Native and Oxidized Low-Density Lipoproteins Increase the Expression of the LDL Receptor and the LOX-1 Receptor, Respectively, in Arterial Endothelial Cells.

Atherosclerotic artery disease is the major cause of death and an immense burden on healthcare systems worldwide. The formation of atherosclerotic plaques is promoted by high levels of low-density lipoproteins (LDL) in the blood, especially in the oxidized form. Circulating LDL is taken up by conventional and non-classical endothelial cell receptors and deposited in the vessel wall. The exact mechanism of LDL interaction with vascular endothelial cells is not fully understood. Moreover, it appears to depend on the type and location of the vessel affected and the receptor involved. Here, we analyze how native LDL (nLDL) and oxidized LDL (oxLDL) modulate the expression of their receptors-classical LDLR and alternative LOX-1-in endothelial cells derived from human umbilical artery (HUAECs), used as an example of a medium-sized vessel, which is typically affected by atherosclerosis. Exposure of HUAECs to nLDL resulted in moderate nLDL uptake and gradual increase in LDLR, but not LOX-1, expression over 24 h. Conversely, exposure of HUAECs to oxLDL, led to significant accumulation of oxLDL and rapid induction of LOX-1, but not LDLR, within 7 h. These activation processes were associated with phosphorylation of protein kinases ERK1/2 and p38, followed by activation of the transcription factor AP-1 and its binding to the promoters of the respective receptor genes. Both nLDL-induced LDLR mRNA expression and oxLDL-induced LOX-1 mRNA expression were abolished by blocking ERK1/2, p-38 or AP-1. In addition, oxLDL, but not nLDL, was capable of inducing LOX-1 through the NF-κB-controlled pathway. These observations indicate that in arterial endothelial cells nLDL and oxLDL signal mainly via LDLR and LOX-1 receptors, respectively, and engage ERK1/2 and p38 kinases, and AP-1, as well as NF-κB transcription factors to exert feed-forward regulation and increase the expression of these receptors, which may perpetuate endothelial dysfunction in atherosclerosis.

Tokishakuyakusan ameliorates lowered body temperature after immersion in cold water through the early recovery of blood flow in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: 'Cold feeling' is a subjective feeling of unusual coldness that aggravates fatigue, stiffness, and other symptoms, thereby reducing quality of life. Tokishakuyakusan (TSS) is a Kampo medicine reported to improve cold feeling and is used to treat symptoms aggravated by cold feeling. However, the mechanism of action of TSS is unclear. Cold feeling may involve reduced blood flow and subsequent inhibition of heat transport. Therefore, elucidating the effects of TSS on blood flow is one of the most important research topics for clarifying the mechanism of action of TSS. AIM OF THE STUDY: We aimed to evaluate the effect of TSS on recovery from lowered body temperature by the immersion of rats in cold water and to clarify the involvement of blood flow in the action of TSS. MATERIALS AND METHODS: After female Wistar rats underwent 9 days of low room temperature stress loading (i.e. room temperature of 18 °C), they were subjected to immersion in cold water (15 °C) for 15 min. Body surface temperature, rectal temperature, and plantar temperature were measured before and after immersion in cold water. Blood flow was measured before and after immersion in cold water without low room temperature stress loading. TSS (0.5 g/kg or 1 g/kg) or the vehicle (i.e. distilled water) was orally administered once daily for 10 days for the measurement of body temperature or once 30 min before immersion in cold water for the measurement of blood flow. In addition, we examined the effect of TSS on calcitonin gene-related peptide (CGRP) release from dorsal root ganglion (DRG) cells, the effect of TSS ingredients on transient receptor potential (TRP) channels, and the effect of TSS ingredients on the membrane potential of vascular smooth muscle cells and evaluated the mechanism of the effects of TSS on blood flow. RESULTS: Body temperature and blood flow decreased after immersion in cold water and then recovered over time. A comparison of body temperature at each timepoint or area under the curve showed that TSS (1 g/kg) accelerated the recovery of body surface temperature, rectal temperature, and blood flow. TSS significantly increased CGRP release from DRG cells, which disappeared after pretreatment with HC-030031 (a transient receptor potential ankyrin 1 [TRPA1] antagonist). The effects of seven TSS ingredients on TRP channels were examined. The agonistic effect on TRPA1 was observed for atractylodin, atractylodin carboxylic acid and levistolide A. Among the TSS ingredients, atractylodin carboxylic acid had significant hyperpolarising effects. CONCLUSIONS: The mechanism by which TSS accelerates the recovery of lowered body temperature in rats after immersion in cold water may involve the acceleration of the recovery of lowered blood flow. Increased CGRP release from DRG cells by TSS, TRPA1 activation by TSS ingredients, and membrane potential changes in vascular smooth muscle cells caused by TSS ingredients are part of the mechanism of action of TSS. These findings may partly contribute to the interpretation of the beneficial effects of TSS on cold feeling.

Circular RNA circ-ARFIP2 regulates proliferation, migration and invasion in human vascular smooth muscle cells via miR-338-3p-dependent modulation of KDR.

Dysfunction of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of intracranial aneurysm (IA). Circular RNAs (circRNAs) have been implicated in the pathogenesis of IA by reducing microRNA (miRNA) activity. In this paper, we investigated the precise roles of circRNA ADP ribosylation factor interacting protein 2 (circ-ARFIP2, circ_0021001) in VSMC dysfunction. The levels of circ-ARFIP2, miR-338-3p and kinase insert domain receptor (KDR) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Ribonuclease (RNase) R and subcellular fractionation assays were used to assess the stability and localization of circ-ARFIP2, respectively. Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay, and cell invasion was measured by transwell assay. Cell proliferation was gauged by 5-Ethynyl-2'-Deoxyuridine (EdU) assay. Cell migration was evaluated by transwell and wound-healing assays. Targeted correlations among circ-ARFIP2, miR-338-3p and KDR were validated by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Circ-ARFIP2 and KDR were underexpressed and miR-338-3p was overexpressed in the arterial wall tissues of IA patients. Overexpression of circ-ARFIP2 in human umbilical artery smooth muscle cells (HUASMCs) showed a significant promotion in cell proliferation, migration and invasion. Mechanistically, circ-ARFIP2 targeted miR-338-3p, and circ-ARFIP2 regulated cell behaviors by miR-338-3p. KDR was a direct and functional target of miR-338-3p. Moreover, KDR was a downstream effector of circ-ARFIP2 function. Circ-ARFIP2 regulated KDR expression by targeting miR-338-3p. Our present findings demonstrated that the increased level of circ-ARFIP2 enhanced HUASMC proliferation, migration and invasion at least in part by the miR-338-3p/KDR axis.

Involvement of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in mPRα (PAQR7)-mediated progesterone induction of vascular smooth muscle relaxation.

Progesterone acts directly on vascular smooth muscle cells (VSMCs) through activation of membrane progesterone receptor α (mPRα)-dependent signaling to rapidly decrease cytosolic Ca2+ concentrations and induce muscle relaxation. However, it is not known whether this progesterone action involves uptake of Ca2+ by the sarco/endoplasmic reticulum (SR) and increased sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity. The present results show that treatment of cultured human VSMCs with progesterone and the selective mPR agonist Org OD-02-0 (OD 02-0) but not with the nuclear PR agonist R5020 increased SERCA protein expression, which was blocked by knockdown of mPRα with siRNA. Moreover, treatments with progesterone and OD 02-0, but not with R5020, increased phospholamban (PLB) phosphorylation, which would result in disinhibition of SERCA function. Progesterone and OD 02-0 significantly increased Ca2+ levels in the SR and caused VSMC relaxation. These effects were blocked by pretreatment with cyclopiazonic acid (CPA), a SERCA inhibitor, and by knockdown of SERCA2 with siRNA, suggesting that SERCA2 plays a critical role in progesterone induction of VSMC relaxation. Treatment with inhibitors of inhibitory G proteins (Gi, NF023), MAP kinase (AZD 6244), Akt/Pi3k (wortmannin), and a Rho activator (calpeptin) blocked the progesterone- and OD 02-0-induced increase in Ca2+ levels in the SR and SERCA expressions. These results suggest that the rapid effects of progesterone on cytosolic Ca2+ levels and relaxation of VSMCs through mPRα involve regulation of the functions of SERCA2 and PLB through Gi, MAP kinase, and Akt signaling pathways and downregulation of RhoA activity.NEW & NOTEWORTHY The rapid effects of progesterone on cytosolic Ca2+ levels and relaxation of VSMCs through mPRα involve regulation of the functions of SERCA2 and PLB through Gi, MAP kinase, and Akt signaling pathways and downregulation of RhoA activity.

Bazedoxifene increases the proliferation of human arterial endothelial cells but does not affect the expression of cyclins A, B, and D1 and of p27Kip1.

OBJECTIVE: Endothelial dysfunction and denudation are considered a first step in atherosclerosis. Endothelial proliferation is key for cellular repair. The effect of bazedoxifene on the vascular endothelium has not been explored. We investigated the effect of bazedoxifene on endothelial cell proliferation. METHODS: Primary cultures from human umbilical artery endothelial cells were used in dose-response experiments (0.1, 1.0, and 10.0 EC50 dose) with bazedoxifene, estradiol, raloxifene and a combination of bazedoxifene and estradiol. Proliferation was assessed with the XTT colorimetric cell-proliferation assay. The possible participation of cyclins A, B, D1 and p27Kip1 was analyzed by the measurement of their expression at both the protein and the gene levels. RESULTS: A significant increase of similar size for cell proliferation was obtained with bazedoxifene, estradiol and raloxifene, but no significant change was observed for the association of bazedoxifene and estradiol. The impact was detected at the first 0.1 EC50 dose and was not dose-dependent. Estradiol achieved a significant increase in the protein expression of cyclin A and p27Kip1, but no change was detected for the other compounds at either the gene or protein level. CONCLUSION: Bazedoxifene demonstrated a proliferative effect of similar size to estradiol in cultured human umbilical artery endothelial cells. The molecular mechanisms need further investigation.

Venous and Arterial Endothelial Cells from Human Umbilical Cords: Potential Cell Sources for Cardiovascular Research.

Although cardiovascular devices are mostly implanted in arteries or to replace arteries, in vitro studies on implant endothelialization are commonly performed with human umbilical cord-derived venous endothelial cells (HUVEC). In light of considerable differences, both morphologically and functionally, between arterial and venous endothelial cells, we here compare HUVEC and human umbilical cord-derived arterial endothelial cells (HUAEC) regarding their equivalence as an endothelial cell in vitro model for cardiovascular research. No differences were found in either for the tested parameters. The metabolic activity and lactate dehydrogenase, an indicator for the membrane integrity, slightly decreased over seven days of cultivation upon normalization to the cell number. The amount of secreted nitrite and nitrate, as well as prostacyclin per cell, also decreased slightly over time. Thromboxane B2 was secreted in constant amounts per cell at all time points. The Von Willebrand factor remained mainly intracellularly up to seven days of cultivation. In contrast, collagen and laminin were secreted into the extracellular space with increasing cell density. Based on these results one might argue that both cell types are equally suited for cardiovascular research. However, future studies should investigate further cell functionalities, and whether arterial endothelial cells from implantation-relevant areas, such as coronary arteries in the heart, are superior to umbilical cord-derived endothelial cells.

Flow with variable pulse frequencies accelerates vascular recellularization and remodeling of a human bioscaffold.

Despite significant advances in vascular tissue engineering, the ideal graft has not yet been developed and autologous vessels remain the gold standard substitutes for small diameter bypass procedures. Here, we explore the use of a flow field with variable pulse frequencies over the regeneration of an ex vivo-derived human scaffold as vascular graft. Briefly, human umbilical veins were decellularized and used as scaffold for cellular repopulation with human smooth muscle cells (SMC) and endothelial cells (EC). Over graft development, the variable flow, which mimics the real-time cardiac output of an individual performing daily activities (e.g., resting vs. exercising), was implemented and compared to the commonly used constant pulse frequency. Results show marked differences on SMC and EC function, with changes at the molecular level reflecting on tissue scales. First, variable frequencies significantly increased SMC proliferation rate and glycosaminoglycan production. These results can be tied with the SMC gene expression that indicates a synthetic phenotype, with a significant downregulation of myosin heavy chain. Additionally and quite remarkably, the variable flow frequencies motivated the re-endothelialization of the grafts, with a quiescent-like structure observed after 10 days of conditioning, contrasting with the low surface coverage and unaligned EC observed under constant frequency (CF). Besides, the overall biomechanics of the generated grafts (conditioned with both pulsed and CFs) evidence a significant remodeling after 55 days of culture, depicted by high burst pressure and Young's modulus. These last results demonstrate the positive recellularization and remodeling of a human-derived scaffold toward an arterial vessel.

Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant clonal disease derived from hematopoietic stem/progenitor cell. Leukemia blasts cause extensive hypoxia of bone marrow (BM), which lead to disorder and remodeling of BM niche, thereby becoming "leukemic niche" to support the development and drug-resistance of AML as well as the maintenance of normal hematopoietic stem cells. In this study, the biological characteristics (such as self-renewal, apoptosis, migration, autocrine) and function (vascularization) of mesenchymal stem cells (MSCs) and human umbilical artery endothelial cells (HUAECs) that make up BM arteriolar niche in simulated hypoxia AML context were investigated. It was found that moderate hypoxia enhanced the viability of the arteriolar niche cells, but severe hypoxia of AML BM resulted in the damage of arteriolar niche cells and the disorder of vascular cytokines C-X-C motif chemokine ligand 6 (CXCL6). The dynamic changes of CXCL6 in the system as well as its anti-apoptotic and promoting angiogenic effects suggested that CXCL6 played an important role in the remodeling of BM arteriolar niche in AML. Taking advantage of CXCL6 can save the damaged MSCs and HUAECs, which is the hope of rescuing arteriolar niche. It is suggested that CXCL6 may be an assistant strategy for microenvironment targeted therapy of AML.

Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth.

The umbilical artery lumen closes rapidly at birth, preventing neonatal blood loss, whereas the umbilical vein remains patent longer. Here, analysis of umbilical cords from humans and other mammals identified differential arterial-venous proteoglycan dynamics as a determinant of these contrasting vascular responses. The umbilical artery, but not the vein, has an inner layer enriched in the hydrated proteoglycan aggrecan, external to which lie contraction-primed smooth muscle cells (SMC). At birth, SMC contraction drives inner layer buckling and centripetal displacement to occlude the arterial lumen, a mechanism revealed by biomechanical observations and confirmed by computational analyses. This vascular dimorphism arises from spatially regulated proteoglycan expression and breakdown. Mice lacking aggrecan or the metalloprotease ADAMTS1, which degrades proteoglycans, demonstrate their opposing roles in umbilical vascular dimorphism, including effects on SMC differentiation. Umbilical vessel dimorphism is conserved in mammals, suggesting that differential proteoglycan dynamics and inner layer buckling were positively selected during evolution.

Removal of an abluminal lining improves decellularization of human umbilical arteries.

The decellularization of long segments of tubular tissues such as blood vessels may be improved by perfusing decellularization solution into their lumen. Particularly, transmural flow that may be introduced by the perfusion, if any, is beneficial to removing immunogenic cellular components in the vessel wall. When human umbilical arteries (HUAs) were perfused at a transmural pressure, however, very little transmural flow was observed. We hypothesized that a watertight lining at the abluminal surface of HUAs hampered the transmural flow and tested the hypothesis by subjecting the abluminal surface to enzyme digestion. Specifically, a highly viscous collagenase solution was applied onto the surface, thereby restricting the digestion to the surface. The localized digestion resulted in a water-permeable vessel without damaging the vessel wall. The presence of the abluminal lining and its successful removal were also supported by evidence from SEM, TEM, and mechanical testing. The collagenase-treated HUAs were decellularized with 1% sodium dodecyl sulfate (SDS) solution under either rotary agitation, simple perfusion, or pressurized perfusion. Regardless of decellularization conditions, the decellularization of HUAs was significantly enhanced after the abluminal lining removal. Particularly, complete removal of DNA was accomplished in 24 h by pressurized perfusion of the SDS solution. We conclude that the removal of the abluminal lining can improve the perfusion-assisted decellularization.

Vitrified Human Umbilical Arteries as Potential Grafts for Vascular Tissue Engineering.

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.

Endothelial specific deletion of FOXO1 alters pericyte coverage in the developing retina.

Pericytes are mural cells that cover small blood vessels. While defects in pericyte coverage are known to be involved in various vessel related pathologies, including diabetic retinopathy, the molecular mechanisms underlying pericyte coverage are not fully understood. In this study, we investigated the contribution of the forkhead transcription factor FOXO1 in endothelial cells to pericyte coverage in the developing retina. We observed retinal pericytes in tamoxifen-inducible endothelium-specific Foxo1 deletion mice. Tamoxifen was injected at postnatal day 1-3 and the retinas were harvested at P21. Our results demonstrated that Foxo1 deletion in the endothelium affected arteriole pericyte morphology without altering pericyte number, proliferation, and apoptosis. We hypothesized that abnormal pericyte morphogenesis in the knockout retina was caused by impaired pericyte differentiation. FOXO1 silencing by siRNA in the primary artery endothelium further revealed that THBS1 (thrombospondin 1), which promotes pericyte differentiation via TGFß activation, was reduced in the FOXO1-deficient endothelium. Immunohistochemistry of FOXO1 knockout mice showed reduced numbers of phospho-Smad3+ arteriole pericytes compared with wild-type mice. In addition, endothelium-pericyte co-culture analysis revealed that pericytes cultured with FOXO1-deficient endothelial cells failed to differentiate sufficiently; this failure was partially rescued by the addition of recombinant THBS1 to the supernatant. The findings suggest that endothelial FOXO1 contributes to pericyte differentiation via regulation of THBS1 expression. This study provides new insights into the molecular mechanism of pericyte coverage in the context of endothelium-derived regulation and highlights a new therapeutic target for pericyte-related pathology.

Human umbilical artery endothelial cells from Large-for-Gestational-Age newborn have increased antioxidant efficiency and gene expression.

Obesity is a public health problem worldwide, and especially in women in reproductive age where more than one in three have obesity. Maternal obesity is associated with an increased maternal, placental, and newborn oxidative stress, which has been proposed as a central factor in vascular dysfunction in large-for-gestational-age (LGA) newborn. However, cellular and molecular mechanisms behind this effect have not been elucidated. Untreated human umbilical artery endothelial cells (HUAEC) from LGA (LGA-HUAEC) presented higher O2- levels, superoxide dismutase activity and heme oxygenase 1 messenger RNA (mRNA) levels, paralleled by reduced GSH:GSSG ratio and NRF2 mRNA levels. In response to an oxidative challenge (hydrogen peroxide), only HUAEC from LGA exhibited an enhanced Glutathione Peroxidase 1 (GPX1) expression, as well as a more efficient antioxidant machinery measured by the biosensor probe, HyPer. An open state of chromatin in the TSS region of GPX1 in LGA-HUAEC was evidenced by the DNase-HS assay. Altogether, our data indicate that LGA-HUAEC have an altered cellular and molecular antioxidant system. We propose that a chronic pro-oxidant intrauterine milieu, as evidenced in pregestational obesity, could induce a more efficient antioxidant system in fetal vascular cells, which could be maintained by epigenetic mechanism during postnatal life.

Decellularization of human umbilical arteries / Descelularización de las arterias umbilicales humanas

SUMMARY: Arterial obstruction in small diameter (<6 mm) vessels are many times treated with grafts, however autologous aren't always available and synthetic have a high rate of complications. Decellularization of umbilical arteries may provide a solution, but the ideal method is debatable. We compare effectiveness between SDS and Triton X-100. Umbilical cords obtained from full term pregnancies with normal development and no evident complications in the newborn, were micro-dissected within 12 h and stored in phosphate buffered saline without freezing. Arteries were then processed for decellularization using 0.1 % and 1 % SDS, and 1 % Triton X100 protocols. Evaluation of cellular and nuclear material, collagen fibers, elastic fibers, and glycosoaminoglycans of the extracellular matrix (ECM) were evaluated as well as morphometric analysis under histological and immunohistochemical techniques. Triton X-100 was ineffective, preserving nuclear remains identified by immunofluorescence, had the most notable damage to elastic fibers, and decrease in collagen. SDS effectively eliminated the nuclei and had a less decrease in elastic fibers and collagen. Laminin was preserved in all groups. No significant differences were identified in luminal diameters; however the middle layer decreased due to decellularization of muscle cells. In conclusion, 0.1 % SDS decellularization was the most effective in eliminating cells and preserving the main components of the ECM.

RESUMEN: La obstrucción arterial en vasos de pequeño diámetro (<6 mm) se trata muchas veces con injertos, sin embargo, los autólogos no siempre están disponibles y los sintéticos tienen una alta tasa de complicaciones. La descelularización de las arterias umbilicales puede proporcionar una solución, pero el método ideal es discutible. Comparamos la efectividad entre los métodos SDS y Triton X-100. Cordones umbilicales obtenidos a partir de embarazos a término con evolución normal y sin complicaciones evidentes del recién nacido, se microdiseccionaron en 12 horas y se almacenaron en solución salina con fosfato sin congelación. Las arterias se procesaron luego para la descelularización usando los protocolos de SDS al 0,1 % y 1 %, y Triton X-100 al 1 %. Se realizó la evaluación de material celular y nuclear, fibras de colágeno, fibras elásticas y glucosoaminoglicanos de la matriz extracelular (MEC), así como el análisis morfométrico bajo técnicas histológicas e inmunohistoquímicas. Triton X-100 fue ineficaz, conservando los restos nucleares identificados por inmunofluorescencia, tuvo el daño más notable a las fibras elásticas y la disminución del colágeno. SDS efectivamente eliminó los núcleos y tuvo una disminución menor en las fibras elásticas y el colágeno. Laminina fue preservado en todos los grupos. No se identificaron diferencias significativas en los diámetros luminales; sin embargo, la capa media disminuyó debido a la descelularización de las células musculares. la descelularización con SDS al 0,1 % fue la más efectiva para eliminar células y preservar los principales componentes de la MEC.

Dengue Virus Infection of Primary Human Smooth Muscle Cells.

Dengue virus (DENV) infection of humans is presently the most important arthropod-borne viral global threat, for which no suitable or reliable animal model exists. Reports addressing the effect of DENV on vascular components other than endothelial cells are lacking. Dengue virus infection of vascular smooth muscle cells, which play a physiological compensatory response to hypotension in arteries and arterioles, has not been characterized, thus precluding our understanding of the role of these vascular components in dengue pathogenesis. Therefore, we studied the permissiveness of primary human umbilical artery smooth muscle cells (HUASMC) to DENV 1-4 infection and compared with the infection in the previously reported primary human umbilical vein endothelial cells (HUVEC) and the classically used, non-transformed, and highly permissive Lilly Laboratories Cell-Monkey Kidney 2 cells. Our results show that HUASMC are susceptible and productive to infection with the four DENV serotypes, although to a lesser extent when compared with the other cell lines. This is the first report of DENV permissiveness in human smooth muscle cells, which might represent an unexplored pathophysiological contributor to the vascular collapse observed in severe human dengue infection.

Arterial Wall Stress Induces Phenotypic Switching of Arterial Smooth Muscle Cells in Vascular Remodeling by Activating the YAP/TAZ Signaling Pathway.

BACKGROUND/AIMS: Increasing wall stress or biomechanical stretch experienced by arteries influences the initiation of atherosclerotic lesions. This initiation is mediated by Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), which are both effectors of the Hippo pathway. In this study, the functional roles of YAP/TAZ proteins in the regulation of the stretch-mediated programing of human umbilical arterial smooth muscle cells (HUASMCs) to a proliferative phenotype were examined. METHODS: HUASMCs were seeded on a Matrigel-coated silicone chamber and subjected to biomechanical stretch for 24 h after 48 h of growth. YAP/TAZ small interfering RNA was used to specifically knockdown YAP/ TAZ expression in HUASMCs. RESULTS: We observed that YAP/TAZ activation via biomechanical stretching is involved in the regulation of critical aspects of the HUASMC phenotypic switch. YAP/TAZ knockdown significantly attenuated the stretch-induced proliferative and pro-inflammatory phenotypes in HUASMCs. Furthermore, treatment with atorvastatin, an anti-atherosclerotic drug, attenuated the stretch-induced phenotypic switch of HUASMCs from the contractile to synthetic state by suppressing YAP/TAZ expression. Additional investigations demonstrated the role of stretch in inhibiting the Hippo pathway, leading to the activation of PI3-kinase (PI3K) and phosphoinositide dependent kinase (PDK1); the key molecule for the regulation of the PDK1 and Hippo complex interaction was Sav1. These results showed the importance of YAP/TAZ activation, induced by biomechanical stretch, in promoting atheroprone phenotypes in HUASMCs. CONCLUSION: Taken together, our findings revealed a mechanism by which YAP/TAZ activation contributes to the pathogenesis of atherosclerosis.

Development of HLA-matched vascular grafts utilizing decellularized human umbilical artery.

Worldwide, there is a great need of small diameter vascular grafts that can be used in human disorders such as cardiovascular and peripheral vascular disease. Until now, severe adverse reactions are caused from the use of synthetic or animal derived grafts, while the use of autologous vessels is restricted only in a small number of patients. The limited availability of the vessels might be resolved by the use of HLA-matched vascular grafts utilizing the decellularized human umbilical arteries. In this study, human umbilical arteries were decellularized and then repopulated with Mesenchymal Stem Cells. The HLA-genotype of the repopulated grafts, analyzed by Next Generation Sequencing technology, indicated their successful production. The HLA-matched vascular grafts could be generated efficiently and might be used in personalized medicine.

Differential HDAC6 Activity Modulates Ciliogenesis and Subsequent Mechanosensing of Endothelial Cells Derived from Pluripotent Stem Cells.

The role of primary cilia in mechanosensation is essential in endothelial cell (EC) shear responsiveness. Here, we find that venous, capillary, and progenitor ECs respond to shear stress in vitro in a cilia-dependent manner. We then demonstrate that primary cilia assembly in human induced pluripotent stem cell (hiPSC)-derived ECs varies between different cell lines with marginal influence of differentiation protocol. hiPSC-derived ECs lacking cilia do not align to shear stress, lack stress fiber assembly, have uncoordinated migration during wound closure in vitro, and have aberrant calcium influx upon shear exposure. Transcriptional analysis reveals variation in regulatory genes involved in ciliogenesis among different hiPSC-derived ECs. Moreover, inhibition of histone deacetylase 6 (HDAC6) activity in hiPSC-ECs lacking cilia rescues cilia formation and restores mechanical sensing. Taken together, these results show the importance of primary cilia in hiPSC-EC mechano-responsiveness and its modulation through HDAC6 activity varies among hiPSC-ECs.

VascuTrainer: A Mobile and Disposable Bioreactor System for the Conditioning of Tissue-Engineered Vascular Grafts.

In vitro tissue engineering of vascular grafts requires dynamic conditioning in a bioreactor system for in vitro tissue maturation and remodeling to receive a mechanically adequate and hemocompatible implant. The goal of the current work was to develop a bioreactor system for the conditioning of vascular grafts which is (i) able to create a wide range of flow, pressure and frequency conditions, including physiological ones; (ii) compact and easy to assemble; (iii) transportable; (iv) disposable. The system is driven by a small centrifugal pump controlled via a custom-made control unit, which can also be operated on batteries to allow for autonomous transportation. To show the potential of the newly developed bioreactor system small-caliber vascular composite grafts (n = 5, internal diameter = 3 mm, length = 12.5 cm) were fabricated using a fibrin scaffold embedding human umbilical artery smooth muscle cells and a polyvinylidene fluoride warp-knitted macroporous mesh. Subsequently, the vascular grafts were endothelialized and mounted in the bioreactor system for conditioning. The conditioning parameters remained within the predefined range over the complete conditioning period and during operation on batteries as tested for up to 25 h. Fabrication and pre-conditioning under arterial pressure and shear stress conditions resulted in robust and hemocompatible tissue-engineered vascular grafts. Analysis of immunohistochemical stainings against extracellular matrix and cell-specific proteins revealed collagen I and collagen III deposition. The luminal surface was confluently covered with endothelial cells. The developed bioreactor system showed cytocompatibility and pH, pO2, pCO2, glucose and lactate stayed constant. Sterility was maintained during the complete fabrication process of the vascular grafts. The potential of a versatile and mobile system and its functionality by conditioning tissue-engineered vascular grafts under physiological pressure and flow conditions could be demonstrated.

Fabrication of a mechanically anisotropic poly(glycerol sebacate) membrane for tissue engineering.

Poly(glycerol sebacate) (PGS) has been used successfully as a scaffolding material for soft tissue engineering. PGS scaffolds, however, are usually mechanically isotropic, which may restrict their use in tissue repairs as many soft tissues in the body have anisotropic mechanical behaviors. Although various methods have been used to fabricate anisotropic scaffolds, it remains challenging to make anisotropic scaffolds from thermoset PGS. Here a new, simple method to fabricate an anisotropic PGS membrane which can then be used to construct thicker three-dimensional anisotropic scaffolds was developed. First, an aligned sacrificial poly(vinyl alcohol) fibrous membrane was prepared by electrospinning. The fibrous membrane was then partially immersed in PGS prepolymer solution, resulting in a composite membrane upon drying. After curing, the sacrificial fibers within the membrane were removed by water, supposedly leaving aligned cylindrical pores in the membrane. Both SEM and AFM illustrated aligned grooves on the surface of the resultant PGS membrane, indicating the successful removal of sacrificial fibers. The PGS membrane was validated to be mechanically anisotropic using uniaxial tensile testing along and perpendicular to the predominant pore direction. The in vitro cytocompatibility of the PGS membrane was confirmed. As a demonstration of its potential application in vascular tissue engineering, a tubular scaffold was constructed by wrapping a stack of two axisymmetric pieces of the anisotropic PGS membranes on a mandrel. The compliance of the scaffold was found to depend on the pitch angle of its double helical structure, imitating the anisotropic mechanical behavior of the arterial media. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 760-770, 2018.