Pneumatically Actuated Microfluidic Platform for Reconstituting 3D Vascular Tissue Compression

The aim of this study is to elucidate the relation of the inelastic expansive deformation of natural blood vessel and the degradation of elasticity to the time pattern of circulating pulsatile pressure flow. When pulsatile pressure amplitude (delta P = Pmax - Pmin) becomes smaller due to Pmin increasing, the blood vessel is subject to creep deformation. In this sense, pulsating pressure will play a role in avoiding creep effect. Fluctuation of maximum pressure Pmax will induce the increase of inelastic deformation and the decrease of rigidity of the blood vessel. The inelastic deformation and the decrease of rigidity in blood vessel is induced at the stage of pressure amplitude rising from a lower one to a higher, but not during pressure amplitude (delta P) kept constant nor at the pressure amplitude decreasing stage. In order to reduce the degradation of mechanical properties of blood vessel, it may be effective to avoid the increase of Pmin and the variability of Pmax.

Metabolism of oxygen by cells can generate potentially dangerous reactive oxygen species (ROS). Endothelial cells lining vascular luminal surface are an important site of signalling molecules and a ROS target during ischemia, inflammation and other pathological conditions. Targeted delivery of ROS modulating enzymes conjugated with antibodies to endothelial surface molecules provide site-specific interventions of endothelial ROS. Excessive ROS cause pathological activation of endothelium including exposure to cell adhesion molecules. Intercellular adhesion molecule-1 (ICAM-1) is a member of the Ig superfamily and is also found on the surface of several other cell types, including endothelial cells. These molecules [e.g., ICAM-1, vascular cell adhesion molecule 1 (VCAM-1) and platelet-endothelial cell adhesion molecule-1 (PECAM-1)] exposed on activated endothelium represent attractive targets for delivery of drugs and imaging probes to pathological sites in the vasculature. This study has investigated the effect of different stereoisomers of the antioxidant thioctic acid on the endothelium of rat kidney vasculature. Twenty-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats were treated for 2 weeks with a daily dose of 12.5 nM/kg racemic (+/-)-, (+)- or (-)-thioctic acid. Kidney vascular microanatomy was Investigated by ICAM-1, VCAM-1 and PECAM-1 immunochemistry and immunohistochemistry. . Treatment with thioctic acid significantly improved renal vascular endothelium status by preventing the ICAM and VCAM adhesion. (+)-Thioctic acid elicited the most sustained effect, (+/-)-thioctic acid was less effective and (-)-thioctic acid worsened activity of the compound. The above data suggest that (+)-thioctic acid, the naturally-occurring enantiomer of the compound, is probably the only form of it able to interfere with endothelial adhesion mechanisms in renal vasculature. The comparatively lower activity of racemic (+/-)- thioctic acid is likely determined by the negative impact of (-)-thioctic acid on the parameters under investigation. Analysis of vascular intercellular adhesion mechanisms could represent an interesting approach for investigating specific vascular disorders and also other pathologies in which vascular involvement plays an important role.

Reactive oxygen species (ROS) are by-products of oxygen metabolism, normally present in low levels inside cells, where they participate in signaling processes. The delicate balance in the continuous cycle of ROS generation and inactivation is maintained by enzymatic and nonenzymatic endogenous systems. Overwhelming production of ROS (by such sources as the mitochondrial electron transport chain, NADPH oxidase, xanthine oxidase, or uncoupled nitric oxide synthase), when inadequately counteracted by destruction through antioxidant systems (such as superoxide dismutase or catalase), leads to a prooxidant state also known as oxidative stress. Increased levels of ROS and markers of oxidative stress have been consistently found in such cardiovascular diseases as atherosclerosis or hypertension, although controversy still exists over the pathophysiological role of oxidative stress in these conditions. ROS can modulate vascular function either by direct oxidative damage or by activating cellular signaling pathways that lead to abnormal contractile, inflammatory, proliferative, or remodeling properties of the blood vessel. Most current research focuses on these processes in arteries, leaving veins, "the other side" of vascular biology, in obscurity. Veins are different structurally and functionally from arteries. Equipped with a smaller smooth muscle layer compared to arteries, but being able to accommodate 70% of the circulating blood volume, veins can modulate cardiovascular homeostasis and contribute significantly to hypertension pathogenesis. Although the reports on the quantitative differences in ROS production in veins compared to arteries had conflicting results, there is a clear qualitative difference in ROS metabolism and utilization between the two vessel types. This review will compare and contrast the current knowledge of ROS metabolism in arteries versus veins in both physiological and pathophysiological conditions. Our understanding of the mechanisms underlying vascular diseases would greatly benefit from a more thorough exploration of the role of veins and venous oxidative stress.

During orthodontic tooth movement (OTM), osteocytes, the most mechanosensitive cells in alveolar bone, suffer the heavy orthodontic force and initiate alveolar bone resorption on the compression side. However, the inherent mechanisms of compressive force-induced osteocyte death are not fully understood. In this study, we established an OTM model on Sprague-Dawley rats by inserting coil springs to investigate osteocyte damage on the compression side of alveolar bone. We then applied compressive force on the MLO-Y4 osteocyte-like cell line in vitro to explore whether the reactive oxygen species (ROS)-mediated endoplasmic reticulum stress (ERS) pathway is involved in compressive force-induced osteocyte death. We found that the orthodontic force caused apparent alveolar bone loss, osteocyte death, and elevated serum sclerostin and receptor activator of NF-κB ligand (RANKL) levels in rats. In vitro, compressive force inhibited cell viability but increased the LDH leakage and loss of mitochondrial membrane potential in MLO-Y4 cells. Simultaneously, it activated protein kinase RNA-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2 (eIF2α), and their downstream pro-apoptotic ERS signaling proteins and caused significant osteocyte apoptosis, which can be blocked by ERS inhibitor salubrinal. Moreover, the compressive force elevated intracellular ROS levels, while the ROS scavenger N-acetyl-L-cysteine (NAC) alleviated ERS and apoptosis in loaded osteocytes. These results suggest that the orthodontic compressive force induced osteocyte apoptosis via the ROS-mediated ERS pathway. This study first proposes the ERS pathway as a new potential pathway for regulating the rate of OTM based on osteocyte death. RESEARCH HIGHLIGHTS: The orthodontic force increases osteocyte death in rat alveolar bone. The compressive force causes osteocyte apoptosis by the endoplasmic reticulum stress (ERS) pathway in vitro. The ROS scavenger NAC blocked compressive force-induced ERS and osteocyte apoptosis.

Blood vessels are permanently subjected to mechanical forces in the form of stretch, encompassing cyclic mechanical strain due to the pulsatile nature of blood flow and shear stress. Significant variations in mechanical forces, of physiological or physiopathological nature, occur in vivo. These are accompanied by phenotypical modulation of smooth muscle cells and endothelial cells, producing structural modifications of the arterial wall. In all the cases, vascular remodelling can be allotted to a modification of the tensional strain or shear, and underlie a trend to reestablish baseline mechanical conditions. Vascular cells are equipped with numerous receptors that allow them to detect and respond to the mechanical forces generated by pressure and shear stress. The cytoskeleton and other structural components have an established role in mechanotransduction, being able to transmit and modulate tension within the cell via focal adhesion sites, integrins, cellular junctions and the extracellular matrix. Mechanical forces also initiate complex signal transduction cascades, including nuclear factor-kappaB and mitogen-activated protein kinase pathways, leading to functional changes within the cell.

Nanotechnology offers unique advantages over small-molecule drugs by delivering a high payload of photosensitizers to the target tissue, providing flexibility in the excitation wavelengths, enabling longer retention in the target tissue, possessing regenerative reactive oxygen speciesproducing capability and stimulating the immune system.

Introduction: Pulmonary arterial hypertension (PAH) is a lethal disease characterized by vascular obstruction. We evaluated the initiating mechanisms for PAH in Fawn-hooded rats (FHR), a strain that spontaneously develops PAH with age. Prior data (Circulation113:2630 – 41, 2006) show that FHR have reduced expression of superoxide dismutase 2 (SOD2), decreased levels of reactive oxygen species (ROS) measured by chemiluminescence, and normoxic activation of hypoxia-inducible factor (HIF-1α)-a pseudohypoxic phenotype. Hypothesis: We hypothesized that ROS are decreased in FHR pulmonary arteries (PA) due to the loss of SOD2 expression. Methods and Results: We measured ROS using Electron Paramagnetic Resonance (EPR) and cell-permeable spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH), a cyclic hydroxylamine derivative. Expression of SOD2 was measured using quantitative PCR. HIF-1α activation was measured using nuclear translocation of the protein on confocal microscopy. ROS levels are 7-fold lower in the pulmonary arteries of FHR (n=3) comparing to weight-matched Sprague-Dawley rats (n=5). The demethylating agent, 5-aza-2′deoxycytidine not only restored expression of SOD2 in FHR pulmonary artery smooth muscle cells (PASMC) but also tended to increase ROS production (3.4-fold increase) in the FHR pulmonary arteries. This agent also decreased activation of HIF-1α as evidenced by a reduced presence of nuclear HIF-1α protein measured by immuno-fluorescence. Conclusions: This spin-probe technique is useful for the measurement of ROS in blood vessels. The data support the hypothesis that methylation-induced silencing of SOD2 accounts for the reduced ROS production in FHR. Loss of ROS signaling molecules activates HIF-1α and may initiate PAH. Since human PAH is also associated with activation of HIF-1α this research may have clinical translational significance. Clinically, decitabine (a commercial name for 5-aza-2′deoxycytidine) has been safely used to treat myeloproliferative disorders in humans.

Combining live imaging with the ability to retrieve individual cells of interest remains a technical challenge. Combining imaging with precise cell retrieval is of particular interest when studying highly dynamic or transient, asynchronous, or heterogeneous cell biological and developmental processes. Here, we present a method to encapsulate live cells in a 3D hydrogel matrix, via hydrogel bead compartmentalisation. Using a small-scale screen, we optimised matrix conditions for the culture and multilineage differentiation of mouse embryonic stem cells. Moreover, we designed a custom microfluidic platform that is compatible with live imaging. With this platform we can long-term culture and subsequently extract individual cells-in-beads by media flow only, obviating the need for enzymatic cell removal from the platform. Specific beads may be extracted from the platform in isolation, without disrupting the adjacent beads. We show that we can differentiate mouse embryonic stem cells, monitor reporter expression by live imaging, and retrieve individual beads for functional assays, correlating reporter expression with functional response. Overall, we present a highly flexible 3D cell encapsulation and microfluidic platform that enables both monitoring of cellular dynamics and retrieval for molecular and functional assays.

Oxidative stress is a key feature in vascular homeostasis. Reactive oxygen species (ROS) are produced by multiple enzymatic sources located in various anatomical structures of the vascular wall, such as the vascular endothelium, the smooth muscle cells and inflammatory cells infiltrating sub-endothelial space and the rest of the vascular wall. Although ROS behave as signaling molecules regulating important aspects of vascular physiology, their excess generation is harmful. Further to the cytotoxic effect of ROS in the vascular wall, they also activate various redox sensitive transcription pathways, regulating the expression of proinflammatory molecules with strong pro-atherogenic effects. The activation of redox-sensitive enzymatic systems in the vascular wall such as matrix metalloproteinases as well as the impairment of endothelial function have a significant impact on vascular elasticity and vascular mechanics in general. The impairment of vascular mechanics has a significant impact on vascular homeostasis, promoting atherogenesis. It is therefore crucial to regulate vascular redox signaling, by developing therapeutic strategies able to target the effectively intracellular ROS bioavailability. Statins, angiotensin converting enzyme inhibitors, thiazolidinediones, folates, tetrahydrobiopterin and other therapeutic strategies seem promising in targeting vascular redox signaling, although it is still unclear which of these treatments have the potential to effectively prevent atherogenesis. Future studies need to define the key redox sensitive pathways in the vascular wall in order to develop effective therapeutic strategies against atherosclerosis.

Human abdominal aortic aneurysm (AAA) is characterized by the induction of intracellular and extracellular inflammatory cytokines and the production of reactive oxygen species (ROS) associated with localized inflammatory responses in the vascular wall. Recent studies have shown that greater circulating levels of the proinflammatory cytokine interleukin-6 (IL-6) are closely associated with AAA presence, suggesting that IL-6 plays an important role in the development of AAA. Previous in vivo studies have indicated that excess activities of NADPH oxidase (NOX), a major oxidase system for ROS production, promote AAA development. Furthermore, long noncoding RNAs (lncRNAs) are involved in the development of AAA. LncRNA MALAT1 has been found closely involved in endothelial cell functions and dysfunctions. In the present study, we explored the effects and the underlying mechanisms of IL-6 and MALAT1 on the expression/activity of NOXs in human aortic endothelial cells (HAOECs). Primary HAOECs with or without overexpression or knockdown of MALTA1 were cultured in the presence of IL-6. We found that IL-6 concentration- and time-dependently elevated the NOX activity as well as the MALAT1 level in HAOECs. Among different NOXs, only NOX2 was induced by IL-6. Overexpression and knockdown of MALAT1 respectively augmented and abolished IL6-induced expression of NOX2, NOX activity/cellular ROS production, and activation of the human NOX2 gene promoter, whereas MALAT1 alone in the absence of IL-6 treatment showed no significant effect. Knockdown of extracellular signal-regulated kinase (ERK) abolished IL6-induced expression of MALAT1. In conclusion, this study provides the first evidence that IL-6 induces expression/activity of NOX2 in HAOECs via inducing MALAT1 by an ERK-dependent mechanism. It adds new insights into the molecular mechanisms underlying AAA development.

NADPH oxidases and atherosclerosis: unraveling the details

Background: Reactive oxygen species (ROS) play a central role in cigarette smoking-induced atherogenesis. The present study aims to assess the smoking-induced acute oxidative stress within vasculatures, and evaluates whether the resveratrol, a natural polyphenol antioxidant, can counteract this ROS production, using a customized, high resolution intravital optical imaging in real-time. Methods and Results: 20-week-old male C57BL/6 mice were divided into four groups according to the preceding administration of resveratrol (R) (25mg/kg via gavage, for 7 days) and exposure to cigarette smoke (CS). To in vivo assess acute oxidative stress in blood vessels, dihydroethidium, which forms a red fluorescence (ethidium, excitation/emission: 520nm/610nm) upon reaction with ROS, was injected intraperitoneally. During CS exposure, temporal changes of fluorescence signals from the mouse cremaster muscle including vasculatures were assessed by intravital optical imaging for 15 minutes. Fluorescence signals were much more pronounced in CS exposed mice than controls (p<0.001). Resveratrol p.o. significantly reduced the CS-induced ROS signals compared to the non-treated group (fluorescence signal to noise ratio, SNR, 2.51±0.09 vs. 12.52±2.116, p=0.0002) (Figure A). Without CS exposure, fluorescence signals in targeted vasculatures were very low showing no difference between groups (SNR, 1.65±0.19 vs. 1.53±0.07, p=0.80) (Figure A). Lipid peroxidation was increased in CS group and significantly attenuated in resveratrol-treated mice (Figure B). Fluorescence microscopy and immunostainings corroborated the in vivo findings. Conclusions: The intravital optical imaging was able to in vivo estimate the dynamic changes of ROS production by CS exposure. Our data demonstrated that even a brief exposure to CS increased oxidative stress in vasculatures promptly, and the resveratrol exerts protective effects against the CS-induced acute oxidative stress.

Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II-mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45(+) inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II-induced vascular smooth muscle cell ROS production. These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II-mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.

193 拍動に伴う血管形状の変形を考慮した2次元超音波計測融合血流解析 : Bモード画像による非定常血管形状抽出

The increased reactive oxygen species (ROS) caused by hyperglycemia (HG) is thought to be a key event in the pathogenesis of diabetic retinopathy. In diabetes, increased vascular endothelial growth factor (VEGF) expression is reported to increase ROS. Using a human RPE cell-line (ARPE-19), we investigated whether HG could promote the expression of VEGF independently of Angiotensin II through the prorenin receptor (PRR), via a NADPH oxidase (Nox)-dependent mechanism. HG significantly induced the expression of PRR, Nox2, Nox4, and VEGF. Also, PRR, Nox2/4, and Rac1 siRNAs, but not the control siRNA, significantly reduced the HG-induction of VEGF. Nox4 overexpression significantly potentiated PRR-induced stimulation of VEGF. Inhibitors of Nox significantly abolished HG-induction of VEGF. Both catalase and superoxide dismutase significantly attenuated VEGF induction by HG, indicative of the involvement of Nox-derived O2– and H2O2 signaling in regulation of VEGF regulation. In summary, Nox4-derived ROS signaling is implicated in the HG-induced increase of VEGF expression through PRR. Nox and redox signaling components can be considered as potential therapeutic targets for treatment of angiogenesis-dependent retinal diseases, including diabetic retinopathy.