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Objective: This article is reporting a method for establishment primary cultures of vascular smooth muscle cells (VSMCs) from the spiral modiolar artery (SMA)., Methods: VSMCs were isolated from guinea pig SMAs. Arterial tissues were cut and enzymatically digested at 37 °C for 20 min using a 0.1% trypsin solution. After digestion, tissue fragments were explanted in a 35-mm culture dish. Contaminated fibroblasts were separated from VSMCs because of their different adhesion abilities. The cells migrated from the explants within 7-10 days and grew to confluence in approximately 4 weeks., Results: We obtained pure and viable VSMCs from the confluent third passage. The morphological and immunofluorescence analyses demonstrated a "hill-and-valley" growth pattern that is characteristics of VSMCs, and the expression of cell type-specific markers (α-smooth muscle actin and myosin), respectively. The change of intracellular calcium concentration induced by angiotensin II and CaCl(2) showed that the VSMCs had good cell viability., Conclusion: We obtained purified VSMCs using this method. All cell cultures expressed smooth muscle markers (α-SM actin, and myosin) and were negative for vWF. This article provides a simple method to obtain VSMCs for in vitro studies of physiology and pathophysiology in the circulation disturbances of the inner ear. In addition, VSMCs are regarded to be an excellent model to evaluate drugs in vitro., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Members of the transforming growth factor-β superfamily play essential roles in various aspects of embryonic development and physiological organ function. Among them, bone morphogenetic protein (BMP) 9 and BMP10 regulate embryonic vascular development by activating their endothelial receptor ALK1 (activin receptor-like kinase 1, also called Acvrl1). ALK1-mediated intracellular signaling is implicated in the etiologies of human diseases, but their downstream functional proteins are largely unknown. In this study, we identified Tmem100, a gene encoding a previously uncharacterized intracellular transmembrane protein, to be an embryonic endothelium-enriched gene activated by BMP9 and BMP10 through the ALK1 receptor. Tmem100 null mice showed embryonic lethality due to impaired differentiation of arterial endothelium and defects of vascular morphogenesis, which phenocopied most of the vascular abnormalities observed with the Acvrl1/Alk1 deficiency. The activity of Notch- and Akt-mediated signaling, which is essential for vascular development, was down-regulated in Tmem100 null mice. Cre-mediated deletion of Tmem100 in endothelial cells was sufficient to recapitulate the null phenotypes. These data indicated that TMEM100 may play indispensable roles downstream of BMP9/BMP10-ALK1 signaling during endothelial differentiation and vascular morphogenesis.

Intrauterine growth restriction is a risk factor for cardiovascular disease in adulthood. We have previously shown that intrauterine growth restriction caused by uteroplacental insufficiency programmes uterine vascular dysfunction and increased arterial stiffness in adult female rat offspring. The aim of this study was to investigate vascular adaptations in growth restricted female offspring when they in turn become pregnant. Uteroplacental insufficiency was induced in WKY rats by bilateral uterine vessel ligation (Restricted) or sham surgery (Control) on day 18 of pregnancy. F0 pregnant females delivered naturally at term. F1 Control and Restricted offspring were mated at 4 months of age and studied on day 20 of pregnancy. Age-matched non-pregnant F1 Control and Restricted females were also studied. Wire and pressure myography were used to test endothelial and smooth muscle function, and passive mechanical wall properties, respectively, in uterine, mesenteric, renal and femoral arteries of all four groups. Collagen and elastin fibres were quantified using polarized light microscopy and qRT-PCR. F1 Restricted females were born 10–15% lighter than Controls (P <0.05). Non-pregnant Restricted females had increased uterine and renal artery stiffness compared with Controls (P <0.05), but this difference was abolished at day 20 of pregnancy. Vascular smooth muscle and endothelial function were preserved in all arteries of non-pregnant and pregnant Restricted rats. Collagen and elastin content were unaltered in uterine arteries of Restricted females. Growth restricted females develop compensatory vascular changes during late pregnancy, such that region-specific vascular deficits observed in the non-pregnant state did not persist in late pregnancy.

Introduction: Off-target effects of novel antiplatelet agents due to their potential clinical benefits are currently an area of intensive investigation. We aimed to compare the effects of different P2Y(12) antagonists on the reactivity of vascular smooth muscle cells., Materials and Methods: Wistar rats (n=30) were pretreated with an investigated drug or placebo. Clopidogrel (50mg/kg, n=7), prasugrel (10mg/kg, n=7), ticagrelor (10mg/kg, n=7) or placebo (n=9) were administered orally 12 and 2 hours before experiments. Constrictions of rat tail arteries induced with a stable analogue of adenosine diphosphate (2-MeS-ADP), phenylephrine and arginine vasopressin were measured as an increase in perfusion pressure. Effects of ticagrelor were assessed in the presence of ticagrelor (1μM/L) added to the perfusion solution as this drug reversibly inhibits the P2Y(12) receptor., Results: Pretreatment with clopidogrel and prasugrel did not inhibit 2-MeS-ADP-induced contraction while ticagrelor did. Experiments employing endothelium-deprived arteries provided similar results. Clopidogrel and prasugrel did not influence concentration-response curves in the presence of neither phenylephrine nor arginine vasopressin. The curves obtained for both vasopressors in the presence of ticagrelor and 2-MeS-ADP were shifted to the right with a significant reduction in the maximal response., Conclusions: Oral administration of ticagrelor, in contrast to clopidogrel and prasugrel, prevents adenosine diphosphate-induced contraction of vascular smooth muscle cells in a rat model. Both the clinical significance and detailed mechanism of our findings warrant further investigation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Our ongoing studies show that vascular endothelial cell growth factor (VEGF)-bound surfaces selectively capture endothelial progenitor cells (EPCs) in vitro and in vivo, and that surface-bound VEGF stimulates intracellular signal transduction pathways over prolonged culture periods, resulting in inductive differentiation of EPCs. In this article, we investigated whether simulated arterial shear stress augments the differentiation of EPCs adhered to a VEGF-bound surface. Human peripheral blood-derived mononuclear cells adhered to a VEGF-bound surface were exposed to 1 day of shear stress (15 dynes/cm(2), corresponding to shear load in arteries). Shear stress suppressed the expression of mRNAs encoding CD34 and CD133, which are markers for EPCs, and augmented the expression of mRNAs encoding CD31 and von Willebrand factor (vWF) as well as vWF protein, which are markers for endothelial cells (ECs). Shear stress enhanced expression of ephrinB2 mRNA, a marker for arterial ECs, but did not significantly change expression of EphB4 mRNA, a marker for venous ECs. Focused protein array analysis showed that mechanotransduction by shear stress activated the p38 and MAPK pathways in EPCs. Thus, arterial shear stress, in concert with surface-bound VEGF, augments the differentiation of EPCs. These results strongly support previous observation of rapid differentiation of EPCs captured on VEGF-bound stents in a porcine model., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We previously demonstrated that human pericytes, which encircle capillaries and microvessels, give rise in culture to genuine mesenchymal stem cells (MSCs). This raised the question as to whether all MSC are derived from pericytes. Pericytes and other cells defined on differential expression of CD34, CD31, and CD146 were sorted from the stromal vascular fraction of human white adipose tissue. Besides pericytes, CD34+ CD31- CD146- CD45- cells, which reside in the outmost layer of blood vessels, the tunica adventitia, natively expressed MSC markers and gave rise in culture to clonogenic multipotent progenitors identical to standard bone marrow-derived MSC. Despite common MSC features and developmental properties, adventitial cells and pericytes retain distinct phenotypes and genotypes through culture. However, in the presence of growth factors involved in vascular remodeling, adventitial cells acquire a pericytes-like phenotype. In conclusion, we demonstrate the co-existence of 2 separate perivascular MSC progenitors: pericytes in capillaries and microvessels and adventitial cells around larger vessels.

Binding of endothelial cell (EC) integrins to extracellular-matrix (ECM) components is one of the key events to trigger intracellular signaling that will ultimately result in proper vascular development. Even within one tissue, the endothelial phenotype differs between arteries and veins. Here, we tested the hypothesis that anchorage dependent processes, such as proliferation, viability, survival and actin organization of venous (VEC) and arterial EC (AEC) differently depend on ECM proteins. Moreover,because of different oxygen tension in AEC and VEC, we tested oxygen as a co-modulator of ECM effects. Primary human placental VEC and AEC were grown in collagens I and IV, fibronectin, laminin, gelatin and uncoated plates and exposed to 12 and 21% oxygen. Our main findings revealed that VEC are more sensitive than AEC to changes in the ECM composition. Proliferation and survival of VEC, in contrast to AEC, were profoundly increased by the presence of collagen I and fibronectin when compared with gelatin or uncoated plates. These effects were reversed by inhibition of focal adhesion kinase (Fak) and modulated by oxygen. VEC were more susceptible to the oxygen dependent ECM effects than AEC. However, no differential ECM effect on actin organization was observed between the two cell types. These data provide first evidence that AEC and VEC from the same vascular loop respond differently to ECM and oxygen in a Fak-dependent manner.

The aim of the present work was to study, by means of immunohistochemical and RT-PCR techniques, the presence and distribution of immunopositivity for orexin A and B (OXA and OXB) and orexin type 1 and 2 receptors (OX(1)R and OX(2)R) in the lacrimal gland of sheep as well as the gene expressions for prepro-orexin (PPOX) and cognate receptors. In serial sections, positive staining for OXA and OXB were localized in the same nervous fibers within the connective tissue septa. Positive staining for OX(1)R was evidenced in the wall of small arteries while that for OX(2)R was observed in the secretory portion of the acinar gland cells with a characteristic localization in the apical cytoplasm. RT-PCR analysis showed the presence of transcripts for PPOX, OX(1)R and OX(2)R in the sheep lacrimal gland; the gene expression of OX(1)R was two-fold greater (p<0.01) than that of OX(2)R. Taken together the present findings raise intriguing questions on the potential role of the orexinergic system in the regulation of lacrimal gland functions that require further investigations., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Ca(2+)-stimulated adenylyl cyclases (ACs) have recently been shown to play important roles in pacemaking in the sino-atrial node. Here we present evidence that Ca(2+)-stimulated ACs are functionally active in guinea-pig atrial myocytes. Basal activity of an AC in isolated atrial myocytes was demonstrated by the observations that MDL 12,330A (10 μm), an AC inhibitor, reduced L-type Ca(2+) current (I(CaL)) amplitude, while inhibition of phosphodiesterases with IBMX (100 μm) increased I(CaL) amplitude. Buffering of cytosolic Ca(2+) by exposure of myocytes to BAPTA-AM (5 μm) reduced I(CaL) amplitude, as did inhibition of Ca(2+) release from the sarcoplasmic reticulum with ryanodine (2 μm) and thapsigargin (1 μm). [Ca(2+)]i-activated calmodulin kinase II (CaMKII) inhibition with KN-93 (1 μm) reduced I(CaL), but subsequent application of BAPTA-AM further reduced I(CaL). This effect of BAPTA-AM, in the presence of CaMKII inhibition, demonstrates that there is an additional Ca(2+)-modulated pathway (not dependent on CaMKII) that regulates I(CaL) in atrial myocytes. The effects of BAPTA could be reversed by forskolin (10 μm), a direct stimulator of all AC isoforms, which would restore cAMP levels. In the presence of BAPTA-AM, the actions of IBMX were reduced. In addition, inclusion of cAMP in the patch electrode in the whole-cell configuration prevented the effects of BAPTA. These effects are all consistent with a role for Ca(2+)-stimulated AC in the regulation of atrial myocyte I(CaL).

For an arterial replacement graft to be effective, it must possess the appropriate strength in order to withstand long-term hemodynamic stress without failure, yet be compliant enough that the mismatch between the stiffness of the graft and the native vessel wall is minimized. The native vessel wall is a structurally complex tissue characterized by circumferentially oriented collagen fibers/cells and lamellar elastin. Besides the biochemical composition, the functional properties of the wall, including stiffness, depend critically on the structural organization. Therefore, it will be crucial to develop methods of producing tissues with defined structures in order to more closely mimic the properties of a native vessel. To this end, we sought to generate cell sheets that have specific ECM/cell organization using micropatterned polydimethylsiloxane (PDMS) substrates to guide cell organization and tissue growth. The patterns consisted of large arrays of alternating grooves and ridges. Adult bovine aortic smooth muscle cells cultured on these substrates in the presence of ascorbic acid produced ECM-rich sheets several cell layers thick in which both the cells and ECM exhibited strong alignment in the direction of the micropattern. Moreover, mechanical testing revealed that the sheets exhibited mechanical anisotropy similar to that of native vessels with both the stiffness and strength being significantly larger in the direction of alignment, demonstrating that the microscale control of ECM organization results in functional changes in macroscale material behavior., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: A variety of inner ear disease is related to microcirculation disturbance of inner ear, but smooth muscle cells (SMC) and endothelial cells (EC) of the spiral modiolar artery (SMA), which is the main blood supply to the inner ear, physiological feature is not very clear., Methods: In this study, two-intracellular microelectrode recording technique and cell staining techniques to study the SMC and EC resting membrane potential characteristics and communication links between cells of SMA., Results: Study found that SMC and EC have high and low resting membrane potential state, two state of the resting membrane potential of cells to ACh and high K+ response is completely different. The different types of cells, EC-EC, SMC-SMC and SMC-EC, can simultaneously record by two-microelectrode, two cell resting membrane potential can also be a double-high RP, double-low RP and one high- and one low- RP. Experiment recorded in one high- and one low- RP are the SMC-EC types, and ECs initial membrane potential are high potential, SMCs membrane potential are low initial potential. The double-high and double-low RP can be SMC-SMC or EC-EC or SMC-EC types., Conclusion: The results show that SMC and EC in the 0.3 - 0.5 mm range, similar type of cells have very good communication, can function together to maintain good and consistent, heterogeneous cell performance is more different.

We report a novel method for estimating fluorescence impulse response function (fIRF) from noise-corrupted time-domain fluorescence measurements of biological tissue. This method is based on the use of high-order Laguerre basis functions and a constrained least-squares approach that addresses the problem of overfitting due to increased model complexity. The new method was extensively evaluated on fluorescence data from simulation, fluorescent standard dyes, ex vivo tissue samples of atherosclerotic plaques and in vivo oral carcinoma. Current results demonstrate that this method allows for rapid and accurate deconvolution of multiple channel fluorescence decays without adaptively adjusting the Laguerre scale parameter. The appropriate choice of the scale parameter is essential for accurate estimation of the fIRF. The method described here is anticipated to play an important role in the development of computational techniques for real-time analysis of time-resolved fluorescence data from biological tissues and to support the advancement of fluorescence lifetime instrumentation for biomedical diagnostics by providing a means for on-line robust analysis of fluorescence decay.

Vascular endothelial growth factor (VEGF) plays a crucial role in developmental and pathological angiogenesis. Expression of VEGF in quiescent adult tissue suggests a potential role in the maintenance of mature blood vessels. We demonstrate, using a Vegf-lacZ reporter mouse model, that VEGF is expressed by arterial but not by venous or capillary endothelial cells (ECs) in vivo. Using an in vitro model, we show that arterial shear stress of human umbilical vein ECs (HUVECs) decreases apoptosis and increases VEGF expression, which is mediated by the induction of Krüppel-like factor 2 (KLF2). Additionally, shear stress stimulates the expression of VEGF receptor 2 (VEGFR2) and is associated with its activation. Knockdown of VEGF in shear stressed HUVECs blocks the protective effect of shear stress, resulting in EC apoptosis equivalent to that in control ECs cultured under static conditions. Similarly, treatment of ECs subjected to arterial shear stress with the VEGF receptor tyrosine kinase inhibitor SU1498, or VEGFR2 neutralizing antiserum, led to increased apoptosis, demonstrating that the mechanoprotection from increased shear is mediated by VEGFR2. Taken together, these studies suggest that arterial flow induces VEGF-VEGFR2 autocrine-juxtacrine signaling, which is a previously unidentified mechanism for vascular EC survival in adult arterial blood vessels.

Interleukin-2 (IL-2) is a multifaceted cytokine with immunostimulatory and immunosuppressive properties. Our laboratory recently demonstrated that the availability of IL-2 is regulated, in part, by association with perlecan, a heparan sulfate proteoglycan. Given the abundance of perlecan in blood vessels, we asked whether IL-2 is present in vessel walls. Our results indicate that IL-2 is associated with endothelial and smooth muscle cells within the human arterial wall. This IL-2 is released by heparanase, and promotes the proliferation of an IL-2-dependent cell line. Given the presence of IL-2 in human arteries, we asked whether the large vessels of IL-2-deficient mice were normal. The aortas of IL-2-deficient mice exhibited a loss of smooth muscle cells, suggesting that IL-2 may contribute to their survival. In their entirety, these results suggest a here-to-fore unrecognized role of IL-2 in vascular biology, and have significant implications for both the immune and cardiovascular systems.

Rationale: A growing body of evidence supports the hypothesis that the Wnt/planar cell polarity (PCP) pathway regulates endothelial cell proliferation and angiogenesis, but the components that mediate this regulation remain elusive., Objective: We investigated the involvement of one of the receptors, Frizzled4 (Fzd4), in this process because its role has been implicated in retinal vascular development., Methods and Results: We found that loss of fzd4 function in mice results in a striking reduction and impairment of the distal small artery network in the heart and kidney. We report that loss of fzd4 decreases vascular cell proliferation and migration and decreases the ability of the endothelial cells to form tubes. We show that fzd4 deletion induces defects in the expression level of stable acetylated tubulin and in Golgi organization during migration. Deletion of fzd4 favors Wnt noncanonical AP1-dependent signaling, indicating that Fzd4 plays a pivotal role favoring PCP signaling. Our data further demonstrate that Fzd4 is predominantly localized on the top of the plasma membrane, where it preferentially induces Dvl3 relocalization to promote its activation and α-tubulin recruitment during migration. In a pathological mouse angiogenic model, deletion of fzd4 impairs the angiogenic response and leads to the formation of a disorganized arterial network., Conclusions: These results suggest that Fzd4 is a major receptor involved in arterial formation and organization through a Wnt/PCP pathway.

Ischemic recruitment of endothelial progenitor cells (EPCs) in involved in compensatory angiogenic in animal models, but this still needs to be substantiated in humans. We enrolled 12 patients, who underwent surgical correction of abdominal aortic aneurysm without atherosclerosis of leg arteries (n = 4) or lower limb atherosclerosis obliterans (AO; n = 8). We measured VEGF, SDF-1, lactate and CD34+ KDR+ EPCs in the arterial and venous circulation of lower limbs. We found that, irrespectively of AO stage and lactate production, there was no consistent arterio-venous gradient of EPC, VEGF and SDF-1. Notably, in 4/8 patients, EPCs were more abundant in the vein than in the artery. EPC gradient was directly correlated with VEGF gradient and inversely correlated with SDF-1 gradient. In conclusion, we failed to show any consistent gradient of EPCs across ischemic limbs in relation to severity of atherosclerosis obliterans, but we speculatively suggest that a bidirectional traffic of EPCs in and out the ischemic tissue might be regulated by VEGF and SDF-1.

After intra-arterial administration of several radiographic contrast media (RCM) a disorder of the downstream microcirculation with regard to blood flow velocity in microvessels and to tissue oxygen partial pressure in the myocardium of the pig heart was described. Iodixanol did not induce such a microcirculatory disorder in the myocardium of the beating heart of pigs. Whether the morphological changes reported in venous endothelial cells after incubation in culture media supplemented with RCM in vitro coincide with a serious endothelial cell dysfunction is not known. In this study we wanted to get information on possible states of dysfunction or perturbation of venous and arterial ECs through the release of prostacyclin, which was shown to follow the perturbation of ECs. Functionally confluent venous endothelial cells on extracellular matrix secreted great amounts of prostacyclin in reaction to the RCMs indicating a clear perturbation of the ECs. This was not the case in arterial EC cultures. The prostacyclin release from arterial ECs exposed to Iodixanol was more than 10-fold higher than that from arterial ECs exposed to Iomeprol. This could be one of the important factors contributing to the undisturbed myocardial microcirculation after injection of Iodixanol despite a slight echinocyte formation.

Recent observations suggest that the adventitial layer of blood vessels exhibits properties resembling a stem/progenitor cell niche. Progenitor cells have been isolated from the adventitia of both murine and human blood vessels with the potential to form endothelial cells, mural cells, osteogenic cells, and adipocytes. These progenitors appear to cluster at or near the border zone between the outer media and inner adventitia. In the mouse, this border zone region corresponds to a localized site of sonic hedgehog signaling in the artery wall. This brief review will discuss the emerging evidence that the tunica adventitia may provide a niche-like signaling environment for resident progenitor cells and will address the role of the adventitia in growth, remodeling, and repair of the artery wall., (Copyright © 2011 S. Karger AG, Basel.)

Platelet-derived growth factor (PDGF)-BB can induce abnormal proliferation and migration of vascular smooth muscle cells (VSMC) that are involved in the development of CVD. In our preliminary study, phytoalexin glyceollins (glyceollins I, II and III) isolated from soyabean seeds cultured with Aspergillus sojae showed strong antioxidant and anti-inflammatory activity. Since antioxidants showed beneficial effects on chronic inflammatory diseases, the purpose of the present study was to examine the effects of glyceollins on PDGF-induced proliferation and migration in human aortic smooth muscle cells (HASMC). Incubation of resting HASMC with glyceollins for 24 h significantly diminished PDGF-increased cell number and DNA synthesis in a dose-dependent manner without any cytotoxicity. In addition to blocking of the PDGF-inducible progression through the G0/G1 to the S phase of the cell cycle, glyceollins down-regulated the expression of cyclin-dependent kinase (CDK)2 and cyclin D1, and up-regulated the expression of CDK inhibitors such as p27kip1 and p53.Glyceollins also effectively inhibited reactive oxygen species generation and phosphorylation of PDGF receptor-β, phospholipase Cγ1, Akt and extracellular signal-regulated kinase 1/2 by PDGF stimulation. Furthermore, glyceollins were found to inhibit PDGF-induced dissociation of actin filaments and cell migration. Thus, the results suggest that glyceollins could become a potent therapeutic agent for regulating VSMC-associated vascular disease such as atherosclerosis and restenosis after angioplasty.

We have recently identified conventional B2 cells as atherogenic and B1a cells as atheroprotective in hypercholesterolemic ApoE(-/-) mice. Here, we examined the development of atherosclerosis in BAFF-R deficient ApoE(-/-) mice because B2 cells but not B1a cells are selectively depleted in BAFF-R deficient mice. We fed BAFF-R(-/-) ApoE(-/-) (BaffR.ApoE DKO) and BAFF-R(+/+)ApoE(-/-) (ApoE KO) mice a high fat diet (HFD) for 8-weeks. B2 cells were significantly reduced by 82%, 81%, 94%, 72% in blood, peritoneal fluid, spleen and peripheral lymph nodes respectively; while B1a cells and non-B lymphocytes were unaffected. Aortic atherosclerotic lesions assessed by oil red-O stained-lipid accumulation and CD68+ macrophage accumulation were decreased by 44% and 50% respectively. B cells were absent in atherosclerotic lesions of BaffR.ApoE DKO mice as were IgG1 and IgG2a immunoglobulins produced by B2 cells, despite low but measurable numbers of B2 cells and IgG1 and IgG2a immunoglobulin concentrations in plasma. Plasma IgM and IgM deposits in atherosclerotic lesions were also reduced. BAFF-R deficiency in ApoE(-/-) mice was also associated with a reduced expression of VCAM-1 and fewer macrophages, dendritic cells, CD4+ and CD8+ T cell infiltrates and PCNA+ cells in lesions. The expression of proinflammatory cytokines, TNF-α, IL1-β and proinflammatory chemokine MCP-1 was also reduced. Body weight and plasma cholesterols were unaffected in BaffR.ApoE DKO mice. Our data indicate that B2 cells are important contributors to the development of atherosclerosis and that targeting the BAFF-R to specifically reduce atherogenic B2 cell numbers while preserving atheroprotective B1a cell numbers may be a potential therapeutic strategy to reduce atherosclerosis by potently reducing arterial inflammation.

We examined the effects of mibefradil, a T-type Ca²⁺ channel inhibitor, on voltage-dependent K⁺ (Kv) channels in rabbit coronary arterial smooth muscle cells using the whole-cell patch clamp technique. Mibefradil reduced the Kv current amplitude in a dose-dependent manner, with an apparent K(d) value of 1.08 μM. Kv current inhibition by mibefradil was highly voltage-dependent over the full activation voltage range (-30 to +10 mV). The decay rate of Kv channel inactivation was accelerated by mibefradil without altering the kinetics of current activation. The rate constants of association and dissociation were 2.23 ± 0.07 μM⁻¹·s⁻¹ and 2.40 ± 0.42 s⁻¹, respectively. Mibefradil had no significant effect on the steady-state activation or inactivation curves. In the presence of mibefradil, the recovery time constant from inactivation was decreased, and the application of train pulses (1 or 2 Hz) increased mibefradil-induced Kv channel inhibition, suggesting that the inhibitory effects of mibefradil were use-dependent. The inhibitory effect of mibefradil on Kv channels was unaffected by extracellular Ca²⁺-free conditions. Moreover, the absence of ATP inside the pipette did not alter the blocking effect of mibefradil. Therefore, we suggest that mibefradil directly inhibited the Kv current, independently of Ca²⁺ channel inhibition.

Paclitaxel-eluting stents dramatically reduce rates of in-stent restenosis; however, paclitaxel is known to lead to endothelial dysfunction. Protective effects of nicorandil on paclitaxel-induced endothelial dysfunction by examining flow-mediated dilation (FMD) were investigated in anesthetized rats. After 7-day osmotic infusion of paclitaxel (5 mg/kg per day), FMD was measured by high-resolution ultrasound in the femoral artery of living rats. Paclitaxel significantly reduced FMD (21.6% ± 3.2% to 7.1% ± 1.7%); this reduction was prevented by co-treatment with nicorandil (15 mg/kg per day), while paclitaxel did not affect nitroglycerin-induced vasodilation. Diazoxide and tempol, but not isosorbide dinitrate, had an effect similar to nicorandil in preventing paclitaxel-induced decrease in FMD. Nicorandil significantly prevented paclitaxel-induced reduction in acetylcholine-induced vasodilation. On the underling mechanisms, paclitaxel increased reactive oxygen species (ROS) production (dihydrorhodamine 123, DCF fluorescence intensity) and NADPH oxidase (p47(phox), gp91(phox) mRNA) in arteries and human coronary artery endothelial cells (HCAECs), while paclitaxel reduced nitric oxide (NO) release (DAF-2 fluorescence intensity), but not endothelial NO synthase (eNOS) phosphorylation in HCAECs. Nicorandil prevented the increased ROS production in arteries and HCAECs, which was 5-hydroxydecanoate (5-HD)-sensitive but 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)-resistant, without significant effect on the reduced NO release. In conclusion, nicorandil prevents paclitaxel-induced endothelial dysfunction, which may be brought by improved NO bioavailability due to the reduction of oxidative stress via K(ATP) channel activation.

Background and Objectives: The macrophage is an important early cellular marker related to risk of future rupture of atherosclerotic plaques. Two-channel two-photon luminescence (TPL) microscopy combined with optical coherence tomography (OCT) was used to detect, and further characterize the distribution of aorta-based macrophages using plasmonic gold nanorose as an imaging contrast agent., Study Design/materials and Methods: Nanorose uptake by macrophages was identified by TPL microscopy in macrophage cell culture. Ex vivo aorta segments (8 × 8 × 2 mm(3) ) rich in macrophages from a rabbit model of aorta inflammation were imaged by TPL microscopy in combination with OCT. Aorta histological sections (5 µm in thickness) were also imaged by TPL microscopy., Results: Merged two-channel TPL images showed the lateral and depth distribution of nanorose-loaded macrophages (confirmed by RAM-11 stain) and other aorta components (e.g., elastin fiber and lipid droplet), suggesting that nanorose-loaded macrophages are diffusively distributed and mostly detected superficially within 20 µm from the luminal surface of the aorta. Moreover, OCT images depicted detailed surface structure of the diseased aorta., Conclusions: Results suggest that TPL microscopy combined with OCT can simultaneously reveal macrophage distribution with respect to aorta surface structure, which has the potential to detect vulnerable plaques and monitor plaque-based macrophages overtime during cardiovascular interventions., (Copyright © 2012 Wiley Periodicals, Inc.)

Objective: The present study was to explore signaling mechanisms underlying nicotine-induced inhibition of large-conductance calcium-activated potassium channels (BK(Ca))., Methods: 8 week male Wistar rats were divided randomly into saline group and nicotine group and received respectively injection with saline or nicotine (Sigma, Shanghai, China) at 2 mg/(kg x d) for 21 days. Coronary vascular smooth muscle cells were dissociated enzymatically. Dissociated smooth muscle cells were interfered with CPT-cAMP (100 micromol/L) or forskolin (10 micromol/L). The signal channel open dwell-time (To), close dwell-time (Tc) and open probability (Po) were recorded., Results: CPT-cAMP or forskolin significantly prolonged To, shorten Tc and increased Po in saline group (P < 0.01). But in nicotine group To, Tc and Po did not been changed., Conclusion: This phenomenon may serve as a physiological mechanism that nicotine inhibits BK(Ca) channel activity to increase via cAMP/PKA-dependent pathway.

Nitric oxide synthase (NOS) is a key regulator of angiogenesis and embryogenesis in the mammalian reproductive process. Here, we attempted to clarify the expression and localization of inducible and endothelial NOS (iNOS and eNOS) in the developing rabbit placenta. Real-time RT-PCR analysis indicated that iNOS mRNA was significantly upregulated till the complete development of the placenta (d18), and then significantly decreased at the end of fetal growth stage (d28) during successful pregnancy. The eNOS mRNA was also enhanced in the pregnant uteri and gradually decreased near the term of pregnancy. Western blot analysis also showed elevation of the iNOS and eNOS protein levels during the course of successful pregnancy till the functional maturation of the placenta (d18). Immunohistochemical study revealed distinct localizations of iNOS along the radial arteries and eNOS at the spiral arteries and arterial sinuses in the developing placenta. This may reflect that iNOS and eNOS participate in pregnancy success through placentation-specific vascular formation and by supporting adequate blood circulation in the rabbit placenta.

Vascular tissue engineering aims to develop implantable blood-vessels, exhibiting biological and biomechanical characteristics close to those of the native vessels. The ultimate goal of our group is to engineer suitable blood vessel substitutes which could be stored for a long time in vascular bank conditions.First attempts tried to develop coating procedures allowing endothelial cells (EC) differentiation, adhesion and retention on current vascular substitutes but the weak in vivo patency of these grafts was related. Since 2003, our group have been evaluated a new surface modification of internal surface of blood vessels based on polyelectrolyte films coating. The layer-by-layer self-assembly and the resulting polyelectrolyte multiplayer films (PEM) is a simple and versatile way to engineer surfaces with highly specific properties. Previous studies indicated that the poly(sodium-4 styrene sulfonate)/poly (allylamine hydrochloride) PSS/PAH multilayered films when ended by PAH, induce strong adhesion and retention of mature EC which spread and keep their phenotype as well on glass, on expanded polytetrafluoroethylene ePTFE and on cryopreserved arteries. The mechanical properties (compliance), leading to early intimal hyperplasia and graft failure, were lost after artery cryopreservation. We have demonstrated that the compliance and elasticity restoration of PEM treated cryopreserved arteries close to native arteries.In other respect, the use of the circulating progenitor which could be differentiated into matures vascular cell offers new opportunities in vascular engineering. Currents protocols, expend at least 1 month to observe both smooth muscle (SMCs) and endothelium (ECs)-like morphology and about two months for confluent monolayer cells. The progenitor cells cultivated on PEM treated glass showed mature and functional vascular cells (SMCs and ECs) development after only 14 days of culture. The morphological appearance, mature and healthy phenotype markers expression and repartition of differentiated cells are close to mature cells.Challenge now is to build up in less a month, an autologous cellularized vascular graft using patient peripheral stem cells.

Of the many models to study vascular biology the avian embryo remains an informative and powerful model system that has provided important insights into endothelial cell recruitment, assembly and remodeling during development of the circulatory system. This review highlights several discoveries in the avian system that show how arterial patterning is regulated using the model of dorsal aortae development along the embryo midline during gastrulation and neurulation. These discoveries were made possible through spatially and temporally controlled gain-of-function experiments that provided direct evidence that BMP signaling plays a pivotal role in vascular recruitment, patterning and remodeling and that Notch-signaling recruits vascular precursor cells to the dorsal aortae. Importantly, BMP ligands are broadly expressed throughout embryos but BMP signaling activation region is spatially defined by precisely regulated expression of BMP antagonists. These discoveries provide insight into how signaling, both positive and negative, regulate vascular patterning. This review also illustrates similarities of early arterial patterning along the embryonic midline in amniotes both avian and mammalians including human, evolutionarily specialized from non-amniotes such as fish and frog., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

There is a need to develop multiscale models of vascular adaptations to understand tissue-level manifestations of cellular level mechanisms. Continuum-based biomechanical models are well suited for relating blood pressures and flows to stress-mediated changes in geometry and properties, but less so for describing underlying mechanobiological processes. Discrete stochastic agent-based models are well suited for representing biological processes at a cellular level, but not for describing tissue-level mechanical changes. We present here a conceptually new approach to facilitate the coupling of continuum and agent-based models. Because of ubiquitous limitations in both the tissue- and cell-level data from which one derives constitutive relations for continuum models and rule-sets for agent-based models, we suggest that model verification should enforce congruency across scales. That is, multiscale model parameters initially determined from data sets representing different scales should be refined, when possible, to ensure that common outputs are consistent. Potential advantages of this approach are illustrated by comparing simulated aortic responses to a sustained increase in blood pressure predicted by continuum and agent-based models both before and after instituting a genetic algorithm to refine 16 objectively bounded model parameters. We show that congruency-based parameter refinement not only yielded increased consistency across scales, it also yielded predictions that are closer to in vivo observations.

Altered endothelial function may underlie human cardiovascular diseases, including hypertension, diabetes and pre-eclampsia. While much is known about endothelial function in small arteries, very little is known about endothelial responses in small veins isolated from humans. Therefore, we assessed endothelium-dependent responses in omental arteries and veins isolated from healthy pregnant women, focussing on endothelium-dependent hyperpolarising (EDH) mechanisms. Human omental arteries and veins were obtained from women undergoing elective caesarean sections and examined using pressure myography. In pressurised vessels, the effects of proposed inhibitors of EDH production/function were examined on responses to bradykinin. The expression of connexins Cx37, 40 and 43 was assessed using immunohistochemistry. Bradykinin caused vasodilatation in human pressurised omental arteries and veins. In both vessels, responses to bradykinin were partially blocked in the presence of the gap junction uncoupler, carbenoxolone, and reduced further with the addition of catalase, which acts to degrade H(2)O(2). The effect of catalase alone was more pronounced in venous preparations. All three connexins were expressed in both arteries and veins, with a similar distribution pattern, where Cx37 and Cx40 were located mainly in the endothelium and Cx43 located mostly in the media. These data show that, in human omental vessels, an EDH mechanism is produced in response to bradykinin that involves gap junction communication and the production of H(2)O(2). These mechanisms may be involved in the haemodynamic alterations that take place during pregnancy, and any aberration in their function could contribute to raised blood pressure in hypertensive disorders of pregnancy, such as pre-eclampsia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The voltage-gated potassium channel, Kv1.3, plays an important role in regulating membrane excitability in diverse cell types ranging from T-lymphocytes to neurons. In the present study, we test the hypothesis that the C-terminal PDZ binding domain modulates the function and localization of Kv1.3. We created a mutant form of Kv1.3 that lacked the last three amino acids of the C-terminal PDZ-binding domain (Kv1.3ΔTDV). This form of Kv1.3 did not bind the PDZ domain containing protein, PSD95. We transfected wild type and mutant Kv1.3 into HEK293 cells and determined if the mutation affected current, Golgi localization, and surface expression of the channel. We found that cells transfected with Kv1.3ΔTDV had greater current and lower Golgi localization than those transfected with Kv1.3. Truncation of the C-terminal PDZ domain did not affect surface expression of Kv1.3. These findings suggest that PDZ-dependent interactions affect both Kv1.3 localization and function. The finding that current and Golgi localization changed without a corresponding change in surface expression suggests that PDZ interactions affect localization and function via independent mechanisms., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The placenta is composed of multiple trophoblast cell types that have diverse endocrine, vascular and nutrient transport functions. We have developed a transgenic system to investigate the developmental and functional roles of specific cell types using conditional expression of a cytotoxin to induce cell ablation in transgenic mice. The Tpbpa gene is expressed in ectoplacental cone cells starting between embryonic days (E) 7.5 and 8.5, and later in the spongiotrophoblast layer of the mature placenta. Tpbpa-positive cells are progenitors of many trophoblast subtypes including three subtypes of trophoblast giant cells (TGCs) and glycogen trophoblast cells. We used a Cre recombinase transgene driven by the Tpbpa promoter to irreversibly activate a diphtheria toxin A (DTA) transgene. Cre/DTA double transgenic placentas showed dramatic reduction of Tpbpa-positive spongiotrophoblast cells by E10.5 and conceptuses died by ~E11.5. The number of cells associated with maternal blood spaces, spiral artery TGCs (SpA-TGCs) and canal TGCs, and glycogen trophoblast cells were reduced. The loss of these specific trophoblast subtypes, especially SpA-TGCs, was correlated with a decrease in maternal spiral artery diameters, indicating a critical role of these cells in modulating the maternal vasculature. In contrast, parietal TGCs were not significantly reduced by progenitor cell ablation, suggesting that there is compensatory growth of this population and indeed a population of Ascl2 (Mash2)-positive/Tpbpa-negative cells was increased in the spongiotrophoblast layer in the Cre/DTA double transgenics. Our work demonstrates that the Tpbpa-positive lineage is essential for placental function and particularly critical for maternal vasculature remodeling., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We tested the hypothesis that reductions in the cellular deacetylase, sirtuin-1 (SIRT-1), contribute to vascular endothelial dysfunction with ageing via modulation of endothelial nitric oxide synthase (eNOS) acetylation/activation-associated nitric oxide (NO) production. In older (30 months, n = 14) vs. young (5-7 months, n = 16) B6D2F1 mice, aortic protein expression of SIRT-1 and eNOS phosphorylated at serine 1177 were lower (both P < 0.05), and acetylated eNOS was 6-fold higher (P < 0.05), whereas total eNOS did not differ (P = 0.65). Acetylcholine (ACh)-induced peak endothelium-dependent dilatation (EDD) was lower in isolated femoral arteries with ageing (P < 0.001). Incubation with sirtinol, a SIRT-1 inhibitor, reduced EDD in both young and older mice, abolishing age-related differences, whereas co-administration with l-NAME, an eNOS inhibitor, further reduced EDD similarly in both groups. Endothelium-independent dilatation to sodium nitroprusside (EID), was not altered by age or sirtinol treatment. In older (64 ± 1 years, n = 22) vs. young (25 ± 1 years, n = 16) healthy humans, ACh-induced forearm EDD was impaired (P = 0.01) and SIRT-1 protein expression was 37% lower in endothelial cells obtained from the brachial artery (P < 0.05), whereas EID did not differ. In the overall group, EDD was positively related to endothelial cell SIRT-1 protein expression (r = 0.44, P < 0.01). Reductions in SIRT-1 may play an important role in vascular endothelial dysfunction with ageing. SIRT-1 may be a key therapeutic target to treat arterial ageing.

In order to address the problem of blood flow over the endothelium in small arteries, the near-endothelial region is here studied in more detail. The method used is a finite-volume discretisation of a Lattice Boltzmann equation over unstructured grids, named unstructured Lattice Boltzmann equation (ULBE). It is a new scheme based on the idea of placing the unknown fields at the nodes of the mesh and evolving them based on the fluxes crossing the surfaces of the corresponding control volumes. The study shows a significant variation and a high sensitivity of wall shear stress to the height of the endothelium corrugation and the presence of erythrocytes. The latter were modelled as deformable, viscous particles within a fluid continuum., (Copyright © 2011 IPEM. Published by Elsevier Ltd. All rights reserved.)

Background: Calcium sparks are ryanodine receptor mediated transient calcium signals that have been shown to hyperpolarize the membrane potential by activating large conductance calcium activated potassium (BK) channels in vascular smooth muscle cells. Along with voltage-dependent calcium channels, they form a signaling unit that has a vasodilatory influence on vascular diameter and regulation of myogenic tone. The existence and role of calcium sparks has hitherto been unexplored in the spiral modiolar artery, the end artery that controls blood flow to the cochlea. The goal of the present study was to determine the presence and properties of calcium sparks in the intact gerbil spiral modiolar artery., Results: Calcium sparks were recorded from smooth muscle cells of intact arteries loaded with fluo-4 AM. Calcium sparks occurred with a frequency of 2.6 Hz, a rise time of 17 ms and a time to half-decay of 20 ms. Ryanodine reduced spark frequency within 3 min from 2.6 to 0.6 Hz. Caffeine (1 mM) increased spark frequency from 2.3 to 3.3 Hz and prolonged rise and half-decay times from 17 to 19 ms and from 20 to 23 ms, respectively. Elevation of potassium (3.6 to 37.5 mM), presumably via depolarization, increased spark frequency from 2.4 to 3.2 Hz. Neither ryanodine nor depolarization changed rise or decay times., Conclusions: This is the first characterization of calcium sparks in smooth muscle cells of the spiral modiolar artery. The results suggest that calcium sparks may regulate the diameter of the spiral modiolar artery and cochlear blood flow.

Vasospasm of the cerebrovasculature is a common manifestation of blast-induced traumatic brain injury (bTBI) reported among combat casualties in the conflicts in Afghanistan and Iraq. Cerebral vasospasm occurs more frequently, and with earlier onset, in bTBI patients than in patients with other TBI injury modes, such as blunt force trauma. Though vasospasm is usually associated with the presence of subarachnoid hemorrhage (SAH), SAH is not required for vasospasm in bTBI, which suggests that the unique mechanics of blast injury could potentiate vasospasm onset, accounting for the increased incidence. Here, using theoretical and in vitro models, we show that a single rapid mechanical insult can induce vascular hypercontractility and remodeling, indicative of vasospasm initiation. We employed high-velocity stretching of engineered arterial lamellae to simulate the mechanical forces of a blast pulse on the vasculature. An hour after a simulated blast, injured tissues displayed altered intracellular calcium dynamics leading to hypersensitivity to contractile stimulus with endothelin-1. One day after simulated blast, tissues exhibited blast force dependent prolonged hypercontraction and vascular smooth muscle phenotype switching, indicative of remodeling. These results suggest that an acute, blast-like injury is sufficient to induce a hypercontraction-induced genetic switch that potentiates vascular remodeling, and cerebral vasospasm, in bTBI patients.

We present a technology of creation of blood vessel connective tissue framework by 2-3-h vessel perfusion with detergents. The technology ensures effective removal of vascular cells without damaging collagen and elastic fibers. The connective tissue frameworks prepared by this method can the used for restoring blood flow in various vascular pathologies. The presented approach attenuates the damaging effect of treatment on the vascular framework due to maximum simplification and shortening of the duration of treatment and is universal for human and animal vessels.

There is a clinical need for better blood vessel substitutes, as current surgical procedures are limited by the availability of suitable autologous vessels and suboptimal behavior of synthetic grafts in small caliber arterial graft (<5  mm) applications. The aim of the present study was to compare the mechanical properties of arterial and venous tissue-engineered vascular constructs produced by the self-assembly approach using cells extracted from either the artery or vein harvested from the same human umbilical cord. The production of a vascular construct comprised of a media and an adventitia (TEVMA) was achieved by rolling a continuous tissue sheet containing both smooth muscle cells and adventitial fibroblasts grown contiguously in the same tissue culture plate. Histology and immunofluorescence staining were used to evaluate the structure and composition of the extracellular matrix of the vascular constructs. The mechanical strength was assessed by uniaxial tensile testing, whereas viscoelastic behavior was evaluated by stepwise stress-relaxation and by cyclic loading hysteresis analysis. Tensile testing showed that the use of arterial cells resulted in stronger and stiffer constructs when compared with those produced using venous cells. Moreover, cyclic loading demonstrated that constructs produced using arterial cells were able to bear higher loads for the same amount of strain when compared with venous constructs. These results indicate that cells isolated from umbilical cord can be used to produce vascular constructs. Arterial constructs possessed superior mechanical properties when compared with venous constructs produced using cells isolated from the same human donor. This study highlights the fact that smooth muscle cells and fibroblasts originating from different cell sources can potentially lead to distinct tissue properties when used in tissue engineering applications.

We previously showed that progesterone (P4) inhibits the proliferation of rat aortic smooth muscle cells (RASMC). Here, we further demonstrate that P4 at physiologic levels (5-500 nM) concentration-dependently inhibited migration of cultured RASMC. The effect is blocked by pretreatment with progesterone receptor (PR) antagonist, RU486. The P4-induced RASMC migration inhibition was through RhoA inactivation induced by cSrc-enhanced RhoA degradation. The P4-induced increases of phosphorylated Src (pSrc) and PR-pSrc complex in RASMC were observed mainly in the membrane fraction. Pre-treatment with a cSrc inhibitor (PP2) or cSrc antisense oligonucleotides prevented the P4-induced decreases of the protein levels of RhoA, phosphorylated FAK (p-FAK) and paxillin phosphorylaton and migration inhibition in RASMC. These findings expend our knowledge of the basis of P4's effect on vascular smooth muscle cell migration and highlight novel pathways of signaling transduction of P4 through PR-mediated nongenomic mechanisms., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Vascular endothelial growth factor (VEGF) inhibits the endothelial apoptosis that is induced by caspases during vascular remodeling; however, the underlying mechanisms have not been completely elucidated., Objectives: We hypothesized that VEGF upregulates poly(ADP-ribose) polymerase-1 (PARP) as a caspase mediator, and sought to investigate the link between apoptosis inhibition by VEGF and PARP regulation in the human vasculature., Methods: Human endothelial cells (primary cells, macrovascular/microvascular lines) were incubated with 100 pg mL(-1) to 1 μg mL(-1) VEGF-A(165) in the absence or presence of PARP small interfering RNA (siRNA). Apoptosis induced by integrin inhibition was measured by flow cytometry, trypan blue exclusion, and nuclear morphology. PARP expression and activity were determined by real-time RT-PCR, Western blot, and ribosylation assay. VEGF receptors and signal transduction were analyzed by inhibitor experiments, enzyme assays, western blot, and immunofluorescence microscopy. Immunohistochemistry was applied to a vascular culture model and to 24 atherosclerotic and 10 normal human arteries., Results: VEGF-A(165) induced resistance to apoptosis caused by caspase activation in endothelial cells in a time-dependent manner. VEGF, but not fibroblast growth factor-2 or transforming growth factor-β, time-dependently and dose-dependently induced PARP expression and activity, involving VEGF receptor-2 colocalized with neuropilin-1 as well as the signal transduction molecules c-Jun N-terminal kinase and Akt. The antiapoptotic effect of VEGF was abrogated by PARP siRNA. The relationship between local VEGF influence and endothelial PARP expression was confirmed in human arteries and the vascular culture model., Conclusions:  VEGF exerts its antiapoptotic effect on the endothelium through the regulation of PARP expression. PARP has been attributed an ambiguous role in apoptosis; here, we show that PARP promotes vascular endothelial integrity in VEGF-associated processes., (© 2011 International Society on Thrombosis and Haemostasis.)

There is an urgent clinical need of tissue-engineering (TE) vascular grafts, so this study was for developing a fast and simple way of producing TE vascular scaffold. The TE vascular scaffold was prepared with pepsin, DNase and RNase enzymatic decellularization and crosslinked with 0.1, 1, 5% glutaraldehyde (GA), respectively. The samples were underwent analyses of burst pressure; suture strength; cytotoxicity; enzymatic degradation in vitro; degradation in vivo; rehydration; biocompatibilities detected with hematoxylin and eosin (H&E), scan electron microscope, immunohistochemistry both in vivo and in vitro; macrophage infiltration and calcification using Von Kossa staining. After being decellularized the scaffold had a complete removal of cellular components, an intact collagen structure. The burst pressure and suture strength were similar to native artery. 0.1% GA crosslinked scaffold showed less cytotoxicity than 1 and 5% GA groups (P < 0.05) and was resistance to enzymatic degradation in vitro. Once being implanted, 0.1% GA group was resistant to degradation and formed endothelium, smooth muscle and adventitia with few macrophages infiltration. However, there appeared calcification in implants compared with that in native artery. This study demonstrated that DVPs producing methods by enzymatic decellularizing and crosslinking with 0.1% GA could be used for clinical TE vascular graft manufacture.

Background: Hypertensive nephrosclerosis alone and in combination with other renal diseases is a leading cause of terminal renal insufficiency. Histologic lesions manifest as benign nephrosclerosis (bN) with arteriolar hyalinosis and later fibrosis. Procoagulant micromilieus have been implicated in fibrosis. Hyalinosis is considered to consist of plasma insudation possibly containing procoagulant factors like von Willebrand factor (VWF). Therefore, it is hypothesized that VWF cleaving protease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type-1 motif, 13) is normally expressed by arteriolar vascular smooth muscle cells (VSMCs) and diminished in bN and that this reduction contributes to fibrosis in bN., Methods: ADAMTS13 expression was examined by immunohistochemistry and quantitative real-time polymerase chain reaction in VSMCs of various human organs. Fifty-four specimens with and seven without bN were immunostained for ADAMTS13, VWF, CD61 and VSMC differentiation markers in arteriolar walls., Results: Expression of ADAMTS13 is confirmed in VSMCs. In bN, ADAMTS13 immunostaining of arterial VSMCs correlated inversely with fibrotic but not hyalinotic lesions. Smooth muscle myosin heavy chain showed an inverse correlation with hyalinotic, as opposed to fibrotic lesions of bN. Smoothelin showed an inverse correlation with both hyalinotic and fibrotic lesions of bN. VWF was absent in normal controls and hyalinotic lesions, but present exclusively in fibrotic lesions in 7/54 (13%) bN cases. CD61 was absent in all arteriolar walls., Conclusions: The present results establish ADAMTS13 as a novel marker of contractile VSMCs that is retained in early hyalinotic bN but partially lost later in fibrotic bN. Loss of ADAMTS13 and accumulation of VWF in fibrotic but not hyalinotic arteriolar walls could further propagate fibrosis in bN.

Objective: Extracellular matrix (ECM) of neointima formed following angioplasty contains elevated levels of versican, loosely arranged collagen, and fragmented deposits of elastin, features associated with lipid and macrophage accumulation. ECM with a low versican content, compact structure, and increased elastic fiber content can be achieved by expression of versican variant V3, which lacks chondroitin sulfate glycosaminoglycans. We hypothesized that V3-expressing arterial smooth muscle cells (ASMC) can be used to form a neointima resistant to lipid and macrophage accumulation associated with hypercholesterolemia., Methods and Results: ASMC transduced with V3 cDNA were seeded into ballooned rabbit carotid arteries, and animals were fed a chow diet for 4 weeks, followed by a cholesterol-enriched diet for 4 weeks, achieving plasma cholesterol levels of 20 to 25 mmol/L. V3 neointimae at 8 weeks were compact, multilayered, and elastin enriched. They were significantly thinner (57%) than control neointimae; contained significantly more elastin (118%), less collagen (22%), and less lipid (76%); and showed significantly reduced macrophage infiltration (85%). Mechanistic studies demonstrated that oxidized low-density lipoprotein stimulated the formation of a monocyte-binding ECM, which was inhibited in the presence of V3 expressing ASMC., Conclusion: These results demonstrate that expression of V3 in vessel wall creates an elastin-rich neointimal matrix that in the presence of hyperlipidemia is resistant to lipid deposition and macrophage accumulation.

Protein tyrosine kinase 7 (PTK7) is a pseudokinase whose precise function in regulating angiogenesis remains unknown. The purpose of this study was to define the mechanisms by which PTK7 promotes vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis in vivo and in vitro. Immunoblotting was used to measure PTK7 expression in several types of vascular endothelial cells. Using both immunoprecipitation and immunoblotting, PTK7 was found to join a receptor complex with Flt-1 (VEGFR1), but not with KDR/Flk-1 (VEGFR2) or with Flt-4 (VEGFR3). By surface plasmon resonance analysis, the interaction between Flt-1 and PTK7 was confirmed and found to be intensified by VEGF-A. Flt-1 phosphorylation and downstream signals of Akt, and focal adhesion kinase (FAK) thus induced were down-regulated by inhibition of PTK7 expression using siRNA. Moreover, PTK7 overexpression in endothelial cells resulted in enhanced angiogenesis in vitro. In contrast, neovascularization induced in vivo by VEGF-A pellets was significantly decreased by injection of siRNA targeting PTK7. These data suggest that PTK7 serves an essential role in Flt-1-mediated angiogenesis.

Enzymes of the phosphodiesterase 3 (PDE3) and PDE4 families each regulate the activities of both protein kinases A (PKAs) and exchange proteins activated by cAMP (EPACs) in cells of the cardiovascular system. At present, the mechanisms that allow selected PDEs to individually regulate the activities of these two effectors are ill understood. The objective of this study was to determine how a specific PDE3 variant, namely PDE3B, interacts with and regulates EPAC1-based signaling in human arterial endothelial cells (HAECs). Using several biochemical approaches, we show that PDE3B and EPAC1 bind directly through protein-protein interactions. By knocking down PDE3B expression or by antagonizing EPAC1 binding with PDE3B, we show that PDE3B regulates cAMP binding by its tethered EPAC1. Interestingly, we also show that PDE3B binds directly to p84, a PI3Kγ regulatory subunit, and that this interaction allows PI3Kγ recruitment to the PDE3B-EPAC1 complex. Of potential cardiovascular importance, we demonstrate that PDE3B-tethered EPAC1 regulates HAEC PI3Kγ activity and that this allows dynamic cAMP-dependent regulation of HAEC adhesion, spreading, and tubule formation. We identify and molecularly characterize a PDE3B-based "signalosome" that integrates cAMP- and PI3Kγ-encoded signals and show how this signal integration regulates HAEC functions of importance in angiogenesis.

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation. The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS) for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

The consumption of cacao-derived (i.e., cocoa) products provides beneficial cardiovascular effects in healthy subjects as well as individuals with endothelial dysfunction such as smokers, diabetics, and postmenopausal women. The vascular actions of cocoa are related to enhanced nitric oxide (NO) production. These actions can be reproduced by the administration of the cacao flavanol (-)-epicatechin (EPI). To further understand the mechanisms behind the vascular action of EPI, we investigated the effects of Ca(2+) depletion on endothelial nitric oxide (NO) synthase (eNOS) activation/phosphorylation and translocation. Human coronary artery endothelial cells were treated with EPI or with bradykinin (BK), a well-known Ca(2+)-dependent eNOS activator. Results demonstrate that both EPI and BK induce increases in intracellular calcium and NO levels. However, under Ca(2+)-free conditions, EPI (but not BK) is still capable of inducing NO production through eNOS phosphorylation at serine 615, 633, and 1177. Interestingly, EPI-induced translocation of eNOS from the plasmalemma was abolished upon Ca(2+) depletion. Thus, under Ca(2+)-free conditions, EPI can stimulate NO synthesis independent of calmodulin binding to eNOS and of its translocation into the cytoplasm. We also examined the effect of EPI on the NO/cGMP/vasodilator-stimulated phosphoprotein (VASP) pathway activation in isolated Ca(2+)-deprived canine mesenteric arteries. Results demonstrate that under these conditions, EPI induces the activation of this vasorelaxation-related pathway and that this effect is inhibited by pretreatment with nitro-L-arginine methyl ester, suggesting a functional relevance for this phenomenon.

S-nitrosoalbumin (SNO-Alb) has been shown to be an efficacious cytoprotective molecule in acute lung injury, as well as ischemia-reperfusion injury in heart and skeletal muscle. Nonetheless, limited information is available on the cellular mechanism of such protection. Accordingly, we investigated the protective effects of SNO-Alb [ and its denitrosated congener, reduced albumin (SH-Alb) ] on tert-butyl hydroperoxide (tBH)-mediated cytotoxicity in cultured rat pulmonary microvascular endothelial cells (RPMEC), as well as hydrogen sulfide (H(2)S)-mediated cytotoxicity in rat pulmonary artery smooth muscle cells (RPASMC). We noted that tBH caused a concentration-dependent necrosis in RPMEC, and pretreatment of RPMEC with SNO-Alb dose-dependently decreased the sensitivity of these cells to tBH. A component of SNO-Alb cytoprotection was sensitive to N(G)-nitro-L-arginine methyl ester and was associated with activation of endothelial nitric oxide synthase (eNOS), phenomena that could be reproduced with pretreatment with SH-Alb. Exogenous H(2)S caused concentration-dependent apoptosis in RPASMC due to activation of ERK1/2 and p38, as well as downregulation of Bcl-2. Pretreatment with SNO-Alb reduced H(2)S-mediated apoptosis in a concentration-dependent manner that was associated with SNO-Alb-mediated inhibition of activation of ERK1/2 and p38. Pretreatment with SNO-Alb reduced toxicity of 1 mM sodium hydrosulfide in an N(G)-nitro-L-arginine methyl ester-sensitive fashion in RPASMC that expressed gp60 and neuronal NOS and was capable of transporting fluorescently labeled SH-Alb. Therefore, SNO-Alb is cytoprotective against models of oxidant-induced necrosis (tBH) and inhibitors of cellular respiration and apoptosis (H(2)S) in both pulmonary endothelium and smooth muscle, respectively, and a component of such protection can be attributed to a SH-Alb-mediated activation of constitutive NOS.

Long-chain acyl-CoA synthetases (ACSLs) catalyze the thioesterification of long-chain FAs into their acyl-CoA derivatives. Purified ACSL4 is an arachidonic acid (20:4)-preferring ACSL isoform, and ACSL4 is therefore a probable regulator of lipid mediator production in intact cells. Eicosanoids play important roles in vascular homeostasis and disease, yet the role of ACSL4 in vascular cells is largely unknown. In the present study, the ACSL4 splice variant expressed in human arterial smooth muscle cells (SMCs) was identified as variant 1. To investigate the function of ACSL4 in SMCs, ACSL4 variant 1 was overexpressed, knocked-down by small interfering RNA, or its enzymatic activity acutely inhibited in these cells. Overexpression of ACSL4 resulted in a markedly increased synthesis of arachidonoyl-CoA, increased 20:4 incorporation into phosphatidylethanolamine, phosphatidylinositol, and triacylglycerol, and reduced cellular levels of unesterified 20:4. Accordingly, secretion of prostaglandin E₂ (PGE₂) was blunted in ACSL4-overexpressing SMCs compared with controls. Conversely, acute pharmacological inhibition of ACSL4 activity resulted in increased release of PGE₂. However, long-term downregulation of ACSL4 resulted in markedly reduced PGE₂ secretion. Thus, ACSL4 modulates PGE₂ release from human SMCs. ACSL4 may regulate a number of processes dependent on the release of arachidonic acid-derived lipid mediators in the arterial wall.

Various micro cell culture systems have recently been developed. However, it is extremely difficult to recover cultured cells from a microchannel because the upper and lower substrates of a microchip are permanently combined. Therefore, we developed a cell culture and recovery system that uses a separable microchip with reversible combining that allows separation between closed and open channels. To realize this system, two problems related to microfluidic control-prevention of leakage and non-invasive recovery of cultured cells from the substrate-must be overcome. In the present study, we used surface chemistry modification to solve both problems. First, octadecyltrimethoxysilane (ODTMS) was utilized to control the Laplace pressure at the liquid/vapor phase interface, such that it was directed toward the microchannels, which suppressed leakage from the slight gap between two substrates. Second, a thermoresponsive polymer poly(N-isopropyl acrylamide) (PNIPAAm) was used to coat the surface of the ODTMS-modified microchannel by UV-mediated photopolymerization. PNIPAAm substrates are well known for controlled cell adhesion/detachment by alteration of temperature. Finally, the ODTMS- and PNIPAAm-modified separable microchips were subjected to patterning, and human arterial endothelial cells (HAECs) were cultured in the resulting microchannels with no leakage. After 96 h of the culture, the HAECs were detached from the microchips by decreasing the temperature and were then recovered from the microchannels. This study is the first to demonstrate the recovery of living cells cultured in a microchannel, and may be useful as a fundamental technique for vascular tissue engineering., (Copyright © 2010 Elsevier Ltd. All rights reserved.)