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Background Arteriovenous fistulae (AVFs) are the gold standard for vascular access in those requiring hemodialysis but may put an extra hemodynamic stress on the cardiovascular system. The complex interactions between the heart, kidney, and AVFs remain incompletely understood. Methods and Results We characterized a novel rat model of five‐sixths partial nephrectomy (NX) and AVFs. NX induced increases in urea, creatinine, and hippuric acid. The addition of an AVF (AVF+NX) further increased urea and a number of uremic toxins such as trimethylamine N‐oxide and led to increases in cardiac index, left and right ventricular volumes, and right ventricular mass. Plasma levels of uremic toxins correlated well with ventricular morphology and function. Heart transcriptomes identified altered expression of 8 genes following NX and 894 genes following AVF+NX, whereas 290 and 1431 genes were altered in the kidney transcriptomes, respectively. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed gene expression changes related to cell division and immune activation in both organs, suppression of ribosomes and transcriptional activity in the heart, and altered renin‐angiotensin signaling as well as chronodisruption in the kidney. All except the latter were worsened in AVF+NX compared with NX. Conclusions Inflammation and organ dysfunction in chronic kidney disease are exacerbated following AVF creation. Furthermore, our study provides important information for the discovery of novel biomarkers and therapeutic targets in the management of cardiorenal syndrome.

Aims During embryonic development, arteriovenous (AV) differentiation ensures proper blood vessel formation and maturation. Defects in arterial or venous identity cause inappropriate fusion of vessels, resulting in atypical shunts, so-called AV malformations (AVMs). Currently, the mechanism behind AVM formation remains unclear, and treatment options are fairly limited. Mammalian AV differentiation is initiated before the onset of blood flow in the embryo; however, this pre-flow mechanism is poorly understood. Here, we aimed to unravel the role of Smad1/5 signalling in pre-flow arterial identity and, in the process, uncovered an unexpected control mechanism of Smad1/5 signalling. Methods and results We establish that despite Notch1 being expressed in the pre-flow mouse embryo, it is not activated, nor is it necessary for the expression of the earliest arterial genes in the dorsal aortae (i.e. Hey1 and Gja4). Furthermore, interrupting blood flow by using the Ncx1 KO model completely prevents the activation of Notch1 signalling, suggesting a strong role of shear stress in maintaining arterial identity. We demonstrate that early expression of Hey1 and Gja4 requires SMAD1/5 signalling. Using embryo cultures, we show that Smad1/5 signalling is activated through the Alk1/Alk5/transforming growth factor (TGF)βR2 receptor complex, with TGFβ1 as a necessary ligand. Furthermore, our findings demonstrate that early arterial gene expression requires the acetylation of Smad1/5 proteins, rendering them more sensitive to TGFβ1 stimulation. Blocking acetyl-CoA production prevents pre-flow arterial expression of Hey1 and Gja4 , while stabilizing acetylation rescues their expression. Conclusion Our findings highlight the importance of the acetyl-CoA production in the cell and provide a novel control mechanism of Smad1/5 signalling involving protein acetylation. As disturbed canonical Smad1/5 signalling is involved in several vascular conditions, our results offer new insights in treatment options for circumventing canonical Smad1/5 signalling. [ABSTRACT FROM AUTHOR]

Abstract Background Cell therapy has been evaluated pre-clinically and clinically as a means to improve wound vascularization and healing. While translation of this approach to clinical practice ideally requires the availability of clinical grade xenobiotic-free cell preparations, studies proving the pre-clinical efficacy of the latter are mostly lacking. Here, the potential of xenobiotic-free human multipotent adult progenitor cell (XF-hMAPC®) preparations to promote vascularization was evaluated. Methods The potential of XF-hMAPC cells to support blood vessel formation was first scored in an in vivo Matrigel assay in mice. Next, a dose-response study was performed with XF-hMAPC cells in which they were tested for their ability to support vascularization and (epi) dermal healing in a physiologically relevant splinted wound mouse model. Results XF-hMAPC cells supported blood vessel formation in Matrigel by promoting the formation of mature (smooth muscle cell-coated) vessels. Furthermore, XF-hMAPC cells dose-dependently improved wound vascularization associated with increasing wound closure and re-epithelialization, granulation tissue formation, and dermal collagen organization. Conclusions Here, we demonstrated that the administration of clinical-grade XF-hMAPC cells in mice represents an effective approach for improving wound vascularization and healing that is readily applicable for translation in humans.

Platelet Endothelial Aggregation Receptor 1 (PEAR1) modulates angiogenesis and platelet contact-induced activation, which play a role in the pathogenesis of colorectal cancer. We therefore tested the association of incident colorectal cancer and genetic and epigenetic variability in PEAR1 among 2532 randomly recruited participants enrolled in the family-based Flemish Study on Environment, Genes and Health Outcomes (51.2% women; mean age 44.8 years). All underwent genotyping of rs12566888 located in intron 1 of the PEAR1 gene; in 926 participants, methylation at 16 CpG sites in the PEAR1 promoter was also assessed. Over 18.1 years (median), 49 colorectal cancers occurred, all in different pedigrees. While accounting for clustering of risk factors within families and adjusting for sex, age, body mass index, the total-to-HDL cholesterol ratio, serum creatinine, plasma glucose, smoking and drinking, use of antiplatelet and nonsteroidal anti-inflammatory drug, the hazard ratio of colorectal cancer contrasting minor-allele (T) carriers vs. major-allele (GG) homozygotes was 2.17 (95% confidence interval, 1.18-3.99; P = 0.013). Bootstrapped analyses, from which we randomly excluded from two to nine cancer cases, provided confirmatory results. In participants with methylation data, we applied partial least square discriminant analysis (PLS-DA) and identified two methylation sites associated with higher colorectal cancer risk and two with lower risk. In-silico analysis suggested that methylation of the PEAR1 promoter at these four sites might affect binding of transcription factors p53, PAX5, and E2F-1, thereby modulating gene expression. In conclusion, our findings suggest that genetic and epigenetic variation in PEAR1 modulates the risk of colorectal cancer in white Flemish. To what extent, environmental factors as exemplified by our methylation data, interact with genetic predisposition and modulate penetrance of colorectal cancer risk is unknown.

Abstract Background Dental pulp represents an easily accessible autologous source of adult stem cells. A subset of these cells, named dental pulp pluripotent-like stem cells (DPPSC), shows high plasticity and can undergo multiple population doublings, making DPPSC an appealing tool for tissue repair or maintenance. Methods DPPSC were harvested from the dental pulp of third molars extracted from young patients. Growth factors released by DPPSC were analysed using antibody arrays. Cells were cultured in specific differentiation media and their endothelial, smooth and skeletal muscle differentiation potential was evaluated. The therapeutic potential of DPPSC was tested in a wound healing mouse model and in two genetic mouse models of muscular dystrophy (Scid/mdx and Sgcb-null Rag2-null γc-null). Results DPPSC secreted several growth factors involved in angiogenesis and extracellular matrix deposition and improved vascularisation in all three murine models. Moreover, DPPSC stimulated re-epithelialisation and ameliorated collagen deposition and organisation in healing wounds. In dystrophic mice, DPPSC engrafted in the skeletal muscle of both dystrophic murine models and showed integration in muscular fibres and vessels. In addition, DPPSC treatment resulted in reduced fibrosis and collagen content, larger cross-sectional area of type II fast-glycolytic fibres and infiltration of higher numbers of proangiogenic CD206+ macrophages. Conclusions Overall, DPPSC represent a potential source of stem cells to enhance the wound healing process and slow down dystrophic muscle degeneration.

Aims: Microvascular dysfunction has been proposed to drive heart failure with preserved ejection fraction (HFpEF), but the initiating molecular and cellular events are largely unknown. Our objective was to determine when microvascular alterations in HFpEF begin, how they contribute to disease progression, and how pericyte dysfunction plays a role herein. Methods and results: Microvascular dysfunction, characterized by inflammatory activation, loss of junctional barrier function, and altered pericyte–endothelial crosstalk, was assessed with respect to the development of cardiac dysfunction, in the Zucker fatty and spontaneously hypertensive (ZSF1) obese rat model of HFpEF at three time points: 6, 14, and 21 weeks of age. Pericyte loss was the earliest and strongest microvascular change, occurring before prominent echocardiographic signs of diastolic dysfunction were present. Pericytes were shown to be less proliferative and had a disrupted morphology at 14 weeks in the obese ZSF1 animals, who also exhibited an increased capillary luminal diameter and disrupted endothelial junctions. Microvascular dysfunction was also studied in a mouse model of chronic reduction in capillary pericyte coverage (PDGF-Bret/ret), which spontaneously developed many aspects of diastolic dysfunction. Pericytes exposed to oxidative stress in vitro showed downregulation of cell cycle-associated pathways and induced a pro-inflammatory state in endothelial cells upon co-culture. Conclusion: We propose pericytes are important for maintaining endothelial cell function, where loss of pericytes enhances the reactivity of endothelial cells to inflammatory signals and promotes microvascular dysfunction, thereby accelerating the development of HFpEF. Graphical Abstract [ABSTRACT FROM AUTHOR]

Background: The heart ejects in the central elastic arteries. No previous study addressed the question whether ECG voltages are more closely associated with central than with peripheral blood pressure (BP). Methods: Using the oscillometric Mobil-O-Graph 24 h PWA monitor, we measured brachial, central BP and central hemodynamics over 24 hours in 177 men (mean age, 29.1 years), and linked to ECG voltages. Results: From wakefulness to sleep, as documented by diaries, systolic/diastolic BP decreased by 11.7/13.1 mmHg peripherally and by 9.3/13.6 mmHg centrally, whereas pulse pressure (PP) increased by 4.3 mmHg. Over 24 hours and the awake and asleep periods, the peripheral-minus-central differences in systolic/diastolic BPs and pulse pressure averaged 11.8/–1.6, 12.7/–1.8 and 10.3/–1.2 mmHg and 13.4, 14.4 and 11.5 mmHg, respectively (P < 0.0001). Cornel voltage and index averaged 1.18 mV and 114.8 mV×ms. The Cornell voltages were 0.104/0.086 and 0.082/0.105 mV higher in relation to brachial 24-h and asleep systolic/diastolic BP (per 1-SD), respectively, and 0.088/0.90 mV and 0.087/0.107 mV higher in relation to central BP. The corresponding estimates for the Cornel indexes were 9.6/8.6 and 8.2/105 mV×ms peripherally and 8.6/8.9 and 8.8/10.7 mV×ms centrally. The regression slopes were similar for brachial and central BP (P≥0.054). Associations of the ECG measurements with awake BP, PP, the augmentation ratio and pressure amplification did not reach significance. Cornell voltage (SV3 + RaVL, mV) Cornell index (Cornell voltage × QRS duration, mV·ms) Peripheral BP Central BP Peripheral BP Central BP Estimate (95% CI) P Estimate (95% CI) P Estimate (95% CI) P Estimate (95% CI) P Systolic BP 24-h 0.104 (0.016 to 0.191) 0.021 0.088 (0.0003 to 0.177) 0.049 9.61 (0.65 to 18.57) 0.036 8.58 (−0.40 to 17.56) 0.061 Awake 0.086 (−0.001 to 0.175) 0.054 0.062 (−0.026 to 0.151) 0.17 7.69 (−1.30 to 16.69) 0.093 5.80 (−3.23 to 14.82) 0.21 Asleep 0.082 (−0.006 to 0.170) 0.068 0.087 (−0.001 to 0.175) 0.053 8.17 (−0.82 to 17.16) 0.075 8.76 (−0.217 to 17.74) 0.056 Diastolic BP 24-h 0.086 (−0.002 to 0.174) 0.056 0.090 (0.002 to 0.178) 0.045 8.57 (−0.41 to 17.55) 0.061 8.93 (−0.04 to 17.90) 0.051 Awake 0.056 (−0.032 to 0.145) 0.21 0.060 (−0.029 to 0.149) 0.18 5.62 (−3.42 to 14.65) 0.22 5.97 (−3.06 to 15.00) 0.19 Asleep BP 0.105 (0.017 to 0.192) 0.020 0.107 (0.019 to 0.194) 0.017 10.53 (1.60 to 19.47) 0.021 10.71 (1.78 to 19.64) 0.019 Pulse pressure 24-h 0.040 (−0.049 to 0.129) 0.38 0.016 (−0.073 to 0.105) 0.72 3.07 (−5.99 to 12.13) 0.50 1.31 (−7.76 to 10.38) 0.77 Awake 0.048 (−0.041 to 0.137) 0.29 0.012 (−0.077 to 0.101) 0.78 3.63 (−5.43 to 12.68) 0.43 0.68 (−8.40 to 9.74) 0.88 Asleep 0.001 (−0.091 to 0.088) 0.98 0.001 (−0.087 to 0.090) 0.98 −0.29 (−9.37 to 8.78) 0.95 0.21 (−8.86 to 9.28) 0.96 ECG refers to electrocardiography. BP stands for blood pressure. Cornell voltage is the voltage sum of S wave in precordial V3 lead (SV3) and R wave in limb aVL lead (ReVL), while Cornell index is the product of QRS duration multiplied by the Cornell voltage. The estimate (95% Confidence Interval, CI) of the association was unadjusted and expressed as 1-SD increase of BP. P value is for significance of the estimate. The association estimates of Cornell voltage (P ≥ 0.054) and index (P ≥ 0.079) with central BP were not significantly different from those estimates with peripheral measurements. TableAssociation of ECG Cornell voltage and indexes with peripheral and central BP. Conclusions: The diurnal rhythm of peripheral and central BP run in parallel. Central BP does not improve the association of Cornell voltage or index with peripheral BP.

Background: Central blood pressure (BP) is a predictor of target organ damage. No previous study addressed the question to what extent central compared with peripheral is related to left ventricular (LV) structure and function in a general population. Methods: In 577 Flemish recruited from the general population (47.8% women; mean age 50.5 years), we assessed the multivariable-adjusted associations of echocardiographic LV structure and systolic and diastolic LV function (Vivid7 Pro device; EchoPac software, version 4.0.4; GE Vingmed, Horten, Norway) with peripheral and central pressure, as recorded by radial applanation tonometry (SphygmoCor software, version 9.0). Association sizes were expressed per 15/10 mmHg increment in peripheral or central systolic/diastolic BP. Results: Peripheral compared with central systolic BP was 10.2 mm Hg higher (P < 0.0001), whereas diastolic BP was similar peripherally and centrally (P = 0.50). Associations were closer (P≤0.020) with central than peripheral systolic BP for LV mass (+0.59 g/m2) and left atrial volume (+0.29 ml/m2) indexed to body surface area, peak A transmitral flow (+0.12 cm/s), peak e′ mitral annular movement (−0.18 cm/s) and the E/A ratio (−0.017). Associations were closer (P≤0.038) with central than peripheral diastolic BP for left atrial volume index (+0.289 ml/m2), e′ (−0.123 cm/s) and E/e′ (+0.094). Ejection fraction and global longitudinal strain were similarly associated with central and peripheral systolic (P≥0.62) and diastolic (P≥0.18) BP. Conclusions: In asymptomatic people recruited from the general population, LV mass and atrial volume indexes and selected haemodynamic measurements reflecting diastolic LV function are slightly but significantly closer associated with central than peripheral BP.

Summary The importance of the blood- and lymph vessels in the transport of essential fluids, gases, macromolecules and cells in vertebrates warrants optimal insight into the regulatory mechanisms underlying their development. Mouse and zebrafish models of lymphatic development are instrumental for gene discovery and gene characterization but are challenging for certain aspects, e.g. no direct accessibility of embryonic stages, or non-straightforward visualization of early lymphatic sprouting, respectively. We previously demonstrated that the Xenopus tadpole is a valuable model to study the processes of lymphatic development. However, a fluorescent Xenopus reporter directly visualizing the lymph vessels was lacking. Here, we created transgenic Tg(Flk1:eGFP) Xenopus laevis reporter lines expressing green fluorescent protein (GFP) in blood- and lymph vessels driven by the Flk1 (VEGFR-2) promoter. We also established a high-resolution fluorescent dye labeling technique selectively and persistently visualizing lymphatic endothelial cells, even in conditions of impaired lymph vessel formation or drainage function upon silencing of lymphangiogenic factors. Next, we applied the model to dynamically document blood and lymphatic sprouting and patterning of the initially avascular tadpole fin. Furthermore, quantifiable models of spontaneous or induced lymphatic sprouting into the tadpole fin were developed for dynamic analysis of loss-of-function and gain-of-function phenotypes using pharmacologic or genetic manipulation. Together with angiography and lymphangiography to assess functionality, Tg(Flk1:eGFP) reporter tadpoles readily allowed detailed lymphatic phenotyping of live tadpoles by fluorescence microscopy. The Tg(Flk1:eGFP) tadpoles represent a versatile model for functional lymph/angiogenomics and drug screening.

BackgroundExperimental studies have demonstrated that lead and cadmium have direct toxic effects on the myocardium, but the few human studies are limited by design, assessment of exposure, and use of heart failure as a late‐stage endpoint. Methods and ResultsIn a prospective population study, we studied the association of left ventricular (LV) function with blood lead (BPb) and 24‐hour urinary cadmium (UCd). In 179 participants randomly recruited from a Flemish population (50.3% women; mean age 39.1 years), geometric mean BPb and UCd at enrollment (1985‐2000) were 0.20 μmol/L and 6.1 nmol, respectively. We assessed systolic and diastolic LV function 11.9 years (median) later (2005‐2010) by using Doppler imaging of the transmitral blood flow and the mitral annular movement and speckle tracking. In multivariable‐adjusted linear regression, LV systolic function decreased with BPb. For a doubling of exposure, estimates were −0.392% for global longitudinal strain (P=0.034), −0.618% and −0.113 s−1 for regional longitudinal strain (P=0.028) and strain rate (P=0.008), and −0.056 s−1 for regional radial strain rate (P=0.050). Regional longitudinal strain rate (−0.066 s−1, P=0.009) and regional radial strain (−2.848%, P=0.015) also decreased with UCd. Models including both exposure indexes did not allow differentiating whether LV dysfunction was predominately related to BPb or UCd. Diastolic LV function was not associated with BPb or UCd (P≥0.159). ConclusionsAlthough effect sizes were small, our results suggest that environmental exposure to lead, cadmium, or both might be a risk factor for systolic LV dysfunction, a condition often proceeding to heart failure.

BackgroundBlood outgrowth endothelial cells (BOECs) mediate therapeutic neovascularization in experimental models, but outgrowth characteristics and functionality of BOECs from patients with ischemic cardiomyopathy (ICMP) are unknown. We compared outgrowth efficiency and in vitro and in vivo functionality of BOECs derived from ICMP with BOECs from age‐matched (ACON) and healthy young (CON) controls. Methods and ResultsWe isolated 3.6±0.6 BOEC colonies/100×106 mononuclear cells (MNCs) from 60‐mL blood samples of ICMP patients (n=45; age: 66±1 years; LVEF: 31±2%) versus 3.5±0.9 colonies/100×106 MNCs in ACON (n=32; age: 60±1 years) and 2.6±0.4 colonies/100×106 MNCs in CON (n=55; age: 34±1 years), P=0.29. Endothelial lineage (VEGFR2+/CD31+/CD146+) and progenitor (CD34+/CD133−) marker expression was comparable in ICMP and CON. Growth kinetics were similar between groups (P=0.38) and not affected by left ventricular systolic dysfunction, maladaptive remodeling, or presence of cardiovascular risk factors in ICMP patients. In vitro neovascularization potential, assessed by network remodeling on Matrigel and three‐dimensional spheroid sprouting, did not differ in ICMP from (A)CON. Secretome analysis showed a marked proangiogenic profile, with highest release of angiopoietin‐2 (1.4±0.3×105 pg/106 ICMP‐BOECs) and placental growth factor (5.8±1.5×103 pg/106 ICMP BOECs), independent of age or ischemic disease. Senescence‐associated β‐galactosidase staining showed comparable senescence in BOECs from ICMP (5.8±2.1%; n=17), ACON (3.9±1.1%; n=7), and CON (9.0±2.8%; n=13), P=0.19. High‐resolution microcomputed tomography analysis in the ischemic hindlimb of nude mice confirmed increased arteriogenesis in the thigh region after intramuscular injections of BOECs from ICMP (P=0.025; n=8) and CON (P=0.048; n=5) over vehicle control (n=8), both to a similar extent (P=0.831). ConclusionsBOECs can be successfully culture‐expanded from patients with ICMP. In contrast to impaired functionality of ICMP‐derived bone marrow MNCs, BOECs retain a robust proangiogenic profile, both in vitro and in vivo, with therapeutic potential for targeting ischemic disease.

Aims: In mice, MEOX2/TCF15 heterodimers are highly expressed in heart endothelial cells and are involved in the transcriptional regulation of lipid transport. We investigated whether coronary heart disease (CHD) in humans is associated with variation in these genes. Methods and results: In 2027 participants enrolled in the Flemish Study on Environment, Genes and Health Outcomes (51.0% women; mean age 43.6 years), we genotyped SNPs in MEOX2 and TCF15, measured baseline cardiovascular risk factors, and recorded CHD incidence. Over 15.2 years (median), CHD occurred in 106 participants. For SNPs, we contrasted minor-allele heterozygotes and homozygotes (variant) vs. major-allele homozygotes (reference) and for haplotypes carriers vs. non-carriers. Sex- and age-standardised CHD rates were higher in MEOX2 rs10777, rs12056299, rs7787043, rs4532497, rs1050290 variants, in MEOX2 GTCCGC haplotype carriers (prevalence, 16.5%), but lower in MEOX2 rs6959056 variants (P ≤ 0.04, adjusted for multiple testing). In multivariable-adjusted analyses, the corresponding hazard ratios were ≥1.50 (P ≤ 0.049), 1.77 (P = 0.0054) and 0.62 (P = 0.025), respectively. None of four TCF15 SNPs was associated with coronary risk (P ≥ 0.29). However, CHD risk associated with MEOX2 rs4532497 was confined to TCF15 rs12624577 variant allele carriers (P for interaction = 0.011). The MEOX2 GTCCGC hap-lotype significantly improved the prediction of CHD over and beyond traditional risk factors and was associated with similar population-attributable risk as smoking (18.7% vs. 16.2%). Conclusions: In randomly recruited Flemish, genetic variation in MEOX2, but not TCF15, is a strong predictor of CHD. Further experimental studies should elucidate the underlying molecular mechanisms.