Endothelial ADAM10 controls cellular response to oxLDL and its deficiency exacerbates atherosclerosis with intraplaque hemorrhage and neovascularization in mice.

Introduction: The transmembrane protease A Disintegrin And Metalloproteinase 10 (ADAM10) displays a "pattern regulatory function," by cleaving a range of membrane-bound proteins. In endothelium, it regulates barrier function, leukocyte recruitment and angiogenesis. Previously, we showed that ADAM10 is expressed in human atherosclerotic plaques and associated with neovascularization. In this study, we aimed to determine the causal relevance of endothelial ADAM10 in murine atherosclerosis development in vivo. Methods and results: Endothelial Adam10 deficiency (Adam10 ecko ) in Western-type diet (WTD) fed mice rendered atherogenic by adeno-associated virus-mediated PCSK9 overexpression showed markedly increased atherosclerotic lesion formation. Additionally, Adam10 deficiency was associated with an increased necrotic core and concomitant reduction in plaque macrophage content. Strikingly, while intraplaque hemorrhage and neovascularization are rarely observed in aortic roots of atherosclerotic mice after 12 weeks of WTD feeding, a majority of plaques in both brachiocephalic artery and aortic root of Adam10ecko mice contained these features, suggestive of major plaque destabilization. In vitro, ADAM10 knockdown in human coronary artery endothelial cells (HCAECs) blunted the shedding of lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1) and increased endothelial inflammatory responses to oxLDL as witnessed by upregulated ICAM-1, VCAM-1, CCL5, and CXCL1 expression (which was diminished when LOX-1 was silenced) as well as activation of pro-inflammatory signaling pathways. LOX-1 shedding appeared also reduced in vivo, as soluble LOX-1 levels in plasma of Adam10ecko mice was significantly reduced compared to wildtypes. Discussion: Collectively, these results demonstrate that endothelial ADAM10 is atheroprotective, most likely by limiting oxLDL-induced inflammation besides its known role in pathological neovascularization. Our findings create novel opportunities to develop therapeutics targeting atherosclerotic plaque progression and stability, but at the same time warrant caution when considering to use ADAM10 inhibitors for therapy in other diseases.

Aging-regulated TUG1 is dispensable for endothelial cell function.

The evolutionary conserved Taurine Upregulated Gene 1 (TUG1) is a ubiquitously expressed gene that is one of the highest expressed genes in human and rodent endothelial cells (ECs). We here show that TUG1 expression decreases significantly in aging mouse carotid artery ECs and human ECs in vitro, indicating a potential role in the aging endothelial vasculature system. We therefore investigated if, and how, TUG1 might function in aging ECs, but despite extensive phenotyping found no alterations in basal EC proliferation, apoptosis, barrier function, migration, mitochondrial function, or monocyte adhesion upon TUG1 silencing in vitro. TUG1 knockdown did slightly and significantly decrease cumulative sprout length upon vascular endothelial growth factor A stimulation in human umbilical vein endothelial cells (HUVECs), though TUG1-silenced HUVECs displayed no transcriptome-wide mRNA expression changes explaining this effect. Further, ectopic expression of the highly conserved and recently discovered 153 amino acid protein translated from certain TUG1 transcript isoforms did not alter angiogenic sprouting in vitro. Our data show that, despite a high expression and strong evolutionary conservation of both the TUG1 locus and the protein sequence it encodes, TUG1 does not seem to play a major role in basic endothelial cell function.

Aging-regulated anti-apoptotic long non-coding RNA Sarrah augments recovery from acute myocardial infarction.

Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.

Atherogenic LOX-1 signaling is controlled by SPPL2-mediated intramembrane proteolysis.

The lectin-like oxidized LDL receptor 1 (LOX-1) is a key player in the development of atherosclerosis. LOX-1 promotes endothelial activation and dysfunction by mediating uptake of oxidized LDL and inducing pro-atherogenic signaling. However, little is known about modulators of LOX-1-mediated responses. Here, we show that the function of LOX-1 is controlled proteolytically. Ectodomain shedding by the metalloprotease ADAM10 and lysosomal degradation generate membrane-bound N-terminal fragments (NTFs), which we identified as novel substrates of the intramembrane proteases signal peptide peptidase-like 2a and b (SPPL2a/b). SPPL2a/b control cellular LOX-1 NTF levels which, following self-association via their transmembrane domain, can activate MAP kinases in a ligand-independent manner. This leads to an up-regulation of several pro-atherogenic and pro-fibrotic targets including ICAM-1 and the connective tissue growth factor CTGF. Consequently, SPPL2a/b-deficient mice, which accumulate LOX-1 NTFs, develop larger and more advanced atherosclerotic plaques than controls. This identifies intramembrane proteolysis by SPPL2a/b as a novel atheroprotective mechanism via negative regulation of LOX-1 signaling.

Long non-coding RNA H19 regulates endothelial cell aging via inhibition of STAT3 signalling.

Aims: Long non-coding RNAs (lncRNAs) have been shown to regulate numerous processes in the human genome, but the function of these transcripts in vascular aging is largely unknown. We aim to characterize the expression of lncRNAs in endothelial aging and analyse the function of the highly conserved lncRNA H19. Methods and results: H19 was downregulated in endothelium of aged mice. In human, atherosclerotic plaques H19 was mainly expressed by endothelial cells and H19 was significantly reduced in comparison to healthy carotid artery biopsies. Loss of H19 led to an upregulation of p16 and p21, reduced proliferation and increased senescence in vitro. Depletion of H19 in aortic rings of young mice inhibited sprouting capacity. We generated endothelial-specific inducible H19 deficient mice (H19iEC-KO), resulting in increased systolic blood pressure compared with control littermates (Ctrl). These H19iEC-KO and Ctrl mice were subjected to hindlimb ischaemia, which showed reduced capillary density in H19iEC-KO mice. Mechanistically, exon array analysis revealed an involvement of H19 in IL-6 signalling. Accordingly, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 were upregulated upon H19 depletion. A luciferase reporter screen for differential transcription factor activity revealed STAT3 as being induced upon H19 depletion and repressed after H19 overexpression. Furthermore, depletion of H19 increased the phosphorylation of STAT3 at TYR705 and pharmacological inhibition of STAT3 activation abolished the effects of H19 silencing on p21 and vascular cell adhesion molecule 1 expression as well as proliferation. Conclusion: These data reveal a pivotal role for the lncRNA H19 in controlling endothelial cell aging.

Endothelial Cell Metabolism in Atherosclerosis.

Atherosclerosis and its sequelae, such as myocardial infarction and stroke, are the leading cause of death worldwide. Vascular endothelial cells (EC) play a critical role in vascular homeostasis and disease. Atherosclerosis as well as its independent risk factors including diabetes, obesity, and aging, are hallmarked by endothelial activation and dysfunction. Metabolic pathways have emerged as key regulators of many EC functions, including angiogenesis, inflammation, and barrier function, processes which are deregulated during atherogenesis. In this review, we highlight the role of glucose, fatty acid, and amino acid metabolism in EC functions during physiological and pathological states, specifically atherosclerosis, diabetes, obesity and aging.

Whole body and hematopoietic ADAM8 deficiency does not influence advanced atherosclerotic lesion development, despite its association with human plaque progression.

Although A Disintegrin And Metalloproteinase 8 (ADAM8) is not crucial for tissue development and homeostasis, it has been implicated in various inflammatory diseases by regulating processes like immune cell recruitment and activation. ADAM8 expression has been associated with human atherosclerosis development and myocardial infarction, however a causal role of ADAM8 in atherosclerosis has not been investigated thus far. In this study, we examined the expression of ADAM8 in early and progressed human atherosclerotic lesions, in which ADAM8 was significantly upregulated in vulnerable lesions. In addition, ADAM8 expression was most prominent in the shoulder region of human atherosclerotic lesions, characterized by the abundance of foam cells. In mice, Adam8 was highly expressed in circulating neutrophils and in macrophages. Moreover, ADAM8 deficient mouse macrophages displayed reduced secretion of inflammatory mediators. Remarkably, however, neither hematopoietic nor whole-body ADAM8 deficiency in mice affected atherosclerotic lesion size. Additionally, except for an increase in granulocyte content in plaques of ADAM8 deficient mice, lesion morphology was unaffected. Taken together, whole body and hematopoietic ADAM8 does not contribute to advanced atherosclerotic plaque development, at least in female mice, although its expression might still be valuable as a diagnostic/prognostic biomarker to distinguish between stable and unstable lesions.

Disease- or Storage-Associated Structural Modifications Are Unlikely to Explain HDL Pro-inflammatory Effects on Macrophages.

Van der Vorst et al. underscore the relevance of HDL quality control, considering HDL source and processing, but argue that disease- or storage-associated structural modifications of HDL cannot explain the observed pro-inflammatory effects on macrophages. Discrepancies between reported effects of HDL in macrophages are probably related to methodological differences.

Adventitial lymphatic capillary expansion impacts on plaque T cell accumulation in atherosclerosis.

During plaque progression, inflammatory cells progressively accumulate in the adventitia, paralleled by an increased presence of leaky vasa vasorum. We here show that next to vasa vasorum, also the adventitial lymphatic capillary bed is expanding during plaque development in humans and mouse models of atherosclerosis. Furthermore, we investigated the role of lymphatics in atherosclerosis progression. Dissection of plaque draining lymph node and lymphatic vessel in atherosclerotic ApoE-/- mice aggravated plaque formation, which was accompanied by increased intimal and adventitial CD3+ T cell numbers. Likewise, inhibition of VEGF-C/D dependent lymphangiogenesis by AAV aided gene transfer of hVEGFR3-Ig fusion protein resulted in CD3+ T cell enrichment in plaque intima and adventitia. hVEGFR3-Ig gene transfer did not compromise adventitial lymphatic density, pointing to VEGF-C/D independent lymphangiogenesis. We were able to identify the CXCL12/CXCR4 axis, which has previously been shown to indirectly activate VEGFR3, as a likely pathway, in that its focal silencing attenuated lymphangiogenesis and augmented T cell presence. Taken together, our study not only shows profound, partly CXCL12/CXCR4 mediated, expansion of lymph capillaries in the adventitia of atherosclerotic plaque in humans and mice, but also is the first to attribute an important role of lymphatics in plaque T cell accumulation and development.

High-Density Lipoproteins Exert Pro-inflammatory Effects on Macrophages via Passive Cholesterol Depletion and PKC-NF-κB/STAT1-IRF1 Signaling.

Membrane cholesterol modulates a variety of cell signaling pathways and functions. While cholesterol depletion by high-density lipoproteins (HDLs) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain elusive. Here we show overt pro-inflammatory effects of HDL-mediated passive cholesterol depletion and lipid raft disruption in murine and human primary macrophages in vitro. These pro-inflammatory effects were confirmed in vivo in peritoneal macrophages from apoA-I transgenic mice, which have elevated HDL levels. In line with these findings, the innate immune responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Expression analysis, ChIP-PCR, and combinatorial pharmacological and genetic intervention studies unveiled that both native and reconstituted HDL enhance Toll-like-receptor-induced signaling by activating a PKC-NF-κB/STAT1-IRF1 axis, leading to increased inflammatory cytokine expression. HDL's pro-inflammatory activity supports proper functioning of macrophage immune responses.

Myeloid A disintegrin and metalloproteinase domain 10 deficiency modulates atherosclerotic plaque composition by shifting the balance from inflammation toward fibrosis.

A disintegrin and metalloproteinase domain 10 (ADAM10) is a metalloprotease involved in cleavage of various cell surface molecules, such as adhesion molecules, chemokines, and growth factor receptors. Although we have previously shown an association of ADAM10 expression with atherosclerotic plaque progression, a causal role of ADAM10 in atherosclerosis has not been investigated. Bone marrow from conditional knockout mice lacking Adam10 in the myeloid lineage or from littermate controls was transplanted into lethally irradiated low density lipoprotein receptor Ldlr(-/-) mice on an atherogenic diet. Myeloid Adam10 deficiency did not affect plaque size, but it increased plaque collagen content. Matrix metalloproteinase 9 and 13 expression and matrix metalloproteinase 2 gelatinase activity were significantly impaired in Adam10-deficient macrophages, whereas their capacity to stimulate collagen production was unchanged. Furthermore, relative macrophage content in advanced atherosclerotic lesions was decreased. In vitro, Adam10-deficient macrophages showed reduced migration toward monocyte chemoattractant protein-1 and transmigration through collagen. In addition, Adam10-deficient macrophages displayed increased anti-inflammatory phenotype with elevated IL-10, and reduced production of proinflammatory tumor necrosis factor, IL-12, and nitric oxide in response to lipopolysaccharide. These data suggest a critical role of Adam10 for leukocyte recruitment, inflammatory mediator production, and extracellular matrix degradation. Thereby, myeloid ADAM10 may play a causal role in modulating atherosclerotic plaque stability.