereas no significant differences were observed between groups in the prevalence of atherosclerotic cardiovascular diseases. In the subset of patients with FCS receiving novel therapies (lomitapide or volanesorsen; 35%), triglyceride levels decreased by 62%, as compared with an 11% reduction in those on conventional treatment. Across the cohort, posttreatment triglyceride levels were 895 mg/dL in FCS, 352 mg/dL in MCS, and 386 mg/dL in variant-negative MCS.

Conclusions: As compared with MCS, patients with FCS showed a more severe phenotype and higher prevalence of LPL variants. Lomitapide and volanesorsen provide better triglyceride control, yet only one-third of FCS were treated with these drugs in the routine clinical practice.

Background: Movement of circulating lipids into tissues and arteries requires transfer across the endothelial cell (EC) barrier. This process allows the heart to obtain fatty acids, its chief source of energy, and apoB-containing lipoproteins to cross the arterial endothelial barrier, leading to cholesterol accumulation in the subendothelial space. Multiple studies have established elevated postprandial TRLs (triglyceride-rich lipoproteins) as an independent risk factor for cardiovascular disease. We explored how chylomicrons affect ECs and transfer their fatty acids across the EC barrier.

Methods: We had reported that media from chylomicron-treated ECs lead to lipid droplet formation in macrophages. To determine the responsible component of this media, we assessed whether removing the extracellular vesicles (EVs) would obviate this effect. EVs from control and treated cells were then characterized by protein, lipid, and microRNA content. We also studied the EV-induced transcription changes in macrophages and ECs and whether knockdown of SR-BI (scavenger receptor-BI) altered these responses. In addition, using chylomicrons labeled with [¹³C]oleate, we studied the uptake and release of this labeled by ECs.

Results: Chylomicron treatment of ECs led to an inflammatory response that included production of EVs that drove macrophage lipid droplet accumulation. The EVs contained little free fatty acids and triglycerides, but abundant phospholipids and diacylglycerols. In concert with this, [¹³]C labeled chylomicron triglycerides exited ECs primarily in phospholipids. EVs from chylomicron-treated versus untreated ECs were larger, more abundant, and contained specific microRNAs. Treatment of macrophages and naive ECs with media from chylomicron-treated ECs increased expression of inflammatory genes.

Conclusions: EC chylomicron metabolism produces EVs that increase macrophage inflammation and create LDs. Media containing these EVs also increases EC inflammation, illustrating an autocrine inflammatory process. Fatty acids within chylomicron triglycerides are converted to phospholipids within EVs. Thus, EC uptake of chylomicrons constitutes an important pathway for vascular inflammation and tissue lipid acquisition.

Background: Inflammation potentiates aortic valve calcification (AVC). ⁶⁸Ga-DOTATATE (gallium-68 DOTA-(Tyr³)-octreotate), a positron emission tomography tracer that binds to somatostatin receptors, provides a measure of tissue inflammation. However, the diagnostic and prognostic values of aortic valve (AV) ⁶⁸Ga-DOTATATE uptake in AVC remain unexplored. Here, we tested whether AV ⁶⁸Ga-DOTATATE uptake predicts the progression of AVC.

Methods: A total of 683 individuals (median age, 63 years; 46% male) underwent clinical ⁶⁸Ga-DOTATATE positron emission tomography/computed tomography imaging from 2017 to 2023; 209 had follow-up imaging (median, 1.3 years interval). AV inflammation was measured as the maximum standardized uptake value of ⁶⁸Ga-DOTATATE uptake within the AV on baseline positron emission tomography/computed tomography. AVC was quantified on baseline and follow-up computed tomography scans (Hounsfield units >130). AVC progression was assessed as the difference between baseline and follow-up AVC. Individuals with a square root difference of annualized AVC change ≥2.5 were characterized as progressors and <2.5 as nonprogressors. Demographic and clinical data were collected from medical records.

Results: Baseline AV ⁶⁸Ga-DOTATATE uptake correlated with baseline AVC (standardized ρ=0.12; P=0.002). Furthermore, baseline AV ⁶⁸Ga-DOTATATE uptake associated with AVC progression (odds ratio [OR], 3.0 [95% CI, 1.4-6.4]; P=0.004) and remained significant after separately adjusting for baseline AVC (OR, 3.1 [95% CI, 1.5-6.6]), sex (OR, 4.0 [95% CI, 1.7-9.0]), hypertension (OR, 2.8 [95% CI, 1.3-6.2]), diabetes (OR, 3.0 [95% CI, 1.4-6.4]), hyperlipidemia (OR, 2.4 [95% CI, 1.1-5.3]), smoking (OR, 3.1 [95% CI, 1.5-6.7]), chronic kidney disease (OR, 2.9 [95% CI, 1.4-6.3]), and body mass index (OR, 3.0 [95% CI, 1.4-6.3]), became insignificant when adjusting to age (OR, 1.9 [95% CI, 0.8-4.3]).

Conclusions: Our study highlights the use of ⁶⁸Ga-DOTATATE for assessing AV inflammation and predicting AVC progression. These findings underscore the role of inflammation in AVC progression and have potential implications for risk assessment and evaluating therapies in AV disease.

Background: Pericytes are vascular mural cells, critical to the formation and maintenance of tightly regulated microvascular networks, including the inner blood-retinal barrier, and their dysfunction is characteristic of many vascular diseases, including diabetic retinopathy. Although donor eye and animal studies have suggested a link between pericyte loss and retinal vascular leakage, this relationship has not been explored in living humans, nor has the role of pericytes in predicting other complications, like albuminuria, in diabetes.

Methods: In this pilot study, we utilized adaptive optics scanning laser ophthalmoscopy to image and quantify retinal capillary pericytes in humans with diabetes. Leakage was manually delineated on fluorescein angiography to calculate macular leakage (%), whereas optical coherence tomography and optical coherence tomography-angiography evaluated retinal thickness and capillary nonperfusion, respectively. In addition, urine albumin/creatinine ratios (mg/g) were extracted from charts, post hoc, to evaluate the relationship between retinal pericytes and albuminuria in a subpopulation (n=14).

Results: The study included 24 eyes from 23 patients with a range of diabetic retinopathy severity spanning from no diabetic retinopathy to proliferative diabetic retinopathy. Notably, capillary pericyte density showed a strong, negative correlation to macular leakage (r=-0.68; P<0.001) and moderate correlation to optical coherence tomography thickness (r=-0.45; P=0.027). Receiver operating characteristics analysis identified pericyte density thresholds (≤13.2 and 13.4 pericytes per 100 µm) that showed high area under the curves for detecting macular leakage (area under the curve, 0.87) and volumetric thickness (area under the curve, 0.80). Interestingly, albumin/creatinine ratios were significantly higher in individuals with ≤13.2 retinal capillary pericyte per 100 µm compared with those above the threshold (n=14, P=0.046).

Conclusions: Retinal capillary pericyte density, quantified in vivo, correlates significantly with macular thickening and angiographic leakage. We identified a threshold of pericyte loss that distinguishes individuals based on macular leakage status and albuminuria, providing important insights into retinal pericytes and their potential utility as a biomarker of vascular permeability.

Background: Venous malformations (VMs) are developmental defects of the vasculature characterized by tremendously enlarged and dysfunctional veins. Gain-of-function somatic mutations in TIE2 (endothelial tyrosine kinase receptor) have been identified as the leading driver of VM pathogenesis. The aim of this study was to determine whether the aberrant venous lumen expansion is caused by recruitment of wild-type (WT) endothelial cells (EC) to the lesion or by TIE2-mutant EC clonal expansion.

Methods: To investigate the contribution of TIE2-mutant EC and WT EC to the aberrant venous lumen expansion, we used a xenograft murine model of VM generated with a combination of TIE2-mutant EC and WT EC. To perform longitudinal studies, we used a 3-dimensional fibrin gel lumen formation assay and a migration assay, both using WT EC in competition or confrontation with TIE2-mutant EC. To investigate the mechanisms implicated in VM lumen expansion, we used RNA-sequencing and short-hairpin RNA silencing in the TIE2-mutant EC.

Results: We demonstrate here that in the VM xenograft model, the aberrant blood vessels were lined almost exclusively by TIE2-mutant EC, and WT EC were rarely found. Functionally, the TIE2-mutant EC exerted a competitive advantage over WT EC by inhibiting WT EC sprouting. In line with these findings, TIE2-mutant EC promoted repulsion of WT EC. Short-hairpin RNA-mediated silencing of Sema (Semaphorin) 3A or Sema3F in TIE2-mutant EC rescued this chemorepellent phenotype and restored the ability of WT EC to migrate, sprout, and form lumens. Furthermore, knockdown of Sema3A or Sema3F in TIE2-mutant EC normalized the blood vessel size in vivo.

Conclusions: Our results demonstrate that WT EC are not recruited to the aberrant veins, suggesting that VM pathogenesis is fueled by clonal expansion of TIE2-mutant EC. Mechanistically, we show that Sema3A and Sema3F are overexpressed in TIE2-mutant EC and play a crucial role in the pathological vascular lumen expansion in VM.

Background: Vascular calcification is an active process driven by osteogenic phenotypic transition of vascular smooth muscle cells (VSMCs) and regulated by a bone-related gene regulatory network. Recent studies showed that FAK (focal adhesion kinase) regulates bone formation by affecting the cellular localization of HDAC (histone deacetylase) 4 and HDAC5. However, it is not known whether FAK exerts effects on vascular calcification in VSMCs through regulating HDACs.

Methods: We used perturbational assays to assess the role of HDAC4, HDAC5, and FAK in VSMC calcification. Pharmacological inhibition and gene silencing of FAK were used to evaluate effects on calcification, cell migration, and the expression of procalcification factors. Leptomycin was used to inhibit the nuclear export of HDACs. In addition, ex vivo cultures of mouse and human arteries were treated with a FAK inhibitor to assess effects on arterial calcification. Single-cell transcriptomic expression of FAK was examined in healthy and diseased human coronary arteries.

Results: HDAC4 and HDAC5 were identified as positive regulators of vascular calcification. Pharmacological inhibition or gene silencing of FAK blocked VSMC calcification, abrogated the osteogenic medium-induced elevation of procalcification factors, and reduced cell migration. FAK inhibition reduced HDAC4 and HDAC5 phosphorylation and enhanced nuclear localization of these HDAC proteins. Inhibition of HDAC4 and HDAC5 nuclear export with leptomycin showed similar effects on calcification as FAK inhibition. Treatment with the FAK inhibitor attenuated the calcification of ex vivo mouse and human arteries. FAK gene expression was dysregulated in human diseased coronary arteries compared with healthy coronary arteries, and, in single-cell analysis of human arterial tissue, FAK expression was highest in VSMCs at an intermediate state between contractile and osteogenic phenotypes.

Conclusions: FAK promotes VSMC calcification, at least in part, via phosphorylation of HDAC4 and HDAC5. Targeted regulation of the activity of FAK, HDAC4, and HDAC5 may be an effective strategy for the treatment of vascular calcification.

The lung lymphatics are formed from lymphatic endothelial cells and play a pivotal role in lung fluid homeostasis and immune trafficking. Though blood vascular function in the lung has long been an area of active investigation, many aspects of lung lymphatic vascular function have only recently been uncovered. In this review, we will discuss our current knowledge of lung lymphatic function and how these vessels differ from lung blood vasculature in their architecture, function, and response to injury in 3 domains: edema, inflammation, and thrombosis. We will review the rich historical anatomic literature that described the lung lymphatics in great detail and elucidated foundational discoveries that continue to shape our current understanding of the lung lymphatics, even in the molecular era. We conclude by highlighting key questions for the field of lung lymphatic biology.

Background: Ischemic diseases have become a major threat to global health, with endothelial cell (EC) damage closely associated with their pathogenesis and progression. Cell therapies targeting endothelial repair have thus become a treatment approach of great interest, yet the procurement of clinically approved ECs for these applications has not been fully established. Modulating the expression of Atf3 (activating transcription factor 3) represents a potential strategy for deriving ECs from stem cells; however, its precise function in the development and differentiation of ECs from stem cells remains elusive. In the present study, we sought to elucidate the potential role of Atf3 in the differentiation of embryonic stem cells into ECs.

Methods: CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) system was used to knockout Atf3 (Atf3KO [Atf3 knockout]) in mouse embryonic stem cells. EC differentiation was initially induced using the hanging drop method to promote embryoid bodies formation, followed by embryoid bodies attachment onto culture slides. The expression changes of EC markers during differentiation were assessed by RNA sequencing, Western blotting, immunofluorescence staining, flow cytometry, and reverse transcription quantitative polymerase chain reaction. Functional comparisons of differentiated ECs were performed by assessing LDL (low-density lipoprotein) uptake and NO production. Potential molecular mechanisms were further explored via bioinformatic analysis of RNA sequencing data.

Results: Atf3KO led to a significant upregulation in the expression levels of progenitor and mesoderm cell markers on days 3 and 6 of differentiation. By day 9, the expression of mature EC markers also exhibited a notable increase. Moreover, Atf3KO enhanced the functional properties of differentiated Atf3KO ECs. In addition, our findings revealed that the activation of the Rap1 (Ras-related protein 1) signaling pathway, triggered by Atf3KO, contributed to ECs development and maturation.

Conclusions: Atf3KO directs embryonic stem cells toward the mesodermal lineage and activates the Rap1 signaling pathway, thereby promoting ECs development. These findings highlight a key role of Atf3 in regulating early stage of vascular endothelial development.