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Competing Interests: Conflict of interest The authors declare that they have no conflict of interests.

Cardiac ischemia/reperfusion(I/R) induced-cardiac vascular endothelial injury is an important pathological process that appears in the early stage of cardiac I/R injury. The autophagy-lysosomal pathway is essential for the maintenance of cellular homeostasis. However, in cardiac I/R injury, the role of the autophagy-lysosomal pathway is controversial. The present study aimed to use oxygen-glucose deprivation/oxygen-glucose resupply(OGD/OGR) in human coronary artery endothelial cells(HCAECs) with I/R injury to assess the role of the autophagy-lysosomal pathway in I/R-induced endothelial injury. The results revealed lysosomal dysfunction and impaired autophagic flux in endothelial cells exposed to OGD/OGR. Meanwhile, our data showed that the levels of cathepsin D(CTSD) decreased time-dependently. Knockdown of CTSD caused lysosomal dysfunction and impaired autophagic flux. Conversely, restoration of CTSD levels protected HCAECs against OGD/OGR induced-defects in autophagy-lysosomal function and cellular damage. Our findings indicated that I/R induced-impaired autophagic flux, rather than excessive autophagic initiation, mediates endothelial cells injury. The maintenance of autophagy-lysosomal function is critical to protect endothelial cells against I/R injury, and CTSD is a key regulator. Thus, strategies focused on restoring CTSD function are potentially novel treatments for cardiac reperfusion injury., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Pharyngeal arch artery (PAA) progenitors undergo proliferative expansion and angioblast differentiation to build vessels connecting the heart with the dorsal aortae. However, it remains unclear whether and how these two processes are orchestrated. Here we demonstrate that Tmem88 is required to fine-tune PAA progenitor proliferation and differentiation. Loss of zebrafish tmem88a/b leads to an excessive expansion and a failure of differentiation of PAA progenitors. Moreover, tmem88a/b deficiency enhances cyclin D1 expression in PAA progenitors via aberrant Wnt signal activation. Mechanistically, cyclin D1-CDK4/6 promotes progenitor proliferation through accelerating the G1/S transition while suppressing angioblast differentiation by phosphorylating Nkx2.5/Smad3. Ectodermal Wnt2bb signaling is confined by Tmem88 in PAA progenitors to ensure a balance between proliferation and differentiation. Therefore, the proliferation and angioblast differentiation of PAA progenitors manifest an inverse relationship and are delicately regulated by cell cycle machinery downstream of the Tmem88-Wnt pathway., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Women are more resistant than men to the development of vascular diseases. However, menopause is a factor leading to deterioration of female vascular integrity, and it is reported that the risk of vascular diseases such as atherosclerosis and abdominal aortic aneurysm is increased in postmenopausal women. Although it is suggested that perivascular adipose tissue (PVAT) is deeply involved in the increased risk of vascular disease development, the effect of menopause on PVAT integrity is unknown. In this study, we aimed to elucidate the effect of menopause on PVAT in ovariectomized (OVX) rats. PVAT was divided into 4 regions based on characteristics. Hypertrophy and increased inflammation of adipocytes in the PVAT were observed in the OVX group, but the effects of OVX were different for each region. OVX induced matrix metalloproteinase (MMP) -9 which degrade extracellular matrix such as elastin and collagen fibers in PVAT. Degeneration of the arterial fibers of the thoracic and abdominal aorta were observed in the OVX group. These results indicate that OVX can cause dysfunction of PVAT which can cause degradation of arterial fibers. Appropriate management of PVAT may play an important role in the prevention and treatment of diseases originating from ovarian hypofunction.

Arteriogenesis is one of the primary physiological means by which the circulatory collateral system restores blood flow after significant arterial occlusion in peripheral arterial disease patients. Vascular smooth muscle cells (VSMCs) are the predominant cell type in collateral arteries and respond to altered blood flow and inflammatory conditions after an arterial occlusion by switching their phenotype between quiescent contractile and proliferative synthetic states. Maintaining the contractile state of VSMC is required for collateral vascular function to regulate blood vessel tone and blood flow during arteriogenesis, whereas synthetic SMCs are crucial in the growth and remodeling of the collateral media layer to establish more stable conduit arteries. Timely VSMC phenotype switching requires a set of coordinated actions of molecular and cellular mediators to result in an expansive remodeling of collaterals that restores the blood flow effectively into downstream ischemic tissues. This review overviews the role of VSMC phenotypic switching in the physiological arteriogenesis process and how the VSMC phenotype is affected by the primary triggers of arteriogenesis such as blood flow hemodynamic forces and inflammation. Better understanding the role of VSMC phenotype switching during arteriogenesis can identify novel therapeutic strategies to enhance revascularization in peripheral arterial disease.

Background The risk of cardiovascular disease is known to increase after menopause. Mitochondria, which undergo quality control via mitochondrial autophagy, play a crucial role in the regulation of cellular senescence. The aim of this study was to investigate whether the effect of estrogen-mediated protection from senescence on arteries is attributed to the induction of mitochondrial autophagy. Methods and Results We used human umbilical vein cells, vascular smooth muscle cells, and 12-week-old female C57BL/6 mice. The administration of 17β-estradiol (E2) to cells inhibited cellular senescence and mitochondrial dysfunction. Furthermore, E2 increased mitochondrial autophagy, maintaining mitochondrial function, and retarding cellular senescence. Of note, E2 did not modulate LC3 (light chain 3), and ATG7 (autophagy related 7) deficiency did not suppress mitochondrial autophagy in E2-treated cells. Conversely, E2 increased the colocalization of Rab9 with LAMP2 (lysosomal-associated membrane protein 2) signals. The E2-mediated effects on mitochondrial autophagy were abolished by the knockdown of either Ulk1 or Rab9. These results suggest that E2-mediated mitochondrial autophagy is associated with Rab9-dependent alternative autophagy. E2 upregulated SIRT1 (sirtuin 1) and activated LKB1 (liver kinase B1), AMPK (adenosine monophosphate-activated protein kinase), and Ulk1, indicating that the effect of E2 on the induction of Rab9-dependent alternative autophagy is mediated by the SIRT1/LKB1/AMPK/Ulk1 pathway. Compared with the sham-operated mice, ovariectomized mice showed reduced mitochondrial autophagy and accelerated mitochondrial dysfunction and arterial senescence; these detrimental alterations were successfully rescued by the administration of E2. Conclusions We showed that E2-induced mitochondrial autophagy plays a crucial role in the delay of vascular senescence. The Rab9-dependent alternative autophagy is behind E2-induced mitochondrial autophagy.

Protein localization in endothelial cells is tightly regulated to create distinct signaling domains within their tight spatial restrictions including luminal membranes, abluminal membranes, and interendothelial junctions, as well as caveolae and calcium signaling domains. Protein localization in endothelial cells is also determined in part by the vascular bed, with differences between arteries and veins and between large and small arteries. Specific protein polarity and localization is essential for endothelial cells in responding to various extracellular stimuli. In this review, we examine protein localization in the endothelium of resistance arteries, with occasional references to other vessels for contrast, and how that polarization contributes to endothelial function and ultimately whole organism physiology. We highlight the protein localization on the luminal surface, discussing important physiological receptors and the glycocalyx. The protein polarization to the abluminal membrane is especially unique in small resistance arteries with the presence of the myoendothelial junction, a signaling microdomain that regulates vasodilation, feedback to smooth muscle cells, and ultimately total peripheral resistance. We also discuss the interendothelial junction, where tight junctions, adherens junctions, and gap junctions all convene and regulate endothelial function. Finally, we address planar cell polarity, or axial polarity, and how this is regulated by mechanosensory signals like blood flow., (© 2021 S. Karger AG, Basel.)

The formation of arteries is thought to occur by the induction of a highly conserved arterial genetic programme in a subset of vessels that will later experience an increase in oxygenated blood flow 1,2 . The initial steps of arterial specification require both the VEGF and Notch signalling pathways 3-5 . Here, we combine inducible genetic mosaics and transcriptomics to modulate and define the function of these signalling pathways in cell proliferation, arteriovenous differentiation and mobilization. We show that endothelial cells with high levels of VEGF or Notch signalling are intrinsically biased to mobilize and form arteries; however, they are not genetically pre-determined, and can also form veins. Mechanistically, we found that increased levels of VEGF and Notch signalling in pre-arterial capillaries suppresses MYC-dependent metabolic and cell-cycle activities, and promotes the incorporation of endothelial cells into arteries. Mosaic lineage-tracing studies showed that endothelial cells that lack the Notch-RBPJ transcriptional activator complex rarely form arteries; however, these cells regained the ability to form arteries when the function of MYC was suppressed. Thus, the development of arteries does not require the direct induction of a Notch-dependent arterial differentiation programme, but instead depends on the timely suppression of endothelial cell-cycle progression and metabolism, a process that precedes arterial mobilization and complete differentiation.

The arterial wall is characterised by a complex microstructure that impacts the mechanical properties of the vascular tissue. The main components consist of collagen and elastin fibres, proteoglycans, Vascular Smooth Muscle Cells (VSMCs) and ground matrix. While VSMCs play a key role in the active mechanical response of arteries, collagen and elastin determine the passive mechanics. Several experimental methods have been designed to investigate the role of these structural proteins in determining the passive mechanics of the arterial wall. Microscopy imaging of load-free or fixed samples provides useful information on the structure-function coupling of the vascular tissue, and mechanical testing provides information on the mechanical role of collagen and elastin networks. However, when these techniques are used separately, they fail to provide a full picture of the arterial micromechanics. More recently, advances in imaging techniques have allowed combining both methods, thus dynamically imaging the sample while loaded in a pseudo-physiological way, and overcoming the limitation of using either of the two methods separately. The present review aims at describing the techniques currently available to researchers for the investigation of the arterial wall micromechanics. This review also aims to elucidate the current understanding of arterial mechanics and identify some research gaps.

In addition to maintaining cellular ER Ca 2+ stores, store-operated Ca 2+ entry (SOCE) regulates several Ca 2+ -sensitive cellular enzymes, including certain adenylyl cyclases (ADCYs), enzymes that synthesize the secondary messenger cyclic AMP (cAMP). Ca 2+ , acting with calmodulin, can also increase the activity of PDE1-family phosphodiesterases (PDEs), which cleave the phosphodiester bond of cAMP. Surprisingly, SOCE-regulated cAMP signaling has not been studied in cells expressing both Ca 2+ -sensitive enzymes. Here, we report that depletion of ER Ca 2+ activates PDE1C in human arterial smooth muscle cells (HASMCs). Inhibiting the activation of PDE1C reduced the magnitude of both SOCE and subsequent Ca 2+ /calmodulin-mediated activation of ADCY8 in these cells. Because inhibiting or silencing Ca 2+ -insensitive PDEs had no such effects, these data identify PDE1C-mediated hydrolysis of cAMP as a novel and important link between SOCE and its activation of ADCY8. Functionally, we showed that PDE1C regulated the formation of leading-edge protrusions in HASMCs, a critical early event in cell migration. Indeed, we found that PDE1C populated the tips of newly forming leading-edge protrusions in polarized HASMCs, and co-localized with ADCY8, the Ca 2+ release activated Ca 2+ channel subunit, Orai1, the cAMP-effector, protein kinase A, and an A-kinase anchoring protein, AKAP79. Because this polarization could allow PDE1C to control cAMP signaling in a hyper-localized manner, we suggest that PDE1C-selective therapeutic agents could offer increased spatial specificity in HASMCs over agents that regulate cAMP globally in cells. Similarly, such agents could also prove useful in regulating crosstalk between Ca 2+ /cAMP signaling in other cells in which dysregulated migration contributes to human pathology, including certain cancers., Competing Interests: Conflict of interest None declared., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Arterial macrophages have different developmental origins, but the association of macrophage ontogeny with their phenotypes and functions in adulthood is still unclear. Here, we combine macrophage fate-mapping analysis with single-cell RNA sequencing to establish their cellular identity during homeostasis, and in response to angiotensin-II (AngII)-induced arterial inflammation. Yolk sac erythro-myeloid progenitors (EMP) contribute substantially to adventitial macrophages and give rise to a defined cluster of resident immune cells with homeostatic functions that is stable in adult mice, but declines in numbers during ageing and is not replenished by bone marrow (BM)-derived macrophages. In response to AngII inflammation, increase in adventitial macrophages is driven by recruitment of BM monocytes, while EMP-derived macrophages proliferate locally and provide a distinct transcriptional response that is linked to tissue regeneration. Our findings thus contribute to the understanding of macrophage heterogeneity, and associate macrophage ontogeny with distinct functions in health and disease.

Ca V 1.2 channel blockers or 5-HT 2 receptor antagonists constitute effective therapy for Raynaud's syndrome. A functional link between the inhibition of 5-HT 2 receptors and Ca V 1.2 channel blockade in arterial smooth muscles has been hypothesized. Therefore, the effects of ritanserin, a nonselective 5-HT 2 receptor antagonist, on vascular Ca V 1.2 channels were investigated through electrophysiological, functional, and computational studies. Ritanserin blocked Ca V 1.2 channel currents (I Ca1.2 ) in a concentration-dependent manner (K r  = 3.61 µM); I Ca1.2 inhibition was antagonized by Bay K 8644 and partially reverted upon washout. Conversely, the ritanserin analog ketanserin (100 µM) inhibited I Ca1.2 by ~50%. Ritanserin concentration-dependently shifted the voltage dependence of the steady-state inactivation curve to more negative potentials (K i  = 1.58 µM) without affecting the slope of inactivation and the activation curve, and decreased I Ca1.2 progressively during repetitive (1 Hz) step depolarizations (use-dependent block). The addition of ritanserin caused the contraction of single myocytes not yet dialyzed with the conventional method. Furthermore, in depolarized rings, ritanserin, and to a lesser extent, ketanserin, caused a concentration-dependent relaxation, which was antagonized by Bay K 8644. Ritanserin and ketanserin were docked at a region of the Ca V 1.2 α 1C subunit nearby that of Bay K 8644; however, only ritanserin and Bay K 8644 formed a hydrogen bond with key residue Tyr-1489. In conclusion, ritanserin caused in vitro vasodilation, accomplished through the blockade of Ca V 1.2 channels, which was achieved preferentially in the inactivated and/or resting state of the channel. This novel activity encourages the development of ritanserin derivatives for their potential use in the treatment of Raynaud's syndrome.

Increasing evidence suggests that T‑cell immunoglobulin and mucin domain 3 (TIM‑3) displays anti‑atherosclerotic effects, but its role in vascular smooth muscle cells (VSMCs) has not been reported. The present study aimed to investigate the function of TIM‑3 and its roles in human artery VSMCs (HASMCs). A protein array was used to investigate the TIM‑3 protein expression profile, which indicated that TIM‑3 expression was increased in the serum of patients with lower extremity arteriosclerosis obliterans disease (LEAOD) compared with healthy individuals. Immunohistochemistry and western blotting of arterial tissue further revealed that TIM‑3 expression was increased in LEAOD artery tissue compared with normal artery tissue. Additionally, platelet‑derived growth factor‑BB (PDGF‑BB) displayed a positive correlation with TIM‑3 expression in HASMCs. TIM‑3 decreased the migration and proliferation of PDGF‑BB‑induced HASMCs, and anti‑TIM‑3 blocked the effects of TIM‑3. The effect of TIM‑3 on the proliferation and migration of HASMCs was further investigated using LV‑TIM‑3‑transduced cells. The results revealed that TIM‑3 also inhibited PDGF‑BB‑induced expression of the inflammatory factors interleukin‑6 and tumor necrosis factor‑α by suppressing NF‑κB activation. In summary, the present study revealed that TIM‑3 displayed a regulatory role during the PDGF‑BB‑induced inflammatory reaction in HASMCs, which indicated that TIM‑3 may display anti‑atherosclerotic effects.

Osteogenesis in human arterial smooth muscle cell (HASMC) is a key feature of uremic vascular calcification (UVC). Concerning pro-oxidant properties of p -cresyl sulfate (PCS), the therapeutic effect of reactive oxygen species (ROS) scavenger on PCS triggered inflammatory signaling transduction in osteogenesis was investigated in this translational research. Based on severity level of chronic kidney disease (CKD), arterial specimens with immunohistochemistry stain were quantitatively analyzed for UVC, oxidative injury and osteogenesis along with PCS concentrations. To mimic human UVC, HASMC model was used to explore whether PCS-induced ROS could trigger mitogen-activated protein kinase (MAPK) pathways with nuclear factor-κB (NF-κB) translocation that drive context-specific gene/protein expression, including Runt-related transcription factor 2 ( Runx2 ) and alkaline phosphatase (ALP). In parallel with PCS accumulation, CKD arteries corresponded with UVC severity, oxidative DNA damage (8-hydroxy-2'-deoxyguanosine), Runx2 and ALP. PCS directly phosphorylated extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK)/P38 (pERK/pJNK/pP38) and modulated NF-κB translocation to promote expressions of Runx2 and ALP in HASMC. Notably, intracellular ROS scavenger attenuated pERK signaling cascade and downstream osteogenic differentiation. Collectively, our data demonstrate PCS induces osteogenesis through triggering intracellular ROS, pERK/pJNK/pP38 MAPK pathways and NF-κB translocation to drive Runx2 and ALP expressions, culminating in UVC. Beyond mineral dysregulation, osteocytic conversion in HASMC could be the stimulation of PCS. Thus PCS may act as a pro-osteogenic and pro-calcific toxin. From the perspective of translational medicine, PCS and intracellular ROS could serve as potential therapeutic targets for UVC in CKD patients.

Micro-optical coherence tomography (µOCT) is a novel imaging approach enabling visualization of the microstructures of biological tissues at a cellular or sub-cellular level. However, it has been challenging to develop a miniaturized flexible endoscopic µOCT probe allowing helical luminal scanning. In this study, we built a flexible endoscopic µOCT probe with an outer diameter of 1.2 mm, which acquires three-dimensional images of the arterial microstructures via helical scanning with an axial and lateral resolutions of 1.83 µm and 3.38 µm in air, respectively. Furthermore, the depth of focus of the µOCT imaging probe was extended two-fold using a binary phase spatial filter. We demonstrated that the present endoscopic µOCT could image cellular level features of a rabbit artery with high-risk atheroma and a bioresorbable scaffold-implanted swine coronary artery. This highly-translatable endoscopic µOCT will be a useful tool for investigating coronary artery disease and stent biology.

Background: The development of a biological based small diameter vascular graft (d < 6 mm), that can be properly stored over a long time period at - 196 °C, in order to directly be used to the patients, still remains a challenge. In this study the decellularized umbilical arteries (UAs) where vitrified, evaluated their composition and implanted to a porcine model, thus serving as vascular graft., Methods: Human UAs were decellularized using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) detergents. Then, vitrified with vitrification solution 55 (VS55) solution, remained for 6 months in liquid nitrogen and their extracellular matrix composition was compared to conventionally cryopreserved UAs. Additionally, total hydroxyproline, sulphated glycosaminoglycan and DNA content were quantified in all samples. Finally, the vitrified umbilical arteries implanted as common carotid artery interposition graft to a porcine animal model., Results: Decellularized and vitrified UAs characterized by proper preservation of extracellular matrix proteins and tissue architecture, whereas conventionally cryopreserved samples exhibited a disorganized structure. Total hydroxyproline content was preserved, although sulphated glycosaminoglycan and DNA contents presented significantly alterations in all samples. Implanted UAs successfully recellularized and remodeled as indicated by the histological analysis., Conclusion: Decellularized and vitrified UAs retained their structure function properties and can be possible used as an alternative source for readily accessible small diameter vascular grafts.

The field of angiogenesis research provides deep understanding regarding this important process, which plays fundamental roles in tissue development and different abnormalities. In vitro models offer the advantages of low-cost high-throughput research of angiogenesis while sparing animal lives, and enabling the use of human cells. Nevertheless, prevailing in vitro models lack stability and are limited to a few days' assays. This study, therefore, examines the hypothesis that closely mimicking the vascular microenvironment can more reliably support longer angiogenesis processes in vitro. To this end, porcine arterial extracellular matrix (paECM)- a key component of blood vessels-was isolated and processed into a thermally induced hydrogel and characterized in terms of composition, structure, and mechanical properties, thus confirming the preservation of important characteristics of arterial extracellular matrix. This unique hydrogel was further tailored into a three-dimensional model of angiogenesis using endothelial cells and supporting cells, in a configuration that allows high-throughput quantitative analysis of cell viability and proliferation, cell migration, and apoptosis, thus revealing the advantages of paECM over frequently used biomaterials. Markedly, when applied with well-known effectors of angiogenesis, the model measures reflected the expected response, hence validating its efficacy and establishing its potential as a promising tool for the research of angiogenesis., (© 2020 Federation of American Societies for Experimental Biology.)

Various decellularization methods for allogenic and xenogenic bioscaffolds have been previously reported; however, decellularization methods for very thin (submillimeter-diameter) vascular tissues have not been discussed well. In this study, rat tail arteries (inner diameter, 0.6 mm) were decellularized with peracetic acid (PAA) and DNase I. PAA treatment is expected not only to disrupt cell membranes which improves the decellularization efficiency in the subsequent DNase treatment, but also to sterilize vascular scaffolds. We succeeded in adequate cell removal by immersing in 0.3% isotonic PAA solution and subsequent washing with DNase solution. For the DNase washing process, the perfusion method was superior in terms of cell removal to the static immersion method. Graft lumen was modified with a peptide composed of a collagen binding sequence and endothelial progenitor cell-binding sequence, (Pro-Hyp-Gly) 7 -Gly-Gly-Gly-Arg-Glu-Asp-Val, as previously reported. They were patent in rat allogeneic transplantation model for 2 weeks, but unexpectedly resulted in graft rupture or tear formation, thereafter, suggesting reduced mechanical strength of the decellularized scaffolds. Histology showed that the thickness of the extracellular matrix (ECM) was decreased by the perfusion of the DNase solution. The method of combination of PAA and DNase was not necessarily optimal for the decellularization of very thin vascular tissues. The decellularization method is a compromise between effective cell removal and maintenance of the ECM nature. Since the acceptability of ECM denaturation by the host tissue highly depends on individual cases, decellularization methods should be carefully selected according to the type of target tissue and its intended use.

Rationale: Significant progress has revealed transcriptional inputs that underlie regulation of artery and vein endothelial cell fates. However, little is known concerning genome-wide regulation of this process. Therefore, such studies are warranted to address this gap., Objective: To identify and characterize artery- and vein-specific endothelial enhancers in the human genome, thereby gaining insights into mechanisms by which blood vessel identity is regulated., Methods and Results: Using chromatin immunoprecipitation and deep sequencing for markers of active chromatin in human arterial and venous endothelial cells, we identified several thousand artery- and vein-specific regulatory elements. Computational analysis revealed that NR2F2 (nuclear receptor subfamily 2, group F, member 2) sites were overrepresented in vein-specific enhancers, suggesting a direct role in promoting vein identity. Subsequent integration of chromatin immunoprecipitation and deep sequencing data sets with RNA sequencing revealed that NR2F2 regulated 3 distinct aspects related to arteriovenous identity. First, consistent with previous genetic observations, NR2F2 directly activated enhancer elements flanking cell cycle genes to drive their expression. Second, NR2F2 was essential to directly activate vein-specific enhancers and their associated genes. Our genomic approach further revealed that NR2F2 acts with ERG (ETS-related gene) at many of these sites to drive vein-specific gene expression. Finally, NR2F2 directly repressed only a small number of artery enhancers in venous cells to prevent their activation, including a distal element upstream of the artery-specific transcription factor, HEY2 (hes related family bHLH transcription factor with YRPW motif 2). In arterial endothelial cells, this enhancer was normally bound by ERG, which was also required for arterial HEY2 expression. By contrast, in venous endothelial cells, NR2F2 was bound to this site, together with ERG, and prevented its activation., Conclusions: By leveraging a genome-wide approach, we revealed mechanistic insights into how NR2F2 functions in multiple roles to maintain venous identity. Importantly, characterization of its role at a crucial artery enhancer upstream of HEY2 established a novel mechanism by which artery-specific expression can be achieved.

Endothelial dysfunction is a critical event in vascular inflammation characterized, in part, by elevated surface expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is heavily N-glycosylated, and like other surface proteins, it is largely presumed that fully processed, complex N-glycoforms are dominant. However, our recent studies suggest that hypoglycosylated or high mannose (HM)-ICAM-1 N-glycoforms are also expressed on the cell surface during endothelial dysfunction, and have higher affinity for monocyte adhesion and regulate outside-in endothelial signaling by different mechanisms. Whether different ICAM-1 N-glycoforms are expressed in vivo during disease is unknown. In this study, using the proximity ligation assay, we assessed the relative formation of high mannose, hybrid and complex α-2,6-sialyated N-glycoforms of ICAM-1 in human and mouse models of atherosclerosis, as well as in arteriovenous fistulas (AVF) of patients on hemodialysis. Our data demonstrates that ICAM-1 harboring HM or hybrid epitopes as well as ICAM-1 bearing α-2,6-sialylated epitopes are present in human and mouse atherosclerotic lesions. Further, HM-ICAM-1 positively associated with increased macrophage burden in lesions as assessed by CD68 staining, whereas α-2,6-sialylated ICAM-1 did not. Finally, both HM and α-2,6-sialylated ICAM-1 N-glycoforms were present in hemodialysis patients who had AVF maturation failure compared to successful AVF maturation. Collectively, these data provide evidence that HM- ICAM-1 N-glycoforms are present in vivo, and at levels similar to complex α-2,6-sialylated ICAM-1 underscoring the need to better understand their roles in modulating vascular inflammation., Competing Interests: Dr. Lee is a consultant for Proteon Therapeutics, Merck, and Boston Scientific. All other authors have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Spiral artery remodeling, which is indispensable for successful pregnancy, is accomplished by endovascular trophoblasts that move upstream along the arterial wall, replace the endothelium, and disrupt the muscular lining. This review outlines the possible factors that could regulate endovascular trophoblast differentiation and invasion. First, high oxygen tension in the spiral artery could initiate endovascular trophoblast invasion. Second, activation of maternal decidual natural killer (dNK) cells could support perivascular invasion of interstitial trophoblasts and consequently could facilitate the endovascular trophoblast invasion. Third, maternal platelets trapped by the endovascular trophoblasts could enhance endovascular trophoblast invasion, which is in part mediated by chemokine CCL5 (C-C motif ligand 5) released from the activated platelets and chemokine receptor CCR1 (C-C chemokine receptor type 1) expressed specifically on the endovascular trophoblasts. The rat, in which trophoblast cells exhibit extensive interstitial and endovascular invasion, could be a suitable model animal for the study of human spiral artery remodeling. Apparently paradoxical results came from the rat study, i.e., exposure to hypoxia or depletion of dNK cells resulted in acceleration of the endovascular trophoblast invasion. This implies the presence of as-yet-undetermined regulator(s) whose effects on endovascular trophoblast invasion surpass the effects of surrounding oxygen tension or maternal dNK cells. In the future, clarification of the molecular differences between human interstitial and endovascular trophoblasts as well as establishment of the pregnant rat model exhibiting shallow endovascular trophoblast invasion and preeclamptic symptoms will contribute to elucidating the mechanism of spiral artery remodeling., Competing Interests: Declarations of competing interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Acute cardiorespiratory responses to O 2 deficiency are essential for physiological homeostasis. The prototypical acute O 2 -sensing organ is the carotid body, which contains glomus cells expressing K + channels whose inhibition by hypoxia leads to transmitter release and activation of nerve fibers terminating in the brainstem respiratory center. The mechanism by which changes in O 2 tension modulate ion channels has remained elusive. Glomus cells express genes encoding HIF2α ( Epas1 ) and atypical mitochondrial subunits at high levels, and mitochondrial NADH and reactive oxygen species (ROS) accumulation during hypoxia provides the signal that regulates ion channels. We report that inactivation of Epas1 in adult mice resulted in selective abolition of glomus cell responsiveness to acute hypoxia and the hypoxic ventilatory response. Epas1 deficiency led to the decreased expression of atypical mitochondrial subunits in the carotid body, and genetic deletion of Cox4i2 mimicked the defective hypoxic responses of Epas1 -null mice. These findings provide a mechanistic explanation for the acute O 2 regulation of breathing, reveal an unanticipated role of HIF2α, and link acute and chronic adaptive responses to hypoxia., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

In this report, we describe a microfluidic vascular-bed (micro-VB) device providing a platform for 3D tissue engineering with vascular network formation. The micro-VB device allows functional connections between endothelial capillaries of heterogeneous sections (5-100 μm in diameter) and artificial plastic tubes or reservoirs (1-10 mm in diameter). Moreover, the micro-VB device can be installed in a standard 100 mm-diameter Petri dish. Endothelial networks in 3D engineered tissues were obtained by cellular self-assembly on the device, after co-culturing of human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs) in fibrin gel. Endothelial capillary connection between vascularized tissues and microfluidic channels, mimicking arteries and veins, was confirmed by perfusion of fluorescent microspheres. The micro-VB devices were compatible with the use of commercially available culture dishes and did not require the involvement of additional equipment. Thus, these micro-VB devices are expected to substantially improve the routine application of 3D tissue engineering to regenerative medicine.

Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30-120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK., Competing Interests: The authors have declared that no competing interests exist.

Background: With the popularity of laparoscopic cholecystectomy, common bile duct injury has been reported more frequently. There is no perfect method for repairing porcine biliary segmental defects., Methods: After the decellularization of human arterial blood vessels, the cells were cultured with GFP + (carry green fluorescent protein) porcine bile duct epithelial cells. The growth and proliferation of porcine bile duct epithelial cells on the human acellular arterial matrix (HAAM) were observed by hematoxylin-eosin (HE) staining, electron microscopy, and immunofluorescence. Then, the recellularized human acellular arterial matrix (RHAAM) was used to repair biliary segmental defects in the pig. The feasibility of it was detected by magnetic resonance cholangiopancreatography, liver function and blood routine changes, HE staining, immunofluorescence, real-time quantitative PCR (RT-qPCR), and western blot., Results: After 4 weeks (w) of co-culture of HAAM and GFP + porcine bile duct epithelial cells, GFP + porcine bile duct epithelial cells grew stably, proliferated, and fused on HAAM. Bile was successfully drained into the duodenum without bile leakage or biliary obstruction. Immunofluorescence detection showed that GFP-positive bile duct cells could still be detected after GFP-containing bile duct cells were implanted into the acellular arterial matrix for 8 w. The implanted bile duct cells can successfully resist bile invasion and protect the acellular arterial matrix until the newborn bile duct is formed., Conclusion: The RHAAM can be used to repair biliary segmental defects in pigs, which provides a new idea for the clinical treatment of common bile duct injury., Competing Interests: Conflicts of interestThe authors have no financial conflicts of interest., (© The Korean Tissue Engineering and Regenerative Medicine Society 2019.)

Key Points: Ageing is associated with changes in the respiratory system including in the lungs, rib cage and muscles. Neural drive to the diaphragm, the principal inspiratory muscle, has been reported to increase during quiet breathing with ageing. We demonstrated that low-threshold motor units of the human diaphragm recruited during quiet breathing have similar discharge frequencies across age groups and shorter discharge times in older age. With ageing, motor unit action potential area increased. We propose that there are minimal functionally significant changes in the discharge properties of diaphragm motor units with ageing despite remodelling of the motor unit in the periphery., Abstract: There are changes in the skeletal, pulmonary and respiratory neuromuscular systems with healthy ageing. During eupnoea, one study has shown relatively higher crural diaphragm electromyographic activity (EMG) in healthy older adults (>51 years) than in younger adults, but these measures may be affected by the normalisation process used. A more direct method to assess neural drive involves the measurement of discharge properties of motor units. Here, to assess age-related changes in neural drive to the diaphragm during eupnoea, EMG was recorded from the costal diaphragm using a monopolar needle electrode in participants from three age groups (n ≥ 7 each): older (65-80 years); middle-aged (43-55 years) and young (23-26 years). In each group, 154, 174 and 110 single motor units were discriminated, respectively. A mixed-effects linear model showed no significant differences between age groups for onset (group mean range 9.5-10.2 Hz), peak (14.1-15.0 Hz) or offset (7.8-8.5 Hz) discharge frequencies during eupnoea. The motor unit recruitment was delayed in the older group (by ∼15% of inspiratory time; p = 0.02 cf. middle-aged group) and had an earlier offset time (by ∼15% of inspiratory time; p = 0.04 cf. young group). However, the onset of multiunit activity was similar across groups, consistent with no global increase in neural drive to the diaphragm with ageing. The area of diaphragm motor unit potentials was ∼40% larger in the middle-aged and older groups (P < 0.02), which indicates axonal sprouting and re-innervation of muscle fibres associated with ageing, even in middle-aged participants., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Efforts to disclose the mechanisms of transcranial therapeutic electro-magnetic fields (EMF) acting on the brain's cells (Marino, Kibleur) and recently immune cells (Gülöksüz) meet unsolved physiological details of blood vessels, exclusively arterial vasomotion or the non-glial-related former g(lia)-lymphatic flow (Iliff; Liu DX) - now replaced by an astrocytic AQP4-pipeline cooling the brain (Nakada 2014). Here within the convergent dyn4TAM-framework, which had suggested the first mast cell behavioral experiment (Fitzpatrick & Morrow 2017), three intertwined physiological concepts are contributed: A) "autocrinicity" - how flushed, thus absent, autocrine signals integrate external fluidics into cellular computations e.g. on motility: EMFs could increase such absences by targeting e.g. dipole-cytokines; B) a new concept of the arterial wall based on a tangible interpretation of the coronal histology of all arteries as a co-axial pulse-dampening engine (Treviranus 2012). In the brain this engine might provide the quickest cerebral outflow via the Cerebral IntraMUral Reverse Arterial Flow (Treviranus 2018b), while transmitting further forces acting upstream to the paravascular spaces; C) some key roles for mast cells in neuro-psychiatry (Silver & Curley 2013) and their interactive lymphatic and non-luminal vascular routes to the brain dictated by peripheral imprinting as to destiny (Csaba 1987) and destination (Treviranus 2013). Within the skull they might advance against para-arterial upstream currents. Some known causal mediators of the effects of transcranially applied EFMs and puzzling results are then put tentatively in perspective with the above "tangible" models, e.g. by aligning probable induced currents with arterial segments or the new direct meningeal-calvario-myeloid channels. RESULTS: The case for a role of mast cells and diverse flows in transcranial electromagnetic brain therapy seems promising.

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([ 18 F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Haematopoietic stem cells are generated from the haemogenic endothelium (HE) located in the floor of the dorsal aorta (DA). Despite being integral to arteries, it is controversial whether HE and arterial endothelium share a common lineage. Here, we present a transgenic zebrafish runx1 reporter line to isolate HE and aortic roof endothelium (ARE)s, excluding non-aortic endothelium. Transcriptomic analysis of these populations identifies Runx1-regulated genes and shows that HE initially expresses arterial markers at similar levels to ARE. Furthermore, runx1 expression depends on prior arterial programming by the Notch ligand dll4. Runx1 -/- mutants fail to downregulate arterial genes in the HE, which remains integrated within the DA, suggesting that Runx1 represses the pre-existing arterial programme in HE to allow progression towards the haematopoietic fate. These findings strongly suggest that, in zebrafish, aortic endothelium is a precursor to HE, with potential implications for pluripotent stem cell differentiation protocols for the generation of transplantable HSCs.

Study Question: Is vascular smooth muscle cell (VSMC) dedifferentiation a feature of uterine spiral artery (SpA) remodelling in early human pregnancy?, Summary Answer: Remodelling of human uterine SpAs is associated with dedifferentiation of VSMCs and can be induced in vitro by uterine natural killer (uNK) cells and extravillous trophoblast cells (EVTs)., What Is Known Already: Uterine SpAs undergo profound morphological changes in normal pregnancy with replacement of the musculoelastic arterial wall structure by fibrinoid containing EVTs. The fate of VSMCs in SpA remodelling is unknown; in guinea pig uterine artery VSMCs dedifferentiate, remain in the vessel wall and differentiate after parturition to restore the arterial wall. There is increasing evidence that uNK cells play a role in SpA remodelling. We hypothesized that SpA remodelling in human pregnancy is associated with VSMC dedifferentiation, initiated by uNK cell-derived growth factors., Study Design, Size, Duration: Formalin fixed, paraffin embedded placental bed biopsies were immunostained for angiogenic growth factor (AGF) receptors and markers of VSMC differentiation. An in vitro model of SpA remodelling using chorionic plate arteries (CPAs) was used to test the effect of different cell types and AGFs on VSMC differentiation., Participants/materials, Setting, Methods: Placental bed biopsies were immunostained for vascular endothelial growth factor receptors 1-3 (VEGF-R1, VEGF-R2, VEGF-R3), transforming growth factor beta 1 receptors I and II (TGF-βRI, TGF-βRII), interferon gamma receptors 1 and 2 (IFN-γR1, IFN-γR2), Tie2, α-smooth muscle actin (α-SMA), H-caldesmon (H-Cal), myosin heavy chain (MyHC), osteopontin and smoothelin. Staining intensity was assessed using a modified quickscore. Expression by VSMCs of the AGF receptors was confirmed by laser capture microdissection and real-time RT-PCR of non-remodelled SpAs, after laser removal of the endothelium. As an in vitro model, VSMC differentiation was assessed in CPAs by immunohistochemistry after culture in uNK cell-conditioned medium (CM), EVT-CM, uNK cell/EVT co-culture CM, Ang-1, Ang-2, IFN-γ, VEGF-A and VEGF-C, and after blocking of both Ang-1 and Ang-2 in uNK-CM., Main Results and the Role of Chance: SpA VSMC expression of Tie-2 (P = 0.0007), VEGF-R2 (P = 0.005) and osteopontin (P = 0.0001) increased in partially remodelled SpAs compared with non-remodelled SpAs, while expression of contractile VSMC markers was reduced (α-SMA P < 0.0001, H-Cal P = 0.03, MyHC P = 0.03, smoothelin P = 0.0001). In the in vitro CPA model, supernatants from purified uNK cell (H-Cal P < 0.0001, MyHC P = 0.03, α-SMA P = 0.02, osteopontin P = 0.03), EVT (H-Cal P = 0.0006, MyHC P = 0.02, osteopontin P = 0.01) and uNK cell/EVT co-cultures (H-Cal P = 0.001, MyHC P = 0.05, osteopontin P = 0.02) at 12-14 weeks, but not 8-10 weeks, gestational age induced reduced expression of contractile VSMC markers and increased osteopontin expression. Addition of exogenous (10 ng/ml) Ang-1 (P = 0.006) or Ang-2 (P = 0.009) also reduced H-Cal expression in the CPA model. Inhibition of Ang-1 (P = 0.0004) or Ang-2 (P = 0.004) in uNK cell supernatants blocked the ability of uNK cell supernatants to reduce H-Cal expression., Limitations, Reasons for Caution: This is an in vitro study and the role of uNK cells, Ang-1 and Ang-2 in SpA remodelling in vivo has not yet been shown., Wider Implications of the Findings: VSMC dedifferentiation is a feature of early SpA remodelling and uNK cells and EVT play key roles in this process by secretion of Ang-1 and Ang-2. This is one of the first studies to suggest a direct role for Ang-1 and Ang-2 in VSMC biology., Study Funding/competing Interest(s): This work was supported by a grant from British Biotechnology and Biosciences Research Council (BB/E016790/1). The authors have no competing interests to declare., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Atherosclerosis, which underlies life-threatening cardiovascular disorders such as myocardial infarction and stroke 1 , is initiated by passage of low-density lipoprotein (LDL) cholesterol into the artery wall and its engulfment by macrophages, which leads to foam cell formation and lesion development 2,3 . It is unclear how circulating LDL enters the artery wall to instigate atherosclerosis. Here we show in mice that scavenger receptor class B type 1 (SR-B1) in endothelial cells mediates the delivery of LDL into arteries and its accumulation by artery wall macrophages, thereby promoting atherosclerosis. LDL particles are colocalized with SR-B1 in endothelial cell intracellular vesicles in vivo, and transcytosis of LDL across endothelial monolayers requires its direct binding to SR-B1 and an eight-amino-acid cytoplasmic domain of the receptor that recruits the guanine nucleotide exchange factor dedicator of cytokinesis 4 (DOCK4) 4 . DOCK4 promotes internalization of SR-B1 and transport of LDL by coupling the binding of LDL to SR-B1 with activation of RAC1. The expression of SR-B1 and DOCK4 is increased in atherosclerosis-prone regions of the mouse aorta before lesion formation, and in human atherosclerotic arteries when compared with normal arteries. These findings challenge the long-held concept that atherogenesis involves passive movement of LDL across a compromised endothelial barrier. Interventions that inhibit the endothelial delivery of LDL into artery walls may represent a new therapeutic category in the battle against cardiovascular disease.

Children on dialysis have a cardiovascular mortality risk equivalent to older adults in the general population, and rapidly develop medial vascular calcification, an age-associated pathology. We hypothesized that premature vascular ageing contributes to calcification in children with advanced chronic kidney disease (CKD). Vessels from children with Stage 5 CKD with and without dialysis had evidence of increased oxidative DNA damage. The senescence markers p16 and p21 were also increased in vessels from children on dialysis. Treatment of vessel rings ex vivo with calcifying media increased oxidative DNA damage in vessels from children with Stage 5 CKD, but not in those from healthy controls. Vascular smooth muscle cells cultured from children on dialysis exhibited persistent DNA damage, impaired DNA damage repair, and accelerated senescence. Under calcifying conditions vascular smooth muscle cells from children on dialysis showed increased osteogenic differentiation and calcification. These changes correlated with activation of the senescence-associated secretory phenotype (SASP), an inflammatory phenotype characterized by the secretion of proinflammatory cytokines and growth factors. Blockade of ataxia-telangiectasia mutated (ATM)-mediated DNA damage signaling reduced both inflammation and calcification. Clinically, children on dialysis had elevated circulating levels of osteogenic SASP factors that correlated with increased vascular stiffness and coronary artery calcification. These data imply that dysregulated mineral metabolism drives vascular "inflammaging" by promoting oxidative DNA damage, premature senescence, and activation of a pro-inflammatory SASP. Drugs that target DNA damage signaling or eliminate senescent cells may have the potential to prevent vascular calcification in patients with advanced CKD., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

The current work reports the functional characterization of human induced pluripotent stem cells (iPSCs)- arterial and venous-like endothelial cells (ECs), derived in chemically defined conditions, either in monoculture or seeded in a scaffold with mechanical properties similar to blood vessels. iPSC-derived arterial- and venous-like endothelial cells were obtained in two steps: differentiation of iPSCs into endothelial precursor cells (CD31 pos /KDR pos /VE-Cad med /EphB2 neg /COUP-TF neg ) followed by their differentiation into arterial and venous-like ECs using a high and low vascular endothelial growth factor (VEGF) concentration. Cells were characterized at gene, protein and functional levels. Functionally, both arterial and venous-like iPSC-derived ECs responded to vasoactive agonists such as thrombin and prostaglandin E2 (PGE 2 ), similar to somatic ECs; however, arterial-like iPSC-derived ECs produced higher nitric oxide (NO) and elongation to shear stress than venous-like iPSC-derived ECs. Both cells adhered, proliferated and prevented platelet activation when seeded in poly(caprolactone) scaffolds. Interestingly, both iPSC-derived ECs cultured in monoculture or in a scaffold showed a different inflammatory profile than somatic ECs. Although both somatic and iPSC-derived ECs responded to tumor necrosis factor-α (TNF-α) by an increase in the expression of intercellular adhesion molecule 1 (ICAM-1), only somatic ECs showed an upregulation in the expression of E-selectin or vascular cell adhesion molecule 1 (VCAM-1).

Several human diseases, include cancer and stroke are characterized by changes in immune system activation and vascular contractility. However, the mechanistic foundation of a vascular immuno-physiology network is still largely unknown. Formyl peptide receptor-1 (FPR-1), which plays a vital role in the function of the innate immune system, is widely expressed in arteries, but its role in vascular plasticity is unclear. We questioned why a receptor that is crucial for immune defense, and cell motility in leukocytes, would be expressed in vascular smooth muscle cells (VSMCs). We hypothesized that activation of FPR-1 in arteries is important for the temporal reorganization of actin filaments, and consequently, changes in vascular function, similar to what is observed in neutrophils. To address our hypothesis, we used FPR-1 knockout and VSMCs lacking FPR-1. We observed that FPR-1 activation induces actin polymerization in wild type VSMCs. Absence of FPR-1 in the vasculature significantly decreased vascular contraction and induced loss of myogenic tone to elevated intraluminal pressures via disruption of actin polymerization. Actin polymerization activator ameliorated these responses. In conclusion, we have established a novel role for FPR-1 in VSMC contractility and motility, similar to the one observed in sentinel cells of the innate immune system. This discovery is fundamental for vascular immuno-pathophysiology, given that FPR-1 in VSMCs not only functions as an immune system receptor, but it also has an important role for the dynamic plasticity of arteries., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mural cells (MCs) are essential for blood vessel stability and function; however, the mechanisms that regulate MC development remain incompletely understood, in particular those involved in MC specification. Here, we investigated the first steps of MC formation in zebrafish using transgenic reporters. Using pdgfrb and abcc9 reporters, we show that the onset of expression of abcc9 , a pericyte marker in adult mice and zebrafish, occurs almost coincidentally with an increment in pdgfrb expression in peri-arterial mesenchymal cells, suggesting that these transcriptional changes mark the specification of MC lineage cells from naïve pdgfrb low mesenchymal cells. The emergence of peri-arterial pdgfrb high MCs required Notch signaling. We found that pdgfrb -positive cells express notch2 in addition to notch3 , and although depletion of notch2 or notch3 failed to block MC emergence, embryos depleted of both notch2 and notch3 lost mesoderm- as well as neural crest-derived pdgfrb high MCs. Using reporters that read out Notch signaling and Notch2 receptor cleavage, we show that Notch activation in the mesenchyme precedes specification into pdgfrb high MCs. Taken together, these results show that Notch signaling is necessary for peri-arterial MC specification., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Background/aims: Hemodialysis causes the systemic inflammatory response, which may affect the function of endothelial cells., Methods: We studied the effect of the serum obtained after a hemodialysis session, compared to serum collected before the start of the treatment, on the gene expression and secretory activity of arterial endothelial cells (AECs) and venous endothelial cells (VECs) in in vitro culture., Results: Serum collected at the end of the hemodialysis session increased expression of the studied genes in VECs, and at the same time decreased their expression in AECs. Secretory activity was increased in VEC: (interleukin-6 [IL-6] +29%, p < 0.05, von Willebrand factor +23%, p < 0.02; tissue plasminogen activator [t-PA] +35%, p < 0.002, t-PA/plasminogen activator inhibitor-1 [PAI-1] ratio + 57%, p < 0.005). In AEC, synthesis of IL-6 and vascular endothelial growth factor were reduced (-36%, p < 0.02, -34%, p < 0.05, respectively) and the tPA/PAI-1 ratio was increased (+22%, p < 0.01)., Conclusions: Hemodialysis induces the inflammatory, procoagulant, and profibrinolytic activity of VEC, whereas suppression of AEC is observed at the same time. Video Journal Club 'Cappuccino with Claudio Ronco' at https://www.karger.com/Journal/ArticleNews/223997?sponsor=52., (© 2018 S. Karger AG, Basel.)

Continuous laminar shear stress increases the process of autophagy, activates endothelial nitric oxide (NO) synthase phosphorylation at serine 1177 (p-eNOS S1177 ), and generates NO in bovine and human arterial endothelial cells (ECs) compared with static controls. However, the translational relevance of these findings has not been explored. In the current study, primary ECs were collected from the radial artery of 7 men using sterile J-wires before (Pre) and after (Post) 60 min of rhythmic handgrip exercise (HG) performed with the same arm. After ECs were identified by positive costaining for vascular endothelial cadherin and 4',6'-diamidino-2-phenylindole, immunofluorescent antibodies were used to assess indices of autophagy, NO generation, and superoxide anion (O 2 ·- ) production. Commercially available primary human arterial ECs were stained and processed in parallel to serve as controls. All end points were evaluated using 75 ECs from each subject. Relative to Pre-HG, HG elevated arterial shear rate ( P < 0.05) ~3-fold, whereas heart rate, arterial pressure, and cardiac output were not altered. Compared with values obtained from ECs Pre-HG, Post-HG ECs displayed increased ( P < 0.05) expression of p-eNOS S1177 , NO generation, O 2 ·- production, BECLIN1, microtubule-associated proteins 1A/1B light chain 3B, autophagy-related gene 3, and lysosomal-associated membrane protein 2A and decreased ( P < 0.05) expression (i.e., enhanced degradation) of the adaptor protein p62/sequestosome-1. These novel findings provide evidence that elevated arterial shear rate associated with functional hyperemia initiates autophagy, activates p-eNOS S1177 , and increases NO and O 2 ·- generation in primary human ECs. NEW & NOTEWORTHY Previously, our group reported in bovine arterial and human arterial endothelial cells (ECs) that shear stress initiates trafficking of the autophagosome to the lysosome and increases endothelial nitric oxide (NO) synthase phosphorylation at serine 1177, NO generation, and O 2 ·- production. Here, the translational relevance of these findings is documented. Specifically, functional hyperemia induced by rhythmic handgrip exercise elevates arterial shear rate to an extent that increases indices of autophagy, NO generation, and O 2 ·- production in primary arterial ECs collected from healthy men.

The increasing prevalence of diabetes has resulted in a global epidemic 1 . Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells 2-4 . Diabetes also impairs the functions of endothelial cells 5 and disturbs the communication between endothelial cells and pericytes 6 . How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.

Mesenchymal stem cells (MSCs) share many properties with other tissue stromal cells, including cell morphology, immunophenotype, differentiation and immunologic properties. In this study, we compared the immunophenotype and the differentiation potential of human vascular wall mesenchymal stem cells (hVW-MSCs) with those of human dermal fibroblasts and myofibroblasts. Cell morphology and surface markers were evaluated by immunofluorescence and flow cytometry; functional assays for immunomodulation, angiogenesis, adipogenesis and osteogenesis were performed, together with the mRNA analysis of the critical differentiation genes. hVW-MSCs, dermal fibroblasts and myofibroblasts were all negative to CD34, whereas the expression of CD44 stemness marker was more intense in hVW-MSCs. As expected, hVW-MSC plasticity was wide and the angiogenic, adipogenic, osteogenic features were confirmed. Fibroblasts were the less effective in terms of immunomodulation, angiogenesis and adipogenic differentiation; differently from fibroblasts, the myofibroblasts showed a poor angiogenic commitment. The mineralization assay was positive in all the three cell types, but ultrastructure interestingly evidenced differential osteogenic patterns among them. Our study supports the higher anti-inflammatory and wound healing repair features of hVW-MSCs, in comparison to the other stromal cells investigated. Moreover, we underline the importance of ultrastructure for investigating the specific osteogenic pattern for each cell type.

Arteries and veins are formed independently by different types of endothelial cells (ECs). In vascular remodeling, arteries and veins become connected and some arteries become veins. It is unclear how ECs in transforming vessels change their type and how fates of individual vessels are determined. In embryonic zebrafish trunk, vascular remodeling transforms arterial intersegmental vessels (ISVs) into a functional network of arteries and veins. Here we find that, once an ISV is connected to venous circulation, venous blood flow promotes upstream migration of ECs that results in displacement of arterial ECs by venous ECs, completing the transformation of this ISV into a vein without trans-differentiation of ECs. Arterial blood flow initiated in two neighboring ISVs prevents their transformation into veins by activating Notch signaling in ECs. Together, different responses of ECs to arterial and venous blood flow lead to formation of a balanced network with equal numbers of arteries and veins.

Dehydroepiandrosterone (DHEA) is an adrenal steroid hormone, which has the highest serum concentration among steroid hormones with DHEA sulfate (DHEAS). DHEA possesses an inhibitory action on glucose-6-phosphate dehydrogenase (G6PD), the first pentose-phosphate pathway enzyme that reduces NADP + to NADPH. DHEA induced relaxation of high K + -induced contraction in rat arterial strips, whereas DHEAS barely induced it. We studied the effects of DHEA on L-type Ca 2+ current ( I Ca,L ) of A7r5 arterial smooth muscle cells and compared the mechanism of inhibition with that produced by the 6-aminonicotinamide (6-AN) competitive inhibitor of G6PD. DHEA moderately inhibited I Ca,L that was elicited from a holding potential (HP) of -80 mV [voltage-independent inhibition (VIDI)] and accelerated decay of I Ca,L during the depolarization pulse [voltage-dependent inhibition (VDI)]. DHEA-induced VDI decreased peak I Ca,L at depolarized HPs. By applying repetitive depolarization pulses from multiple HPs, novel HP-dependent steady-state inactivation curves ( f ∞ -HP) were constructed. DHEA shifted f ∞ -HP to the left and inhibited the window current, which was recorded at depolarized HPs and obtained as a product of current-voltage relationship and f ∞ -HP. The IC 50 value of I Ca,L inhibition was much higher than serum concentration. DHEA-induced VDI was downregulated by the dialysis of guanosine 5'- O-(2-thiodiphosphate), which shifted f ∞ -voltage to the right before the application of DHEA. 6-AN gradually and irreversibly inhibited I Ca,L by VIDI, suggesting that the inhibition of G6PD is involved in DHEA-induced VIDI. In 6-AN-pretreated cells, DHEA induced additional inhibition by increasing VIDI and generating VDI. The inhibition of G6PD underlies DHEA-induced VIDI, and DHEA additionally induces VDI as described for Ca 2+ channel blockers. NEW & NOTEWORTHY Dehydroepiandrosterone, the most abundantly released adrenal steroid hormone with dehydroepiandrosterone sulfate, inhibited L-type Ca 2+ current and its window current in aortic smooth muscle cells. The IC 50 value of inhibition decreased with the depolarization of holding potential to 15 µM at -20 mV. The inhibition occurred in a voltage-dependent manner as described for Ca 2+ channel blockers and in a voltage-independent manner because of the inhibition of glucose-6-phosphate dehydrogenase.

The transcription factor RUNX1 is required in the embryo for formation of the adult hematopoietic system. Here, we describe the seminal findings that led to the discovery of RUNX1 and of its critical role in blood cell formation in the embryo from hemogenic endothelium (HE). We also present RNA-sequencing data demonstrating that HE cells in different anatomic sites, which produce hematopoietic progenitors with dissimilar differentiation potentials, are molecularly distinct. Hemogenic and non-HE cells in the yolk sac are more closely related to each other than either is to hemogenic or non-HE cells in the major arteries. Therefore, a major driver of the different lineage potentials of the committed erythro-myeloid progenitors that emerge in the yolk sac versus hematopoietic stem cells that originate in the major arteries is likely to be the distinct molecular properties of the HE cells from which they are derived. We used bioinformatics analyses to predict signaling pathways active in arterial HE, which include the functionally validated pathways Notch, Wnt, and Hedgehog. We also used a novel bioinformatics approach to assemble transcriptional regulatory networks and predict transcription factors that may be specifically involved in hematopoietic cell formation from arterial HE, which is the origin of the adult hematopoietic system., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Secretoneurin (SN) is a neuropeptide derived from specific proteolytic processing of the precursor secretogranin II (SgII). In zebrafish and other teleosts, there are two paralogs named sgIIa and sgIIb. Our results showed that neurons expressing sgIIb were aligned with central arteries in the hindbrain, demonstrating a close neurovascular association. Both sgIIb-/- and sgIIa-/-/sgIIb-/- mutant embryos were defective in hindbrain central artery development due to impairment of migration and proliferation of central artery cells. Further study revealed that sgIIb is non-cell autonomous and required for central artery development. Hindbrain arterial and venous network identities were not affected in sgIIb-/- mutant embryos, and the mRNA levels of Notch and VEGF pathway-related genes were not altered. However, the activation of MAPK and PI3K/AKT pathways was inhibited in sgIIb-/- mutant embryos. Reactivation of MAPK or PI3K/AKT in endothelial cells could partially rescue the central artery developmental defects in the sgIIb mutants. This study provides the first in vivo evidence that sgIIb plays a critical role in neurovascular modeling of the hindbrain. Targeting the SgII system may, therefore, represent a new avenue for the treatment of vascular defects in the central nervous system.

The microstructure of arteries, consisting, in particular, of collagen, elastin, and vascular smooth muscle cells, plays a very significant role in their biomechanical response during a cardiac cycle. In this article, we highlight the microstructure and the contributions of each of its components to the overall mechanical behavior. We also describe the changes of the microstructure that occur as a result of abdominal aortic aneurysms and disease, such as atherosclerosis. We also focus on how the passive and active constituents are incorporated into a mathematical model without going into detail of the mathematical formulation. We conclude by mentioning open problems toward a better characterization of the biomechanical aspects of arteries that will be beneficial for a better understanding of cardiovascular pathophysiology.

Conferring antithrombogenicity to tissue-engineered vascular grafts remains a major challenge, especially for urgent bypass grafting that excludes approaches based on expanding autologous endothelial cells (ECs) that requires weeks of cell culture. Adipose-derived stem cells (ASCs) are available from most patients in sufficient number for coronary bypass graft seeding and may be effective as allogeneic cells. We thus compared the adhesion and platelet binding of human ASCs that were shear conditioned with constant and pulsatile shear stress (SS) after seeding the cells on a biologically engineered matrix suitable for arterial grafts. A monolayer of cells was maintained up to 15 dyn/cm 2 constant SS and up to 15 dyn/cm 2 mean pulsatile SS for 6 days of shear flow. Platelet binding was reduced from 83% to 6% of surface area and nitric oxide production was increased 23-fold with 7.5-15 dyn/cm 2 constant SS, but not pulsatile SS, relative to cells cultured statically on the matrix for 6 days. The reduction in platelet binding varied from no reduction to maximum reduction over a constant shear range of ∼2 to 4 dyn/cm 2 , respectively. Collectively, the study supports the potential use of ASCs to seed the luminal surface of a vascular graft made from this biologically engineered matrix to confer an antithrombogenic surface during the development of an endothelium from the seeded cells or the surrounding blood and tissue.

It has been thought that incretin signaling prevents arteriosclerosis, and very recently anti-arteriosclerotic effects through GLP-1 receptor were finally demonstrated in clinical human study. The purpose of this study was to investigate how vascular GLP-1 receptor expression is influenced in human subjects. First, we evaluated GLP-1 receptor expression in human arteries in immunostaining. Next, we separated the artery into the intima and media, and evaluated gene expression levels of various factors. We divided the subjects into obesity and non-obesity group and compared their expression levels between them. Finally, we evaluated which factors determine vascular GLP-1 receptor expression. GLP-1 receptor expression in intima and media was lower in obesity group compared to non-obesity group which was correlated with the alteration of TCF7L2 expression. Multiple regression analyses showed that BMI was an independent determining factor for GLP-1 receptor expression in the intima and media. Furthermore, using small interfering RNA method and TCF7L2-EGFP adenovirus, we showed that TCF7L2 was involved in GLP-1 receptor expression in human vascular cells. Taken together, vascular GLP-1 receptor and TCF7L2 expression was significantly down-regulated in human subjects with obesity. In addition, it is likely that TCF7L2 functions as a modulator of vascular GLP-1 receptor expression.

Human cell-based 3D tissue constructs play an increasing role in disease modeling and drug screening. Inflammation, atherosclerosis, and many autoimmune disorders involve the interactions between immune cells and blood vessels. However, it has been difficult to image and model these interactions under realistic conditions. In this study, we fabricated a perfusion and imaging chamber to allow the real-time visualization of leukocyte perfusion, adhesion, and migration inside a tissue-engineered blood vessel (TEBV). We monitored the elevated monocyte adhesion to the TEBV wall and transendothelial migration (TEM) as the TEBV endothelium was activated by the inflammatory cytokine TNF-α. We demonstrated that treatment with anti-TNF-α or an NF-kB signaling pathway inhibitor would attenuate the endothelium activation and reduce the number of leukocyte adhesion (>74%) and TEM events (>87%) close to the control. As the first demonstration of real-time imaging of dynamic cellular events within a TEBV, this work paves the way for drug screening and disease modeling in TEBV-associated microphysiological systems.

TMEM16A is essential for Ca 2+ -activated Cl - conductance in vascular smooth muscle. The importance of TMEM16A for agonist-induced vascular constriction and blood pressure control is, however, under debate. Previous studies suggested that TMEM16A might have a complex cellular function beyond being essential for the Ca 2+ -activated Cl - conductance, for example modulation of Ca 2+ channel expression. Mice with constitutive, smooth muscle-specific expression of siRNA directed against Tmem16a (transgenic mice, TG) were generated. Isometric constrictions of isolated aorta, mesenteric, femoral and tail arteries from TG mice were compared with wild-types. Protein expression was analysed by Western blots. Blood pressure and heart rate were studied telemetrically. Significant TMEM16A down-regulation was seen in aorta and tail arteries, while no changes were detected in mesenteric and femoral arteries. Contractile responses of mesenteric and femoral arteries from TG and wild-type mice were not different. Aorta from TG mice showed reduced agonist-induced constriction, while their responses to elevated K + were unchanged. Tail arteries from TG mice also constricted less to adrenergic stimulation than wild-types. Surprisingly, tail arteries from TG mice constricted less to elevated K + too and were more sensitive to nifedipine-induced relaxation. Consistently, TMEM16A down-regulation in tail arteries was associated with reduction in CACNA1C protein (i.e. vascular L-type Ca 2+ channel) expression. No differences in blood pressure and heart rate between the groups were seen. This study suggests a complex contribution of TMEM16A in vascular function. We suggest that TMEM16A modulates arterial contractility, at least in part, indirectly via regulation of CACNA1C expression., (© 2018 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Arteries and veins are specified by antagonistic transcriptional programs. However, during development and regeneration, new arteries can arise from pre-existing veins through a poorly understood process of cell fate conversion. Here, using single-cell RNA sequencing and mouse genetics, we show that vein cells of the developing heart undergo an early cell fate switch to create a pre-artery population that subsequently builds coronary arteries. Vein cells underwent a gradual and simultaneous switch from venous to arterial fate before a subset of cells crossed a transcriptional threshold into the pre-artery state. Before the onset of coronary blood flow, pre-artery cells appeared in the immature vessel plexus, expressed mature artery markers, and decreased cell cycling. The vein-specifying transcription factor COUP-TF2 (also known as NR2F2) prevented plexus cells from overcoming the pre-artery threshold by inducing cell cycle genes. Thus, vein-derived coronary arteries are built by pre-artery cells that can differentiate independently of blood flow upon the release of inhibition mediated by COUP-TF2 and cell cycle factors.

Endothelial cells (ECs) contribute to haematopoietic stem cell (HSC) maintenance in bone marrow, but the differential contributions of EC subtypes remain unknown, owing to the lack of methods to separate with high purity arterial endothelial cells (AECs) from sinusoidal endothelial cells (SECs). Here we show that the combination of podoplanin (PDPN) and Sca-1 expression distinguishes AECs (CD45 - Ter119 - Sca-1 bright PDPN - ) from SECs (CD45 - Ter119 - Sca-1 dim PDPN + ). PDPN can be substituted for antibodies against the adhesion molecules ICAM1 or E-selectin. Unexpectedly, prospective isolation reveals that AECs secrete nearly all detectable EC-derived stem cell factors (SCF). Genetic deletion of Scf in AECs, but not SECs, significantly reduced functional HSCs. Lineage-tracing analyses suggest that AECs and SECs self-regenerate independently after severe genotoxic insults, indicating the persistence of, and recovery from, radio-resistant pre-specified EC precursors. AEC-derived SCF also promotes HSC recovery after myeloablation. These results thus uncover heterogeneity in the contribution of ECs in stem cell niches.