Abstract P374: Functional Impairment Of Cardiac Endothelial Cells In Diabetic Failing Heart Via Dysregulated Exosomal Mirnas

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Prabhat Ranjan ◽  
Sumanta K Goswami ◽  
Prasanna Krishnamurthy ◽  
Gangjian Qin ◽  
Suresh Verma
Keyword(s):  
Endothelial Cells ◽  
Functional Impairment ◽  
Cardiac Fibrosis ◽  
Tube Formation ◽  
Diabetic Heart ◽  
Boyden Chamber ◽  
Growth Media ◽  
Ang Ii ◽  
Diabetic Mice ◽  
Failing Heart

Introduction: Cardiac endothelial cells (ECs) and fibroblasts (FBs) together maintain cardiac homeostasis. Their functional impairment aggravates complications in the heart. In diabetes, acute inflammation leads to cardiac FBs activation, which predisposes the diabetic myocardium to severe fibrosis. Further, inflammation-related vascular dysfunction is a major end-organ complication in diabetics. However, it is not known, whether myofibroblast regulates ECs function in a diseased diabetic heart. Therefore, we hypothesized that “myofibroblast in diabetic heart secretes exosomes packed with antiangiogenic/profibrotic factors, which impede EC function and exaggerate pathological remodelling in pressure-overloaded (PO) myocardium. Methods: Exosomes were isolated from diabetic mice plasma and FB condition media by ultracentrifugation and characterized by nanosight & electron microscopy. We cultured mouse primary heart ECs in growth media and treated with exosomes derived from FBs (treated with 25mM glucose or 500nM Angiotensin II (AngII) or both) for 48 hr. Mannitol (25mM) served as control. Results: Ang II and glucose significantly activate FBs as shown by qPCR (fibronectin, collagenase1α1) and western blot (pSmad2, p-p38). Exosomes derived from diabetic Ang II treated FBs significantly impaired ECs function as shown by Matrigel tube formation and Boyden chamber migration assays. Interestingly, ECs markers (eNOS, VEGF, CD31) genes and proteins expression were significantly inhibited in ECs treated with exosomes-derived from glucose and Ang II treated FBs. We, further, checked the effect of diabetic mice plasma exosomes on ECs function and found significantly impaired as shown by tube formation and migration data. Finally, microRNA (miR) array and qPCR analysis revealed that miR-216a-5p, miR-26a-5p and miR7a-5p were highly upregulated in exosomes derived from FBs co-treated with glucose and AngII. Conclusions: Taken together, this study demonstrates that glucose and AngII co-treated FBs-derived exosomes are enriched in pro-fibrotic factors and can lead to EC dysfunction and promotes cardiac fibrosis in PO myocardium. In future studies, we will modulate the target miRs in diabetic FBs to see whether it rescue reparative function of ECs and inhibits fibrosis in failing heart.

Abstract 34: Human Cardiac Mesenchymal Cell Derived Extracellular Vesicles Promote Angiogenesis Through Angiopoietin-TIE2 Signaling

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Asif Pathan ◽  
Talha Farid ◽  
Abdur Rahman Khan ◽  
Marjan Nasr ◽  
Marcin Wysoczynski ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Embryonic Stem ◽  
Tube Formation ◽  
Right Atrial ◽  
Boyden Chamber ◽  
Human Umbilical Cord ◽  
Angiogenic Activity ◽  
Cell Based Therapy

Cell-based therapy is considered a promising approach to treat the damaged heart due to myocardial infarction. Although the mechanisms for their beneficial action are not yet clear, exosome/extracellular vesicles (EVs) secreted by these cells may be involved in their reparative paracrine signaling. Previous studies have suggested that EVs isolated from several cell types (e.g. cardiosphere-derived cells, embryonic stem cell, CD34+ stem cells) induce angiogenic activity both in vitro and in vivo . Here, we investigated whether EVs secreted by adult human cardiac mesenchymal cells (hCMCs) exhibit pro-angiogenic activity, and if so, what signaling molecules are involved in this process. hCMCs were isolated from right atrial appendage of patients undergoing cardiac procedures and were characterized by the expression of classical mesenchymal markers- CD29 (99.1%), CD73 (99.0%), CD90 (20.4%), CD105 (99.3%), CD 31 (16.8%), CD34 (0.9%) and CD45 (0.1%). EVs isolated from serum-free 24-hour hCMC conditioned media using PEG4000-based precipitation technique exhibited two distinct population of particles with size range of 10-60nm and 100-500nm in diameter; expressed characteristic exosomal markers- CD63, HSP70, Flotillin-1 and were negative for cellular organelle markers- calreticulin (ER and apoptotic bodies), prohibitin (mitochondria), GM130 (Golgi), Lamin B (nuclear protein), β-actin (cytoskeleton) and PMP70 (peroxisomes) as determined by immunoblotting. In vitro assays revealed that hCMC EVs promote human umbilical cord endothelial cells (HUVECs) proliferation, transwell migration in Boyden chamber and tube formation on Matrigel, indicative of enhanced angiogenesis. Angiogenic proteomic array identified that angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2) proteins are highly enriched in EVs secreted by hCMCs. Furthermore, hCMC EV mediated HUVEC migration and tube formation was inhibited by TIE2 kinase inhibitor. Overall, these findings suggest that ANG-1 and ANG-2 are the key component of hCMC secreted EVs and they promote angiogenesis by activating TIE2 receptor in endothelial cells.

scholarly journals MicroRNA-133a impairs perfusion recovery after hindlimb ischemia in diabetic mice

2018 ◽  
Vol 38 (4) ◽  
Author(s):  
Lingdan Chen ◽  
Chunli Liu ◽  
Dejun Sun ◽  
Tao Wang ◽  
Li Zhao ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Endothelial Cells ◽  
Arterial Disease ◽  
Tube Formation ◽  
Cgmp Level ◽  
Diabetic Mice ◽  
Rt Pcr ◽  
Patients With Diabetes ◽  
Peripheral Arterial ◽  
Ischemic Muscle

Objective: Peripheral arterial disease (PAD) patients with diabetes mellitus suffer from impaired neovascularization after ischemia which results in poorer outcomes. MicroRNA (miR)-133a is excessively expressed in endothelial cells under diabetic conditions. Here, we test whether diabetes-induced miR-133a up-regulation is involved in the impaired capability of neovascularization in experimental PAD models. Methods and results: MiR-133a level was measured by quantitative RT-PCR and showed a higher expression level in the ischemic muscle from diabetic mice when compared with nondiabetic mice. Knockdown of miR-133a using antagomir improved perfusion recovery and angiogenesis in experimental PAD model with diabetes day 21 after HLI. On the other hand, overexpression of miR-133a impaired perfusion recovery. Ischemic muscle was harvested day 7 after experimental PAD for biochemical test, miR-133a antagonism resulted in reduced malondialdehyde, and it increased GTP cyclohydrolase 1 (GCH1), and cyclic guanine monophosphate (cGMP) levels. In cultured endothelial cells, miR-133a antagonism resulted in reduced reactive oxygen species level, and it increased tube formation, nitric oxide (NO), and cGMP level. Moreover, miR-133a antagonism-induced angiogenesis was abolished by GCH1 inhibitor. In contrary, miR-133a overexpression impairs angiogenesis and it reduces GCH1, NO, and cGMP levels in nondiabetic models. Conclusion: Diabetes mellitus-induced miR-133a up-regulation impairs angiogenesis in PAD by reducing NO synthesis in endothelial cells. MiR-133a antagonism improves postischemic angiogenesis.

scholarly journals Atherogenic Cytokines Regulate VEGF-A-Induced Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells into Endothelial Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Izuagie Attairu Ikhapoh ◽  
Christopher J. Pelham ◽  
Devendra K. Agrawal
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Mesenchymal Stem Cells ◽  
Tube Formation ◽  
Ang Ii ◽  
Induced Differentiation ◽  
Endothelial Tube Formation ◽  
Marker Expression

Coronary artery stenting or angioplasty procedures frequently result in long-term endothelial dysfunction or loss and complications including arterial thrombosis and myocardial infarction. Stem cell-based therapies have been proposed to support endothelial regeneration. Mesenchymal stem cells (MSCs) differentiate into endothelial cells (ECs) in the presence of VEGF-Ain vitro. Application of VEGF-A and MSC-derived ECs at the interventional site is a complex clinical challenge. In this study, we examined the effect of atherogenic cytokines (IL-6, TNFα, and Ang II) on EC differentiation and function. MSCs (CD44+, CD73+, CD90+, CD14−, and CD45−) were isolated from the bone marrow of Yucatan microswine. Naïve MSCs cultured in differentiation media containing VEGF-A (50 ng/mL) demonstrated increased expression of EC-specific markers (vWF, PECAM-1, and VE-cadherin), VEGFR-2 and Sox18, and enhanced endothelial tube formation. IL-6 or TNFαcaused a dose-dependent attenuation of EC marker expression in VEGF-A-stimulated MSCs. In contrast, Ang II enhanced EC marker expression in VEGF-A-stimulated MSCs. Addition of Ang II to VEGF-A and IL-6 or TNFαwas sufficient to rescue the EC phenotype. Thus, Ang II promotes but IL-6 and TNFαinhibit VEGF-A-induced differentiation of MSCs into ECs. These findings have important clinical implications for therapies intended to increase cardiac vascularity and reendothelialize coronary arteries following intervention.

scholarly journals Angiotensin II-induced process of angiogenesis is mediated by spleen tyrosine kinase via VEGF receptor-1 phosphorylation

2011 ◽  
Vol 301 (3) ◽  
pp. H1043-H1055 ◽  
Author(s):  
Cuneyt K. Buharalioglu ◽  
Chi Young Song ◽  
Fariborz A. Yaghini ◽  
Hafiz U. B. Ghafoor ◽  
Mustafa Motiwala ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinase ◽  
Egf Receptor ◽  
Umbilical Vein ◽  
Underlying Mechanism ◽  
Tube Formation ◽  
Vegf Receptor ◽  
Ang Ii ◽  
Spleen Tyrosine Kinase ◽  
Vegf Expression

Spleen tyrosine kinase (Syk), expressed in endothelial cells, has been implicated in migration and proliferation and in vasculogenesis. This study was conducted to determine the contribution of Syk and the underlying mechanism to the angiogenic effect of ANG II and VEGF. Angiogenesis was determined by tube formation from the endothelial cell line EA.hy926 (EA) and human umbilical vein endothelial cells (HUVECs) and microvessel sprouting in rat aortic rings. ANG II (10 nM), EGF (30 ng/ml), and VEGF (50 ng/ml) stimulated EA cells and HUVECs to form tubular networks and increased aortic sprouting; these effects were blocked by VEGF receptor-1 and Flt-1 antibody (Flt-1/Fc) but not by the VEGF receptor-2 (Flk-1) antagonist SU-1498. ANG II increased the phosphorylation of Flt-1 but not Flk-1, whereas VEGF increased the phosphorylation of both receptors in EA cells and HUVECs. VEGF expression elicited by ANG II was not altered by Flt-1/Fc or SU-1498. EGF stimulated tube formation from EA cells and HUVECs and Flt-1 phosphorylation and aortic sprouting, which were blocked by the EGF receptor antagonist AG-1478 and Flt-1/Fc but not by SU-1498. ANG II-, EGF-, and VEGF-induced tube formation and aortic sprouting were attenuated by the Syk inhibitor piceatannol and by Syk short hairpin interfering (sh)RNA and small interfering RNA, respectively. ANG II, EGF, and VEGF increased Syk phosphorylation, which was inhibited by piceatannol and Syk shRNA in EA cells and HUVECs. Neither piceatannol nor Syk shRNA altered ANG II-, EGF-, or VEGF-induced phosphorylation of Flt-1. These data suggest that ANG II stimulates angiogenesis via transactivation of the EGF receptor, which promotes the phosphorylation of Flt-1 and activation of Syk independent of VEGF expression.

Abstract 1301: NFATc- And SRF-induced Early Growth Response (Egr)-3 Is Essential For VEGF-mediated Endothelial Cell Migration And Tube Formation

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Jun-ichi Suehiro ◽  
Mai Miura ◽  
Tatsuhiko Kodama ◽  
Takashi Minami
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Growth Response ◽  
Cell Activation ◽  
Early Growth ◽  
Tube Formation ◽  
Endothelial Cell Migration ◽  
Boyden Chamber ◽  
Early Growth Response

Endothelium is a dynamic cell layer constantly responding to changes in the various extracellular mediators. Responses are usually beneficial to host, but oversustained or dysregulated responses can result in vascular dysfunction, leading to the initiation of atherosclerosis, tumor growth, and inflammation. Such endothelial cell activation and dysfunction are mediated in large part by alterations in gene expression. Here we show, using comprehensive transcriptome analyses, that VEGF, thrombin and TNF-α each induces a dramatic and rapid up-regulation of early growth response (Egr)-3 in human umbilical vein endothelial cells (HUVEC). The effect of VEGF on Egr-3 was similar in human coronary artery, pulmonary artery, and dermal microvascular endothelial cells. In chemical inhibitor studies, VEGF-mediated induction of Egr-3 (mRNA peak 300-fold at 45 min) depended on MEK1/2, JNK, PI3K, PKA, and Ca-calcineurin. Egr-3 promoter luciferase and electrophoretic mobility shift analysis revealed that the 5′-flanking region at −57 to −130 was necessary and sufficient for transducing of VEGF-mediated Egr-3 upregulation and that region could bind NFATc1, c2, and SRF. Co-transfection assays with Egr-3 reporter and either NFATc or SRF expression plasmids resulted in 4 and 2 -fold Egr-3 promoter activation, respectively. In DNA microarray studies, HUVEC treated with VEGF for 1 and 4 hours in the presence of two independent siRNAs against Egr-3, 25 and 70 VEGF-inducible genes were strikingly downregulated, respectively. The pro-angiogenesis factors Ets-1, CXCL1, and tissue factor were among those genes downregu-lated. SiRNA knockdown of Egr-3 markedly impaired VEGF-mediated cell migration and tube formation, as determined by in vitro wound healing, boyden chamber, and collagen gel assays. Moreover in aortic ring assays, VEGF-stimulated neo-angiogenesis from the extracted mice aorta was completely abolished by administration of adenoviral-transferred miRNA against Egr-3. Collectively, these findings suggest that NFATc and SRF cooperatively upregulate Egr-3. Egr-3 might have an important function as a signal transducer in VEGF-mediated angiogenesis in activated endothelium.

scholarly journals Exosomes derived from adipose-derived stem cells overexpressing glyoxalase-1 protect endothelial cells and enhance angiogenesis in type 2 diabetic mice with limb ischemia

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xing Zhang ◽  
Yihong Jiang ◽  
Qun Huang ◽  
Zhaoyu Wu ◽  
Hongji Pu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Limb Ischemia ◽  
Tube Formation ◽  
Adipose Derived Stem Cells ◽  
Diabetic Mice ◽  
Type 2 Diabetic

Abstract Background Diabetic limb ischemia is a clinical syndrome and refractory to therapy. Our previous study demonstrated that adipose-derived stem cells (ADSCs) overexpressing glyoxalase-1 (GLO-1) promoted the regeneration of ischemic lower limbs in diabetic mice, but low survival rate, difficulty in differentiation, and tumorigenicity of the transplanted cells restricted its application. Recent studies have found that exosomes secreted by the ADSCs have the advantages of containing parental beneficial factors and exhibiting non-immunogenic, non-tumorigenic, and strong stable characteristics. Methods ADSCs overexpressing GLO-1 (G-ADSCs) were established using lentivirus transfection, and exosomes secreted from ADSCs (G-ADSC-Exos) were isolated and characterized to coculture with human umbilical vein endothelial cells (HUVECs). Proliferation, apoptosis, migration, and tube formation of the HUVECs were detected under high-glucose conditions. The G-ADSC-Exos were injected into ischemic hindlimb muscles of type 2 diabetes mellitus (T2DM) mice, and the laser Doppler perfusion index, Masson’s staining, immunofluorescence, and immunohistochemistry assays were adopted to assess the treatment efficiency. Moreover, the underlying regulatory mechanisms of the G-ADSC-Exos on the proliferation, migration, angiogenesis, and apoptosis of the HUVECs were explored. Results The G-ADSC-Exos enhanced the proliferation, migration, tube formation, and anti-apoptosis of the HUVECs in vitro under high-glucose conditions. After in vivo transplantation, the G-ADSC-Exo group showed significantly higher laser Doppler perfusion index, better muscle structural integrity, and higher microvessel’s density than the ADSC-Exo and control groups by Masson’s staining and immunofluorescence assays. The underlying mechanisms by which the G-ADSC-Exos protected endothelial cells both in vitro and in vivo might be via the activation of eNOS/AKT/ERK/P-38 signaling pathways, inhibition of AP-1/ROS/NLRP3/ASC/Caspase-1/IL-1β, as well as the increased secretion of VEGF, IGF-1, and FGF. Conclusion Exosomes derived from adipose-derived stem cells overexpressing GLO-1 protected the endothelial cells and promoted the angiogenesis in type 2 diabetic mice with limb ischemia, which will be a promising clinical treatment in diabetic lower limb ischemia.

scholarly journals NPC-EXs Alleviate Endothelial Oxidative Stress and Dysfunction through the miR-210 Downstream Nox2 and VEGFR2 Pathways

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Hua Liu ◽  
Jinju Wang ◽  
Yusen Chen ◽  
Yanfang Chen ◽  
Xiaotang Ma ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Endothelial Cells ◽  
Angiotensin Ii ◽  
Neural Progenitor Cells ◽  
Tube Formation ◽  
Oxygen Species ◽  
Protective Effects ◽  
Ang Ii

We have demonstrated that neural progenitor cells (NPCs) protect endothelial cells (ECs) from oxidative stress. Since exosomes (EXs) can convey the benefit of parent cells through their carried microRNAs (miRs) and miR-210 is ubiquitously expressed with versatile functions, we investigated the role of miR-210 in the effects of NPC-EXs on oxidative stress and dysfunction in ECs. NPCs were transfected with control and miR-210 scramble/inhibitor/mimic to generate NPC-EXscon, NPC-EXssc, NPC-EXsanti-miR-210, and NPC-EXsmiR-210. The effects of various NPC-EXs on angiotensin II- (Ang II-) induced reactive oxygen species (ROS) overproduction, apoptosis, and dysfunction, as well as dysregulation of Nox2, ephrin A3, VEGF, and p-VEGFR2/VEGFR2 in ECs were evaluated. Results showed (1) Ang II-induced ROS overproduction, increase in apoptosis, and decrease in tube formation ability, accompanied with Nox2 upregulation and reduction of p-VEGFR2/VEGFR2 in ECs. (2) Compared to NPC-EXscon or NPC-EXssc, NPC-EXsanti-miR-210 were less whereas NPC-EXsmiR-210 were more effective on attenuating these detrimental effects induced by Ang II in ECs. (3) These effects of NPC-EXsanti-miR-210 and NPC-EXsmiR-210 were associated with the changes of miR-210, ephrin A3, VEGF, and p-VEGFR2/VEGFR2 ratio in ECs. Altogether, the protective effects of NPC-EXs on Ang II-induced endothelial injury through miR-210 which controls Nox2/ROS and VEGF/VEGFR2 signals were studied.

Abstract 339: Ang-(1-7) Counteracts Ang II in Regulating Cerebrovascular Endothelial Cell Function and Gene Expression

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Ji Chen ◽  
Xiang Xiao ◽  
Shuzhen Chen ◽  
Cheng Zhang ◽  
Jinju Wang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Function ◽  
Tube Formation ◽  
Cell System ◽  
Cell Culture Supernatant ◽  
Complete Medium ◽  
Ang Ii ◽  
Gene Expressions ◽  
Endothelial Cell Function ◽  
Tnf Α

Cerebrovascular endothelial cells (cECs) play an important role in maintaining the health of cerebral vasculatures. In this study, we investigated the role of angiotensin (Ang)-(1-7) in counteracting Ang II-induced effects on cECs. The 3rd-5th passages of brain microvascular endothelial cells (BMECs, Cell system) cultured in CSC complete medium (Cell system) were used for this study. BMECs were treated with Ang II (100 nM), Ang-(1-7) (100 nM) and A-779 (100 nM, Mas receptor antagonist). After treatment for 24 hrs, cell apoptosis was determined by flow cytometery. BMEC tube formation ability was determined using a commercial kit. The levels of tumor necrosis factor- α (TNF-α), monocyte chemoattractant protein 1 (MCP-1) and interleukin (IL-8) in the cell culture supernatant were determined by ELISA. Gene expressions of NFκB, inhibitor of kappa B (IκB) were determined by western blot. To verify the involvement of NFκB pathway, the inhibitor (BAY11-7082, 10 uM) was applied. We found (Figure): 1) Treatment with Ang-(1-7) decreased Ang II-induced apoptosis (22 ± 1.5% vs. 14 ± 1.2%, Ang II vs. Ang II + Ang-(1-7), P<0.01). 2) Ang-(1-7) improved BMEC tube formation ability compromised by Ang II, which was accompanied by up-regulation of IκB expression, the down-regulation of NFκB, and decrease of TNF-α, MCP-1 and IL-8 production of BMECs. 3) These effects of Ang-(1-7) were totally abolished by A-779 and partially blocked by BAY 11-7082. In conclusion, Ang-(1-7)/Mas activation counteracts Ang II-induced BMEC apoptosis and dysfunction, partially via the NFκB dependent inflammation pathway.

Abstract 11989: Cardiac 12-Lipoxygenase-Induced Oxidative Stress Promotes the Development of Diabetic Cardiomyopathy

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Yosuke Kayama ◽  
Hirofumi Suzuki ◽  
Masaya Sakamoto ◽  
Ippei Shimizu ◽  
Tomohisa Nagoshi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Diabetic Cardiomyopathy ◽  
Mitochondrial Membrane ◽  
Cardiac Fibrosis ◽  
Diabetic Heart ◽  
Diabetic Mice ◽  
12 Lipoxygenase ◽  
Glucose Condition

Diabetes mellitus affects cardiac structure and function, and it has been suggested that diabetes leads to cardiomyopathy. Arachidonic acid (AA) metabolism was thought to be a potential mediator of cardiac fibrosis and heart failure associated with oxidative stress. 12-lipoxygenase (12-LOX) is a key lipid peroxidizing enzyme of the AA cascade that plays an important role in the development of atherogenesis and neurodegenerative disease. However, the role of 12-LOX in diabetic cardiomyopathy has not been examined. To determine whether 12-LOX is a key molecule in the development of diabetic cardiomyopathy, we created streptozotocin (STZ)-induced diabetic mice (WT-STZ) and compared to control mice. Cardiac expression of 12-LOX pathway was up-regulated after induction of diabetes. Histological analysis revealed that expression of 12-LOX was specifically up-regulated in cardiomyocytes but not vascular cells and fibroblast cells. Cardiac fibrosis was increased after induction of diabetes. We next created STZ-induced diabetic mice using 12-LOX KO mice (KO-STZ) and compared them to WT-STZ. Cardiac dysfunction and fibrosis in WT-STZ were significantly inhibited in KO-STZ. We next examined the relationship between 12-LOX and cardiac oxidative stress in the diabetic heart. Cardiac expression of 4-hydroxy-2-nonenal (4-HNE) and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) were up-regulated in the diabetic heart. This increase was inhibited by disruption of 12-LOX. To further investigate the subcellular mechanism of the increase reactive oxygen species (ROS) in the diabetic heart, intracellular ROS levels in cardiomyocytes were estimated under high glucose condition (HG) and normal glucose condition (LG) by fluorimetry and using MitoTracker® Red in vitro. Cardiomyocytes under HG showed the enhancement of fluorescence intensity of DCF-DA and loss of mitochondrial membrane potential. Treatment with 12-LOX inhibitor (CDC) improved the enhancement of DCF-DA and mitochondrial membrane potential under HG condition. These in vivo and in vitro results suggest that 12-LOX pathway is important in the process of production of ROS and oxidative stress in diabetic heart and promotes the development of diabetic cardiomyopathy.

Abstract 3654: Semaphorin3E is a Novel Angiogenic Regulator

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Junji Moriya ◽  
Tohru Minamino ◽  
Kaoru Tateno ◽  
Masayuki Orimo ◽  
Hideyuki Miyauchi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Umbilical Vein ◽  
Therapeutic Angiogenesis ◽  
Tube Formation ◽  
Diabetic Mice ◽  
Vitro Assay ◽  
Novel Strategy ◽  
Umbilical Vein Endothelial Cells

Semaphorin3E (sema3E) and its specific receptor plexinD1 are known to regulate the patterning of vessels during embryogenesis. However, it remains unclear whether these molecules are involved in postnatal angiogenesis. To elucidate the role of sema3E/plexinD1, we performed in vitro assay using human umbilical vein endothelial cells (HUVECs). Treatment with vascular endothelial growth factor (VEGF) increased proliferation and tube formation and this increase was significantly inhibited by sema3E. Moreover, treatment with the plexinD1-Fc fusion protein antagonized this anti-angiogenic activity of sema3E. Western blot analyses revealed that sema3E suppressed VEGF-induced phosphorylation of VEGFR2, suggesting that sema3E negatively regulates angiogenesis by inhibiting the VEGF signaling pathway. Expression of sema3E and plexinD1 was markedly upregulated in ischemic limbs. Immunohistochemistry showed that sema3E was expressed by the arterioles, myocytes, and capillary endothelial cells in ischemic tissue. Introduction of the plexinD1-Fc gene into ischemic limbs led to significant improvement of blood flow recovery and an increase in the number of CD31-positive cells. It has been reported that other members of the sema3 family are transcriptionally regulated by p53, a tumor suppressor protein that inhibits neovascularization in tumors. Consistent with these reports, forced expression of p53 was found to upregulate sema3E expression in HUVECs. We also found that the expression of p53 was markedly increased in ischemic limbs and that this increase was further enhanced in ischemic tissues of diabetic mice. Consequently, expression of sema3E was significantly higher in ischemic limbs of diabetic mice than in control mice, and the blood flow recovery after ischemia was strongly impaired in these mice even though treated with VEGF. In contrast, treatment with both VEGF and PlexinD1-Fc markedly improved blood flow recovery in diabetic mice. These results indicate that sema3E/plexinD1 negatively regulates postnatal angiogenesis under the regulation of p53 and suggest that inhibition of sema3E would be a novel strategy for therapeutic angiogenesis, especially when VEGF treatment is ineffective.

Sign in / Sign up

Export Citation Format

Share Document