Publications by authors named "Paul H A Quax"

163 Publications

The Cortisol Response of Male and Female Choroidal Endothelial Cells: Implications for Central Serous Chorioretinopathy.

J Clin Endocrinol Metab 2021 Sep 21. Epub 2021 Sep 21.

Department of Medicine, Division of Endocrinology and Metabolism, Leiden University Medical Center, Leiden, the Netherlands.

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 has a striking prevalence in males of 80-90% 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.

Design And Objective: We aimed to reveal the potential steroidal contribution to CSC. 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.
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http://dx.doi.org/10.1210/clinem/dgab670DOI Listing
September 2021

In Vivo Matrigel Plug Assay as a Potent Method to Investigate Specific Individual Contribution of Angiogenesis to Blood Flow Recovery in Mice.

Int J Mol Sci 2021 Aug 18;22(16). Epub 2021 Aug 18.

Department of Surgery, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

Neovascularization restores blood flow recovery after ischemia in peripheral arterial disease. The main two components of neovascularization are angiogenesis and arteriogenesis. Both of these processes contribute to functional improvements of blood flow after occlusion. However, discriminating between the specific contribution of each process is difficult. A frequently used model for investigating neovascularization is the murine hind limb ischemia model (HLI). With this model, it is difficult to determine the role of angiogenesis, because usually the timing for the sacrifice of the mice is chosen to be optimal for the analysis of arteriogenesis. More importantly, the occurring angiogenesis in the distal calf muscles is probably affected by the proximally occurring arteriogenesis. Therefore, to understand and subsequently intervene in the process of angiogenesis, a model is needed which investigates angiogenesis without the influence of arteriogenesis. In this study we evaluated the in vivo Matrigel plug assay in genetic deficient mice to investigate angiogenesis. Mice deficient for and underwent the in vivo Matrigel model. Histological analysis of the Matrigel plugs showed an increased angiogenesis in mice deficient of , and , and a decreased angiogenesis in deficient mice. Our results also suggest an involvement of in angiogenesis. Comparing our results with results of the HLI model found in the literature suggests that the in vivo Matrigel plug assay is superior in evaluating the angiogenic response after ischemia.
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http://dx.doi.org/10.3390/ijms22168909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8396178PMC
August 2021

PFKFB3 gene deletion in endothelial cells inhibits intraplaque angiogenesis and lesion formation in a murine model of venous bypass grafting.

Angiogenesis 2021 Aug 25. Epub 2021 Aug 25.

Laboratory of Physiopharmacology, University of Antwerp, Universiteitsplein 1, B-2610, Antwerp, Belgium.

Vein grafting is a frequently used surgical intervention for cardiac revascularization. However, vein grafts display regions with intraplaque (IP) angiogenesis, which promotes atherogenesis and formation of unstable plaques. Graft neovessels are mainly composed of endothelial cells (ECs) that largely depend on glycolysis for migration and proliferation. In the present study, we aimed to investigate whether loss of the glycolytic flux enzyme phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) in ECs inhibits IP angiogenesis and as such prevents unstable plaque formation. To this end, apolipoprotein E deficient (ApoE) mice were backcrossed to a previously generated PFKFB3 Cdh5 mouse strain. Animals were injected with either corn oil (ApoEPFKFB3) or tamoxifen (ApoEPFKFB3), and were fed a western-type diet for 4 weeks prior to vein grafting. Hereafter, mice received a western diet for an additional 28 days and were then sacrificed for graft assessment. Size and thickness of vein graft lesions decreased by 35 and 32%, respectively, in ApoEPFKFB3 mice compared to controls, while stenosis diminished by 23%. Moreover, vein graft lesions in ApoEPFKFB3 mice showed a significant reduction in macrophage infiltration (29%), number of neovessels (62%), and hemorrhages (86%). EC-specific PFKFB3 deletion did not show obvious adverse effects or changes in general metabolism. Interestingly, RT-PCR showed an increased M2 macrophage signature in vein grafts from ApoEPFKFB3 mice. Altogether, EC-specific PFKFB3 gene deletion leads to a significant reduction in lesion size, IP angiogenesis, and hemorrhagic complications in vein grafts. This study demonstrates that inhibition of endothelial glycolysis is a promising therapeutic strategy to slow down plaque progression.
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http://dx.doi.org/10.1007/s10456-021-09816-3DOI Listing
August 2021

Phosphorylcholine antibodies restrict infarct size and left ventricular remodelling by attenuating the unreperfused post-ischaemic inflammatory response.

J Cell Mol Med 2021 Aug 30;25(16):7772-7782. Epub 2021 Jun 30.

Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands.

Phosphorylcholine is a pro-inflammatory epitope exposed on apoptotic cells, and phosphorylcholine monoclonal immunoglobulin (Ig)G antibodies (PC-mAb) have anti-inflammatory properties. In this study, we hypothesize that PC-mAb treatment reduces adverse cardiac remodelling and infarct size (IS) following unreperfused transmural myocardial infarction (MI). Unreperfused MI was induced by permanent ligation of the left anterior descending (LAD) coronary artery in hypercholesterolaemic APOE*3-Leiden mice. Three weeks following MI, cardiac magnetic resonance (CMR) imaging showed a reduced LV end-diastolic volume (EDV) by 21% and IS by 31% upon PC-mAb treatment as compared to the vehicle control group. In addition, the LV fibrous content was decreased by 27% and LV wall thickness was better preserved by 47% as determined by histological analysis. Two days following MI, CCL2 concentrations, assessed by use of ELISA, were decreased by 81% and circulating monocytes by 64% as assessed by use of FACS analysis. Additionally, local leucocyte infiltration determined by immunohistological analysis showed a 62% decrease after three weeks. In conclusion, the local and systemic inflammatory responses are limited by PC-mAb treatment resulting in restricted adverse cardiac remodelling and IS following unreperfused MI. This indicates that PC-mAb holds promise as a therapeutic agent following MI limiting adverse cardiac remodelling.
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http://dx.doi.org/10.1111/jcmm.16662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8358891PMC
August 2021

Post-ischemic Myocardial Inflammatory Response: A Complex and Dynamic Process Susceptible to Immunomodulatory Therapies.

Front Cardiovasc Med 2021 28;8:647785. Epub 2021 Apr 28.

Department of Surgery, Leiden University Medical Center, Leiden, Netherlands.

Following acute occlusion of a coronary artery causing myocardial ischemia and implementing first-line treatment involving rapid reperfusion, a dynamic and balanced inflammatory response is initiated to repair and remove damaged cells. Paradoxically, restoration of myocardial blood flow exacerbates cell damage as a result of myocardial ischemia-reperfusion (MI-R) injury, which eventually provokes accelerated apoptosis. In the end, the infarct size still corresponds to the subsequent risk of developing heart failure. Therefore, true understanding of the mechanisms regarding MI-R injury, and its contribution to cell damage and cell death, are of the utmost importance in the search for successful therapeutic interventions to finally prevent the onset of heart failure. This review focuses on the role of innate immunity, chemokines, cytokines, and inflammatory cells in all three overlapping phases following experimental, mainly murine, MI-R injury known as the inflammatory, reparative, and maturation phase. It provides a complete state-of-the-art overview including most current research of all post-ischemic processes and phases and additionally summarizes the use of immunomodulatory therapies translated into clinical practice.
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http://dx.doi.org/10.3389/fcvm.2021.647785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113407PMC
April 2021

Endoglin/CD105-Based Imaging of Cancer and Cardiovascular Diseases: A Systematic Review.

Int J Mol Sci 2021 Apr 30;22(9). Epub 2021 Apr 30.

Department of Gastroenterology and Hepatology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

Molecular imaging of pathologic lesions can improve efficient detection of cancer and cardiovascular diseases. A shared pathophysiological feature is angiogenesis, the formation of new blood vessels. Endoglin (CD105) is a coreceptor for ligands of the Transforming Growth Factor-β (TGF-β) family and is highly expressed on angiogenic endothelial cells. Therefore, endoglin-based imaging has been explored to visualize lesions of the aforementioned diseases. This systematic review highlights the progress in endoglin-based imaging of cancer, atherosclerosis, myocardial infarction, and aortic aneurysm, focusing on positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), near-infrared fluorescence (NIRF) imaging, and ultrasound imaging. PubMed was searched combining the following subjects and their respective synonyms or relevant subterms: "Endoglin", "Imaging/Image-guided surgery". In total, 59 papers were found eligible to be included: 58 reporting about preclinical animal or in vitro models and one ex vivo study in human organs. In addition to exact data extraction of imaging modality type, tumor or cardiovascular disease model, and tracer (class), outcomes were described via a narrative synthesis. Collectively, the data identify endoglin as a suitable target for intraoperative and diagnostic imaging of the neovasculature in tumors, whereas for cardiovascular diseases, the evidence remains scarce but promising.
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http://dx.doi.org/10.3390/ijms22094804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8124553PMC
April 2021

The Inflammatory Profile of CTEPH-Derived Endothelial Cells Is a Possible Driver of Disease Progression.

Cells 2021 03 26;10(4). Epub 2021 Mar 26.

Department of Cell and Chemical Biology, Laboratory for CardioVascular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a form of pulmonary hypertension characterized by the presence of fibrotic intraluminal thrombi and causing obliteration of the pulmonary arteries. Although both endothelial cell (EC) dysfunction and inflammation are linked to CTEPH pathogenesis, regulation of the basal inflammatory response of ECs in CTEPH is not fully understood. Therefore, in the present study, we investigated the role of the nuclear factor (NF)-κB pro-inflammatory signaling pathway in ECs in CTEPH under basal conditions. Basal mRNA levels of interleukin (IL)-8, IL-1β, monocyte chemoattractant protein-1 (MCP-1), C-C motif chemokine ligand 5 (CCL5), and vascular cell adhesion molecule-1 (VCAM-1) were upregulated in CTEPH-ECs compared to the control cells. To assess the involvement of NF-κB signaling in basal inflammatory activation, CTEPH-ECs were incubated with the NF-κB inhibitor Bay 11-7085. The increase in pro-inflammatory cytokines was abolished when cells were incubated with the NF-κB inhibitor. To determine if NF-κB was indeed activated, we stained pulmonary endarterectomy (PEA) specimens from CTEPH patients and ECs isolated from PEA specimens for phospho-NF-κB-P65 and found that especially the vessels within the thrombus and CTEPH-ECs are positive for phospho-NF-κB-P65. In summary, we show that CTEPH-ECs have a pro-inflammatory status under basal conditions, and blocking NF-κB signaling reduces the production of inflammatory factors in CTEPH-ECs. Therefore, our results show that the increased basal pro-inflammatory status of CTEPH-ECs is, at least partially, regulated through activation of NF-κB signaling and potentially contributes to the pathophysiology and progression of CTEPH.
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http://dx.doi.org/10.3390/cells10040737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067175PMC
March 2021

Endothelial Barrier Function and Leukocyte Transmigration in Atherosclerosis.

Biomedicines 2021 Mar 24;9(4). Epub 2021 Mar 24.

Department of Vascular Surgery, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands.

The vascular endothelium is a highly specialized barrier that controls passage of fluids and migration of cells from the lumen into the vessel wall. Endothelial cells assist leukocytes to extravasate and despite the variety in the specific mechanisms utilized by different leukocytes to cross different vascular beds, there is a general principle of capture, rolling, slow rolling, arrest, crawling, and ultimately diapedesis via a paracellular or transcellular route. In atherosclerosis, the barrier function of the endothelium is impaired leading to uncontrolled leukocyte extravasation and vascular leakage. This is also observed in the neovessels that grow into the atherosclerotic plaque leading to intraplaque hemorrhage and plaque destabilization. This review focuses on the vascular endothelial barrier function and the interaction between endothelial cells and leukocytes during transmigration. We will discuss the role of endothelial dysfunction, transendothelial migration of leukocytes and plaque angiogenesis in atherosclerosis.
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http://dx.doi.org/10.3390/biomedicines9040328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8063931PMC
March 2021

BMP Receptor Inhibition Enhances Tissue Repair in Endoglin Heterozygous Mice.

Int J Mol Sci 2021 Feb 18;22(4). Epub 2021 Feb 18.

Department of Cell and Chemical Biology, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands.

Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a severe vascular disorder caused by mutations in the TGFβ/BMP co-receptor . Endoglin haploinsufficiency results in vascular malformations and impaired neoangiogenesis. Furthermore, HHT1 patients display an impaired immune response. To date it is not fully understood how haploinsufficient immune cells contribute to HHT1 pathology. Therefore, we investigated the immune response during tissue repair in mice, a model for HHT1. +/- mice exhibited prolonged infiltration of macrophages after experimentally induced myocardial infarction. Moreover, there was an increased number of inflammatory M1-like macrophages (Ly6C/CD206) at the expense of reparative M2-like macrophages (Ly6C/CD206). Interestingly, HHT1 patients also showed an increased number of inflammatory macrophages. In vitro analysis revealed that TGFβ-induced differentiation of monocytes into M2-like macrophages was blunted. Inhibiting BMP signaling by treating monocytes with LDN-193189 normalized their differentiation. Finally, LDN treatment improved heart function after MI and enhanced vascularization in both wild type and mice. The beneficial effect of LDN was also observed in the hind limb ischemia model. While blood flow recovery was hampered in vehicle-treated animals, LDN treatment improved tissue perfusion recovery in mice. In conclusion, BMPR kinase inhibition restored HHT1 macrophage imbalance in vitro and improved tissue repair after ischemic injury in mice.
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http://dx.doi.org/10.3390/ijms22042010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7922601PMC
February 2021

P300/CBP Associated Factor (PCAF) Deficiency Enhances Diet-Induced Atherosclerosis in ApoE3Leiden Mice via Systemic Inhibition of Regulatory T Cells.

Front Cardiovasc Med 2020 15;7:604821. Epub 2021 Jan 15.

Department of Surgery, Leiden University Medical Center, Leiden, Netherlands.

Inflammatory stimuli induced by NF-kB drive atherosclerotic lesion formation. The epigenetic P300/CBP associated factor (PCAF) post-transcriptionally acetylates FoxP3, which is required for regulatory T-cell (Treg) differentiation and immune modulation. We hypothesize that PCAF deficiency affects atherosclerosis via regulation of regulatory Tregs. ApoE3Leiden ( = 13) and ApoE3LeidenxPCAF ( = 13) were fed a high-fat diet (HFD) containing 1.25% cholesterol. Systemic FoxP3 T cells were measured every 4 weeks by flow cytometry ( = 6). After 5-months of HFD, mice were euthanized, and hearts and blood were collected. IL-6 and TNFα concentrations were measured in plasma to identify systemic inflammatory responses. Compositional and morphometrical analyses were performed on the atherosclerotic lesions in the aortic sinuses. After 5 months of HFD, plasma cholesterol concentrations were not different for ApoE3LeidenxPCAF compared to ApoE3Leiden mice. Expression of FoxP3 by systemic CD4 T cells decreased 1.8 fold in ApoE3LeidenxPCAF after 5 months HFD and remained significantly reduced after 5 months of HFD. Systemic TNFα and IL-6 concentrations were comparable, whereas the atherosclerotic lesion size in ApoE3LeidenxPCAF mice was increased by 28% compared to ApoE3Leiden mice. In atherosclerotic lesions, no differences were observed in macrophage differentiation or VSMC content, although a small increase in collagen was identified. Our data show that PCAF deficiency resulted in a decrease in circulatory FoxP3 regulatory T cells and ameliorated atherosclerotic lesions with no differences in systemic inflammation or macrophage differentiation in the atherosclerotic lesions. This suggests that PCAF regulates atherosclerosis via modulation of FoxP3 regulatory T cell differentiation.
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http://dx.doi.org/10.3389/fcvm.2020.604821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874080PMC
January 2021

Atorvastatin pleiotropically decreases intraplaque angiogenesis and intraplaque haemorrhage by inhibiting ANGPT2 release and VE-Cadherin internalization.

Angiogenesis 2021 08 7;24(3):567-581. Epub 2021 Feb 7.

Department of Vascular Surgery, Leiden University Medical Center, Leiden, The Netherlands.

Objective: Statins pleiotropically provide additional benefits in reducing atherosclerosis, but their effects on intraplaque angiogenesis (IPA) and hemorrhage (IPH) remain unclear. Therefore, we discriminated statin's lipid-lowering dependent and independent effects on IPA and IPH.

Approach And Results: ApoE3*Leiden mice are statin-responsive due to ApoE and LDLR presence, but also allow to titrate plasma cholesterol levels by diet. Therefore, ApoE3*Leiden mice were fed a high-cholesterol-inducing-diet (HCD) with or without atorvastatin (A) or a moderate-cholesterol-inducing-diet (MCD). Mice underwent vein graft surgery to induce lesions with IPA and IPH. Cholesterol levels were significantly reduced in MCD (56%) and HCD + A (39%) compared to HCD with no significant differences between MCD and HCD + A. Both MCD and HCD + A have a similar reduction in vessel remodeling and inflammation comparing to HCD. IPA was significantly decreased by 30% in HCD + A compared to HCD or MCD. Atorvastatin treatment reduced the presence of immature vessels by 34% vs. HCD and by 25% vs. MCD, resulting in a significant reduction of IPH. Atorvastatin's anti-angiogenic capacity was further illustrated by a dose-dependent reduction of ECs proliferation and migration. Cultured mouse aortic-segments lost sprouting capacity upon atorvastatin treatment and became 30% richer in VE-Cadherin expression and pericyte coverage. Moreover, Atorvastatin inhibited ANGPT2 release and decreased VE-Cadherin(Y685)-phosphorylation in ECs.

Conclusions: Atorvastatin has beneficial effects on vessel remodeling due to its lipid-lowering capacity. Atorvastatin has strong pleiotropic effects on IPA by decreasing the number of neovessels and on IPH by increasing vessel maturation. Atorvastatin improves vessel maturation by inhibiting ANGPT2 release and phospho(Y658)-mediated VE-Cadherin internalization.
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http://dx.doi.org/10.1007/s10456-021-09767-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292290PMC
August 2021

Endothelial Dysfunction in Pulmonary Hypertension: Cause or Consequence?

Biomedicines 2021 Jan 9;9(1). Epub 2021 Jan 9.

Department of Cell and Chemical Biology, Laboratory for CardioVascular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

Pulmonary arterial hypertension (PAH) is a rare, complex, and progressive disease that is characterized by the abnormal remodeling of the pulmonary arteries that leads to right ventricular failure and death. Although our understanding of the causes for abnormal vascular remodeling in PAH is limited, accumulating evidence indicates that endothelial cell (EC) dysfunction is one of the first triggers initiating this process. EC dysfunction leads to the activation of several cellular signalling pathways in the endothelium, resulting in the uncontrolled proliferation of ECs, pulmonary artery smooth muscle cells, and fibroblasts, and eventually leads to vascular remodelling and the occlusion of the pulmonary blood vessels. Other factors that are related to EC dysfunction in PAH are an increase in endothelial to mesenchymal transition, inflammation, apoptosis, and thrombus formation. In this review, we outline the latest advances on the role of EC dysfunction in PAH and other forms of pulmonary hypertension. We also elaborate on the molecular signals that orchestrate EC dysfunction in PAH. Understanding the role and mechanisms of EC dysfunction will unravel the therapeutic potential of targeting this process in PAH.
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http://dx.doi.org/10.3390/biomedicines9010057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827874PMC
January 2021

Phosphorylcholine Antibodies Preserve Cardiac Function and Reduce Infarct Size by Attenuating the Post-Ischemic Inflammatory Response.

JACC Basic Transl Sci 2020 Dec 2;5(12):1228-1239. Epub 2020 Dec 2.

Department of Surgery, Leiden University Medical Center, Leiden, the Netherlands.

Phosphorylcholine monoclonal immunoglobulin G antibody attenuates the immediate post-ischemic inflammatory response by reducing the proinflammatory chemokine (C-C motif) ligand 2 chemokine and circulating Ly-6C monocytes. This subsequently enhances the post-ischemic repair process, resulting in limited adverse cardiac remodeling and preservation of cardiac function. Therefore, phosphorylcholine monoclonal immunoglobulin G antibody therapy may be a valid therapeutic approach against myocardial ischemia-reperfusion injury.
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http://dx.doi.org/10.1016/j.jacbts.2020.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775955PMC
December 2020

Circulating tRNA Fragments as a Novel Biomarker Class to Distinguish Acute Stroke Subtypes.

Int J Mol Sci 2020 Dec 24;22(1). Epub 2020 Dec 24.

Department of Vascular Surgery, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

Early blood biomarkers to diagnose acute stroke could drastically reduce treatment delays. We investigated whether circulating small non-coding RNAs can serve as biomarkers to distinguish between acute ischemic stroke (IS), intracerebral hemorrhage (ICH) and stroke mimics (SM). In an ongoing observational cohort study, we performed small RNA-sequencing in plasma obtained from a discovery cohort of 26 patients (9 IS, 8 ICH and 9 SM) presented to the emergency department within 6 h of symptom onset. We validated our results in an independent dataset of 20 IS patients and 20 healthy controls. ICH plasma had the highest abundance of ribosomal and tRNA-derived fragments, while microRNAs were most abundant in plasma of IS patients. Combinations of four to five tRNAs yielded diagnostic accuracies (areas under the receiver operating characteristics curve) up to 0.986 (ICH vs. IS and SM) in the discovery cohort. Validation of the IS and SM models in the independent dataset yielded diagnostic accuracies of 0.870 and 0.885 to distinguish IS from healthy controls. Thus, we identified tRNA-derived fragments as a promising novel class of biomarkers to distinguish between acute IS, ICH and SM, as well as healthy controls.
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http://dx.doi.org/10.3390/ijms22010135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7796003PMC
December 2020

Assessment of Microvessel Permeability in Murine Atherosclerotic Vein Grafts Using Two-Photon Intravital Microscopy.

Int J Mol Sci 2020 Dec 3;21(23). Epub 2020 Dec 3.

Department of Surgery, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands.

Plaque angiogenesis and plaque hemorrhage are major players in the destabilization and rupture of atherosclerotic lesions. As these are dynamic processes, imaging of plaque angiogenesis, especially the integrity or leakiness of angiogenic vessels, can be an extremely useful tool in the studies on atherosclerosis pathophysiology. Visualizing plaque microvessels in 3D would enable us to study the architecture and permeability of adventitial and intimal plaque microvessels in advanced atherosclerotic lesions. We hypothesized that a comparison of the vascular permeability between healthy continuous and fenestrated as well as diseased leaky microvessels, would allow us to evaluate plaque microvessel leakiness. We developed and validated a two photon intravital microscopy (2P-IVM) method to assess the leakiness of plaque microvessels in murine atherosclerosis-prone ApoE3*Leiden vein grafts based on the quantification of fluorescent-dextrans extravasation in real-time. We describe a novel 2P-IVM set up to study vessels in the neck region of living mice. We show that microvessels in vein graft lesions are in their pathological state more permeable in comparison with healthy continuous and fenestrated microvessels. This 2P-IVM method is a promising approach to assess plaque angiogenesis and leakiness. Moreover, this method is an important advancement to validate therapeutic angiogenic interventions in preclinical atherosclerosis models.
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http://dx.doi.org/10.3390/ijms21239244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730593PMC
December 2020

Periprocedural Hydrogen Sulfide Therapy Improves Vascular Remodeling and Attenuates Vein Graft Disease.

J Am Heart Assoc 2020 11 4;9(22):e016391. Epub 2020 Nov 4.

Department of Surgery and the Heart and Vascular Center Brigham & Women's Hospital Harvard Medical School Boston MA.

Background Failure rates after revascularization surgery remain high, both in vein grafts (VG) and arterial interventions. One promising approach to improve outcomes is endogenous upregulation of the gaseous transmitter-molecule hydrogen sulfide, via short-term dietary restriction. However, strict patient compliance stands as a potential translational barrier in the vascular surgery patient population. Here we present a new therapeutic approach, via a locally applicable gel containing the hydrogen sulfide releasing prodrug (GYY), to both mitigate graft failure and improve arterial remodeling. Methods and Results All experiments were performed on C57BL/6 (male, 12 weeks old) mice. VG surgery was performed by grafting a donor-mouse cava vein into the right common carotid artery of a recipient via an end-to-end anastomosis. In separate experiments arterial intimal hyperplasia was assayed via a right common carotid artery focal stenosis model. All mice were harvested at postoperative day 28 and artery/graft was processed for histology. Efficacy of hydrogen sulfide was first tested via GYY supplementation of drinking water either 1 week before VG surgery (pre-GYY) or starting immediately postoperatively (post-GYY). Pre-GYY mice had a 36.5% decrease in intimal/media+adventitia area ratio compared with controls. GYY in a 40% Pluronic gel (or vehicle) locally applied to the graft/artery had decreased intimal/media area ratios (right common carotid artery) and improved vessel diameters. GYY-geltreated VG had larger diameters at both postoperative days 14 and 28, and a 56.7% reduction in intimal/media+adventitia area ratios. Intimal vascular smooth muscle cell migration was decreased 30.6% after GYY gel treatment, which was reproduced in vitro. Conclusions Local gel-based treatment with the hydrogen sulfide-donor GYY stands as a translatable therapy to improve VG durability and arterial remodeling after injury.
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http://dx.doi.org/10.1161/JAHA.120.016391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763704PMC
November 2020

Effects on cardiac function, remodeling and inflammation following myocardial ischemia-reperfusion injury or unreperfused myocardial infarction in hypercholesterolemic APOE*3-Leiden mice.

Sci Rep 2020 10 6;10(1):16601. Epub 2020 Oct 6.

Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands.

Many novel therapies to treat myocardial infarction (MI), yielding promising results in animal models, nowadays failed in clinical trials for several reasons. The most used animal MI model is based on permanent ligation of the left anterior descending (LAD) coronary artery in healthy mice resulting in transmural MI, while in clinical practice reperfusion is usually accomplished by primary percutaneous coronary interventions (PCI) limiting myocardial damage and inducing myocardial ischemia-reperfusion (MI-R) injury. To evaluate a more similar murine MI model we compared MI-R injury to unreperfused MI in hypercholesterolemic apolipoprotein (APO)E*3-Leiden mice regarding effects on cardiac function, left ventricular (LV) remodeling and inflammation. Both MI-R and MI resulted in significant LV dilation and impaired cardiac function after 3 weeks. Although LV dilation, displayed by end-diastolic (EDV) and end-systolic volumes (ESV), and infarct size (IS) were restricted following MI-R compared to MI (respectively by 27.6% for EDV, 39.5% ESV, 36.0% IS), cardiac function was not preserved. LV-wall thinning was limited with non-transmural LV fibrosis in the MI-R group (66.7%). Two days after inducing myocardial ischemia, local leucocyte infiltration in the infarct area was decreased following MI-R compared to MI (36.6%), whereas systemic circulating monocytes were increased in both groups compared to sham (130.0% following MI-R and 120.0% after MI). Both MI-R and MI models against the background of a hypercholesterolemic phenotype appear validated experimental models, however reduced infarct size, restricted LV remodeling as well as a different distributed inflammatory response following MI-R resemble the contemporary clinical outcome regarding primary PCI more accurately which potentially provides better predictive value of experimental therapies in successive clinical trials.
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http://dx.doi.org/10.1038/s41598-020-73608-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7538581PMC
October 2020

bFGF blockade reduces intraplaque angiogenesis and macrophage infiltration in atherosclerotic vein graft lesions in ApoE3*Leiden mice.

Sci Rep 2020 09 29;10(1):15968. Epub 2020 Sep 29.

Department of Vascular Surgery, D6-33, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands.

Intraplaque angiogenesis increases the chance of unstable atherosclerotic plaque rupture and thrombus formation leading to myocardial infarction. Basic Fibroblast Growth Factor (bFGF) plays a key role in angiogenesis and inflammation and is involved in the pathogenesis of atherosclerosis. Therefore, we aim to test K5, a small molecule bFGF-inhibitor, on remodelling of accelerated atherosclerotic vein grafts lesions in ApoE3*Leiden mice. K5-mediated bFGF-signalling blockade strongly decreased intraplaque angiogenesis and intraplaque hemorrhage. Moreover, it reduced macrophage infiltration in the lesions by modulating CCL2 and VCAM1 expression. Therefore, K5 increases plaque stability. To study the isolated effect of K5 on angiogenesis and SMCs-mediated intimal hyperplasia formation, we used an in vivo Matrigel-plug mouse model that reveals the effects on in vivo angiogenesis and femoral artery cuff model to exclusively looks at SMCs. K5 drastically reduced in vivo angiogenesis in the matrigel plug model while no effect on SMCs migration nor proliferation could be seen in the femoral artery cuff model. Moreover, in vitro K5 impaired endothelial cells functions, decreasing migration, proliferation and tube formation. Our data show that K5-mediated bFGF signalling blockade in hypercholesterolemic ApoE3*Leiden mice reduces intraplaque angiogenesis, haemorrhage and inflammation. Therefore, K5 is a promising candidate to stabilize advanced atherosclerotic plaques.
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http://dx.doi.org/10.1038/s41598-020-72992-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7525538PMC
September 2020

Adenosine-to-Inosine Editing of Vasoactive MicroRNAs Alters Their Targetome and Function in Ischemia.

Mol Ther Nucleic Acids 2020 Sep 21;21:932-953. Epub 2020 Jul 21.

Department of Surgery, Leiden University Medical Center, 2333 ZA Leiden, the Netherlands; Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 2333 ZA Leiden, the Netherlands; Department of Laboratory Medicine, Medical University of Vienna, 1090 Vienna, Austria; Department of Internal Medicine II, Medical University of Vienna, 1090 Vienna, Austria. Electronic address:

Adenosine-to-inosine (A-to-I) editing in the seed sequence of microRNAs can shift the microRNAs' targetomes and thus their function. Using public RNA-sequencing data, we identified 35 vasoactive microRNAs that are A-to-I edited. We quantified A-to-I editing of the primary (pri-)microRNAs in vascular fibroblasts and endothelial cells. Nine pri-microRNAs were indeed edited, and editing consistently increased under ischemia. We determined mature microRNA editing for the highest expressed microRNAs, i.e., miR-376a-3p, miR-376c-3p, miR-381-3p, and miR-411-5p. All four mature microRNAs were edited in their seed sequence. We show that both ADAR1 and ADAR2 (adenosine deaminase acting on RNA 1 and RNA 2) can edit pri-microRNAs in a microRNA-specific manner. MicroRNA editing also increased under ischemia in vivo in a murine hindlimb ischemia model and ex vivo in human veins. For each edited microRNA, we confirmed a shift in targetome. Expression of the edited microRNA targetomes, not the wild-type targetomes, was downregulated under ischemia in vivo. Furthermore, microRNA editing enhanced angiogenesis in vitro and ex vivo. In conclusion, we show that microRNA A-to-I editing is a widespread phenomenon, induced by ischemia. Each editing event results in a novel microRNA with a unique targetome, leading to increased angiogenesis.
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http://dx.doi.org/10.1016/j.omtn.2020.07.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452086PMC
September 2020

Bis(maltolato)oxovanadium(IV) Induces Angiogenesis via Phosphorylation of VEGFR2.

Int J Mol Sci 2020 Jun 30;21(13). Epub 2020 Jun 30.

Department of Surgery, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

VEGFR2 and VEGF-A play a pivotal role in the process of angiogenesis. VEGFR2 activation is regulated by protein tyrosine phosphatases (PTPs), enzymes that dephosphorylate the receptor and reduce angiogenesis. We aim to study the effect of PTPs blockade using bis(maltolato)oxovanadium(IV) (BMOV) on in vivo wound healing and in vitro angiogenesis. BMOV significantly improves in vivo wound closure by 45% in C57BL/6JRj mice. We found that upon VEGFR2 phosphorylation induced by endogenously produced VEGF-A, the addition of BMOV results in increased cell migration (45%), proliferation (40%) and tube formation (27%) in HUVECs compared to control. In a mouse ex vivo, aortic ring assay BMOV increased the number of sprouts by 3 folds when compared to control. However, BMOV coadministered with exogenous VEGF-A increased ECs migration, proliferation and tube formation by only 41%, 18% and 12% respectively and aortic ring sprouting by only 1-fold. We also found that BMOV enhances VEGFR2 Y951 and p38MAPK phosphorylation, but not ERK1/2. The level of phosphorylation of these residues was the same in the groups treated with BMOV supplemented with exogenous VEGF-A and exogenous VEGF-A only. Our study demonstrates that BMOV is able to enhance wound closure in vivo. Moreover, in the presence of endogenous VEGF-A, BMOV is able to stimulate in vitro angiogenesis by increasing the phosphorylation of VEGFR2 and its downstream proangiogenic enzymes. Importantly, BMOV had a stronger proangiogenic effect compared to its effect in coadministration with exogenous VEGF-A.
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http://dx.doi.org/10.3390/ijms21134643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370103PMC
June 2020

Three-Dimensional Imaging of Intraplaque Neovascularization in a Mouse Model of Advanced Atherosclerosis.

J Vasc Res 2020 1;57(6):348-354. Epub 2020 Jul 1.

Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium,

Multiple lines of evidence suggest that intraplaque (IP) neovascularization promotes atherosclerotic plaque growth, destabilization, and rupture. However, pharmacological inhibition of IP neovascularization remains largely unexplored due to the limited number of animal models that develop IP neovessels and the lack of reliable methods for visualizing IP angiogenesis. Here, we applied 3D confocal microscopy with an optimized tissue-clearing process, immunolabeling-enabled three-dimensional imaging of solvent-cleared organs, to visualize IP neovessels in apolipoprotein E-deficient (ApoE-/-) mice carrying a heterozygous mutation (C1039+/-) in the fibrillin-1 gene. Unlike regular ApoE-/- mice, this mouse model is characterized by the presence of advanced plaques with evident IP neovascularization. Plaques were stained with antibodies against endothelial marker CD31 for 3 days, followed by incubation with fluorescently labeled secondary antibodies. Subsequent tissue clearing with dichloromethane (DCM)/methanol, DCM, and dibenzyl ether allowed easy visualization and 3D reconstruction of the IP vascular network while plaque morphology remained intact.
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http://dx.doi.org/10.1159/000508449DOI Listing
January 2021

The Role of Immunomodulation in Vein Graft Remodeling and Failure.

J Cardiovasc Transl Res 2021 02 16;14(1):100-109. Epub 2020 Jun 16.

Department of Vascular Surgery, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands.

Obstructive arterial disease is a major cause of morbidity and mortality in the developed world. Venous bypass graft surgery is one of the most frequently used revascularization strategies despite its considerable short and long time failure rate. Due to vessel wall remodeling, inflammation, intimal hyperplasia, and accelerated atherosclerosis, vein grafts may (ultimately) fail to revascularize tissues downstream to occlusive atherosclerotic lesions. In the past decades, little has changed in the prevention of vein graft failure (VGF) although new insights in the role of innate and adaptive immunity in VGF have emerged. In this review, we discuss the pathophysiological mechanisms underlying the development of VGF, emphasizing the role of immune response and associated factors related to VG remodeling and failure. Moreover, we discuss potential therapeutic options that can improve patency based on data from both preclinical studies and the latest clinical trials. This review contributes to the insights in the role of immunomodulation in vein graft failure in humans. We describe the effects of immune cells and related factors in early (thrombosis), intermediate (inward remodeling and intimal hyperplasia), and late (intimal hyperplasia and accelerated atherosclerosis) failure based on both preclinical (mouse) models and clinical data.
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http://dx.doi.org/10.1007/s12265-020-10001-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892738PMC
February 2021

Arteriogenesis and Therapeutic Angiogenesis in Its Multiple Aspects.

Cells 2020 06 10;9(6). Epub 2020 Jun 10.

Department of Surgery, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

Arteriogenesis, also frequently called collateral formation or even therapeutic angiogenesis, comprises those processes that lead to the formation and growth of collateral blood vessels that can act as natural bypasses to restore blood flow to distal tissues in occluded arteries [...].
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http://dx.doi.org/10.3390/cells9061439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349222PMC
June 2020

From Increased Fluid Shear Stress to Natural Bypass Growth.

Int J Mol Sci 2020 May 25;21(10). Epub 2020 May 25.

Department of Surgery, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

This Special Issue enqueues a series of publications dealing with arteriogenesis, which is the growth of a natural bypass from pre-existing arteriolar connections, as defined by Wolfgang Schaper, Werner Risau and Ramon Munoz-Chapuli in the late nineties of the last century [...].
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http://dx.doi.org/10.3390/ijms21103707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279397PMC
May 2020

Myostatin Inhibits Vascular Smooth Muscle Cell Proliferation and Local 14q32 microRNA Expression, But Not Systemic Inflammation or Restenosis.

Int J Mol Sci 2020 May 15;21(10). Epub 2020 May 15.

Department of Surgery, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

Myostatin is a negative regulator of muscle cell growth and proliferation. Furthermore, myostatin directly affects the expression of 14q32 microRNAs by binding the 14q32 locus. Direct inhibition of 14q32 microRNA miR-495-3p decreased postinterventional restenosis via inhibition of both vascular smooth muscle cell (VSMC) proliferation and local inflammation. Here, we aimed to investigate the effects of myostatin in a mouse model for postinterventional restenosis. In VSMCs in vitro, myostatin led to the dose-specific downregulation of 14q32 microRNAs miR-433-3p, miR-494-3p, and miR-495-3p. VSMC proliferation was inhibited, where cell migration and viability remained unaffected. In a murine postinterventional restenosis model, myostatin infusion did not decrease restenosis, neointimal area, or lumen stenosis. Myostatin inhibited expression of both proliferation marker PCNA and of 14q32 microRNAs miR-433-3p, miR-494-3p, and miR-495-3p dose-specifically in cuffed femoral arteries. However, 14q32 microRNA expression remained unaffected in macrophages and macrophage activation as well as macrophage influx into lesions were not decreased. In conclusion, myostatin did not affect postinterventional restenosis. Although myostatin inhibits 14q32 microRNA expression and proliferation in VSMCs, myostatin had no effect on macrophage activation and infiltration. Our findings underline that restenosis is driven by both VSMC proliferation and local inflammation. Targeting only one of these components is insufficient to prevent restenosis.
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http://dx.doi.org/10.3390/ijms21103508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278907PMC
May 2020

Therapeutic Antibody Against Phosphorylcholine Preserves Coronary Function and Attenuates Vascular F-FDG Uptake in Atherosclerotic Mice.

JACC Basic Transl Sci 2020 Apr 25;5(4):360-373. Epub 2020 Mar 25.

Turku PET Centre, University of Turku, Turku, Finland.

This study showed that treatment with a therapeutic monoclonal immunoglobulin-G1 antibody against phosphorylcholine on oxidized phospholipids preserves coronary flow reserve and attenuates atherosclerotic inflammation as determined by the uptake of F-fluorodeoxyglucose in atherosclerotic mice. The noninvasive imaging techniques represent translational tools to assess the efficacy of phosphorylcholine-targeted therapy on coronary artery function and atherosclerosis in clinical studies.
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http://dx.doi.org/10.1016/j.jacbts.2020.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188869PMC
April 2020

Loss of Endothelial Glycocalyx Hyaluronan Impairs Endothelial Stability and Adaptive Vascular Remodeling After Arterial Ischemia.

Cells 2020 03 29;9(4). Epub 2020 Mar 29.

The Einthoven laboratory for Vascular and Regenerative Medicine, Department of Internal Medicine, Division of Nephrology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands.

We recently reported that loss of hyaluronan (HA) from the endothelial glycocalyx leads to loss of vessel stability in specific microcirculatory vascular beds. Here we hypothesized that such derangements in the glycocalyx may also impair the adaptive response to vascular ischemia. Endothelial specific conditional hyaluronan synthase 2-KO (Has2-cKO) mice revealed reduced endothelial HA expression and lower hindlimb perfusion at baseline compared to control mice. After a single ligation of the common femoral artery in these mice, we observed dysregulated angiogenesis in the gastrocnemius muscle which did not restore capillary perfusion. Mechanistically, decreased endothelial binding of the pericyte-derived molecule angiopoietin1 (Ang1) could be observed in the Has2-cKO mouse. In vitro angiogenesis assays with an endothelial cell-pericyte coculture confirmed such disturbed Ang1-TIE2 signaling resulting in excessive angiogenesis upon loss of HA. These data could be of relevance to diabetes patients, where we confirm loss of endothelial HA in the microcirculation of muscle tissue, indicating that this may contribute to the known disturbed adaptation to ischemia in these patients. In summary, loss of endothelial HA results in impaired microvascular perfusion and endothelial stability in ischemic gastrocnemius muscle. Endothelial HA is a potential target to improve angiogenic therapy in diabetic patients with critical limb ischemia.
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http://dx.doi.org/10.3390/cells9040824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226746PMC
March 2020

Local Mast Cell Activation Promotes Neovascularization.

Cells 2020 03 12;9(3). Epub 2020 Mar 12.

Department of Surgery, Leiden University Medical Center, 2300RC Leiden, The Netherlands.

Mast cells have been associated with arteriogenesis and collateral formation. In advanced human atherosclerotic plaques, mast cells have been shown to colocalize with plaque neovessels, and mast cells have also been associated with tumor vascularization. Based on these associations, we hypothesize that mast cells promote angiogenesis during ischemia. In human ischemic muscle tissue from patients with end-stage peripheral artery disease, we observed activated mast cells, predominantly located around capillaries. Also, in mouse ischemic muscles, mast cells were detected during the revascularization process and interestingly, mast cell activation status was enhanced up to 10 days after ischemia induction. To determine whether mast cells contribute to both arteriogenesis and angiogenesis, mast cells were locally activated immediately upon hind limb ischemia in C57Bl/6 mice. At day 9, we observed a 3-fold increase in activated mast cell numbers in the inguinal lymph nodes. This was accompanied by an increase in the amount of Ly6C inflammatory monocytes. Interestingly, local mast cell activation increased blood flow through the hind limb (46% at day 9) compared to that in non-activated control mice. Histological analysis of the muscle tissue revealed that mast cell activation did not affect the number of collaterals, but increased the collateral diameter, as well as the number of CD31 capillaries. Together, these data illustrate that locally activated mast cell contribute to arteriogenesis and angiogenesis.
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http://dx.doi.org/10.3390/cells9030701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140680PMC
March 2020

Prolonged Hyperoxygenation Treatment Improves Vein Graft Patency and Decreases Macrophage Content in Atherosclerotic Lesions in ApoE3*Leiden Mice.

Cells 2020 02 1;9(2). Epub 2020 Feb 1.

Department of surgery; Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

Unstable atherosclerotic plaques frequently show plaque angiogenesis which increases the chance of rupture and thrombus formation leading to infarctions. Hypoxia plays a role in angiogenesis and inflammation, two processes involved in the pathogenesis of atherosclerosis. We aim to study the effect of resolution of hypoxia using carbogen gas (95% O, 5% CO) on the remodeling of vein graft accelerated atherosclerotic lesions in ApoE3*Leiden mice which harbor plaque angiogenesis. Single treatment resulted in a drastic decrease of intraplaque hypoxia, without affecting plaque composition. Daily treatment for three weeks resulted in 34.5% increase in vein graft patency and increased lumen size. However, after three weeks intraplaque hypoxia was comparable to the controls, as were the number of neovessels and the degree of intraplaque hemorrhage. To our surprise we found that three weeks of treatment triggered ROS accumulation and subsequent Hif1a induction, paralleled with a reduction in the macrophage content, pointing to an increase in lesion stability. Similar to what we observed in vivo, in vitro induction of ROS in bone marrow derived macrophages lead to increased Hif1a expression and extensive DNA damage and apoptosis. Our study demonstrates that carbogen treatment did improve vein graft patency and plaque stability and reduced intraplaque macrophage accumulation via ROS mediated DNA damage and apoptosis but failed to have long term effects on hypoxia and intraplaque angiogenesis.
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http://dx.doi.org/10.3390/cells9020336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072413PMC
February 2020
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