Gene Therapy With the Angiogenic Cytokine Secretoneurin Induces Therapeutic Angiogenesis by a Nitric Oxide–Dependent Mechanism

Abstract

Key Words: angiogenesis Ⅲ endothelium Ⅲ gene therapy Ⅲ hypoxia Ⅲ peripheral vascular disease C ardiovascular diseases represent the leading cause of death worldwide. 1 In the case of peripheral arterial disease, a substantial number of patients who develop critical limb ischemia are not eligible for surgical or interventional revascularization and, despite optimal medical therapy, have reduced quality of life and life expectancy. 2,3 This group of patients would require new therapeutic methods to increase collateral blood flow to areas of decreased tissue perfusion distal to arterial occlusion.Angiogenesis, the growth of new blood vessels from the preexisting vasculature, plays an important role in wound healing, tumor growth, diabetic retinopathy, tissue ischemia, and inflammatory diseases. 4 Cytokines mediating this process include vascular endothelial growth factor (VEGF) 5 and basic fibroblast growth factor (b-FGF). 6 For stability of newly formed vessels, recruitment of pericytes and smooth muscle cells (SMCs) is important; such recruitment is known as arteriogenesis, which is mediated by cytokines such as monocyte chemoattractant protein-1 and platelet-derived growth factor (PDGF)-BB. 7,8 Postnatal vasculogenesis, which is the mobilization and incorporation of bone marrow-derived endothelial progenitor cells (EPCs), also plays an important role in the growth of new vessels. 9 Recently, application of endothelial cytokines as proteins or genes has been of scientific and clinical interest to treat limb or myocardial ischemia, a procedure called therapeutic angiogenesis. 10 -14 We recently demonstrated that the neuropeptide secretoneurin (SN) induces angiogenesis and postnatal vasculogen- MethodsAn expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Construction of the Human SN Expression PlasmidSynthetic oligonucleotides encoding SN and a signal peptide were cloned into the expression vector pAAV-MCS (Stratagene). The plasmid vector is described in the Online Data Supplement. Animals, Mouse Hindlimb Ischemia Model, SN Gene Therapy, and Bone Marrow Transplantation ModelMice at ages between 12 and 18 months were subjected to unilateral hindlimb surgery and injected with plasmid DNA expressing SN or green fluorescent protein (GFP) into thigh and calf muscles immediately after surgery.Bone marrow transplantation was performed as described. 19 After hindlimb ischemia, injection of SN plasmid or saline was performed. Blood Flow Measurement and In Vivo Assessment of Limb Function and Ischemic DamageBlood flow measurements were performed using a laser-Doppler perfusion imaging (LDPI) analyzer. Blood perfusion is expressed as LDPI index representing the ratio of left (ie, operated, ischemic leg) versus right (ie, unoperated, not-ischemic leg) limb blood flow. Movement score and analysis of necrosis are described in the Online Data Supplement. Immunofluorescence and Fluorescence-Activated Cell Sorting AnalysisECs were stained for CD31, and arteries/arteriole...

The Neuropeptide Catestatin Acts As a Novel Angiogenic Cytokine via a Basic Fibroblast Growth Factor–Dependent Mechanism

“…These findings indicate that this peptide may also induce postnatal vasculogenesis. Consistent with this, we found that CST increases incorporation of EPCs in ischemic hindlimbs in GFP BMT mice, which is a wellcharacterized model to study these cells, 16,17 findings that were also confirmed by confocal microscopy. We also found increased numbers of arteries/arterioles in CST treated ischemic hindlimbs, consistent with induction of arteriogenesis.…”

“…16 Several angiogenic cytokines have been used to treat patients experiencing peripheral arterial or coronary heart disease 32 and CST, as well as SN, is emerging as a promising novel candidate in the therapy of these diseases. …”

“…10 Some Matrigel assays were performed with a mixture of 5000 HUVECs and 3000 DiI LDL-labeled EPCs. 16 The association of EPC to capillary structures was quantified using Image J. …”

Neuron-derived Neurotrophic Factor Functions as a Novel Modulator That Enhances Endothelial Cell Function and Revascularization Processes

“…Furthermore, secretoneurin is a neuropeptide that promotes the angiogenic signaling pathways, including Akt, and stimulates the proliferation and network formation of endothelial cells in vitro (46). Secretoneurin has also been reported to stimulate the revascularization responses in a mouse model of hind limb ischemia by a nitric oxide-dependent mechanism (47). Similar to these neuron-derived factors or neuropeptides, NDNF exerts prosurvival and proangiogenic properties in vitro and in vivo.…”

Approaches in Gene Therapy of Cancer and Cardiovascular Diseases

“…Authors were able to detect functionally significant angiogenesis that clearly improved the pathological consequences of disease induction. The model of hind limb ischemia is actually a commonly used model to prove the efficiency of proangiogenic viral and non-viral gene delivery in ischemic disease (Schgoer et al, 2009). Bosch-Marce et al, have used this model to test adenovirus vector carrying the gene encoding for constitutively active HIF-1 , resulting in a sufficient induction of reperfusion of the affected areas even in older animals (Bosch-Marce et al, 2007).…”