C/D box small nucleolar RNAs (snoRNAs) of the DLK1-DIO3 locus are associated with vascular remodeling and cardiovascu-lar disease. None of these snoRNAs has any known targets yet except for one,... Show moreC/D box small nucleolar RNAs (snoRNAs) of the DLK1-DIO3 locus are associated with vascular remodeling and cardiovascu-lar disease. None of these snoRNAs has any known targets yet except for one, AF357425/SNORD113-6. We previously showed that this snoRNA targets mRNAs of the integrin signaling pathway and affects arterial fibroblast function. Here, we aimed to identify whether AF357425/SNORD113-6 can also target small RNAs. We overexpressed or inhibited AF357425 in murine fibroblasts and performed small RNA sequencing. Expression of transfer (t)RNA fragments (tRFs) was predominantly regulated. Compared with overexpression, AF357425 knockdown led to an overall decrease in tRFs but with an enrichment in smaller tRFs (<30 nucleotides). We focused on tRNA leucine anti-codon TAA (tRNALeu(TAA)), which has a conserved predicted binding site for AF357425/ SNORD113-6. Adjacent to this site, the tRNA is cleaved to form tRFLeu 47-64 in both primary murine and human fibro-blasts and in intact human arteries. We show that AF357425/ SNORD113-6 methylates tRNALeu(TAA) and thereby prevents the formation of tRFLeu 47-64. Exposing fibroblasts to oxidative or hypoxic stress increased AF357425/SNORD113-6 and tRNALeu(TAA) expression, but AF357425/SNORD113-6 knockdown did not increase tRFLeu 47-64 formation under stress even further. Thus, independent of cellular stress, AF357425/SNORD113-6 protects against site-specific frag-mentation of tRNALeu(TAA) via 2'O-ribose-methylation. Show less
Ewing, M.; Karper, J.C.; Vries, M.R. de; Quax, P.H.A. 2022
Vein grafts (VGs) are used to bypass atherosclerotic obstructions and arteriovenous fistulas (AVFs) as vascular access for hemodialysis. Vascular remodeling governs post-interventional... Show moreVein grafts (VGs) are used to bypass atherosclerotic obstructions and arteriovenous fistulas (AVFs) as vascular access for hemodialysis. Vascular remodeling governs post-interventional arterialization, but may also induce VG and AVF failure. Although the endpoint characteristics of vascular remodeling are known, the in vivo process and the role of blood flow dynamics has not been fully studied. Therefore, here we non-invasively quantify vascular remodeling and blood flow alterations over time in murine VG and AVF models. C57BL/6J (n = 7, chow diet) and atherosclerosis-prone ApoE3*Leiden (n = 7) mice underwent VG surgery. Ultrasound imaging was performed at 3, 7, 14, 21, and 28 days post-surgery. C57BL/6J mice (n = 8) received AVF surgery. Ultrasound imaging was performed at 7 and 14 days post-surgery. The luminal volume increased by 42% in the VGs of C57BL/6J and 38% in the VGs of ApoE3*Leiden mice at 28 days relative to 3 days post-surgery. Longitudinally, an 82% increase in wall volume and 76% increase in outward remodeling was found in the ApoE3*Leiden mice, with a constant wall size in C57BL/6J mice. Proximally, the pulsatility index, resistive index, and peak systolic velocity decreased longitudinally in both groups. Distally, the maximum acceleration increased with 56% in C57BL/6J VGs. Among the AVFs, 50% showed maturation after 7 days, based on a novel flow-criterium of 23 mL/min. Distinct flow patterns were observed at the anastomotic site and inflow artery of the AVFs relative to the control carotid arteries. Vascular remodeling can be quantified by ultra-high-frequency ultrasound imaging over time in complex animal models, via three-dimensional structural parameters and site-specific hemodynamic indices. Show less
Phosphorylcholine (PC) is one of the main oxLDL epitopes playing a central role in atherosclerosis, due to its atherogenic and proinflammatory effects. PC can be cleared by natural IgM antibodies... Show morePhosphorylcholine (PC) is one of the main oxLDL epitopes playing a central role in atherosclerosis, due to its atherogenic and proinflammatory effects. PC can be cleared by natural IgM antibodies and low levels of these antibodies have been associated with human vein graft (VG) failure. Although PC antibodies are recognized for their anti-inflammatory properties, their effect on intraplaque angiogenesis (IPA) and intraplaque hemorrhage (IPH)-interdependent processes contributing to plaque rupture-are unknown. We hypothesized that new IgG phosphorylcholine antibodies (PC-mAb) could decrease vulnerable lesions in murine VGs.Therefore, hypercholesterolemic male ApoE3*Leiden mice received a (donor) caval vein interposition in the carotid artery and weekly IP injections of (5 mg/kg) PCmAb (n = 11) or vehicle (n = 12) until sacrifice at day 28. We found that PCmAb significantly decreased vein graft media (13%), intima lesion (25%), and increased lumen with 32% compared to controls. PCmAb increased collagen content (18%) and decreased macrophages presence (31%). PCmAb resulted in 23% decreased CD163+ macrophages content in vein grafts whereas CD163 expression was decreased in Hb:Hp macrophages. PCmAb significantly lowered neovessel density (34%), EC proliferation and migration with/out oxLDL stimulation. Moreover, PCmAb enhanced intraplaque angiogenic vessels maturation by increasing neovessel pericyte coverage in vivo (31%). Together, this resulted in a 62% decrease in IPH. PCmAb effectively inhibits murine atherosclerotic lesion formation in vein grafts by reducing IPA and IPH via decreased neovessel density and macrophages influx and increased neovessel maturation. PC-mAb therefore holds promise as a new therapeutic approach to prevent vein graft disease. Show less
Purpose of Review: Small non-coding RNAs regulate gene expression and are highly implicated in heart failure. Recently, an additional level of post-transcriptional regulation has been identified,... Show morePurpose of Review: Small non-coding RNAs regulate gene expression and are highly implicated in heart failure. Recently, an additional level of post-transcriptional regulation has been identified, referred to as the epitranscriptome, which encompasses the body of post-transcriptional modifications that are placed on RNA molecules. In this review, we summarize the current knowledge on the small non-coding RNA epitranscriptome in heart failure. Recent Findings: With the rise of new methods to study RNA modifications, epitranscriptome research has begun to take flight. Over the past 3 years, the number of publications on the epitranscriptome in heart failure has significantly increased, and we expect many more highly relevant publications to come out over the next few years. Currently, at least six modifications on small non-coding RNAs have been investigated in heart failure-relevant studies, namely N6-adenosine, N5-cytosine and N7-guanosine methylation, 2'-O-ribose-methylation, adenosine-to-inosine editing, and isomiRs. Their potential role in heart failure is discussed. Show less
Cytotoxic CD4+ T cells have previously been found in peripheral blood of patients with coronary artery disease (1), however their occurrence in atherosclerotic plaques and their association with... Show moreCytotoxic CD4+ T cells have previously been found in peripheral blood of patients with coronary artery disease (1), however their occurrence in atherosclerotic plaques and their association with the pathophysiology of atherosclerosis has not been established. Single-cell RNA sequencing was performed on human carotid atherosclerotic plaques of 18 patients to identify specific T cell populations (2). Next, human femoral and carotid atherosclerotic plaques (n=95) and matched blood samples (n=49) were analyzed by flow cytometry for the presence of CD4+GZMB+ T cells. Plaque morphology was assessed by Movat’s Pentachrome staining. A distinct cytotoxic GZMB+ PRF1+ CD28- CD4+ T cell cluster was identified using single-cell RNA sequencing. Furthermore, flow cytometry analysis showed that the percentage of GZMB+CD4+ T cells was significantly elevated in plaque compared to blood (Blood: 12.17±2.0 vs. Plaque: 17.40±1.0; P=0.0002). Moreover, a significant positive correlation was observed between the percentage of GZMB+CD4+ T cells in blood versus plaque (P=0.031). In line with the proinflammatory character of these cells, we found a positive association of GZMB+CD4+ T cells (P=0.036) with necrotic core size, whereas no correlation was found with this subtype in the circulation. In this study we have shown an enrichment of cytotoxic CD4+ T cells in atherosclerotic lesions, which positively correlate with necrotic core size. Future studies are aimed at elucidating the role of these cells in advanced atherosclerosis. Show less
Fernandes, H.; Zonnari, A.; Abreu, R.; Aday, S.; Barao, M.; Albino, I.; ... ; Ferreira, L. 2022
Endothelial cell (EC) activity is essential for tissue regeneration in several (patho)physiological contexts. However, our capacity to deliver in vivo biomolecules capable of controlling EC fate is... Show moreEndothelial cell (EC) activity is essential for tissue regeneration in several (patho)physiological contexts. However, our capacity to deliver in vivo biomolecules capable of controlling EC fate is relatively limited. Here, we screened a library of microRNA (miR) mimics and identified 25 miRs capable of enhancing the survival of ECs exposed to ischemia-mimicking conditions. In vitro, we showed that miR-425-5p, one of the hits, was able to enhance EC survival and migration. In vivo, using a mouse Matrigel plug assay, we showed that ECs transfected with miR-425-5p displayed enhanced survival compared with scramble-transfected ECs. Mechanistically, we showed that miR-425-5p modulated the PTEN/PI3K/AKT pathway and inhibition of miR-425-5p target genes (DACH1, PTEN, RGS5, and VASH1) phenocopied the pro-survival. For the in vivo delivery of miR-425-5p, we modulated small extracellular vesicles (sEVs) with miR-425-5p and showed, in vitro, that miR-425-5pmodulated sEVs were (1) capable of enhancing the survival of ECs exposed to ischemia-mimic conditions, and (2) efficiently internalized by skin cells. Finally, using a streptozotocininduced diabetic wound healing mouse model, we showed that, compared with miR-scrambled-modulated sEVs, topical administration of miR-425-5p-modulated sEVs significantly enhanced wound healing, a process mediated by enhanced vascularization and skin re-epithelialization. Show less
The choroid is a key player in maintaining ocular homeostasis and plays a role in a variety of chorioretinal diseases, many of which are poorly understood. Recent advances in the field of single... Show moreThe choroid is a key player in maintaining ocular homeostasis and plays a role in a variety of chorioretinal diseases, many of which are poorly understood. Recent advances in the field of single-cell RNA sequencing have yielded valuable insights into the properties of choroidal endothelial cells (CECs). Here, we review the role of the choroid in various physiological and pathophysiological mechanisms, focusing on the role of CECs. We also discuss new insights regarding the phenotypic properties of CECs, CEC subpopulations, and the value of measuring transcriptomics in primary CEC cultures derived from post-mortem eyes. In addition, we discuss key phenotypic, structural, and functional differences that distinguish CECs from other endothelial cells such as retinal vascular endothelial cells. Understanding the specific clinical and molecular properties of the choroid will shed new light on the pathogenesis of the broad clinical range of chorioretinal diseases such as age-related macular degeneration, central serous chorioretinopathy and other diseases within the pachychoroid spectrum, uveitis, and diabetic choroidopathy. Although our knowledge is still relatively limited with respect to the clinical features and molecular pathways that underlie these chorioretinal diseases, we summarise new approaches and discuss future directions for gaining new insights into these sight-threatening diseases and highlight new therapeutic strategies such as pluripotent stem cell-based technologies and gene therapy. Show less
Bezhaeva, T.; Karper, J.; Quax, P.H.A.; Vries, M.R. de 2022
Activation of Toll like receptors (TLR) plays an important role in cardiovascular disease development, progression and outcomes. Complex TLR mediated signaling affects vascular and cardiac function... Show moreActivation of Toll like receptors (TLR) plays an important role in cardiovascular disease development, progression and outcomes. Complex TLR mediated signaling affects vascular and cardiac function including tissue remodeling and repair. Being central components of both innate and adaptive arms of the immune system, TLRs interact as pattern recognition receptors with a series of exogenous ligands and endogenous molecules or so-called danger associated molecular patterns (DAMPs) that are released upon tissue injury and cellular stress. Besides immune cells, a number of structural cells within the cardiovascular system, including endothelial cells, smooth muscle cells, fibroblasts and cardiac myocytes express TLRs and are able to release or sense DAMPs. Local activation of TLR-mediated signaling cascade induces cardiovascular tissue repair but in a presence of constant stimuli can overshoot and cause chronic inflammation and tissue damage. TLR accessory molecules are essential in guiding and dampening these responses toward an adequate reaction. Furthermore, accessory molecules assure specific and exclusive TLR-mediated signal transduction for distinct cells and pathways involved in the pathogenesis of cardiovascular diseases. Although much has been learned about TLRs activation in cardiovascular remodeling, the exact role of TLR accessory molecules is not entirely understood. Deeper understanding of the role of TLR accessory molecules in cardiovascular system may open therapeutic avenues aiming at manipulation of inflammatory response in cardiovascular disease. The present review outlines accessory molecules for membrane TLRs that are involved in cardiovascular disease progression. We first summarize the up-to-date knowledge on TLR signaling focusing on membrane TLRs and their ligands that play a key role in cardiovascular system. We then survey the current evidence of the contribution of TLRs accessory molecules in vascular and cardiac remodeling including myocardial infarction, heart failure, stroke, atherosclerosis, vein graft disease and arterio-venous fistula failure. Show less
Chronic thromboembolic pulmonary hypertension (CTEPH) and pulmonary arterial hypertension (PAH) are two forms of pulmonary hypertension (PH) characterized by obstructive vasculopathy. Endothelial... Show moreChronic thromboembolic pulmonary hypertension (CTEPH) and pulmonary arterial hypertension (PAH) are two forms of pulmonary hypertension (PH) characterized by obstructive vasculopathy. Endothelial dysfunction along with metabolic changes towards increased glycolysis are important in PAH pathophysiology. Less is known about such abnormalities in endothelial cells (ECs) from CTEPH patients. This study provides a systematic metabolic comparison of ECs derived from CTEPH and PAH patients. Metabolic gene expression was studied using qPCR in cultured CTEPH-EC and PAH-EC. Western blot analyses were done for HK2, LDHA, PDHA1, PDK and G6PD. Basal viability of CTEPH-EC and PAH-EC with the incubation with metabolic inhibitors was measured using colorimetric viability assays. Human pulmonary artery endothelial cells (HPAEC) were used as healthy controls. Whereas PAH-EC showed significant higher mRNA levels of GLUT1, HK2, LDHA, PDHA1 and GLUD1 metabolic enzymes compared to HPAEC, CTEPH-EC did not. Oxidative phosphorylation associated proteins had an increased expression in PAH-EC compared to CTEPH-EC and HPAEC. PAH-EC, CTEPH-EC and HPAEC presented similar HOXD macrovascular gene expression. Metabolic inhibitors showed a dose-dependent reduction in viability in all three groups, predominantly in PAH-EC. A different metabolic profile is present in CTEPH-EC compared to PAH-EC and suggests differences in molecular mechanisms important in the disease pathology and treatment. Show less
Jong, A. de; Sier, V.Q.; Peters, H.A.B.; Schilder, N.K.M.; Jukema, J.W.; Goumans, M.J.T.H.; ... ; Vries, M.R. de 2022
Aims: Vein grafts are frequently used to bypass coronary artery occlusions. Unfortunately, vein graft disease (VGD) causes impaired patency rates. ALK1 mediates signaling by TGF-beta via TGF beta... Show moreAims: Vein grafts are frequently used to bypass coronary artery occlusions. Unfortunately, vein graft disease (VGD) causes impaired patency rates. ALK1 mediates signaling by TGF-beta via TGF beta R2 or BMP9/10 via BMPR2, which is an important pathway in fibrotic, inflammatory, and angiogenic processes in vascular diseases. The role of the TGF-beta pathway in VGD is previously reported, however, the contribution of ALK1 signaling is not known. Therefore, we investigated ALK1 signaling in VGD in a mouse model for vein graft disease using either genetic or pharmacological inhibition of the Alk1 signaling. Methods and Results: Male ALK1 heterozygous (ALK1(+/-)), control C57BL/6, as well as hypercholesterolemic ApoE3*Leiden mice, underwent vein graft surgery. Histologic analyses of ALK1(+/-) vein grafts demonstrated increased outward remodeling and macrophage accumulation after 28 days. In hypercholesterolemic ApoE3*Leiden mice receiving weekly ALK1-Fc injections, ultrasound imaging showed 3-fold increased outward remodeling compared to controls treated with control-Fc, which was confirmed histologically. Moreover, ALK1-Fc treatment reduced collagen and smooth muscle cell accumulation, increased macrophages by 1.5-fold, and resulted in more plaque dissections. No difference was observed in intraplaque neovessel density. Flow cytometric analysis showed increased systemic levels of Ly6C(High) monocytes in ALK1-Fc treated mice, supported by in vitro increased MCP-1 and IL-6 production of LPS-stimulated and ALK1-Fc-treated murine monocytes and macrophages. Conclusion: Reduced ALK1 signaling in VGD promotes outward remodeling, increases macrophage influx, and promotes an unstable plaque phenotype. Translational Perspective: Vein graft disease (VGD) severely hampers patency rates of vein grafts, necessitating research of key disease-driving pathways like TGF-beta. The three-dimensional nature of VGD together with the multitude of disease driving factors ask for a comprehensive approach. Here, we combined in vivo ultrasound imaging, histological analyses, and conventional in vitro analyses, identifying the ambiguous role of reduced ALK1 signaling in vein graft disease. Reduced ALK1 signaling promotes outward remodeling, increases macrophage influx, and promotes an unstable plaque phenotype in murine vein grafts. Characterization of in vivo vascular remodeling over time is imperative to monitor VGD development and identify new therapies. Show less
Brinks, J.; Dijk, E.H.C. van; Kielbasa, S.M.; Mei, H.L.; Veen, I. van der; Peters, H.A.B.; ... ; Meijer, O.C. 2022
Context: Central serous chorioretinopathy (CSC) is a severe ocular disease characterized by fluid accumulation under the retina and abnormalities in the underlying vascular layer, the choroid. CSC... Show moreContext: Central serous chorioretinopathy (CSC) is a severe ocular disease characterized by fluid accumulation under the retina and abnormalities in the underlying vascular layer, the choroid. CSC has a striking prevalence in males of 80% to 90% of total patients. Corticosteroids are the most pronounced extrinsic risk factor for CSC. Choroidal endothelial cells (CECs) are important for the vascular integrity of the choroid, but the effects of corticosteroid effects in these cells are unknown.Objective: We aimed to reveal the potential steroidal contribution to CSC.Method: We characterized the expression of the glucocorticoid, mineralocorticoid, and androgen receptor in the human choroid using immunohistochemistry. Using RNA-sequencing, we describe the cortisol response in human CECs derived from 5 male and 5 female postmortem donors.Results: The glucocorticoid receptor was highly expressed in the human choroid, whereas no to minimal expression of the mineralocorticoid and androgen receptors was observed. The extensive transcriptional response to cortisol in human primary cultured CECs showed interindividual differences but very few sex differences. Several highly regulated genes such as ZBTB16(log2 fold change males 7.9; females 6.2) provide strong links to choroidal vascular regulation.Conclusions: The glucocorticoid receptor predominantly mediates the response to cortisol in human CECs. Interindividual differences are an important determinant regarding the cortisol response in human cultured CECs, whereas intrinsic sex differences appear less pronounced. The marked response of particular target genes in endothelial cells to cortisol, such as ZBTB16, warrants further investigation into their potential role in the pathophysiology of CSC and other vascular conditions. Show less
Homberg, D.A.L. van den; Kwast, R.V.C.T. van der; Quax, P.H.A.; Nossent, A.Y. 2022
N-6-methyladenosine (m6A) is the most prevalent post-transcriptional RNA modification in eukaryotic cells. The modification is reversible and can be dynamically regulated by writer and eraser... Show moreN-6-methyladenosine (m6A) is the most prevalent post-transcriptional RNA modification in eukaryotic cells. The modification is reversible and can be dynamically regulated by writer and eraser enzymes. Alteration in the levels of these enzymes can lead to changes in mRNA stability, alternative splicing or microRNA processing, depending on the m6A-binding proteins. Dynamic regulation of mRNA m6A methylation after ischemia and hypoxia influences mRNA stability, alternative splicing and translation, contributing to heart failure. In this study, we studied vasoactive microRNA m6A methylation in fibroblasts and examined the effect of hypoxia on microRNAs methylation using m6A immunoprecipitation. Of the 19 microRNAs investigated, at least 16 contained m6A in both primary human fibroblasts and a human fibroblast cell line, suggesting vasoactive microRNAs are commonly m6A methylated in fibroblasts. More importantly, we found that mature microRNA m6A levels increased upon subjecting cells to hypoxia. By silencing different m6A writer and eraser enzymes followed by m6A immunoprecipitation, we identified METTL4, an snRNA m6A methyltransferase, to be predominantly responsible for the increase in m6A modification. Moreover, by using m6A-methylated microRNA mimics, we found that microRNA m6A directly affects downstream target mRNA repression efficacy. Our findings highlight the regulatory potential of the emerging field of microRNA modifications. Show less
