Maarten M. Brandt

MicroRNA-132/212 family enhances arteriogenesis after hindlimb ischaemia through modulation of the Ras-MAPK pathway

Arteriogenesis is a complicated process induced by increased local shear‐and radial wall‐stress, leading to an increase in arterial diameter. This process is enhanced by growth factors secreted by both inflammatory and endothelial cells in response to physical stress. Although therapeutic promotion of arteriogenesis is of great interest for ischaemic diseases, little is known about the modulation of the signalling cascades via microRNAs. We observed that miR‐132/212 expression was significantly upregulated after occlusion of the femoral artery. miR‐132/212 knockout (KO) mice display a slower perfusion recovery after hind‐limb ischaemia compared to wildtype (WT) mice. Immunohistochemical analysis demonstrates a clear trend towards smaller collateral arteries in KO mice. Although Ex vivo aortic ring assays score similar number of branches in miR‐132/212 KO mice compared to WT, it can be stimulated with exogenous miR‐132, a dominant member of the miR‐132/212 family. Moreover, in in vitro pericyte‐endothelial co‐culture cell assays, overexpression of miR‐132 and mir‐212 in endothelial cells results in enhanced vascularization, as shown by an increase in tubular structures and junctions. Our results suggested that miR‐132/212 may exert their effects by enhancing the Ras‐Mitogen‐activated protein kinases MAPK signalling pathway through direct inhibition of Rasa1, and Spred1. The miR‐132/212 cluster promotes arteriogenesis by modulating Ras‐MAPK signalling via direct targeting of its inhibitors Rasa1 and Spred1.

Distinct endothelial cell responses in the heart and kidney microvasculature characterize the progression of heart failure with preserved ejection fraction in the obese ZSF1 rat with cardiorenal metabolic syndrome

Background—The combination of cardiac and renal disease driven by metabolic risk factors, referred to as cardiorenal metabolic syndrome (CRMS), is increasingly recognized as a critical pathological entity. The contribution of (micro)vascular injury to CRMS is considered to be substantial. However, mechanistic studies are hampered by lack of in vivo models that mimic the natural onset of the disease. Here, we evaluated the coronary and renal microvasculature during CRMS development in obese diabetic Zucker fatty/Spontaneously hypertensive heart failure F1 hybrid (ZSF1) rats. Methods and Results—Echocardiographic, urine, and blood evaluations were conducted in 3 groups (Wistar-Kyoto, lean ZSF1, and obese ZSF1) at 20 and 25 weeks of age. Immunohistological evaluation of renal and cardiac tissues was conducted at both time points. At 20 and 25 weeks, obese ZSF1 rats showed higher body weight, significant left ventricular hypertrophy, and impaired diastolic function compared with all other groups. Indices of systolic function did not differ between groups. Obese ZSF1 rats developed hyperproliferative vascular foci in the subendocardium, which lacked microvascular organization and were predilection sites of inflammation and fibrosis. In the kidney, obese ZSF1 animals showed regression of the peritubular and glomerular microvasculature, accompanied by tubulointerstitial damage, glomerulosclerosis, and proteinuria. Conclusions—The obese ZSF1 rat strain is a suitable in vivo model for CRMS, sharing characteristics with the human syndrome during the earliest onset of disease. In these rats, CRMS induces microvascular fibrotic responses in heart and kidneys, associated with functional impairment of both organs.

Absence of Chemokine (C-X-C Motif) Ligand 10 Diminishes Perfusion Recovery After Local Arterial Occlusion in Mice

Objective— In arteriogenesis, pre-existing anastomoses undergo enlargement to restore blood flow in ischemic tissues. Chemokine (C-X-C motif) ligand 10 (CXCL10) is secreted after Toll-like receptor activation. Toll-like receptors are involved in arteriogenesis; however, the role of CXCL10 is still unclear. In this study, we investigated the role for CXCL10 in a murine hindlimb ischemia model. Approach and Results— Unilateral femoral artery ligation was performed in wild-type (WT) and CXCL10−/− knockout (KO) mice and perfusion recovery was measured using laser-Doppler perfusion analysis. Perfusion recovery was significantly lower in KO mice compared with WT at days 4 and 7 after surgery (KO versus WT: 28±5% versus 81±13% at day 4; P=0.003 and 57±12% versus 107±8% at day 7; P=0.003). Vessel measurements of &agr;-smooth muscle actin–positive vessels revealed increasing numbers in time after surgery, which was significantly higher in WT when compared with that in KO. Furthermore, &agr;-smooth muscle actin–positive vessels were significantly larger in WT when compared with those in KO at day 7 (wall thickness, P<0.001; lumen area, P=0.003). Local inflammation was assessed in hindlimb muscles, but this did not differ between WT and KO. Chimerization experiments analyzing perfusion recovery and histology revealed an equal contribution for bone marrow–derived and circulating CXCL10. Migration assays showed a stimulating role for both intrinsic and extrinsic CXCL10 in vascular smooth muscle cell migration. Conclusions— CXCL10 plays a causal role in arteriogenesis. Bone marrow–derived CXCL10 and tissue-derived CXCL10 play a critical role in accelerating perfusion recovery after arterial occlusion in mice probably by promoting vascular smooth muscle cell recruitment and maturation of pre-existing anastomoses.

THSD1 preserves vascular integrity and protects against intraplaque haemorrhaging in ApoE−/− mice

AIMS Impairment of the endothelial barrier leads to microvascular breakdown in cardiovascular disease and is involved in intraplaque haemorrhaging and the progression of advanced atherosclerotic lesions that are vulnerable to rupture. The exact mechanism that regulates vascular integrity requires further definition. Using a microarray screen for angiogenesis-associated genes during murine embryogenesis, we identified thrombospondin type I domain 1 (THSD1) as a new putative angiopotent factor with unknown biological function. We sought to characterize the role of THSD1 in endothelial cells during vascular development and cardiovascular disease. METHODS AND RESULTS Functional knockdown of Thsd1 in zebrafish embryos and in a murine retina vascularization model induced severe haemorrhaging without affecting neovascular growth. In human carotid endarterectomy specimens, THSD1 expression by endothelial cells was detected in advanced atherosclerotic lesions with intraplaque haemorrhaging, but was absent in stable lesions, implying involvement of THSD1 in neovascular bleeding. In vitro, stimulation with pro-atherogenic factors (3% O2 and TNFα) decreased THSD1 expression in human endothelial cells, whereas stimulation with an anti-atherogenic factor (IL10) showed opposite effect. Therapeutic evaluation in a murine advanced atherosclerosis model showed that Thsd1 overexpression decreased plaque vulnerability by attenuating intraplaque vascular leakage, subsequently reducing macrophage accumulation and necrotic core size. Mechanistic studies in human endothelial cells demonstrated that THSD1 activates FAK-PI3K, leading to Rac1-mediated actin cytoskeleton regulation of adherens junctions and focal adhesion assembly. CONCLUSION THSD1 is a new regulator of endothelial barrier function during vascular development and protects intraplaque microvessels against haemorrhaging in advanced atherosclerotic lesions.

Activation of CECR1 in M2-like TAMs promotes paracrine stimulation-mediated glial tumor progression

BACKGROUND The majority of glioma-associated microglia/macrophages have been identified as M2-type macrophages with immune suppressive and tumor supportive action. Recently, the extracellular adenosine deaminase protein Cat Eye Syndrome Critical Region Protein 1 (CECR1) was shown to regulate macrophage maturation. In this study, we investigate the role of CECR1 in the regulation of the glioma-associated macrophage response. METHODS Expression of CECR1 was assessed in human glioma samples. CECR1-mediated macrophage response was studied in vitro, using donor derived CD14+ monocytes and the THP-1 monocytic cell line. The response of the human glioma cell line U87 to conditioned medium of macrophages preconditioned with recombinant human CECR1 or CECR1 silencing was also assessed. RESULTS CECR1 was strongly expressed in high-grade gliomas (P < .001) and correlated positively with the M2 phenotype markers in tumor-associated microglia/macrophages (TAMs) (overall, P < .05). In vitro studies confirmed the presence of a significantly higher level of CECR1 expression in M2-like macrophages exposed to U87 conditioned medium (P < .001). CECR1 knockdown or stimulation of macrophages affected differentiation toward the M2-like phenotype. Stimulation of U87 cells with conditioned medium of CECR1 knockdown or stimulated macrophages affected tumor cell proliferation and migration, coinciding with altered intracellular signaling of mitogen-activated protein kinase (MAPK). In glioma tissue samples, CECR1 expression correlated with Ki67 and MAPK signaling protein. CONCLUSIONS CECR1 is a potent regulator of TAM polarization and is consistently highly expressed by M2-type TAMs, particularly in high-grade glioma. Paracrine effects induced by CECR1 in M2-like TAMs activate MAPK signaling and stimulate the proliferation and migration of glioma cells.Background The majority of glioma-associated microglia/macrophages have been identified as M2-type macrophages with immune suppressive and tumor supportive action. Recently, the extracellular adenosine deaminase protein Cat Eye Syndrome Critical Region Protein 1 (CECR1) was shown to regulate macrophage maturation. In this study, we investigate the role of CECR1 in the regulation of the glioma-associated macrophage response. Methods Expression of CECR1 was assessed in human glioma samples. CECR1-mediated macrophage response was studied in vitro, using donor derived CD14+ monocytes and the THP-1 monocytic cell line. The response of the human glioma cell line U87 to conditioned medium of macrophages preconditioned with recombinant human CECR1 or CECR1 silencing was also assessed. Results CECR1 was strongly expressed in high-grade gliomas (P < .001) and correlated positively with the M2 phenotype markers in tumor-associated microglia/macrophages (TAMs) (overall, P < .05). In vitro studies confirmed the presence of a significantly higher level of CECR1 expression in M2-like macrophages exposed to U87 conditioned medium (P < .001). CECR1 knockdown or stimulation of macrophages affected differentiation toward the M2-like phenotype. Stimulation of U87 cells with conditioned medium of CECR1 knockdown or stimulated macrophages affected tumor cell proliferation and migration, coinciding with altered intracellular signaling of mitogen-activated protein kinase (MAPK). In glioma tissue samples, CECR1 expression correlated with Ki67 and MAPK signaling protein. Conclusions CECR1 is a potent regulator of TAM polarization and is consistently highly expressed by M2-type TAMs, particularly in high-grade glioma. Paracrine effects induced by CECR1 in M2-like TAMs activate MAPK signaling and stimulate the proliferation and migration of glioma cells.

Additional Candidate Genes for Human Atherosclerotic Disease Identified Through Annotation Based on Chromatin OrganizationCLINICAL PERSPECTIVE

Background— As genome-wide association efforts, such as CARDIoGRAM and METASTROKE, are ongoing to reveal susceptibility loci for their underlying disease—atherosclerotic disease—identification of candidate genes explaining the associations of these loci has proven the main challenge. Many disease susceptibility loci colocalize with DNA regulatory elements, which influence gene expression through chromatin interactions. Therefore, the target genes of these regulatory elements can be considered candidate genes. Applying these biological principles, we used an alternative approach to annotate susceptibility loci and identify candidate genes for human atherosclerotic disease based on circular chromosome conformation capture followed by sequencing. Methods and Results— In human monocytes and coronary endothelial cells, we generated 63 chromatin interaction data sets for 37 active DNA regulatory elements that colocalize with known susceptibility loci for coronary artery disease (CARDIoGRAMplusC4D) and large artery stroke (METASTROKE). By circular chromosome conformation capture followed by sequencing, we identified a physical 3-dimensional interaction with 326 candidate genes expressed in at least 1 of these cell types, of which 294 have not been reported before. We highlight 16 genes based on expression quantitative trait loci. Conclusions— Our findings provide additional candidate-gene annotation for 37 disease susceptibility loci for human atherosclerotic disease that are of potential interest to better understand the complex pathophysiology of cardiovascular diseases.

CMTM3 (CKLF-Like Marvel Transmembrane Domain 3) Mediates Angiogenesis by Regulating Cell Surface Availability of VE-Cadherin in Endothelial Adherens Junctions.

Objective— Decrease in VE-cadherin adherens junctions reduces vascular stability, whereas disruption of adherens junctions is a requirement for neovessel sprouting during angiogenesis. Endocytosis plays a key role in regulating junctional strength by altering bioavailability of cell surface proteins, including VE-cadherin. Identification of new mediators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we assessed the function of CMTM3 (CKLF-like MARVEL transmembrane domain 3), which we have previously identified as highly expressed in Flk1+ endothelial progenitor cells during embryonic development. Approach and Results— Using a 3-dimensional coculture of human umbilical vein endothelial cells-GFP (green fluorescent protein) and pericytes-RFP (red fluorescent protein), we demonstrated that siRNA-mediated CMTM3 silencing in human umbilical vein endothelial cells impairs angiogenesis. In vivo CMTM3 inhibition by morpholino injection in developing zebrafish larvae confirmed that CMTM3 expression is required for vascular sprouting. CMTM3 knockdown in human umbilical vein endothelial cells does not affect proliferation or migration. Intracellular staining demonstrated that CMTM3 colocalizes with early endosome markers EEA1 (early endosome marker 1) and Clathrin+ vesicles and with cytosolic VE-cadherin in human umbilical vein endothelial cells. Adenovirus-mediated CMTM3 overexpression enhances endothelial endocytosis, shown by an increase in Clathrin+, EEA1+, Rab11+, Rab5+, and Rab7+ vesicles. CMTM3 overexpression enhances, whereas CMTM3 knockdown decreases internalization of cell surface VE-cadherin in vitro. CMTM3 promotes loss of endothelial barrier function in thrombin-induced responses, shown by transendothelial electric resistance measurements in vitro. Conclusions— In this study, we have identified a new regulatory function for CMTM3 in angiogenesis. CMTM3 is involved in VE-cadherin turnover and is a regulator of the cell surface pool of VE-cadherin. Therefore, CMTM3 mediates cell–cell adhesion at adherens junctions and contributes to the control of vascular sprouting.

Abstract 2348: Expression of CECR1 by activated M2-type macrophages in glioma

Objectives Glial neoplasms harbor macrophages which are mainly of the M2 type and secrete Th2 cytokines. These M2 macrophages are not only involved in immune suppression but also serve functions in angiogenesis. Recent studies have shown that the adenosine deaminase CECR1 is involved in the maintenance of vascular homeostasis and M2-macrophage differentiation. Here we aim to identify the expressional levels of CECR1 in M1 and M2 macrophage populations in gliomas and investigate the role of these cells in glioma development. Methods To identify the relation between CECR1 expression and the macrophage subtypes, tissue samples of human glioma and normal brain controls were used for Real Time PCR and immunostaining. For in vitro functional studies, we generated M1 and M2-like macrophages by differentiating CD14+ monocytes isolated from human peripheral blood in GM-CSF and M-CSF medium. With and without stimulation of GBM conditioned medium, CECR1 level was measured by RT-PCR and western blot. CECR1 protein was added to the macrophage culture medium and the expression level of the M1 marker CD80 and the M2 marker CD163 were measured by flow cytometry and confocal microscopy. Results The macrophages were predominantly present in perivascular spaces, but also in the tumor tissue and neuropil of the normal brain. At the mRNA level the expression of CECR1 was positively correlated with the M2 markers CD204 and IL-10. Immunohistochemical investigation revealed that CECR1 is localized in microglia and macrophages in low-grade gliomas and control brain and it overlaps and co-localizes with the M2-markers CD204 and CD163, but not the M1-marker CD80. In vitro experiments showed that CECR1 expression is higher in the M-CSF-induced M2-macrophages than in their GM-CSF-induced M1 counterparts. Under the stimulation of GBM conditioned medium, increased CECR1 is detected in both M1 and M2-like macrophages. In addition, exogenous CECR1 greatly affects both M-CSF and GM-CSF induced macrophages’ response by up-regulating CD163 in M1-like macrophages and increasing CD163 positive cells in M2-like macrophages. As a result, CECR1 skews macrophage differentiation towards the M2 phenotype. Conclusion This study is the first to show that the M2-macrophage, not the M1-macrophage in glioma, is the main source of CECR1. In addition, CECR1 is shown to be a potent regulator of M2-macrophages. High levels of CECR1 is able to skew GM-CSF driven M1 macrophage differentiation towards an M2 macrophage phenotype. We propose that CECR1 expression by macrophages promotes M2-macrophage differentiation under the influence of an adenosine-rich glioma microenvironment to support tumor growth and tumor angiogenesis. The findings may well direct the search for new anti-angiogenic target molecules for the treatment of glioma. Citation Format: Changbin Zhu, Marcel M. van der Weiden, Adrea Scchetti, Thierry P.P. van den Bosch, Ihsan Chrifi, Maarten M. Brandt, Dana A.M. Mustafa, Caroline Cheng, Johan M. Kros. Expression of CECR1 by activated M2-type macrophages in glioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2348. doi:10.1158/1538-7445.AM2015-2348

Folic acid reduces doxorubicin-induced cardiomyopathy by modulating endothelial nitric oxide synthase

The use of doxorubicin (DOXO) as a chemotherapeutic drug has been hampered by cardiotoxicity leading to cardiomyopathy and heart failure. Folic acid (FA) is a modulator of endothelial nitric oxide (NO) synthase (eNOS), which in turn is an important player in diseases associated with NO insufficiency or NOS dysregulation, such as pressure overload and myocardial infarction. However, the role of FA in DOXO‐induced cardiomyopathy is poorly understood. The aim of this study was to test the hypothesis that FA prevents DOXO‐induced cardiomyopathy by modulating eNOS and mitochondrial structure and function. Male C57BL/6 mice were randomized to a single dose of DOXO (20 mg/kg intraperitoneal) or sham. FA supplementation (10 mg/day per oral) was started 7 days before DOXO injection and continued thereafter. DOXO resulted in 70% mortality after 10 days, with the surviving mice demonstrating a 30% reduction in stroke volume compared with sham groups. Pre‐treatment with FA reduced mortality to 45% and improved stroke volume (both P < 0.05 versus DOXO). These effects of FA were underlain by blunting of DOXO‐induced cardiomyocyte atrophy, apoptosis, interstitial fibrosis and impairment of mitochondrial function. Mechanistically, pre‐treatment with FA prevented DOXO‐induced increases in superoxide anion production by reducing the eNOS monomer:dimer ratio and eNOS S‐glutathionylation, and attenuated DOXO‐induced decreases in superoxide dismutase, eNOS phosphorylation and NO production. Enhancing eNOS function by restoring its coupling and subsequently reducing oxidative stress with FA may be a novel therapeutic approach to attenuate DOXO‐induced cardiomyopathy.