Objective—: Dyslipidemia is one of the key factors behind coronary heart disease. Blood and lymphatic vessels play pivotal roles in both lipoprotein metabolism and development of atherosclerotic plaques. Recent studies have linked members of VEGF (vascular endothelial growth factor) family to lipid metabolism, but the function of VEGF-D has remained unexplored. Here, we investigated how the deletion of VEGF-D affects lipid and lipoprotein metabolism in atherogenic LDLR ^−/− ApoB ^100/100 mice. Approach and Results—: Deletion of VEGF-D (VEGF-D ^−/− LDLR ^−/− ApoB ^100/100 ) led to markedly elevated plasma cholesterol and triglyceride levels without an increase in atherogenesis. Size distribution and hepatic lipid uptake studies confirmed a delayed clearance of large chylomicron remnant particles that cannot easily penetrate through the vascular endothelium. Mechanistically, the inhibition of VEGF-D signaling significantly decreased the hepatic expression of SDC1 (syndecan 1), which is one of the main receptors for chylomicron remnant uptake when LDLR is absent. Immunohistochemical staining confirmed reduced expression of SDC1 in the sinusoidal surface of hepatocytes in VEGF-D deficient mice. Furthermore, hepatic RNA-sequencing revealed that VEGF-D is also an important regulator of genes related to lipid metabolism and inflammation. The lack of VEGF-D signaling via VEGFR3 (VEGF receptor 3) led to lowered expression of genes regulating triglyceride and cholesterol production, as well as downregulation of peroxisomal β-oxidation pathway. Conclusions—: These results demonstrate that VEGF-D, a powerful lymphangiogenic and angiogenic growth factor, is also a major regulator of chylomicron metabolism in mice.

The 9p21.3 genomic locus is a hot spot for disease-associated single-nucleotide polymorphisms (SNPs), and its strongest associations are with coronary artery disease (CAD). The disease-associated SNPs are located within the sequence of a long noncoding RNA ANRIL, which potentially contributes to atherogenesis by regulating vascular cell stress and proliferation, but also affects pancreatic β-cell proliferation. Altered expression of a neighboring gene, CDKN2B, has been also recognized to correlate with obesity and hepatic steatosis in people carrying the risk SNPs. In the present study, we investigated the impact of 9p21.3 on obesity accompanied by hyperlipidemia in mice carrying a deletion of the murine ortholog for the 9p21.3 (Chr4^Δ70/Δ70) risk locus in hyperlipidemic Ldlr^−/−ApoB^100/100 background. The Chr4^Δ70/Δ70 mice showed decreased mRNA expression of insulin receptors in white adipose tissue already at a young age, which developed into insulin resistance and obesity by aging. In addition, the Sirt1-Ppargc1a-Ucp2 pathway was downregulated together with the expression of Cdkn2b, specifically in the white adipose tissue in Chr4^Δ70/Δ70 mice. These results suggest that the 9p21.3 locus, ANRIL lncRNA, and their murine orthologues may regulate the key energy metabolism pathways in a white adipose tissue-specific manner in the presence of hypercholesterolemia, thus contributing to the pathogenesis of metabolic syndrome.

Genetic variation in the 9p21.3 chromosomal region has one of the strongest associations known for coronary artery disease (CAD) that often leads to myocardial infarction (MI). This risk locus encodes a long noncoding RNA, ANRIL, which has been suggested to regulate the neighboring cyclin‐dependent kinase inhibitors 2A and B (Cdkn2A/B), the key regulators of cell proliferation. In this study, we aimed to clarify the role of the 9p21.3 risk locus in acute and chronic myocardial ischemia in mice. Mice carrying a deletion equivalent to the human CAD risk interval (Chr4^{Δ70kb/Δ70kb}) and wild type mice were exposed to MI and followed until 5 days or 4 weeks. In the wild type mice, expression of a lncRNA, Ak148321, was increased after MI, and Cdkn2a was upregulated in chronic ischemia. Chr4^{Δ70kb/Δ70kb} downregulated both Cdkn2a/b, but this did not affect the survival or cardiac pathology after MI. These results suggest that the 9p21.3 locus is activated in response to cardiac ischemia. However, deficiency in the risk locus does not play a role in the cardiac pathophysiology in mice, supporting the studies suggesting the risk locus being more involved in the development of CAD, rather than the subsequent MI.

To develop a T _1ρ ‐weighted quantitative susceptibility mapping (QSM) technique for simultaneous imaging of T _1ρ and T ₂ * relaxation and magnetic susceptibility in ex vivo mouse hearts after myocardial infarction (MI). MI was induced in C75Bl/6JOlaHsd mice, and their hearts ( n _MI = 7, n _CTRL = 6) were imaged ex vivo by combining adiabatic T _1ρ preparation with a 3‐D multi‐echo gradient recalled echo (GRE) sequence for simultaneous T _1ρ ‐weighted QSM. As a reference, 2‐D adiabatic T _1ρ mapping and QSM without preparation were used. T ₂ * and susceptibility maps were calculated for both T _1ρ –QSM and QSM measurements. Additionally, T _1ρ maps were calculated from T _1ρ –T ₂ *–QSM. Region of interest analysis was performed using American Heart Association (AHA) segments. Correlation and Bland–Altman bias between 2‐D/3‐D T _1ρ and T _1ρ –T ₂ *–QSM/QSM segments were assessed, respectively. Statistical significance between the segments was tested using Mann–Whitney U test. Histological validation was conducted using Picro Sirius red (SR), hematoxylin and eosin (HE), and Perls' Prussian Blue (PPB) stainings. T _1ρ relaxation times were elevated significantly in basal AHA segments 1, 2, and 6, compared to the corresponding control segments. T ₂ * relaxation times decreased significantly in basal and apical segments 4, 6, 13, and 14. QSM revealed visible heterogeneity within the same segments, and greater variance in basal, mid, and apical segments 6, 7, 11, 12, and 13 compared to controls. Histology confirmed the formation of myocardial fibrosis and hemosiderin‐laden macrophages within the corresponding MRI segments with minimal residual blood in MI hearts. Quantitative analysis showed a moderate agreement between 2‐D and 3‐D T _1ρ mapping (Spearman's r = 0.71). Bland–Altman analysis between T _1ρ –T ₂ *–QSM and reference QSM showed minimal bias (−7.6 · 10 ⁻⁴ ) and Pearson's correlation coefficient r _p = 0.69. For T ₂ * (T _1ρ –T ₂ *–QSM) and QSM, the Bland–Altman analysis bias was −0.84 and r _p = 0.77. The developed T _1ρ ‐weighted QSM technique enabled simultaneous acquisition of T _1ρ , T ₂ *, and susceptibility maps in ex vivo mouse hearts. All imaging contrasts showed good correlation with reference methods, and MI regions were visible using either T _1ρ, T ₂ *, or susceptibility mapping. The identified MI areas were in line with histological findings, supporting the accuracy of the proposed imaging approach.