Angiogenic Integrity Preservation/Restoration: Hit for Vascular Endothelial Growth Factor Prone Therapeutic Coronary Revascularization

Review Article

Austin J Vasc Med. 2014;1(2): 9.

Angiogenic Integrity Preservation/Restoration: Hit for Vascular Endothelial Growth Factor Prone Therapeutic Coronary Revascularization

Anita A. Mehta* and Kiranj K Chaudagar

Department of Pharmacology, L. M. College of Pharmacy, India

*Corresponding author: Anita A. Mehta, Department of Pharmacology, L. M. College of Pharmacy, Opp Gujarat University, Navarangpura, Ahmedabad-380 009, India

Received: November 11, 2014; Accepted: December 04, 2014; Published: December 05, 2014

Abstract

Various mediators such as acetylcholine, bradykinin, and Vascular Endothelial Growth Factor (VEGF) are widely known for endothelial cells dependent Nitric Oxide (NO) release. However, the VEGF-A165/VEGFR-2 binding stimulated NO is associated with induction of angiogenesis. This axis is disturbed in coronary endothelial cells of heart failure patients, and VEGF prone therapeutic revascularization establishes the integrity of such axis in preclinical studies but not in clinical trials. The present review discusses possible approaches that can be useful for angiogenic integrity restoration, and preservation to succeed the clinical trials of VEGF.

Keywords: Vascular Endothelial Growth Factor; Angiogenic integrity; Therapeutic revascularization; Nitric oxide; Heart failure

Introduction

Heart Failure (HF) is a prominent cause of mortality and morbidity worldwide. It is characterized by loss of blood pumping capacity of heart to compensate the demand of body tissue. Approximately, 5.7 million people have been suffering from HF in the US. It is predicated that half of these patient to be die within 5 years of diagnosis [1]. General Practice Research Database of UK suggested that 800,000 people are suffering from HF. It is more prevalent in male than female, and elders than younger [2].The high prevalence rate of HF have been predicted in developing countries India, however, there is no specific epidemiological data due to lack of surveillance program [3,4]. It is exceptionally more prevalent in Indian young age population than other developed countries [5]. Ultimately, HF is a threatening issue for both developed and developing countries.

Angiogenesis is a process of new blood vessel formation from pre-existing endothelial cells. It involves simultaneous coordination of parent vessel vasodilation, basement membrane degradation-reformation, endothelial migration-proliferation, lumen formation, loop development, and pericytes incorporation [6]. The cardiac contractility is maintained in the acute phase HF by cardiac hypertrophy and angiogenesis [7].The co-ordination of such hypertrophy and angiogenesis require to fulfill the oxygen demand of heart [8]. The disturbance of coordination due to loss of angiogenesis transits cardiac insults into heart failure in late phase [7,9,10]. This is indicated by absence of coronary collateralization (anastomotic connections between portions of the same coronary artery and between different coronary arteries without an intervening capillary bed which is developed by angiogenesis) in HF patients [11,12]. The collateral vessels formation is a phenomenon of compensatory physiological revascularization that preserves the heart in late phase of chronic injury. However, it is not fully combat the worsening inside the heart. Therefore, there is need of therapeutic revascularization (cells or growth factor induced coronary angiogenesis).

Many angiogenesis inducers such as fibroblast growth factor (FGF), Transforming Growth Factors (TGF) [13], angiopoietin [14], Hepatocyte Growth Factor (HGF) [15], interleukin-8 (IL-8) [16], angiogenin [17] and Vascular Endothelial Growth Factor (VEGF) have been reported [18]. One of them, VEGF is only an endothelial cell selective potent mitogen (ED50=2-10pM). It stimulates the proliferation of micro-macrovascular endothelial cells (EC) of arteries, veins, and lymphatic vessels [19-21]. VEGF possess dose (10-100ng/mL) dependent anti-apoptotic effects on endothelial cells [22], and promote survival rate in serum starved ECs [23]. It require for survival of EC during neonatal life until vessels are rich in the supporting cells such as pericyte [24,25]. It is a key player for tumor induced angiogenesis. VEGF is also known as a Vascular Permeability Factor (VPF), and increase permeability at concentration less than 1nmol/L in Miles assay [26]. It involve in tumor dependent ascites [27,28]. Other than EC proliferation, survival, it also auto-regulates the response of cells for the angiogenesis. The angiogenic integrity of Endothelial Cells (EC) means proliferative, migratory, adhesive, organization response of EC to VEGF.

The hypertrophy induced coronary angiogenesis is mediated through activation of mammalian target of rapamycin (mTOR) dependent myocardial VEGF expression. The decoy of VEGF receptor or loss of angiogenic integrity decreases capillary density, induces hypoxia, contractile dysfunction, interstitial fibrosis, maladaptive cardiac hypertrophy, and heart failure [7,9]. The angiogenic integrity of EC is modulated by various phenomena such as hypercholesterolemia, diabetes, oxidative stress, shear stress, drug treatment and preconditioning [29,30]. Various preclinical experiments of VEGF induced therapeutic angiogenesis has been indicated promising data of increase in cardiac contractility, collateralization and perfusion volume. However, the clinical trials are failure to show benefit similar to preclinical studies. Because preclinical studies are well control experiments on normal or pathology-possessing animals whereas clinical trials include the patients with disturbed EC due to more than one disease, and they are also vary in VEGF responsiveness. This review focuses VEGF signaling, angiogenic integrity, outcome of VEGF prone therapeutic angiogenesis, and approaches for the preservation/restoration of angiogenic integrity to fill the gap between preclinical and clinical outcomes.

VEGF family

Molecular cloning revealed first time that multiple forms of VEGF such as 206, 186, 165 and 121 (VEGF206, VEGF186, VEGF165, and VEGF121) arose due to alternative splicing of RNA transcript in vascular smooth muscle cells [31]. Transfection experiment on human embryonic kidney 293 cells showed that VEGF189, VEGF206 was predominately cell associated and only very poorly secreted whereas VEGF121 and VEGF165 were efficiently exported from the cell. Vascular permeability activity was detected in the medium of 293 cells transfected with all four forms of VEGF but endothelial cell mitogenic activity was present only with VEGF121 and VEGF165 [32]. Immunostaining of VEGF isoform in transfected human embryonic kidney CEN4 cells revealed that VEGF189 or VEGF206 localized in the subepithelial Extracellular Matrix (ECM). ECM bound VEGF released into a soluble and bioactive form by heparin or plasmin that possessed molecular masses consistent with the intact polypeptides. This study also showed that VEGF165 possessed intermediate ECM binding properties [33]. Plasmin mediated cleavage of VEGF generated two smaller fragments, an amino terminal homodimeric protein containing receptor binding determinants and a carboxyl-terminal polypeptide which bound heparin. The various isoforms of VEGF (165, 165/110, 110 and 121) bound soluble kinase domain region receptor with similar affinity (approximately 30pM) whereas bound soluble Flt-1 receptor with different affinities (10, 30, 120, and 200pM, respectively). VEGF110 and VEGF121 showed 100 fold less endothelial cell mitogenic potencies as compared to VEGF165 [34]. VEGF164 and VEGF188 lacking mice possessed defect in myocardial angiogenesis but not due to VEGF120 [35]. Thus, VEGF165 is a most potent angiogenesis inducing isoform of VEGF family. Currently, the VEGF family has seven members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PlGF. VEGF-A, VEGF-B, VEGF-C and VEGF-D are found in human. VEGF121, 165, 189 and 206 are sub members of VEGF-A [36].

VEGF signalling and angiogenic integrity

VEGF interact with tyrosine kinase receptor and coreceptor on the endothelial cells. VEGF receptor-1 (VEGFR-1/ Fms like tyrosine kinase-1-Flt-1), VEGFR-2 (Kinase Domain Containing Receptor (KDR)/ Fetal liver kinase-1 (Flk-1)) and VEGFR-3 (Flt-4) are receptors and Neuropilins-1 (NP-1) and NP-2 are coreceptors [37]. VEGFR-1 and VEGFR-2 deficient mice died in utero between 8.5 and 9.5 day due to vascular disorganization [38,39]. In VEGFR-1 deficient mice, vascular disorganization was related to excessive angioblast activity whereas proliferating endothelial cells were present [40]. In VEGFR-2 deficient mice, vascular disorganization was related to loss of blood islands and proliferating endothelial cells [38]. Flt-1 tyrosine kinase domain deficient mice showed normal development of vasculature [41]. VEGFR-2 selectively expressed in vascular endothelial cells and possessed lower binding affinity (Kd= 400-800pM) to VEGF-A as compared to VEGFR-1 [42,43]. Binding of VEGF to VEGFR-2 resulted in activation of phosphoinositide 3 kinase (PI3K)/Akt-dependent integrins such as αvβ3, αvβ5, α5β1 and α2β1, activation of Protein Kinase C (PKC)-dependent-Ras-independent Raf/ MEK/ Mitogen Activated Protein Kinase (MAPK) and induction of angiogenesis cascade such as endothelial cell proliferation, migration, adhesion and tube formation [44-47]. VEGFR-3 selectively expressed on lymphatic endothelial cells and bound only with VEGF-C and VEGF-D but not with other forms of VEGF [48,49]. Defect in VEGF-C dependent VEGFR-3 activation caused severe embryonic lethality due to loss of embryonic lymphatic system and embryonic tissue oedema [50]. Neuropilin (NP) coexpressed with VEGFR-2 in endothelial cells, enhanced the binding of VEGF165 to VEGFR-2 and increased effectiveness of VEGF165-VEGFR-2-mediated signal transduction [51]. Therefore, VEGF165-VEGFR-2 mediated signalling is a determinant of angiogenic integrity.

VEGF prone therapeutic revascularization

The angiogenesis appears at the border zone of the infracted area for 3 day immediately after insults. After 7 days, it gradually disappears in various experiment model of ischemia reperfusion injury. Early phase angiogenesis is regulated by VEGF-A and VEGFR level which are downregulated in the late phase. The VEGF-A/VEGFR expression is not affected in the noninfarcted area [10]. However, the VEGF liposomes showed improvement in systolic function and increase myocardial perfusion due to 21% increase in collateralization and 74% increase in number of perfused vessels in the MI region [52]. Other preclinical studies has reported the similar results due to VEGF therapy [53,54]. According to such preclinical reports, VEGF administration is itself restore and preserve the angiogenic integrity.

In angina patients, the intramyocardial administration of plasmid based VEGF165 (phVEGF165) gene increased plasma VEGF level that returned to baseline on 90th day. The 13 out of 17 patients were angina free at the end of 6 months. There was no report on adverse events related to gene delivery [55]. The 14 coronary artery disease patients (who were not candidates for mechanical revascularization) treated with low-dose (0.005 and 0.017mg/kg, intracoronary, for 20min) and high-dose (0.05 and 0.167mg/kg, intracoronary, for 20min) of recombinant human VEGF (rhVEGF) showed improvement in myocardial perfusion dose dependently under stress and rest conditions visual analysis using Single Photon Emission CT (SPECT) at 60th day after treatment of rhVEGF. This visual improvement was not significant as per statistical analysis [56]. In the first placebo-controlled phase-II trial of VEGF (Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis-VIVA), high-dose rhVEGF (50ng/kg/min, intracoronary infusion on 0day for 20min, followed by 4hr intravenous infusions on 3, 6, and 9day) significantly ameliorated angina class on 120day but did not show significant increase in ETT time and angina frequency. Whereas low-dose treatment of rhVEGF (17ng/kg/min) did not show any significant benefit as compared to placebo [57]. Direct intramyocardial injections of adenovirus mediated VEGF121 (AdVEGF121) and plasmid VEGF165 DNA in anginal patients with optimal medical therapy showed site specific delivery of gene but did not show significant improvement in treadmill testing as compared to patients treated with only medical [58,59]. Ultimately, the failure of clinical trials on VEGF induced cardiac angiogenesis compared to preclinical studies is related to loss of angiogenic integrity restoration and preservation by VEGF. Therefore, there is need of approaches that fulfills the gap of VEGF prone therapeutic revascularization in preclinical, and clinical studies by restoration and preservation of angiogenic integrity.

Angiogenic integrity preservation/restoration

Endothelial cells, cover luminal side of vessels, have been prominently reported for regulating vascular homeostasis [60]. The angiogenic integrity of EC is autoregulated by VEGF/KDR/ PKC+PI3K/eNOS/NO pathway [61]. There are mainly three approaches, such as cell therapy, drug treatment, and food supplementation for recoupling or preservation of VEGF dependent nitric oxide releasing axis in endothelial cells (Figure 1).

Citation: Mehta AA and Chaudagar KK. Angiogenic Integrity Preservation/Restoration: Hit for Vascular Endothelial Growth Factor Prone Therapeutic Coronary Revascularization. Austin J Vasc Med. 2014;1(2): 9.