scholarly journals Therapeutic Angiogenesis by a “Dynamic Duo”: Simultaneous Expression of HGF and VEGF165 by Novel Bicistronic Plasmid Restores Blood Flow in Ischemic Skeletal Muscle

Pharmaceutics ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1231
Author(s):  
Ekaterina Slobodkina ◽  
Maria Boldyreva ◽  
Maxim Karagyaur ◽  
Roman Eremichev ◽  
Natalia Alexandrushkina ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Blood Flow ◽  
Growth Factor ◽  
Umbilical Vein ◽  
Limb Ischemia ◽  
Bidirectional Promoter ◽  
Capillary Density ◽  
Therapeutic Angiogenesis ◽  
Angiogenic Growth Factors ◽  
Umbilical Vein Endothelial Cells

Therapeutic angiogenesis is a promising strategy for relief of ischemic conditions, and gene delivery was used to stimulate blood vessels’ formation and growth. We have previously shown that intramuscular injection of a mixture containing plasmids encoding vascular endothelial growth factor (VEGF)165 and hepatocyte growth factor (HGF) leads to restoration of blood flow in mouse ischemic limb, and efficacy of combined delivery was superior to each plasmid administered alone. In this work, we evaluated different approaches for co-expression of HGF and VEGF165 genes in a panel of candidate plasmid DNAs (pDNAs) with internal ribosome entry sites (IRESs), a bidirectional promoter or two independent promoters for each gene of interest. Studies in HEK293T culture showed that all plasmids provided synthesis of HGF and VEGF165 proteins and stimulated capillary formation by human umbilical vein endothelial cells (HUVEC), indicating the biological potency of expressed factors. Tests in skeletal muscle explants showed a dramatic difference and most plasmids failed to express HGF and VEGF165 in a significant quantity. However, a bicistronic plasmid with two independent promoters (cytomegalovirus (CMV) for HGF and chicken b-actin (CAG) for VEGF165) provided expression of both grow factors in skeletal muscle at an equimolar ratio. Efficacy tests of bicistronic plasmid were performed in a mouse model of hind limb ischemia. Intramuscular administration of plasmid induced significant restoration of perfusion compared to an empty vector and saline. These findings were supported by increased CD31+ capillary density in animals that received pHGF/VEGF. Overall, our study reports a first-in-class candidate gene therapy drug to deliver two pivotal angiogenic growth factors (HGF and VEGF165) with properties that provide basis for future development of treatment for an unmet medical need—peripheral artery disease and associated limb ischemia.

Role of the small GTPase Rap1 for integrin activity regulation in endothelial cells and angiogenesis

Blood ◽  
2009 ◽  
Vol 113 (2) ◽  
pp. 488-497 ◽  
Author(s):  
Guillaume Carmona ◽  
Stephan Göttig ◽  
Alessia Orlandi ◽  
Jürgen Scheele ◽  
Tobias Bäuerle ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Umbilical Vein ◽  
Critical Role ◽  
Limb Ischemia ◽  
Small Gtpase ◽  
And Migration ◽  
Umbilical Vein Endothelial Cells ◽  
Endothelial Growth Factor

Abstract Ras-associated protein 1 (Rap1), a small GTPase, attracted attention because of its involvement in several aspects of cell adhesion, including integrin- and cadherin-mediated adhesion. Yet, the role of Rap1 genes and of Rap1 effectors for angiogenesis has not been investigated. Human umbilical vein endothelial cells (HUVECs) express Rap1a and Rap1b mRNA. To determine the contribution of Rap1 activity for angiogenesis, we overexpressed Rap1GAP1, a GTPase-activating protein that inhibits Rap1 activity. Overexpression of Rap1GAP1 significantly blocked angiogenic sprouting and tube-forming activity of HUVECs as well as migration and integrin-dependent adhesion. Silencing of Rap1a, Rap1b, or both significantly blocked HUVECs sprouting under basal and basic fibroblast growth factor-stimulated conditions and reduced HUVEC migration and integrin-dependent adhesion. We found that Rap1a and Rap1b are essential for the conformational activation of β1-integrins in endothelial cells. Furthermore, silencing of Rap1a and Rap1b prevented phosphorylation of tyrosine 397 in focal adhesion kinase (FAK) and vascular endothelial growth factor-induced Akt1-activation. Rap1a−/−-deficient and Rap1a+/− heterozygote mice displayed reduced neovascularization after hind limb ischemia compared with wild-type mice. Silencing of RAPL significantly blocked the Rap1-induced sprouting of HUVECs, suggesting that the angiogenic activity of Rap1 is partly mediated by RAPL. Our data demonstrate a critical role of Rap1 in the regulation of β1-integrin affinity, adhesion, and migration in endothelial cells and in postnatal neovascularization.

scholarly journals Tetragonia tetragonoides (Pall.) Kuntze Restores Blood Perfusion from Hind-Limb Ischemic Mice

2020 ◽  
Vol 10 (23) ◽  
pp. 8562
Author(s):  
Hyun Yang ◽  
Dong Ho Jung ◽  
Hye Won Lee ◽  
Dongoh Lee ◽  
Byoung Seob Ko
Keyword(s):  
Blood Flow ◽  
Mouse Model ◽  
Hind Limb ◽  
Umbilical Vein ◽  
Blood Perfusion ◽  
Blood Flow Rate ◽  
Capillary Density ◽  
Peripheral Circulation ◽  
Tube Formation ◽  
Umbilical Vein Endothelial Cells

Tetragonia tetragonoides (Pall.) Kuntze (TTK) is grown for the edible leaves, and can be used as food. And which commonly called Beonhaengcho in Republic of Korea. TTK is found along the seaside of the Jeju-Island and it has long been consumed as a food for women’s health. We investigated the effects of TTK on peripheral circulation disorder during menopausal transition and/or menopause in a hind-limb ischemic (HLI) mouse model. Chemotactic motility and tube formation of vascular epithelial cells were evaluated in human umbilical vein endothelial cells (HUVECs). Female C57BL/6 mice were fed a TTK (150 or 450 mg/kg/day) for four weeks and the rate of blood flow was assessed using a laser Doppler after HLI. TTK treatment significantly increased cell migration and the branch interval value of tubular structure in a dose-dependently. In the TTK treatment group, blood flow rate was significant induced at 7, 14, and 28 days after HLI, compared with the vehicle. TTK treatment also an increase in capillary density, and the highest levels of pERK(1/2), pAkt, pPLCγ1 and pFAK proteins compared to the vehicle control. These results suggest that extract of TTK may ameliorate the blood flow via improvement of peripheral angiogenesis under hind-limb ischemic stress in a menopausal mouse model.

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.

Platelet-Derived Exosomes Affect the Proliferation and Migration of Human Umbilical Vein Endothelial Cells Via miR-126

2019 ◽  
Vol 17 (4) ◽  
pp. 379-387 ◽  
Author(s):  
Yan Sun ◽  
Xiao-li Liu ◽  
Dai Zhang ◽  
Fang Liu ◽  
Yu-jing Cheng ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Real Time ◽  
Umbilical Vein ◽  
Angiogenic Factors ◽  
Healthy Donors ◽  
And Migration ◽  
Umbilical Vein Endothelial Cells ◽  
Tgf Β1 ◽  
Proliferation And Migration

Background:Intraplaque angiogenesis, the process of generating new blood vessels mediated by endothelial cells, contributes to plaque growth, intraplaque hemorrhage, and thromboembolic events. Platelet-derived Exosomes (PLT-EXOs) affect angiogenesis in multiple ways. The ability of miR-126, one of the best-characterized miRNAs that regulates angiogenesis, carried by PLT-EXOs to influence angiogenesis via the regulation of the proliferation and migration of endothelial cells is unknown. In this study, we aimed to investigate the effects of PLT-EXOs on angiogenesis by Human Umbilical Vein Endothelial Cells (HUVECs).Methods:We evaluated the levels of miR-126 and angiogenic factors in PLT-EXOs from Acute Coronary Syndrome (ACS) patients and healthy donors by real-time Polymerase Chain Reaction (PCR) and western blotting. We incubated HUVECs with PLT-EXOs and measured cell proliferation and migration with the Cell Counting Kit-8 assay and scratch assay, respectively. We also investigated the expression of miR-126 and angiogenic factors in HUVECs after exposure to PLT-EXOs by western blotting and real-time PCR.Results:PLT-EXOs from ACS patients contained higher levels of miR-126 and angiogenic factors, including Vascular Endothelial Growth Factor (VEGF), basic Fibroblast Growth Factor (bFGF), and Transforming Growth Factor Beta 1 (TGF-β1), than those from healthy donors (p<0.05). Moreover, the levels of exosomal miR-126 and angiogenic factors were increased after stimulation with thrombin (p<0.01). HUVEC proliferation and migration were promoted by treatment with activated PLT-EXOs (p<0.01); they were accompanied by the over-expression of miR-126 and angiogenic factors, including VEGF, bFGF, and TGF-β1 (p<0.01).Conclusion:Activated PLT-EXOs promoted the proliferation and migration of HUVECs, and the overexpression of miR-126 and angiogenic factors, thereby elucidating potential new therapeutic targets for intraplaque angiogenesis.

Insulin-mediated changes in leg blood flow are coupled to capillary density in skeletal muscle in healthy 70-year-old men

Metabolism ◽  
2001 ◽  
Vol 50 (9) ◽  
pp. 1078-1082 ◽  
Author(s):  
Anu Hedman ◽  
Per-Erik Andersson ◽  
Richard Reneland ◽  
Hans O. Lithell
Keyword(s):  
Skeletal Muscle ◽  
Blood Flow ◽  
Capillary Density ◽  
Leg Blood Flow ◽  
Old Men

Isolation and sequencing of a cDNA clone homologous to the v-sis oncogene from human endothelial cells

1986 ◽  
Vol 6 (8) ◽  
pp. 3018-3022
Author(s):  
B D Tong ◽  
S E Levine ◽  
M Jaye ◽  
G Ricca ◽  
W Drohan ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Cdna Library ◽  
Cdna Clone ◽  
Umbilical Vein ◽  
Platelet Derived Growth Factor ◽  
Human Endothelial Cells ◽  
A Chain ◽  
Umbilical Vein Endothelial Cells ◽  
Reading Frames

A clone containing the 3' end of the mRNA for the human c-sis gene (homologous to the B chain of platelet-derived growth factor) was isolated from a cDNA library derived from human umbilical vein endothelial cells and then sequenced. The analysis of possible translation products in all three reading frames indicated that the A chain of platelet-derived growth factor was not coded for within the 3' end of the c-sis mRNA. The 3' end of the mRNA for c-sis is contained in or adjacent to exon 6.

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