Microvascular Reviews and Communications

The 39^th Annual Meeting of Japanese Society for Microcirculation

Released: March 20, 2014

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Clinical application of huma iPS cells for cardiovascular Medicine

Keiichi Fukuda

Released: May 21, 2014

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Although heart transplantation can drastically improve the survival, shortage of the donor heart is a serious problem. The regenerative medicine of the failing heart had been long awaited. To address this question, we had developed novel methods to induce human iPS cells from circulating human T lymphocytes using Sendai virus containing Yamanaka 4 factors. We had screened the factor that were expressed in future heart forming area of the early mouse embryo, found several growth factors and cytokines that can induce cardiomyocytes differentiation and proliferation, and applied them to human iPS cells. We performed transcriptome of the metabolic enzymes and fluxome analysis using ¹³glucose and 13lactic acid on ES/iPS cells and cardiomyocytes, and found that their metabolic pathways were completely different. Based on these findings, we purified cardiomyocytes using glucose-free lactate-supplemented medium. Purity of the cardiomyocytes was > 99%, and they did not make teratoma formation. The transplanted cardiomyocytes using our technique can survive in the heart with more than 90%, and can show physiological growth after transplantation. We expect the combination of these techniques can achieve future heart regeneration. We also developed human disease model cardiomyocytes using human iPS cells from the patients with long QT syndrome and other hereditary heart disease. These disease model cardiomyocytes represented the phenotype of the disease, and might be helpful for drug screening and pathophysiological analysis.

Cancer Stem Cell of Digestive Organs

Masaki Mori

Released: May 21, 2014

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Recent studies supported the notion that a small population, which mimics norm al adult stem cells in the dormant phase of the cell cycle, plays a role in th e biological behaviors of tumors. Indeed such distinct cells, i.e., cancer ste m cells are resistant to toxic injuries and chemoradiation therapy in vitro an d in vivo. After possible involvement was indicated in leukemia, we were able to report cancer stem cells in gastrointestinal tumors. Our exploration of new screening for surface markers were supposed to be beneficial to identify gast rointestinal cancer stem cells, followed by characterization of chemoresistanc e and tumorigenicity, indicating that several cell surface markers including CD13/APN play a role in biological function of cancer stem cells. Furthermore, we examined the possible effects of cellular reprogramming by induction or inh ibition of cancer-related genes and immature status-related genes including th at of induced pluripotent stem (iPS) cell genes, whose alterations have been r eported in gastrointestinal cancer cells. Introduction of iPS cell genes but a lso several microRNAs, including miR302 was necessary for inducing the express ion of immature status-related proteins and the possible expression of morphol ogical patterns and showed slow proliferation and were sensitized to different iation-inducing treatment, and in vivo tumorigenesis was reduced in nonobese d iabetic mice with severe combined immunodeficiency. Taken together the present study indicates that the combination of traditional therapies with targeted c ancer stem cell-specific agents may target the whole tumors and may offer a pr omising strategy for lasting treatment and even cure.

Balancing angiogenic pathways in solid tumors

Dai Fukumura

Released: May 21, 2014

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Intravital microscopy techniques have provided unprecedented insight into tumor angiogenesis, microcirculation and microenvironment. Tumor microvasculature has an abnormal organization, structure, and function. Tumor vessels are leaky. Blood flow is heterogeneous and often compromised. Lymphatic vessels are either defective or not functional inside tumors and together with leaky blood vessels elevate interstitial fluid pressure in solid tumors. All of these abnormalities hinder the delivery of therapeutic agents to tumors and also induce a hostile microenvironment characterized by hypoxia and acidosis. The abnormal microenvironment fuels malignancies of tumors and further lowers the effectiveness of anti-tumor treatments such as radiation therapy, chemotherapy and novel molecularly targeting therapies.
However, one can also exploit aberrant microenvironment in tumors for selective treatment of tumors. Enhanced permeability and retention effect of relatively large size particles in tumors is the major basis of nanomedicine. It not only increases therapeutic index but also allows delivering toxic agents and hydrophobic drugs to tumors otherwise prohibited for clinical use due to normal tissue toxicity. Unfortunately, crucial drawback of this approach is diffusion hindrance of the large nanoparticles. These nanotherapeutics cannot advance into tumor tissues after the extravasation from tumor vessels. To solve this dichotomy we proposed a multistage nanoparticle delivery system. We have developed a relatively large nanoparticle that can release small size nanoparticles upon exposure to enzymes uniquely present in tumor tissues and demonstrated superior intratumoral diffusion of these multistage nanoparticles.
Alternatively, one may try to tame abnormal tumor microenvironment. For example, host-tumor interactions regulate expression of pro- and anti-angiogenic factors. Imbalance of these factors results in above-mentioned pathophysiological features in the tumor. In a physiological setting, angiogenic vessels eventually become mature and stable vessels that represent long-lasting functional units. Restoring tissue balance of these factors in tumors may “normalize” tumor vasculature and thus, improve its function. Administration of cytotoxic therapy during the vascular normalization can enhance its efficacy. We have demonstrated a number of approaches to normalize tumor vasculature and microenvironment that improve a variety of anti-tumor therapies.

Role of ribosomal protein S19 oligomer-C5a receptor system in acute inflammation resolution

Tetsuro Yamamoto

Released: May 21, 2014

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Ribosomal protein S19 (RP S19) is a component of the small ribosome subunit and essential for ribosome biogenesis. RP S19 is also present in blood plasma, forming a complex with prothrombin. Cellular RP S19 is inter-molecularly cross-linked by an intracellular transglutaminase during apoptosis, and plasma RP S19 is similarly cross-linked by activated coagulation factor XIII during blood coagulation, forming an isopeptide bond between Lys122 and Gln137 in both cases. The cross-linked RP S19 oligomers thus formed gain a ligand capacity to the C5a receptor and express various kinds of extra-ribosomal functions.
The cells undergoing apoptosis de novo synthesize the C5a receptor. The RP S19 oligomers liberated by the apoptotic cells hasten the apoptosis execution on one hand and recruit phagocytic macrophages on the other, completing the prompt clearance of the apoptotic cells. Isolated neutrophils spontaneously undergo apoptosis and generate the RP S19 oligomers. The RP S19 oligomers do not elicit chemotactic response of neutrophils but rather speed up the apoptotic process of the cells, while these molecules induce chemotactic migration of monocytes/macrophages. We currently made a hypothesis that this would be a crucial mechanism in resolution of acute inflammation. This hypothesis has been experimentally supported. For instance, when the RP S19 oligomers were immunologically neutralized in a carrageenan-induced mouse pleurisy model, neutrophil number in the pleural exudate greatly increased and the inflammation spread to lung parenchyma. Similar phenomena were observed in the carrageenan pleurisy induced in Gln137Glu-RP S19 knock-in mice without the neutralization.
Regarding the discrimination by the RP S19 oligomers but not by complement C5a between neutrophils and monocytes/macrophages, we made a hypothesis that a molecule(s) that disconnects the RP S19 oligomer/C5a receptor complex but not the C5a/C5a receptor complex from the intracellular signal transduction pathway is present near C5a receptor in neutrophils but not in monocytes/macrophages. To examine the hypothesis and identify the disconnecter molecule(s), we prepared a recombinant C5a/RP S19 chimeric protein which reproduces the functions of RP S19 oligomers as a monomeric protein. Delta-lactoferrin (δ-Lf) was co-separated with C5a receptor when ligated by C5a/RP S19 but not by C5a in neutrophils. δ-Lf is an intracellular protein, and it is not synthesized by monocytes/macrophages. When δ-Lf mRNA translation was blocked, HL-60-derived neutrophil-like phenotypes changed to chemotactically respond to C5a/RPS19. δ-Lf seems to be the disconnecter molecule.

Sibasaburou Kitasato and the Kitasato Institute

Kanji Tsuchimoto

Released: May 21, 2014

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Dr.Shibasaburo Kitasato officially established the Kitasato Institute in 1914, but the long history exists before then.
Shibasaburo Kitasato was born in 1853 in Kumamoto Prefecture. He received strict home discipline and instruction from Constant George van Mansveldt at Kumamoto Medical School. After graduating from the University of Tokyo in 1883, he went to Robert Koch’s laboratory in 1886 and achieved in the field of preventive medicine, especially immunology, where he successfully grew a pure culture of tetanus bacilli, followed by his discovery of the serotherapy used to treat that disease. After returning from Germany in 1892, he established Japan’s first private medical research facility for infectious diseases supported by Yukichi Fukuzawa, the founder of Keio University and others both materially and spiritually. This institute made great progress and was placed under the control of the Japan Hygiene Society in 1899. In 1914, as the government transferred the Institute under the University of Tokyo, Kitasato and his followers resigned and started the Kitasato Institute.
The Spirit of Kitasato, which he developed over a life time - to investigate with a pioneering spirit, be appreciative in your dealings with people, possess wisdom and be a person of practical science, as well as to persist with an unwavering spirit - has been continuously handed down from generation to generation at the Kitasato Institute · Kitasato University and Keio University.
Now approaching our centennial of the founding of the Institute, a landmark moment, we take the Spirit to heart once more and I am sure the Institute will evolve eternally.

Nicotine ameliorates colonic inflammation via down-regulation of MAdCAM-1 expression on high endotherial venule like vessel.

Koji Maruta, Hideaki Hozumi, Ryota Hokari, Yuichi Yasutake, Hirokazu Sato, Kazuyuki Narimatsu, Chie Kurihara, Yoshikiyo Okada, Shingo Usui, Chikako Watanabe, Shunsuke Komoto, Kengo Tomita, Shigeaki Nagao, Soichiro Miura

Released: May 21, 2014

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Background: Ulcerative colitis (UC) is an intractable colonic disease. Lymphocytes migration to colonic mucosa through endotherial venule like vessel is considered to be involved in pathophysiology of this disease. Anti-adhesion molecule therapy targeting MAdCAM-1 on high endotherial venule like vessel is one of the promising therapy. Smoking has been reported to have a beneficial effect on UC. Nevertheless, pathophysiology of nicotine on activity of UC is still to be elucidated. This time, we investigated the involvement of nicotine in the colonic inflammation using murine colitis model.

Method: In murine study, tissue samples were obtained from colon of C57BL/6J mouse provided with drinking water containing dextran sulfate sodium (DSS). Degree of mRNA expression of TNF-α and MAdCAM-1 was determined by using quantitative RT-PCR. The inhibitory effects of nicotine on activity of colitis and mRNA expression were determined. To induce high endothelial venules in vitro, bEnd3 cell line was treated with TNF-alpha. Effect of nicotine on MAdCAM-1 expression on high endothelial venule (HEV) like vessel was also measured by using quantitative RT-PCR.

Results: In murine colitis model, administration of nicotine ameliorated DSS colitis. Administration of nicotine also significantly decreased degree of expression of MAdCAM-1 mRNA on HEV-like vessel.

Conclusion: Nicotine ameliorates DSS colitis possibly via down regulation of MAdCAM-1 expression on HEV-like vessel, and accordingly, inhibition of aberrant lymphocyte migration in colonic mucosa.

VEGFR1 signaling facilitates diabetic skin wound healing in mice

Shin-ichiro Okizaki, Yoshiya Ito, Hirotoki Okubo, Ken Kojyou, Kazuhito Ohba, Shichiri Masayoshi, Masataka Majima

Released: May 21, 2014

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Aims: Signaling of vascular endothelial growth factor receptor 1 (VEGFR1) is suggested to involve in angiogenesis and lymphangiogenesis. The objective of the present study was to examine the role of VEGFR1 signaling in angiogenesis/lymphangiogenesis during diabetic skin wound healing.

Methods: VEGFR1-tyrosine kinase knockout mice (KO) or their wild counterparts (WT) were treated with streptozotosin (STZ) or vehicle (Veh). Full-thickness skin wounds were created on the backs of mice.

Results: Compared with non-diabetic mice (Veh/WT), wound healing and angiogenesis were suppressed in diabetic mice (STZ/WT) and non-diabetic KO mice (Veh/KO), with reduced expression of VEGF-A and CD31 in wound granulation tissues. Formation of lymphatic vessels was inhibited with reduced expression of VEGF-C, VEGF-D and VEGFR3. Accumulated VEGFR1-positive macrophages with VEGF-C or VEGF-D-expressing cells in granulation tissues were decreased. This was associated with attenuated expression of mannose receptor (MR) and transforming growth factor-beta (TGF β). Diabetic KO (STZ/KO) showed further delayed wound healing and wound-induced angiogenesis/lymphangiogenesis. Exaggerated reduction in recruitment of VEGFR1-positive macrophages and in expression of MR and TGF β was also demonstrated.

Conclusions: These results indicate that VEGFR1 signaling plays a role in angiogenesis/lymphangiogenesis through recruitment of VEGFR1-positive macrophages during diabetic wound healing.

Post-stroke administration of cilostazol changes metabolic profile in transsulfuration pathway of ischemic brain in a mouse model

Yasoo Sugiura, Mayumi Kajimura, Tsuyoshi Nakanishi, Takayuki Morikawa, Takako Hishiki, Makoto Suematsu

Released: May 21, 2014

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Cilostazol, an inhibitor of phosphodiesterase3 (PDE3), has been suggested to minimize post-stroke cognitive impairment. However, mechanisms underlining these beneficial effects remain elusive. We, therefore, examined effects of cilostazol on biochemical characteristics of cerebral metabolism using mouse cerebral ischemia model in vivo. To decipher multifold mechanisms whereby cilostazol changes metabolic dynamics in different regions of the brain, we conducted metabolome analysis to target metabolic pathways responding to the cilostazol treatment. To this end, focal ischemia was induced by a left middle cerebral artery occlusion. Right after the induction of ischemia, either the cilostazol (30 mg/kg or 100 mg/kg) or vehicle was administered orally. At 60 min after the occlusion, metabolic processes were rapidly suspended by the in situ freezing to minimize autolytic changes. Metabolites were extracted and measured with high-throughput capillary electrophoresis mass spectrometry. We then conducted cluster analysis to compare and contrast changes in 90 metabolites extracted from contralateral (CL) and ipsilateral (IL) hemispheric brains. In both CL and IL, the cilostazol treatment tended to increase cystathionine, taurine, cysteine, and the reduced form of glutathione, suggesting that the treatment alters sulfur amino acid metabolism and the transsulfuration pathway. Such an observation led us to hypothesize that cilostazol controls the activity of cystathionine β-synthase (CBS) which catalyzes the first committed step of the transsulfuration pathway. When primary cultured astrocytes which endogenously express CBS were treated with cilostazol, CBS expression increased as judged by Western blot analysis. These results indicate that cilostazol treatment could achieve neuroprotection via controlling CBS activity. Alteration of metabolites in the transsulfulation pathway induced by cilostazol oral administration may lead to beneficial therapeutic stratagem in cerebrovascular diseases.

Role of leukotriene B4 receptor 1 (BLT1) signaling in liver repair after hepatic ischemia reperfusion injury

Hirotoki Ohkubo, Yoshiya Ito, Ken Kojo, Masahiko Watanabe, Masataka Majima

Released: May 21, 2014

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Aims: Leukotriene B4 (LTB4) is a potent chemoattractant for macrophages, and recruited macrophages play a critical role in liver repair and recovery from acute liver injury. The objective of the present study was to examine the role of LTB4 receptor 1 (BLT1) signaling in liver repair after hepatic ischemia/reperfusion (I/R) injury.

Methods: BLT1knockout mice (BLT1-/-) and wild-type mice (WT) were subjected to 60 min of partial (70%) hepatic warm ischemia followed by reperfusion. The process of liver repair after hepatic I/R was determined.

Results: In WT, ALT levels peaked at 6h, and then declined to controls at 96h. In BLT1-/-, ALT levels also peaked at 6h, but those at 48 and 96h (recovery phase) were 2-fold higher than WT. The necrotic area in WT peaked at 24h, and reduced gradually, while that in BLT1-/- was remained high until 96h. In BLT1-/-, the expression of proliferating cell nuclear antigen (PCNA) was delayed, which was associated with reduced levels of hepatic mRNA expression of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and VEGF receptor 1 (VEGFR1). Recruitment of VEGFR1-positive macrophages expressing EGF in injured liver from BLT1-/- was attenuated. Treatment of WT mice with an EGF-neutralizing antibody delayed liver repair and reduced macrophage recruitment, compared with control immunoglobulin G (IgG). BLT1 signaling enhanced the expression of VEGF, VEGFR1, and EGF in isolated peritoneal macrophages in vitro.

Conclusions: BLT1 signaling plays an important role in liver repair after hepatic I/R through enhanced EGF expression in recruited macrophages.

3, 4-dihydroxyl-phenyl lactic acid restores NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10 expression to ameliorate cardiac reperfusion injury

Ke He, Xiao-Yuan Yang, Na Zhao, Yu-Ying Liu, Bai-He Hu, Kai Sun, Xin Chang, Xiao-Hong Wei, Jing-Yu Fan, Jing-Yan Han

Released: May 21, 2014

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Background: Protection of ischemia/reperfusion (I/R) induced myocardial injury remains a challenge for clinician. 3, 4-dihydroxyl-phenyl lactic acid (DLA) is a major ingredient of cardiotonic pills®, a undergoing phase Ⅲ clinical trials drug for treatment of cardiovascular diseases in FDA in USA. However whether DLA exerts protective role against I/R and the intracellular target for DLA action remains unclear.

Methods and Results: Male Spragu-Dawley (SD) rats were subjected to left descending artery occlusion for 30 min, followed by reperfusion with or without DLA administration for 90 min. Results showed DLA reduced infarct size, diminished myocardial apoptosis and ameliorated impaired cardiac function and myocardial blood flow (MBF) after I/R. The results of 2-D fluorescence difference gel electrophoresis and activity assay kit revealed that DLA prevented from decrease in NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 10 (NDUFA10) expression, one of the subunits of Complex Ⅰ, blunted the impairment of Complex Ⅰ activity and mitochondrial function. To find the target of DLA, the binding affinity of Sirtuin 1 (SIRT1) to DLA and DLA derivatives with replaced two phenolic hydroxyls were detected using surface plasmon resonance and bilayer interferometry. The observed results demonstrated DLA was able to bind to SIRT1, depending on phenolic hydroxyl.

Conclusions: The present study demonstrated the capability of DLA to bind to and activate SIRT1, which plays an essential role in the cadioprotective effects of DLA. Preserved SIRT1 activity by DLA is responsible for the restored NDUFA10 protein and improved mitochondrial function, eventually leading to repressed infarct size and apoptosis, preserved cardiac function and MBF after I/R.

Comparison of peripheral vascular resistance based on macro- and micro-circulatory responses by Poilleuille’s law

Kazuhiro Yokokawa, Saki Hamashima, Masahiro Shibata

Released: May 21, 2014

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The total peripheral vascular resistance (TPR) is essential index in the cardiovascular system, since both the systemic blood pressure and blood flow could be determined by the changes of TPR. Such important index, the TPR cannot be measured directly, so Darcy’s law would be applied to determine TPR. On the other hand, vascular flow resistance would be mainly controlled by the contraction or dilation of small arteries and arterioles, existing at the upstream of capillaries. Regarding the single small artery and the arteriole, the vascular flow resistance (R) could be represented as R=8µL/πr⁴, called Poilleuille’s law (µ: viscosity, r: vessel radius L: vessel length=constant). In addition, the major contribution of these vascular resistances would be caused by the resistance vessels in the skeletal muscle, since the blood flow in skeletal muscle dramatically changes from resting to excise, approximately 20 times increases. These facts suggest the TPR would be determined by the levels of contraction and dilation in skeletal muscle arterioles. In the present study, we tried to investigate in macro- and microcirculation whether the TPR can be estimated from the diameter changes of single arteriole in the skeletal muscle using Dalcy and Poilleuille’s laws. Wister rats (180 - 400g b.w.) were anesthetized, and carotid artery and vein were canulated for the blood pressure measurement and administration of L-NAME, inhibiter of NOS production, respectively. The observation of microcirculation was carried out in the cremaster muscle by intravitalmicroscopy. The TPR was calculated by the changes in the blood pressure during L-NAME caused vasoconstriction based on the Dalcy’s law, while the R was calculated by the changes in the arteriolar diameter based on the Poillleulle’s law. The TPR and R were increased 23.9±7.7% and 23.5±8.7% from control to L-NAME caused vasocontraction, respectively. These results suggest the Poilleulle’s law can apply to estimate the TPR in vivo microcirculation. Furthermore, it has been confirmed the TPR would be regulated mainly by the contraction and dilation of the skeletal muscle arterioles.

Roles of Prostanoids in Regulation of Angiogenesis and Lymphatic Tissue Remodeling

Masataka Majima

Released: May 21, 2014

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Inflammation influences the pathogenesis of cancers by induction of genome damage, proliferation in stromal cells, and generation of inflammatory mediators. Angiogenesis is also a critical step for development and metastasis of cancers. Proinflammatory mediators, such as prostaglandins (PGs) may have cell-autonomous effects on tumor cells in autocrine fashion, however, our results from tumor implantation models in knockout mice which lack the host receptor signaling clarified that host stromal signaling of a G-protein coupled PGE receptor, EP3 has a crucial role in tumor-associated angiogenesis through the induction of proaniogenic growth factors, and exhibited the landscaping effects on tumor cells. An EP3 antagonist inhibited tumor-associated angiogenesis in wild type mice, but not in EP3 knockout mice, suggesting that the blockade of host EP3 receptor signaling is important in prevention of tumor-associated angiogenesis. Further, bone marrow transplantation experiment revealed that recruitment of bone marrow cells which express EP3 is critical for angiogenesis in vivo. Our recent results also suggested that lymphangiogenesis observed in chronic inflammation and wound healing was regulated by an inducible cyclooxygenase, COX-2 and EP signaling. Further, we recently clarified that lymph node metastasis is enhanced by COX-2 and EP signaling via tissue remodeling of the regional lymph nodes to form premetastatic niche in the subcapsular regions. Thus, control of EP signaling as well as COX-2 in the tumor microenvironment is likely to be a therapeutic approach against cancers.

New Trends in therapeutic strategies against ischemia/reperfusion injury; Postconditioning and pharmacological intervention in acute myocardial infarction

Megumi Shimada, Takashi Koyama, Akiyasu Baba, Rie Kosugi, Makoto Akaishi

Released: May 21, 2014

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Despite better outcomes with early coronary artery reperfusion for the treatment of acute myocardial infarction (AMI), morbidity and mortality from AMI remain significant, and myocardial reperfusion injury is a critical contributor to the final infarct size. In the past decade, several pharmacological treatments applied at early reperfusion have been tested in experimental models and in the clinical setting. Unfortunately, efforts at reducing reperfusion injury by several studies have largely been unsuccessful. There is a need to provide better cardioprotective therapy that reduces the amount of necrosis that may be coupled with better clinical outcomes.
Postconditioning: Ischemic postconditioning, defined as brief periods of ischemia immediately after the onset of reperfusion, has been recently shown to be one of the novel strategies of cardioprotection against reperfusion injury. However, recent clinical trials have not elucidated the protective effects of postconditioning. The protective effect of postconditioning is thought to result from delayed recovery from intracellular acidosis during the reperfusion period. It is generally accepted that lactate accumulation is responsible for intracellular acidosis during ischemia. As a higher extracellular lactate concentration impedes lactate transport from inside the cells, reperfusion with lactate-enriched blood should protect myocardial cell against reperfusion injury through prolonged intracellular acidification. We therefore modified the original postconditioning protocol by using lactated Ringer’s solution to achieve controlled reperfusion with tissue oxygenation and minimal lactate washout from the cells. Ischemic postconditioning with lactate-enriched blood consistently suppressed the various detrimental effects of reperfusion and preserved myocardial viability well. Given the excellent microcirculation recovery consistently observed in this series, the modified ischemic postconditioning protocol might be a promising approach to effectively suppress myocardial reperfusion injury.
Pharmacological intervention: Recent clinical trials of cardiovascular disease have demonstrated that carperitide, a synthetic alpha-human atrial natriuretic peptide (ANP), improve survival in patients with acute myocardial infarction due to their cardioprotective effects. On the other hand, Rho kinase (ROCK) activation plays a major role as a mediator of irreversible injury in reperfused myocardium. We hypothesized that ROCK is activated specifically after ischemia-reperfusion (I-R) and that suppression of ROCK activity during I-R by ANP limits infarct size. A rat model of myocardial I-R injury was investigated by ligating the left descending coronary artery for 30 min and then reperfusing for 180 min. Continuous infusion of ANP (0.1 ug/kg/min) was started 5 min after the ligation and lasting for 175 min. Phosphorylation of the ROCK substrate protein myosin phosphatase targeting subunit (MYPT)-1 assessed by western blotting was used as a marker of ROCK activation. The myocardial infarct size and the area at risk of ischemia were measured by staining with triphenyltetrazolium chloride (TTC). The results showed that I-R injury induced ROCK activation significantly, and ANP reduced infarct size compared to control (9.4 ± 4.3 vs. 35.9 ± 3.5%, ANP vs. control, mean±SD, p < 0.05). Interestingly, the cardioprotective effect of ANP was abolished by 5-Hydroxydecanoate (5-HD), a putative mitochondrial KATP (mKATP) channel inhibitor (32.6±2.9% infarction). In Western blot analysis, attenuation of ROCK activation by ANP was reversed by 5HD, L-NAME, but not wortmannin, an inhibitor of phosphatidylinositol-3-kinase/Akt signaling. In conclusion, inhibition of ROCK activation by ANP limits infarct size via an opening of mKATP/NO-dependent mechanism.

Perfusion fixation method is critical for immunoelectron microscopy and ultrastructural evaluation on changes of caveolin-1 and caveolae relates with capillarization of liver sinusoidal endothelial cells in human cirrhotic liver

Hiroaki Yokomori, Jing-Yan Han, Masaya Oda

Released: May 21, 2014

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Backgrounds and aims: Most vascular endothelial cells are continuously exposed to shear stress in vivo. Caveolae, omega-shaped membrane invaginations on endothelial cell (EC), also are plasmalemmal domain enriched in cholesterol, caveolins, and signaling molecules. Previous studies have proposed a role for caveolin(CAV)-1 in the regulation of angiogenesis and sinusoidal differentiation. This study was designed to elucidate the ultrastructural localization and change in CAV-1 expression on human liver sinusoidal endothelial cells (LSECs) during the progression of cirrhosis, using sections prepared by perfusion fixation method.

Methods: Normal control and Child-Pugh A and C cirrhotic liver specimens by surgical procedure were studied. CAV-1 protein and gene expression was examined by immunohistochemistry, Western blotting, laser-capture microdissection (LCM)-PCR. For immunoelectron microscopy, CAV-1 expressions in sinusoid was examined by perfusion fixed liver tissue.

Results: In control liver tissue, CAV-1 was localized on caveolae mainly in arterial and portal endothelial cells of the portal tract, and was also found on vesicles and some fenestrae in LSECs around the central vein. In cirrhotic liver tissue, aberrant CAV-1 expression was observed on caveolae-like structures and a few vesicles in LSECs. Significant overexpressions of CAV-1 at protein and mRNA level in cirrhotic liver was demonstrated by Western blotting and LCM-PCR (p<0.01 Child-Pugh A and C vs control, p<0.01 Child-Pugh A versus C).

Conclusion: CAV-1 was strongly expressed on caveolae-like structures and vesicles on LSECs in the sinusoids of cirrhotic liver, suggesting an association of CAV-1 with angiogenesis and differentiation of LSECs in cirrhosis

Brain-derived neurotrophic factor promotes angiogenesis via oxidative stress in human vascular endothelial cells: Implication for atherogenesis?

Hideyuki Yamawaki

Released: May 21, 2014

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Aim: Brain-derived neurotrophic factor (BDNF), a major type of neurotrophins, promotes synaptic plasticity and neuronal cell survival, which contribute to the maintenance of structure and function of neuronal cells. Recent studies also indicate a possible involvement of BDNF in the atherogenesis. However, the detailed mechanisms for this remain to be fully clarified. We hypothesized that BDNF may at least partly play a role in the atherosclerotic plaque development through the promotion of angiogenesis. To gain mechanistic insights, we examined whether BDNF causes angiogenesis and underlying mechanisms with focusing on reactive oxygen species (ROS) and related intracellular signals in human cultured vascular endothelial cells (ECs).

Methods and results: In vascular ECs, BDNF increased ROS generation as measured fluorometrically using 2’ 7’-dichlorofluorescein diacetate as well as NADPH oxidase (NOX) activity as determined by a chemiluminescent measurement. BDNF-increased ROS generation and NOX activity were inhibited by K252a, an inhibitor of tropomyosin-related kinase B (TrkB) receptor. BDNF caused phosphorylation of p47 phox, a regulatory component of NOX, which was inhibited by K252a as determined by Western blotting. In matrigel, BDNF caused angiogenic tube formation of ECs, which was inhibited by K252a or gp91ds-tat, a specific inhibitor of NOX. BDNF induced phosphorylation of Akt but not ERK in ECs, which was inhibited by K252a or gp91ds-tat. It was further confirmed that small interfering RNA (siRNA) against TrkB inhibited BDNF-induced ROS generation and tube formation.

Conclusion: The present results for the first time showed that BDNF promotes angiogenesis through NOX-derived ROS generation via the activation of p47 phox in a TrkB receptor-dependent manner.

Cilostazol inhibits leukocyte-endothelial cell interactions in murine microvessels after transient bilateral common carotid artery occlusion

Takuya Fukuoka, Takeshi Hayashi, Makiko Hirayama, Hajime Maruyama, Norio Tanahashi

Released: May 21, 2014

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Leukocyte behavior in the cerebral microvasculature following vessel occlusion has not been fully elucidated. The purpose of this study was to investigate the effects of cilostazol on leukocyte behavior (rolling and adhesion) in murine cerebral microvessels following transient bilateral carotid artery occlusion using intravital fluorescence microscopy. Four groups of mice were assigned: a sham group (n=16); an ischemia (induced by 15-min occlusion of bilateral common carotid arteries) and reperfusion (I/R) group (n=13); I/R+cilostazol (I/R+CZ3mg/kg) group (I/R after oral administration of cilostazol at 3mg/kg) (n=8) and I/R+cilostazol (I/R+CZ30mg/kg) group (I/R after oral administration of cilostazol at 30mg/kg) (n=12). Leukocytes labeled with 0.05% acridine orange were administered intravenously and their behavior was investigated at 3 and 6 h after reperfusion. Numbers of rolling or adherent leukocytes were expressed as the count per square millimeter per 30s. Numbers of rolling and adherent leukocytes at 3 and 6h after reperfusion were significantly higher in the I/R group than in the sham or I/R+CZ30mg/kg groups in both pial veins (P<0.05) and pial arteries (P<0.05). Cilostazol (30mg/kg) inhibited leukocyte-endothelial interactions following cerebral ischemia and reperfusion.

Impairment of CO₂ reactivity in RBC velocity and CBF after cortical spreading depression in anesthetized mice

Miyuki Unekawa, Yutaka Tomita, Haruki Toriumi, Takashi Osada, Kazuto Masamoto, Hiroshi Kawaguchi, Yoshiaki Itoh, Iwao Kanno, Norihiro Suzuki

Released: May 21, 2014

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Background: We previously reported that cortical spreading depression (CSD) drastically suppresses red blood cell (RBC) velocity and alters cerebral blood flow (CBF) and vessel diameter in cortical arteriole. It has been reported that CSD induces disruption of neurovascular and neurometabolic coupling.
Objective: To further understand mechanisms involved in the disturbance of microcirculation, reactivity to carbon dioxide (CO₂) in RBC velocity flowing in intraparenchymal capillaries and CBF was measured before and after CSD passage.
Methods: To visualize blood vessels, we used Tie2-GFP transgenic mice (N=10), in which specifically vascular endothelial cells emit fluorescence. Under urethane anesthesia and artificial ventilation, RBC velocity was measured using a confocal laser-scanning microscope with high-speed camera (125 fps) and an original image analyzing system of KEIO-IS2 working on MATLAB through a cranial window installed on the temporo-parietal region of the cerebral cortex, along with CBF by laser Doppler flowmeter. CO₂ reactivity was measured with 5% CO₂ inhalation for 1min. CSD was induced by microapplication of 1M KCl through a tiny cranial hole posterior to the cranial window.
Results: RBC velocity was measured in 4 to 21 capillaries in each mouse. CO₂ inhalation increased partial pressure of arterial CO₂ by 14.1±3.9mmHg. During hypercapnia, CBF and RBC velocity averaged in each mouse increased by 14.1±11.3% and 17.7±19.0% with significant correlation between the increases (r=0.79, n=8). After CSD passage, increase in CBF and RBC velocity were reduced to 7.3±21.8% and 11.6±22.9%, respectively, and the correlation was lost (r= -0.15, n=11).
Conclusion: CSD attenuated CO₂ reactivity in CBF and RBC velocity by different mechanism, probably due to impairment of neurovascular and neurometabolic coupling.

EXPLORE THE ANGIOGENESIS OF AUTOLOGOUS TRANSPLANTED BRAIN TISSUES IN RABBITS

Jin Xuelong

Released: May 21, 2014

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Objective: This study intends to discuss about the methodology of rabbit’s brain tissue transplantation, including the way of operation and the law of microcirculatory formation.

Methods: 20 male Japanese white rabbits (1.2˜1.3 kg) were chosen for an intracerebral transplantation test, which were anaesthetized by 3% pentobarbital sodium in vein before receiving an intracerebral transplantation operation. A window was opened on their parietal bone and the cortical brain tissues on the symmetrical areas on the left and right side of the rabbits’ parietal cortex areas were exchanged and transplanted. Gentamycin sulfate was injected each day to resist infection. Ten and twenty days later， an observation was made as to the survival of the transplanted area and host brain tissue. A microcirculation color camera system was used to analyze the pictures of angiogenesis. With regard to the survival of transplanted brain tissues, their changes in micromorphology were observed. Besides, pathological sections were also prepared to determine their surviving conditions on a cell level.

Result:
(1) Surgical operation has contributed to a satisfactory morphological anastomosis between transplanted brain tissues and host brain tissues.
(2) Analysis of the pathological sections of the transplanted brain tissues showed traces of surviving neural cell.

Conclusions: Under the given conditions, transplanted brain tissues can maintain neuron’s survival, and can be nourished by angiogenesis and characteristic microcirculation connections with host brain tissues.

HO-2/CO system protects against metabolic disorders following acute cerebral ischemia

Takayuki Morikawa, Mayumi Kajimura, Tsuyoshi Nakanishi, Yoshinori Yukutake, Makoto Suematsu

Released: May 21, 2014

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Although it has been known that brain generates carbon monoxide (CO) via heme oxygenase (HO) catalyzed reactions, physiologic roles of CO in the central nervous system remain elusive. Previous study showed that HO-2 generates CO in an O₂-dependent manner. By acting as an acute O₂ sensor within the neurovascular unit, HO-2 contributes to the maintenance of cerebral ATP levels against acute global hypoxia (Morikawa et al., PNAS, 109, 1293-1298). In this study, we examined if the deletion of HO-2 exacerbates cerebral metabolism upon acute focal brain ischemia. We compared contents of 87 metabolites extracted from contralateral- and ipsilateral hemispheres after a left middle cerebral artery occlusion (MCAO) between wild-type- and HO-2-null mice. With hierarchical clustering analysis, we found that, in ipsilateral hemispheres, there was no obvious difference in patterns of metabolic alteration between wild-type- and HO-2-null mice. On the other hand, in the contralateral hemispheres, we found the clusters showed striking difference in patterns of metabolic alteration between two groups during MCAO. Such a cluster included high energy phosphonucleotides; e.g., ATP, UTP and CTP. These data indicate that nucleotide degradation after MCAO is more severe in the HO-2-null mice than that in wild-type mice. Furthermore, our results indicate that HO-2 contributes to the improvement of metabolic disorders during cerebral ischemia in contralateral hemisphere rather than in ipsilateral hemisphere. This is the first report showing the potency of HO-2/CO system to diminish the remote metabolic insults of acute focal cerebral ischemia.

The blood cell flow and the vascular responses in arterioles and capillaries after subarachnoid hemorrhage

Mami Ishikawa, Mayumi Kajimura, Takayuki Morikawa, Tomomi Nakamura, Yuichi Tanaka, Eiju Watanabe, Makoto Suematsu

Released: May 21, 2014

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Purpose: Immediately after subarachnoid hemorrhage (SAH), brain injury begins and determines the acute phase mortality and the long-term prognosis, but its mechanism is not well understood. When SAH at the skull base induces platelet-leukocyte-endothelial cell interactions in venules, the cerebral blood flow is kept well at the cerebral surface¹⁾. We investigated cerebral microcirculation through a mouse cranial window using two-photon laser scanning microscopy at a depth of about 100µm^2,3), after SAH was induced at the skull base.
Methods: Tracheotomy was performed and femoral artery was cannulated in mice (FVB/N-Tｇ (GFAP GFP) 14Mes/j). Q-dot 655 nanocrystal (Q21021MP; Invitrogen) or rhodamine-6G was injected from the cannulated femoral vein, after a craniotomy at the parietal bone without cutting dura matter. SAH was induced at a prone position by using the endovascular perforation model4). Immediately and one hour after SAH, blood cell velocities were measured with a line scan method in precapillary and capillary using two-photon laser scanning microscopy.
Results: A penetrating arteriole branched into a precapillary arteriole at the depth of 85.9 +/- 21.0µm (n=7). Arterioles dilated immediately after SAH and then gradually constricted (n=5/7) and the blood flow disappeared immediately after SAH in the others (n=2/7). The blood cell velocity of the precapillary arteriole decreased from 10.7 +/- 3.0 mm/s before SAH to 0.9 +/- 0.4 mm/s after SAH. The capillary-velocities of blood cells (red blood cells, platelets and leukocytes) also decreased, and rolling and adherent leukocytes prevented blood cells from flowing in capillaries.
Conclusion: The cerebral blood flow decreases in arterioles and capillaries, when the SAH is induced.

c-Met interaction with Angiogenesis and Stem Cell in Helicobacter heilmannii-induced gastric MALT lymphoma: Interaction with VASH-2

Masahiko Nakamura, Hidenori Matsui, Tetsufumi Takahashi, Shinichi Takahashi, Toshifumi Hibi, K. Tsuchimoto

Released: May 21, 2014

p.35a

We established a low-grade MALT lymphoma model in C57BL/6 mouse infection of Helicobacter heilmannii obtained from cynomolgus monkey (Infect. Immun. 75 (3): 1214-1222, 2007). After long-term infection, we found the MALT lymphoma formation in the liver and lung in addition to gastric MALT lymphoma. Recently, c-MET, the tyrosine kinase receptor for hepatocyte growth factor (HGF) has attracted attention as one of the key players in survival and proliferation of B-cell malignancies.
Thus, we have planned to clarify the difference of c-MET, HGF and HGF activator (HGFA) expression as well as VEGF and its receptors, Flt-1, Flk-1 and vasohibin-2 (VASH2) in gastric, hepatic and pulmonary lesions in the MALT lymphoma by immunohistochemistry. The effect of c-MET antibodies or inhibitor, PHA-665752 (10 mg/kg b.w.) on the formation of liver and lung lesion was also investigated.
As a result, Nine months after the infection, small lymphocyte aggregates mostly composed of B cells were observed in the portal area of the liver and the peribronchial area of the lung as well as the gastric MALT lymphoma in approximately 50% of the infected mice. These lymphocytes were mostly centrocyte-like cells, and lymphoepithelial lesions characteristic of MALT lymphoma were also recognized. PCR and in situ hybridization analysis showed the existence of Helicobacter heilmannii not only in the fundic mucosa but in the lung and liver. Twelve and eighteen months after the infection, approximately 100% of infected mice had hepatic and pulmonary lesions. c-MET immunoreactivity was found in the lymphocytes composing the MALT lymphoma, and HGF immunoreactivity was recognized mostly in the endothelial cells and macrophages. HGFA was localized on mesenchymal cells other than the lymphocytes. The administration of antibodies against c-MET or a c-Met inhibitor to the infected mice induced the significant suppression of hepatic and pulmonary lesions as well as the gastric MALT lymphoma, while VASH2 immunoreactivity rather increased within the tumor.
In conclusion, HGF and c-MET pathway were suggested to contribute to the lymphomagenesis and the VASH2 has a compensatory effect in the liver and lung after Helicobacter heilmannii infection.

Visualisation of drug delivery by using high resolution microscopic mass spectrometry

Masahiro Yasunaga, Masaru Furuta, Koretsugu Ogata, Yoshikatsu Koga, Yoshiyuki Yamamoto, Misato Takigahira, Yasuhiro Matsumura

Released: May 21, 2014

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Background: Pharmacokinetic (PK) and pharmacodynamic (PD) studies are important to evaluate the efficacy and toxicity of the drugs. In these analyses, tissue homogenates are generally used for the quantification by high-performance liquid chromatography (HPLC) or liquid chromatography mass spectrometry (LC-MS). However, they lack the drug distribution in a specific anatomical area. The precise information about the distribution allows the researchers to optimize the drug design enabling more efficient targeted delivery.

Purpose: We studied the tissue distribution of paclitaxel (PTX) and its micellar formulation (NK105) using a microscopic mass spectroscopy (MMS).

Method: A MMS in which a microscope is coupled with an atmospheric pressure matrix-assisted laser desorption/ionization (MALDI) and quadruple ion trap time-of-flight (TOF) analyser was used. The matrix-coated drug sample is ionised and then separated on the basis of its mass-to-charge ratio (m/z). Images were acquired from imaging mass spectrometry (IMS) or tandem mass spectrometry (MS/MS) data.

Result: (1) We established the drug imaging system with enhanced resolution and sensitivity. In the analysis, MS and MS/MS were used for quantification and validation, respectively. (2) NK105 showed much stronger antitumor effects on a human pancreatic cancer BxPC3 xenograft than PTX. In the drug imaging, we demonstrated that NK105 delivered more PTX to the whole tumor tissue (including the center lesion). In the mouse model, PTX caused the peripheral neurotoxicity but NK105 did not. Multiple high drug-signal areas surrounding and inside the caudal nerve were observed in the case of PTX, whereas the signals after NK105 injection were significantly low.

Conclusion: We succeeded in corroborating the EPR effect using MMS. The data obtained by the drug imaging may be useful for facilitating DDS-drug design.

Salvianolic acid B binds to Src and ameliorates mesenteric venules hyperpermeability in endotoxmia rats

Chun-Shui Pan, Ying-Hua Liu, Yu-Ying Liu, Yu Zhang, Ke He, Xiao-Yuan Yang, Bai-He Hu, Xin Chang, Xiao-Hong Wei, Jing-Yu Fan, Jing-Yan Han

Released: May 21, 2014

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Background: Microvascular hyperpermeability is a crucial contributor to the gastrointestinal injury, for which the current clinical therapy remains unsatisfied. Src regulates the hyperpermeability-related proteins, such as caveolin-1, VE-cadherin and ZO-1. This study aimed to evaluate whether salvianolic acid B (SalB) binds to Src to regulate caveolin-1, VE-cadherin and ZO-1, to ameliorate mesenteric venules hyperpermeability in endotoxmia rats.

Methods: The male Wistar rats were challenged by infusion of LPS (2mg/kg/h) for 90 min, with or without SalB (5mg/kg/h). Human umbilical vein endothelial cells (HUVECs) were incubated with LPS or/and SalB. Microcirculation was assessed by intravital microscopy, caveolae in microvascular endothelial cells by electron microscopy, endothelial cell junctional proteins, caveolin-1 and Src by Western blot and confocal microscopy, and the interaction of Src and SalB by Surface Plasmon Resonance (SPR) and BioLayer Interferometry (BLI).

Results: SPR and BLI demonstrated that SalB was able to bind to Src in a dose-dependent manner, further to inhibit the phosphorylation of Src, caveolin-1 and vascular endothelial cadherin increased in human umbilical vein endothelial cells, to restore the distribution of Zonula occluden-1 and VE-cadherin degradated in cells exposed to LPS. Furthermore, SalB alleviated the inclement of caveolae in microvascular venules, and the evoked albumin leakage from venules in endotoxmia rat mesentery.

Conclusions: SalB prevents endothelial barrier dysfunction and hyperpermeability via binding to Src to inhibit the activity of Src. These findings identify SalB as a promising approach to permeability, and indicate Src as novel target for hyperpermeability treatment.

RhoJ defines angiogenic endothelial cell motility by integrating VEGF and Sema3E signals

Akiyoshi Uemura, Yoko Fukushima, Koichi Nishiyama, Yuichiro Ogura, Shin-Ichi Nishikawa

Released: May 21, 2014

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During tissue morphogenesis, cells migrate in response to diverse extrinsic cues. For angiogenic endothelial cells (ECs), vascular endothelial growth factor (VEGF) and semaphorin 3E (Sema3E) are a pivotal attractant and repellent, respectively. However, it is still unclear how individual ECs integrate these opposite signals to determine their migratory behaviors. Here, we show that the small GTPase RhoJ is an EC-intrinsic integrator of VEGF and Sema3E signals. In its GTP-bound state, RhoJ bound to the cytoplasmic domain of PlexinD1. Upon Sema3E stimulation, RhoJ was released from PlexinD1 and directly induced cell contraction. Upon VEGF stimulation, RhoJ facilitated VEGFR2-PlexinD1 association, thereby preventing VEGFR2 degradation, prolonging downstream signal transduction events, and promoting directional EC movements. Consequently, RhoJ deficiency, even in a single allele, led to variable morphogenetic defects in retinal vascular patterning. Our results indicate that RhoJ may be a novel therapeutic target to manipulate EC motility in disease or tissue regeneration.

C-peptide Effects on Glomerular Filtration

Hiroshi Nakamoto, Kazuhiko Nakayama, Noriaki Emoto, Toyotaka Yada, Yasuo Ogasawara

Released: May 21, 2014

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In our previous visualisation study, we reported that there is a leakage of proteins at the level of glomeruli on the early stage of diabetes when proteinuria was not present and that C-peptide ameliorated this leakage. C-peptide, a byproduct of insulin secretion was once considered biologically inactive. Since as early as the 1980s, studies have provided direct evidence that C-peptide is a biologically active endogenous peptide hormone. It is now known that administration of physiologically relevant doses of C-peptide reduces diabetes-induced glomerular hyperfiltration, decreases albuminuria, and reduces renal hypertrophy. We hypothesised that C-peptide might work on the structure of glomerular filtration barrier to show its effects.
We used streptozotocin-induced rats as diabetic animal models (50mg/kg). Part of both control and diabetic rats were given C-peptide continuously one hour prior to sacrifice (50pmol/kg/min). Extracted kidney samples were examined by fluorescence antibody technique and electron microscopy. Of glomerular slit membrane components, podocin, nephrin and CD2AP were stained and glomerular section images were binalised. The distribution of slit membrane proteins were analysed by area ratio (ratio for coexisting area of two component proteins). Inter-footprocess spaces were measured as podocytic structural change.
There was a loose negative correlation between the diabetic duration and the area ratio of either two out of three protein components (r=-0.24 to -0.59). There was not a significant change in area ratio after C-peptide administration in both control and diabetic rats. The inter-footprocess spaces were significantly different (p<0.01), 8.17±0.44nm, 9.72±0.44nm for control and diabetic rats respectively. C-peptide administration widened the spaces to 10.39±0.65nm (p<0.01) and to 15.45±0.96nm (p<0.01) for control and diabetic rats respectively.
C-peptide did not change the structure of glomerular slit membranes but widened the inter-footprocess spaces. C-peptide effects on ameliorating glomerular leakage of protein was considered to be due to other effects than causing structural changes to glomerular filtration barrier.

Measurement of blood flow velocity profiles in retinal arterioles and venules using spectral-domain doppler optical coherence tomography in healthy subjects

Taiji Nagaoka, Tomofumi Tani, Akihiro Ishibazawa, Kenji Sogawa, Seigo Nakabayashi, Tsuneaki Omae, Akitoshi Yoshida

Released: May 21, 2014

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Purpose: To evaluate the reproducibility of retinal blood flow (RBF) velocity profiles using Doppler optical coherence tomography (OCT) in healthy young volunteers.

Methods: RBF velocity profiles were measured over one cardiac cycle using Doppler OCT in six healthy volunteers. The measured vessels were chosen at the straight portion 1 to 2 disc diameters from the optic disc. Two-dimensional velocity profiles in the venules and arterioles were extracted, and the maximal (V_max) and average (Vave) velocities were calculated automatically. The ratio of V_max and V_ave was set to characterize the flow conditions in the vessels.

Results: The ratios of V_max to V_ave were about 1.6 to 1.8 for the arterioles and venules in most cases. The ratio in the arterioles was unchanged during the cardiac cycle. In addition, this ratio has constant for one cardiac cycle at the straight portion.

Conclusions: The current study showed that Doppler OCT enables accurate and reproducible measurements of the blood velocity profiles in the entire lumen of the retinal arterioles and venules in healthy young volunteers.

Clinical characteristics of peripheral type of diabetic retinopathy diagnosed with ultra-wide field fluorescein angiography

Shuichiro Hirahara, Taneto Tomiyasu, Miho Nozaki, Munenori Yoshida, Yuichiro Ogura

Released: May 21, 2014

p.38a

Purpose: Fluorescein angiography with Optos® 200Tx is useful to visualize the microcirculations of peripheral retina. The purpose of this study is to evaluate the feature of the patients with peripheral type of diabetic retinopathy (DR) diagnosed by using Ultra-wide field FA (UWFA) with Optos® 200Tx.

Methods: The UWFA was performed on 154 eyes of 77 patients with DR (62 male, 15 female, average age : 60.2 years). Fifteen eyes (9.7%) were found to have the capillary non-perfusion area only in the far-periphery without any evidences of non-perfusion in the posterior pole and the mid-periphery. Eight eyes of 6 patients were non-treated cases with peripheral type of DR (4 male, 12 female, average age : 66.3 years). We evaluated the FA findings, HbA1c and disease duration of these patients with peripheral type of DR.

Results: All of the non-treated patients with peripheral type of DR have been having diabetes for more than 20 years (average of 26.2 years). The control of blood sugar was poor (HbA1c 6.7˜10.1%, average 8.1%). One of these cases, whose blood sugar was good (HbA1c 6.7%), had diabetes for as long as 40 years. It was reported that most cases of peripheral type of DR progress slowly. However, we experienced a case that represented vitreous hemorrhage caused by retinal neovascularization existed in only periphery.

Conclusions: Peripheral type of DR was considered to progress slowly. Most of them were found in elder patients. It is not rare to see the cases in which peripheral capillary occlusion exist even though posterior pole is intact, especially in the patients with long history of diabetes.

H₂O₂-induced Vasodilatation Compensates Diabetes-induced Microvascular Endothelial Dysfunction during Acute Coronary Occlusion in Canine Coronary Native Collateral Microvessels in Vivo

Toyotaka Yada, Hiroaki Shimokawa, Osamu Hiramatsu, Hiroshi Nakamoto, Masami Goto, Yasuo Ogasawara, Fumihiko Kajiya

Released: May 21, 2014

p.39

Background: It has been previously demonstrated that endothelial Cu, Zn-SOD and caveolin-1 play crucial roles of hydrogen peroxide (H₂O₂) production as an endothelium-derived hyperpolarizing factor (EDHF) in mouse mesenteric arteries. We thus examined whether this mechanism is involved in the EDHF-mediated responses in diabetes mellitus (DM) during acute coronary occlusion.

Methods: Canine subepicardial coronary collateral small arteries (CSA>100µm) and arterioles (CA <100µm) were visually traced by an intravital microscope between left anterior descending artery (LAD) and left circumflex coronary artery (LCX) with an injection of indocyanine green. We examined bradykinin-induced vasodilatation of native coronary collaterals before and after myocardial ischemia by proximal LAD occlusion (90min) under the following conditions (n=6 each); (i) control and (ii) DM (alloxan, 1 week prior to study, IV) with cyclooxygenase blockade (ibuprofen, 12.5 mg/kg, IV) before the onset of the ischemia. Myocardial levels of Cu,Zn-SOD, caveolin-1 and H₂O₂ (Amplex Red) and plasma levels of 8-OHdG, as a marker of oxidative stress and tetrahydrobiopterin in the coronary sinus were measured by ELISA and high-performance liquid chromatography.

Results: Although the levels of Cu, Zn-SOD, tetrahydrobiopterin and caveolin-1 in the LAD area were comparable between the control and DM groups, caveolin-1 levels were greater in coronary microvessels than in coronary conduit arteries in the control group. Nitric oxide (NO)-mediated coronary vasodilatation of CSA by bradykinin significantly decreased in DM compared with control, and was restored by compensation of EDHF/H₂O₂ in CA with H₂O₂ production for the loss of NO. Oxidative stress (8-OHdG) was significantly increased in DM compared with control.

Conclusions: NO-mediated, endothelium-dependent vasodilatations of CSA during acute coronary occlusion are impaired in DM and are compensated by EDHF/H₂O₂ in dogs in vivo.

Nailfold microcapillary findings reveal early stage of congestion of right ventricle of the heart.

Ichiro Miura, Masato Matsuo, Tsuyoshi Konta, Katsuya Nagayama, Masami Miyazaki

Released: May 21, 2014

p.39a

Back groud and aims: Nailfold capillaroscopy is usefull for determine the state of peripheral microvessels in various disease, especially rheumatic disease. While capillary loss is well known in essential hypertension as findings of the cardiovascular disease, clinical significance of horizontal capillaries is not well known. We have examined the relationship between serum concentration of brain natriuretic peptide (BNP) and horizontal vessels in nailfold capillary microscopy.

Method: The study examined 78 patients 60 years of age or older. Serum BNP concentration was measured for all cases, and we examined the statistical significant difference of the cases with and without findings of transverse blood vessels by the nailfold capillary microscopy.

Result: Serum BNP concentration increased in many cases with a horizontal vessels and significant difference was observed statistically. BNP concentration was enough to slightly more than normal values.

Discussion: Using the nailfold capillary microscopy, deformation of the vessel in the vertical direction is most notable. On the other hand, the meaning of the transverse blood vessels has received less attention. Evaluated the observation of blood flow of many finger tips, it was considered that the blood flow in the horizontal direction is venous blood flow. We estimated that horizontal blood flow means the decrease of venous return, namely expresses the early state of congestion of the right ventricle of the heart. Since the BNP rise and this finding had a significant difference statistically, we considered nailfold microcapillary findings can reveal early stage of congestion of right ventricle of the heart.

Ma-Xing-Shi-Gan-Tang, a traditional Chinese medicine, attenuates lipopolysaccharide-induced pulmonary microcirculatory disturbance and lung edema in rats

Li-Qian Ma, Kai Sun, Chun-Shui Pan, Yu-Ying Liu, Li Yan, Jing-Yu Fan, Jing-Yan Han

Released: May 21, 2014

p.40

Background: Ma-Xing-Shi-Gan-Tang (MXSGT) is a compound traditional Chinese medicine which has been widely used in clinic for treatment of acute upper respiratory tract infection in China. However, whether MXSGT could improve LPS-induced pulmonary microcirculatory disturbance and lung edema is still not clear.

Objective: The aim of this study is to investigate the ameliorating effect and potential mechanism of MXSGT on the LPS-induced pulmonary microcirculatory disturbance and lung edema.

Method: Male Sprague-Dawley rats (weight in 200±20g) were injected with LPS (7mg/kg) intraperitoneally to induce lung microcirculatory disturbance and lung edema. MXSGT (2.6075g/kg) was given by gavage 6 hours after LPS exposure. Examinations were undertaken at 6 and 12 hours after LPS exposure, respectively.

Result: The number of leukocytes adhering to pulmonary venules and the expression of ICAM-1 in lung tissue were increased at 6 hours and 12 hours after LPS exposure. In addition, LPS increased pulmonary tissue W/D ratio and decreased the expression of junctional adhesion molecule-1 and claudin-5 in lung tissue. Post-treatment with MXSGT significantly suppressed these changes induced by LPS exposure.

Conclusion: This study demonstrated that MXSGT ameliorated rat pulmonary microcirculation disturbance and lung edema induced by LPS, the underlying mechanism may involve protection of leukocytes from adhering to pulmonary venule and up -regulation of junctional adhesion molecule-1 and claudin-5 in lung tissue.

The protective effects of rapamycin on intestinal ischemia/reperfusion induced remote lung injury in mice

Takaya Iida, Yuji Naito, Tomohisa Takagi, Kazuhiro Katada, Katsura Mizushima, Kazuhiro Kamada, Kazuhiko Uchiyama, Osamu Handa, Nobuaki Yagi, Yoshito Ito, Toshikazu Yoshikawa

Released: May 21, 2014

p.40a

Introduction: The intestinal ischemia/reperfusion (I/R) injury occurs in various clinical settings, such as mesenteric artery occlusion, trauma, and intestinal transplantation. Intestinal I/R injury elicits inflammatory responses within the intestine, including mucosal epithelial cell damage, loss of barrier function, proinflammatory cytokine production, and increased microvascular permeability. These changes develop multiple organ failure, and become fatal. However, the cure is not fully established. The mammalian target of rapamycin (mTOR) plays an important role in cellular proliferation and survival. It has been shown to be focused on a therapeutic target for various diseases such as cancer and inflammation. Herein, this study was designed to determine whether the mTOR inhibitor, rapamycin, had protective effects on intestinal I/R injury in mice.

Methods: The small intestine of C57BL/6 mice was challenged with ischemia by occluding superior mesenteric artery for 1h. Rapamycin was administered intraperitoneally 1h before the induction of ischemia. In mice following intestinal I/R, the survival rate, inflammatory responses and adhesion molecules in the small intestine and lung were assessed. Bacterial cultures were performed using the homogenate samples from lungs. Phagocytic capacity in the alveolar macrophages and the activation of NF-κB in the lung were also assessed.

Results: Treatment with rapamycin significantly improved survival rate after intestinal I/R. Inflammatory markers (TNF-alpha and MPO activity) in the intestinal tissue of rapamycin-treated mice were unchanged but these assays in the lung were attenuated in rapamycin-treated mice along with decreased bacterial culture and increased phagocytic capacity. The activation of NF-κB and downstream expression of adhesion molecules in the lung of rapamycin-treated mice were downregulated.

Conclusion: Treatment with rapamycin improved survival rate via attenuation of intestinal I/R-induced remote lung injury.

Volume And Issue List
Microvascular Reviews and Communications

Microvascular Reviews and Communications Vol. 7(2014) No. 1

The 39^th Annual Meeting of Japanese Society for Microcirculation

Clinical application of huma iPS cells for cardiovascular Medicine

Cancer Stem Cell of Digestive Organs

Balancing angiogenic pathways in solid tumors

Role of ribosomal protein S19 oligomer-C5a receptor system in acute inflammation resolution

Sibasaburou Kitasato and the Kitasato Institute

Nicotine ameliorates colonic inflammation via down-regulation of MAdCAM-1 expression on high endotherial venule like vessel.

VEGFR1 signaling facilitates diabetic skin wound healing in mice

Post-stroke administration of cilostazol changes metabolic profile in transsulfuration pathway of ischemic brain in a mouse model

Role of leukotriene B4 receptor 1 (BLT1) signaling in liver repair after hepatic ischemia reperfusion injury

3, 4-dihydroxyl-phenyl lactic acid restores NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10 expression to ameliorate cardiac reperfusion injury

Comparison of peripheral vascular resistance based on macro- and micro-circulatory responses by Poilleuille’s law

Roles of Prostanoids in Regulation of Angiogenesis and Lymphatic Tissue Remodeling

New Trends in therapeutic strategies against ischemia/reperfusion injury; Postconditioning and pharmacological intervention in acute myocardial infarction

Perfusion fixation method is critical for immunoelectron microscopy and ultrastructural evaluation on changes of caveolin-1 and caveolae relates with capillarization of liver sinusoidal endothelial cells in human cirrhotic liver

Brain-derived neurotrophic factor promotes angiogenesis via oxidative stress in human vascular endothelial cells: Implication for atherogenesis?

Cilostazol inhibits leukocyte-endothelial cell interactions in murine microvessels after transient bilateral common carotid artery occlusion

Impairment of CO₂ reactivity in RBC velocity and CBF after cortical spreading depression in anesthetized mice

EXPLORE THE ANGIOGENESIS OF AUTOLOGOUS TRANSPLANTED BRAIN TISSUES IN RABBITS

HO-2/CO system protects against metabolic disorders following acute cerebral ischemia

The blood cell flow and the vascular responses in arterioles and capillaries after subarachnoid hemorrhage

c-Met interaction with Angiogenesis and Stem Cell in Helicobacter heilmannii-induced gastric MALT lymphoma: Interaction with VASH-2

Visualisation of drug delivery by using high resolution microscopic mass spectrometry

Salvianolic acid B binds to Src and ameliorates mesenteric venules hyperpermeability in endotoxmia rats

RhoJ defines angiogenic endothelial cell motility by integrating VEGF and Sema3E signals

C-peptide Effects on Glomerular Filtration

Measurement of blood flow velocity profiles in retinal arterioles and venules using spectral-domain doppler optical coherence tomography in healthy subjects

Clinical characteristics of peripheral type of diabetic retinopathy diagnosed with ultra-wide field fluorescein angiography

H₂O₂-induced Vasodilatation Compensates Diabetes-induced Microvascular Endothelial Dysfunction during Acute Coronary Occlusion in Canine Coronary Native Collateral Microvessels in Vivo

Nailfold microcapillary findings reveal early stage of congestion of right ventricle of the heart.

Ma-Xing-Shi-Gan-Tang, a traditional Chinese medicine, attenuates lipopolysaccharide-induced pulmonary microcirculatory disturbance and lung edema in rats

The protective effects of rapamycin on intestinal ischemia/reperfusion induced remote lung injury in mice

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Microvascular Reviews and Communications Vol. 7(2014) No. 1

System Maintenance

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Microvascular Reviews and Communications