eISSN: 2084-9869
ISSN: 1233-9687
Polish Journal of Pathology
Current issue Archive Manuscripts accepted About the journal Supplements Editorial board Abstracting and indexing Subscription Contact Instructions for authors Ethical standards and procedures
SCImago Journal & Country Rank
4/2017
vol. 68
 
Share:
Share:
more
 
 
Original paper

Role of vascular endothelial growth factor in inducing production of angiopoetin-1 – in vivo study in Fisher rats

Piotr Barć
,
Tomasz Płonek
,
Dagmara Baczyńska
,
Artur Pupka
,
Wojciech Witkiewicz
,
Agnieszka Mastalerz-Migas
,
Artur Milnerowicz
,
Maciej Antkiewicz
,
Agnieszka Hałoń
,
Jan P. Skóra

Pol J Pathol 2017; 68 (4): 326-329
Online publish date: 2018/03/06
Article file
- Role.pdf  [0.24 MB]
Get citation
ENW
EndNote
BIB
JabRef, Mendeley
RIS
Papers, Reference Manager, RefWorks, Zotero
AMA
APA
Chicago
Harvard
MLA
Vancouver
 
PlumX metrics:
 

Introduction

Peripheral artery disease (PAD) affects over 25 mln people in Europe and USA [1]. Many patients do not qualify for standard surgery or endovascular treatment. In those patients, optimal palliative medical therapy is the only available treatment. Such patients still suffer from chronic pain, ulcerations and often require limb amputations [2].
Hence, alternative methods of treatment, including therapeutic angiogenesis, are sought [3, 4]. One therapeutic concept is based on the use of genes encoding angiogenic factors delivered to the ischaemic muscle tissues [5, 6]. The clinical benefits (reduction of pain, accelerated healing of ischaemic ulcerations, reduction in the incidence of limb amputation) of such a therapy supports its use in patients suffering from critical limb ischemia and with no other therapeutic options [7, 8]. The administration of genes does not require surgical intervention and is considered safe for the patients [9].
Several studies that assessed the efficiency of factors and genes in promoting angiogenesis, i.e. vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and angiopoetin 1 (ANGPT1) are available. An increased vascularization, extracellular matrix remodelling and an inhibition of apoptosis and inflammatory processes were reported [9, 10, 11, 12, 13, 14, 15].
Angiopoetin 1 mainly acts in the late phase of angiogenesis and is responsible for the stabilization and maturing of the vessels [11]. In contrast to VEGF, ANGPT1 does not show mitogenic features [12]. Angiopoetin1 stimulates the capillary endothelial cells to bind through the Tie2 receptor and stabilizes them [13]. Furthermore, it promotes the migration of the endothelial cells towards damaged vessels and promotes vessel regeneration.
The aim of this study is to investigate how an intramuscular injection of plasmids with genes coding various pro-angiogenic factors (plAPT1, pIRES/ANGPT1/VEGF165 and pIRES/VEGF165/HGF) influences the production of angiopoetin 1 (ANGPT1).

Material and methods

The RNA was extracted from human heart tissue using an BIoTEC EZNA RNA isolation Kit. The plasmids pIRES/ANGPT1/VEGF165 and pIRES/VEGF165/HGF were prepared according to a previously described protocol [16]. In order to produce monogene plANGPT1, a cDNA fragment was digested using EcoRI and XhoI and subsequently cloned into a pcDNA3 vector (Invitrogen, USA) using T4 Ligase (Sigma, USA). The apyrogenecity of the plasmids was verified using a Limulus amebocyte lysate assay, Pyrochrome Chromogenic Test Kit (Charles River). Each sample contained less than 10EU of endotoxin per 1mg of DNA.
Forty healthy Fischer rats were used in the study (weight: 200-250 g). All rats grew under controlled conditions (temperature 23oC and 12 hour light/dark cycles). The rats were divided into four equal groups: ANGPT1, APGPT1/VEGF, VEGF/HGF and the control group. Each animal received four consecutive intramuscular injections into the right hind limb containing: 4mg of the plANGPT1 plasmid in the ANGPT1 group, 4 mg of the pIRES/ANGPT1/VEGF165 plasmid in the ANGPT1/VEGF group, 4 mg of the pIRES/VEGF165/HGF plasmid in the VEGF/HGF group and 4mg of a naked plasmid in the control group. After 12 weeks, the rats were euthanasied using 200 mg/kg of sodium pentobarbital solution. The tissues were extracted from the injection areas and sent for a histological and immunohistochemical analysis.
The muscle samples were divided in 5-µm slices and stained using hematoxylin and eosin. The number of the vessels stained with the anti-ANGPT1 antibodies were counted in each group in order to assess the induction of the production of angiopoetin-1. The immunohistochemical reactions were performed using the anti-angiopoietin 1 antibody (rabbit polyclonal to agiopoietin 1, catalogue number ab8451, ABCAM, dilution 1 : 100). The immunochemically marked samples were assessed under the Olympus BX41 light microscope with computer microscopic image analysis software (AnalySIS DOCU). The ANGPT1 protein expression was evaluated by recording multiple random fields of view using the modified Weidner’s method [17]. Microscopic images of the slices were analyzed digitally at a 200× magnification. Five randomly selected fields of view with the highest density of positive (brown) stained foci that marked blood vessels were selected automatically. Each vessel or concentration of endothelial cells (regardless of the presence or absence of a full lumen) was counted as an individual micro-vessel. The average number of vessels was calculated for each sample.
The data were analysed using Statistica 10.0 (StatSoft, Inc. Tulsa, USA). Prior to any further statistical analyses, the distribution of the variables was estimated using the Shapiro-Wilk’s W test. The normality test failed and data were analysed using nonparametric tests. The Kruskal-Wallis ANOVA on ranks with post-hoc test was used for multi-group comparisons (independent samples). Statistical significance was determined as p < 0.05.
The study was approved by the local Ethical Committee.

Results

All the rats survived the 12 week experiment period. Their development was normal. All the animals were similar in appearance and behaviour. Post mortem analysis showed no morphological pathologies in the muscles or the internal organs. The histological assessment of all of the harvested muscle specimens confirmed as the presence of normal skeletal striated muscles.
The anti-ANGPT1 antibodies stained the vessels in all the groups. There were on average 14.1 ±2.3 vessels in the ANGPT1 group , 32.5 ±10.5 in the ANGPT1/VEGF group and 30.8 ±13.3 vessels in the HGV/VEGF group. There were on average 7.3 ±2.3 vessels in the control group (p < 0.0001). The results are shown in Fig. 1. The microscopic images with the stained vessels are presented in Fig. 2.

Discussion

Angiogenesis is a complex process that involves the extracellular matrix, the endothelial progenitor cells and cytokines. The extracellular matrix acts as a storage of cytokines, whichare released through the activation of matrix metaloproteinases (MMP). Angiogenesis is regulated mainly by interactions between cytokines, such asthe vascular endothelial growth factor (VEGF), angiopoietin-1 (ANGPT1) and the hepatocyte growth factor (HGF) [4, 5, 6].
VEGF is the critical initiating factor and a mediator of the inflammatory process and vascular permeability [18]. HGF inhibits vascular permeability and pro-inflammatory effects [19, 20]. Bicistronic plasmids coding VEGF with HGF seems to be effective during the first stage of the neoangiogenesis gene therapy [21, 22].
ANGPT1 is an important factor in the late stage of angiogenesis. The function of this cytokine acts in the acute and chronic phase. During the acute phase, the expression of APT1 reduces the ischemic damage due to its anti-apoptotic functions [11]. It acts on the endothelial cells through Tie-2 receptors, improving their survival and maturation [13]. ANGPT 1 also suppresses VEGF-induced permeability and inflammation in endothelial cells [23, 24]. In the chronic phase, it promotes the creation of new vessels from stem/progenitor-like cells and induces the reconstruction of the basal membrane [25]. In vivo studies confirmed that ANGPT1 is largely responsible for capillary maturation and the chemotactic response, the formation of blood vessels and their stability but and does not promote mitotic activity [11, 12, 13].
The number of anti-ANGPT1-stained vessels was higher in the ANGPT1/VEGF group than in the ANGPT1 group. Surprisingly, the expression of angiopoetin 1 increased in the VEGF/HGF group, which did not receive the ANGPT1 gene. Moreover, the administration of the ANGPT1 plasmid did not induce a significant angiopoetin 1 expression compared to the control group that received a naked plasmid. This indicates that the expression of angiopoetin 1 is increased only in the late phase of angiogenesis and the administration of the plasmids encoding angiopoetin 1 alone do not promote sufficient angiogenesis. To increase the production of ANGPT1, additional factors such as VEGF, HGF, which act in the early phases of angiogenesis, are necessary. Hence, an optimal therapeutic set of genes to accelerate angiogenesis should include late mediators of angiogenesis as well as factors that induce this process.
In summary, an administration of the plasmid encoding the ANGPT1 gene did not significantly increase the production of angiopoetin 1 compared to the control group in Fisher rats. Additional factors acting in the early phase of angiogenesis (VEGF, HGF) were necessary to accelerate the production of angiopoetin 1.
This study is part of the Project “WroVasc – Integrated Cardiovascular Centre”, co-financed by the European Regional Development Fund, within the Innovative Economy Operational Program, 2007-2013, realized in the Regional Specialist Hospital, Research and Development Center in Wroclaw. “European Funds – for the development of innovative economy”. The project is supported by the Wroclaw Centre of Biotechnology, and the 2014-2018 Leading National Research Centre (KNOW) programme.

The authors declare no conflict of interest.

References

1. Iida O, Nakamura M, Yamauchi Y, et al. Endovascular treatment for infrainguinal vessels in patients with critical limb ischemia: Olive registry, a prospective, multicenter study in japan with 12-month follow-up. Circ Cardiovasc Interv 2013; 6: 68-76.
2. Rowlands TE, Donnelly R. Medical therapy for intermittent claudication. Eur J Vasc Endovasc Surg 2007; 34: 314-321.
3. Belch J, Hiatt WR, Baumgartner I, et al. Effect of fibroblast growth factor NV1FGF on amputation and death: a randomised placebo-controlled trial of gene therapy in critical limb ischaemia. Lancet 2011; 377: 1929-1937.
4. Hee Ko S, Bandyk DF. Therapeutic angiogenesis for critical limb ischemia. Semin Vasc Surg 2014; 27: 23-31.
5. Losordo DW, Dimmeler S. Therapeutic angiogenesis and vasculogenesis for ischemic disease, part I: angiogenic cytokines. Circulation 2004; 109: 2487-2491.
6. Losordo DW, Dimmeler S. Therapeutic angiogenesis and vasculogenesis for ischemic disease, part II: cell-based therapies. Circulation 2004; 109: 2692-2697.
7. Skóra J, Barć P, Dawiskiba T, et al Angiogenesis after plasmid VEGF165 gene transfer in animals model. Centr Eur J Immunol 2013; 38: 305-309.
8. Sadakierska-Chudy A, Skóra J, Barć P, et al. Angiogenic therapy for clinical lower limb ischemia. Adv Clin Exp Med 2010; 19: 347-359.
9. Gupta R, Tongers J, Losordo DW. Human studies of angiogenic gene therapy. Circ Res 2009; 105: 724-736.
10. Shyu KG, Chang H, Wang BW, Kuan P. Intramuscular vascular endothelial growth factor gene therapy in patients with chronic critical leg ischemia. Am J Med 2003; 114: 85-92.
11. Suri Ch, McClain J, Thurston G, et al. Increased Vascularization in Mice Overexpressing Angiopoietin-1. Science 1998; 282: 468-471.
12. Gamble JR, Drew J, Trezise L, et al. Angiopoietin-1 is an antipermeability and anti-inflammatory agent in vitro and targets cell junctions. Circ Res 2000; 87:603-607.
13. Davis S, Aldrich TH, Jones PF et al. Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell 1996; 87: 1161-1169.
14. Rubin JS, Chan AM, Bottaro DP, et al. A broad-spectrum human lung fibroblast-derived mitogen is a variant of hepatocyte growth factor. Proc Natl Acad Sci U S A 1991; 88: 415-419.
15. Kan M, Zhang GH, Zarnegar R, et al. Hepatocyte growth factor/hepatopoietin A stimulates the growth of rat kidney proximal tubule epithelial cells (RPTE), rat nonparenchymal liver cells, human melanoma cells, mouse keratinocytes and stimulates anchorage-independent growth of SV-40 transformed RPTE. Biochem Biophys Res Commun 1991; 174: 331-337.
16. Barc P, Plonek T, Baczynska D, et al. A combination of VEGF165/HGF genes is more effective in blood vessels formation than ANGPT1/VEGF165 genes in an in vivo rat model. Int J Clin Exp Med 2016; 9: 12737-12744.
17. Sengupta S, Gherardi E, Sellers LA, et al. Hepatocyte growth factor/scatter factor can induce angiogenesis independently of vascular endothelial growth factor. Arterioscler Thromb Vasc Biol 2003; 23: 69-75.
18. Ylä-Herttuala S, Rissanen TT, Vajanto I, Hartikainen J. Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine. J Am Coll Cardiol 2007; 49: 1015-1026.
19. Shimamura M, Sato N, Oshima K, et al. Novel therapeutic strategy to treat brain ischemia: overexpression of hepatocyte growth factor gene reduced ischemic injury without cerebral edema in rat model. Circulation 2004; 109: 424-431.
20. Min JK, Lee YM, Kim JH, et al. Hepatocyte growth factor suppresses vascular endothelial growth factor-induced expression of endothelial ICAM-1 and VCAM-1 by inhibiting the nuclear factor-kappaB pathway. Circ Res 2005; 96: 300-307.
21. Jadczyk T, A Faulkner A, Madeddu P. Stem cell therapy for cardiovascular disease: the demise of alchemy and rise of pharmacology. Br J Pharmacol 2013; 169: 247-268.
22. Suzuki H, Iso Y. Clinical Application of Vascular Regenerative Therapy for Peripheral Artery Disease. Biomed Res Int 2013; 2013: 179730.
23. Kim I, Moon SO, Park SK, et al. Angiopoietin-1 reduces VEGF-stimulated leukocyte adhesion to endothelial cells by reducing ICAM-1, VCAM-1, and E-selectin expression. Circ Res 2001; 89: 477-479.
24. Chae JK, Kim I, Lim ST, et al. Coadministration of angiopoietin-1 and vascular endothelial growth factor enhances collateral vascularization. Arterioscler Thromb Vasc Biol 2000; 20: 2573-2578.
25. Skóra J, Biegus J, Pupka A, et al. Molecular basics of angiogenesis. Postepy Hig Med Dosw 2006; 60: 410-415.

Address for correspondence

Agnieszka Mastalerz-Migas
Family Medicine Department
Wroclaw Medical University
Syrokomli 1
51-141 Wroclaw, Poland
e-mail: agnieszka.migas@gmail.com
Copyright: © 2018 Polish Association of Pathologists and the Polish Branch of the International Academy of Pathology This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
Quick links
© 2022 Termedia Sp. z o.o. All rights reserved.
Developed by Bentus.