Gene Therapy With the Angiogenic Cytokine Secretoneurin Induces Therapeutic Angiogenesis by a Nitric Oxide–Dependent Mechanism
Rationale: The neuropeptide secretoneurin induces angiogenesis and postnatal vasculogenesis and is upregulated by hypoxia in skeletal muscle cells.
Objective: We sought to investigate the effects of secretoneurin on therapeutic angiogenesis.
Methods and Results: We generated a secretoneurin gene therapy vector. In the mouse hindlimb ischemia model secretoneurin gene therapy by intramuscular plasmid injection significantly increased secretoneurin content of injected muscles, improved functional parameters, reduced tissue necrosis, and restored blood perfusion. Increased muscular density of capillaries and arterioles/arteries demonstrates the capability of secretoneurin gene therapy to induce therapeutic angiogenesis and arteriogenesis. Furthermore, recruitment of endothelial progenitor cells was enhanced by secretoneurin gene therapy consistent with induction of postnatal vasculogenesis. Additionally, secretoneurin was able to activate nitric oxide synthase in endothelial cells and inhibition of nitric oxide inhibited secretoneurin-induced effects on chemotaxis and capillary tube formation in vitro. In vivo, secretoneurin induced nitric oxide production and inhibition of nitric oxide attenuated secretoneurin-induced effects on blood perfusion, angiogenesis, arteriogenesis, and vasculogenesis. Secretoneurin also induced upregulation of basic fibroblast growth factor and platelet-derived growth factor-B in endothelial cells.
Conclusions: In summary, our data indicate that gene therapy with secretoneurin induces therapeutic angiogenesis, arteriogenesis, and vasculogenesis in the hindlimb ischemia model by a nitric oxide–dependent mechanism.
Cardiovascular 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 vasculogenesis in a cornea neovascularization assay.15,16 In vitro, SN induced capillary tube formation in a Matrigel assay, exerted proliferative, chemotactic and antiapoptotic effects on endothelial cells (ECs) and EPCs, and stimulated protein kinase B/Akt and mitogen-activated protein kinase pathways in these cells. We also could show that SN is upregulated in skeletal muscle cells by hypoxia via an indirect, b-FGF– and hypoxia-inducible factor 1-α–dependent pathway.17 In this work, we also demonstrated that inhibition of SN worsened the physiological angiogenic response in the hindlimb ischemia model. Recently, a beneficial effect of SN was reported in murine stroke models, resulting in antiapoptotic effects of SN on neuronal cells mediated by the Jak/Stat pathway, resulting from effects on stem cells and induction of angiogenesis.18
We, therefore, hypothesized that SN might be a promising new candidate to induce therapeutic angiogenesis and show in this work that SN gene therapy mediates angiogenesis, arteriogenesis, and vasculogenesis in the mouse hindlimb ischemia model.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Construction of the Human SN Expression Plasmid
Synthetic 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 Model
Mice 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 Damage
Blood 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 Analysis
ECs were stained for CD31, and arteries/arterioles were stained with a mouse monoclonal α-smooth muscle actin (α-SMA) antibody (Ab) as described.15 Vessels were counted as arterioles/arteries when 1 or more cell layer(s) were positive for α-SMA staining surrounded the whole circumference of the vessel.
Labeling of functional vessels and of EPCs in mice that underwent bone marrow transplantation was performed as described19 by conventional and confocal microscopy. Details of fluorescence-activated cell sorting (FACS) analysis are described in the Online Data Supplement.
Effects of SN Gene Therapy in the Mouse Hindlimb Ischemia Model: Blood Perfusion, Functional Outcome, and Tissue Necrosis
To evaluate the effect of SN gene therapy (50 μg) on blood perfusion, mice were subjected to LDPI measurements. In mice injected with GFP plasmid blood flow recovers after 28 days to a ratio ischemic/nonischemic limb of 0.69±0.05, whereas in SN plasmid injected mice LDPI ratio after 4 weeks was 1.05±0.07 (P<0.01 versus GFP; n=12), indicating complete recovery of blood perfusion in SN treated animals (Figure 1a, gray and black lines). Also after 2 and 3 weeks, SN-treated animals exerted a significantly higher blood perfusion (P<0.05 for both time points SN versus GFP group; n=12; Figure 1a). Representative LDPI pictures after 4 weeks are shown in Figure 1a for GFP- and SN-treated mice.
Intramuscular application of an intermediate dose (25 μg; Figure 1a, blue line) and a low dose (5 μg; Figure 1a, red line) of SN plasmid revealed different effects: whereas the group receiving 5 μg did not show any significant effect on blood perfusion compared to GFP (LDPI ratios: week 4, 0.69±0.04 for GFP; 0.75±0.07 for SN; P=NS; n=7), mice treated with 25 μg showed complete recovery after 4 weeks (LDPI ratios: week 4, 0.69±0.04 for GFP; 0.99±0.04 for SN; P<0.01; n=7).
Mice were also analyzed for functional recovery by observation of movement behavior using a movement score described previously.8 Whereas movement behavior of control mice improved only to some extent, SN-injected mice performed significantly better after 3 and 4 weeks (4 week movement score: 2.3±0.42 for GFP; 3.3±0.22 for SN; P<0.05; n=12; Figure 1b). In terms of clinical outcome, necrosis of ischemic limbs was evaluated. Percentage of mice with necrosis increased during the observational period especially in the GFP group (Figure 1c). At 4 weeks, 56% of GFP plasmid treated animals showed signs of necrosis, whereas only in 24% of SN-treated mice, tissue defects were observed (P<0.05 for SN treatment using Fischer’s exact test; n=12; Figure 1c).
Effects of SN Gene Therapy in the Mouse Hindlimb Ischemia Model: Angiogenesis and Arteriogenesis
To investigate the effect of SN gene therapy on angiogenesis, we performed staining of capillaries by CD-31 immunofluorescence (Figure 2a). Capillary counts (expressed as capillaries/mm2 or as capillaries/muscle fiber in CD-31–stained sections) were significantly higher in SN plasmid–treated mice compared to GFP-treated animals (capillaries/mm2: control [not ischemic] side, 270±70; GFP-treated ischemic side, 380±70; SN-treated ischemic side, 670±80; capillaries/muscle fiber GFP, 0.92±0.07; SN, 1.89±0.13; P<0.05 GFP versus SN; n=8).
We also observed an increase of α-SMA–positive vessels in animals treated by SN gene therapy, consistent with induction of arteriogenesis: α-SMA–positive vessels/mm2: control (not ischemic) side, 4±2.1; GFP-treated ischemic side, 6.4±1.7; SN-treated ischemic side, 14.3±3.0; P<0.05 GFP versus SN; n=8 (Figure 2a).
Adjacent ischemic hindlimb sections of an SN treated mouse were stained for CD-31 and α-SMA and merged image shows arterioles/arteries with layers of SMCs surrounding the vessel wall (Figure 2b, arrowheads).
Effects of SN Gene Therapy on Vasculogenesis in the Actin–GFP Bone Marrow–Transplanted Mouse
We used the actin-GFP bone marrow–transplanted mouse19 to study vasculogenesis in the hindlimb ischemia model after SN gene therapy. Three weeks after ischemia operation, rhodamine-labeled BS1 lectin was injected IV; double-positive cells (GFP-positive cells from bone marrow and rhodamine-BS1–positive host ECs) were considered as EPCs and counted in frozen sections of ischemic muscles: EPCs per high powered field (×100 magnification) were 3.5±0.4 cells for control mice and 7.6±0.5 cells for SN-treated mice (P<0.05, n=8; Figure 3a).
We also performed confocal fluorescence microscopy studies in hindlimb ischemia sections after SN gene therapy to improve colocalization of GFP+ bone marrow–derived cells and host ECs labeled by lectin (Figure 3b). Cells were additionally stained for Hoechst to show cell nuclei. Merging respective images (Figure 3b, right images) shows cells double positive for GFP and rhodamine–BS1 lectin (yellow cells labeled by arrows).
FACS analysis of muscle extracts 3 weeks after hindlimb ischemia and gene therapy showed that SN induced a significant increase of Sca1+ and Flk1+ cells of the Lin−/c-Kit+ cell population compared to saline (SN, 0.87±0.1%; saline, 0.38±0.04%; P<0.05; n=5; Figure 3c).
SN-Induced Angiogenesis Is Attenuated by Inhibition of the NO Pathway
The NO pathway plays an important role in angiogenesis. Therefore we tested if SN-induced angiogenesis is dependent on NO. We could show that SN induces activation (ie, phosphorylation) of endothelial NO synthase (eNOS) in human umbilical vein endothelial cell (HUVEC) by Western blotting with a maximum at 10 and 40 minutes (Figure 4a). SN-induced HUVEC migration was blocked by the NO-inhibitor l-NG-monomethyl-arginine (L-NMMA) (chemotactic index relative to control medium: SN [10 ng/mL]: 1.7±0.15; SN+L-NMMA [500 μmol/L]: 1.1±0.12; SN+d-NG-monomethyl-arginine [D-NMMA] [500 μmol/L]: 1.8±0.2; P<0.05: SN versus control, SN+L-NMMA; P=NS: SN versus SN+D-NMMA; n=3) (Figure 4b). SN-induced in vitro angiogenesis was performed in Matrigel, and capillary tubes were counted and normalized to control medium (relative capillary tube formation: SN [10 ng/mL]: 1.8±0.2; SN+L-NMMA [500 μmol/L]: 0.85±0.15; SN+D-NMMA [500 μmol/L]: 1.7±0.2; P<0.05: SN versus control, SN+L-NMMA; P=NS: SN versus SN+D-NMMA; n=3). Coculture of HUVECs with EPCs revealed that SN significantly induced association and incorporation of EPCs with vascular tubes and that this association could be blocked by L-NMMA (percentage of EPCs associated with tubes: control, 35.9±1.8%; SN, 62.8±2.6%; L-NMMA, 35.3±1.7%; D-NMMA, 34±0.9%; SN+L-NMMA, 40±1.8%; SN+D-NMMA, 63.2±2.3%; P<0.05, SN versus control and SN+L-NMMA; n=3; Figure 4c, right graph).
To extend these findings to in vivo angiogenesis we performed hindlimb ischemia operation and gene therapy and studied mice treated by GFP, SN and SN+L-NAME (Nω-nitro-l-arginine methyl ester, 0.5 mg/mL in drinking water). The group of mice treated with GFP plasmid and L-NAME could not be further studied because of high autoamputation rate (>80%). Blood perfusion, as measured by LDPI, was improved by SN gene therapy, but the effects of SN were inhibited by L-NAME (LDPI ratio 3 weeks after operation: GFP-treated mice, 0.58±0.05; SN-treated mice, 0.78±0.06; SN+L-NAME—treated mice, 0.52±0.04; P<0.05, SN versus GFP and SN+L-NAME; n=7; Figure 5a). L-NAME also inhibited SN-induced increase in capillary and arteriole density (CD-31–positive capillaries/mm2: ischemic side: GFP-treated, 295±21; SN-treated, 515±25; SN+L-NAME, 253±19; CD-31–positive capillaries/muscle fiber: GFP, 0.89±0.07; SN, 1.25±0.08; SN+L-NAME, 0.75±0.06; SMA-positive arterioles/mm2: ischemic side: GFP-treated, 6.1±0.7; SN-treated, 10.3±0.4; SN+L-NAME, 5.8±1; P<0.05, SN versus GFP and SN versus SN+L-NAME; n=7; Figure 5b and 5c).
We further studied if inhibition of NO might also inhibit EPC recruitment in vivo and treated bone marrow–transplanted mice after hindlimb ischemia operation with SN gene therapy and L-NAME. EPCs were analyzed as described above (cells double positive for GFP and rhodamine-labeled BS1 lectin). L-NAME treatment decreased EPCs in muscle sections to control levels (EPCs per high powered field [hpf]: control, 3.5±0.4 cells/hpf; SN, 7.6±0.5 cells/hpf; SN+L-NAME, 4.0±0.8 cells/hpf; P<0.05, SN versus control and versus SN+L-NAME; n=8 for control and SN; n=5 for SN+L-NAME; Figure 5d).
To evaluate whether SN gene therapy stimulates NO in vivo, muscle extracts were studied for NO content 3 days after induction of ischemia and SN or GFP plasmid injection. We found that SN gene therapy significantly increased NO in ischemic muscles (GFP, 8.2±0.4 μmol NO/g protein; SN, 12.3±0.4 μmol NO/g protein; P<0.05, n=4; Figure 5e).
Regulation of Angiogenic Cytokines in HUVECs by SN
HUVECs were treated with SN to evaluate regulation of other angiogenic cytokines by this factor. We observed that SN time- and dose-dependently upregulated mRNAs of b-FGF and PDGF-B. As shown by semiquantitative polymerase chain reaction (PCR), b-FGF was increased by SN with a maximum at 7 hours and at a concentration of 10 and 100 ng/mL (Figure 6a). Real-time PCR revealed significant increase in b-FGF mRNA (1.87±0.2 relative b-FGF RNA/18S RNA versus control; n=3, P<0.05; Figure 6b). ELISA of b-FGF showed significant increase of b-FGF protein by SN treatment (pg/106 cells: control, 8.1±1.5; SN, 17.6±1; n=4, P<0.05, Figure 6c). Other angiogenic factors, such as VEGF and angiopoietin 1 and 2, were not regulated by SN (data not shown).
PDGF-B was increased at 10 ng/mL after 24 hours of incubation and at 100 ng/mL after 12 and 24 hours of incubation (Figure 6d). Real-time PCR revealed significant increase in PDGF-B mRNA (2.35±0.24 relative PDGF-B RNA/18S RNA versus control; n=3, P<0.05; Figure 6e). To evaluate whether PDGF-B increased by SN mediates SN-induced effects, we performed EC chemotaxis (Figure 6f) and in vitro angiogenesis assays (Figure 6g) in the presence of a neutralizing PDGF-BB Ab. We observed that PDGF-BB Ab inhibited PDGF-BB–mediated chemotaxis and capillary tube formation in vitro but not SN-mediated effects (relative chemotaxis index: SN, 1.86±0.04; SN+PDGF-BB Ab, 1.83±0.05; PDGF-BB, 1.86±0.06; PDGF-BB+PDGF-BB Ab, 1.24±0.04; relative capillary tube formation: SN, 1.98±0.03; SN+PDGF-BB Ab, 1.85±0.05; PDGF-BB, 2.04±0.04; PDGF-BB+PDGF-BB Ab, 1.01±0.03; P<0.05, control versus SN, SN+PDGF-BB Ab, PDGF-BB; P<0.05, PDGF-BB versus PDGF-BB+PDGF-BB Ab; P=NS, SN versus SN+PDGF-BB Ab; n=3 to 4 independent experiments).
The main findings of our work are that gene therapy with a plasmid vector expressing the angiogenic cytokine SN induces therapeutic angiogenesis in the hindlimb ischemia model, is capable of recruiting EPCs to sites of neovascularization, and increases the number of α-SMA surrounded arteries, consistent with induction of arteriogenesis.
Several angiogenic cytokines, including VEGF, b-FGF, kinins, stromal cell–derived factor-1, or the morphogen sonic hedgehog, have been shown to improve tissue perfusion in the hindlimb ischemia model.10,20–23 In particular, gene therapy with these factors has been proven to be an effective therapy in the hindlimb and other models of ischemia.10,21,22,24 Also, treatment with neuropeptides, such as adrenomedullin or neuropeptide Y, or with nerve growth factor25–27 exerted beneficial effects. Therefore, we designed a plasmid vector for the neuropeptide SN, which was shown before to induce angiogenesis,15,16,18 and demonstrated that cells transfected with this vector secrete a functionally active SN molecule (Online Figures I and II). After intramuscular injection of SN plasmid into ischemic hindlimb muscles, we found significantly higher expression levels of SN for mRNA by PCR (maximum at 1 week) and for peptide by radioimmunoassay (after 1 week), indicating that recombinant SN is overexpressed in the ischemic muscle after SN plasmid injection (Online Figure II). This finding is consistent with reports using other plasmid vectors, eg, VEGF plasmid,28 which was detectable up to 2 weeks.10
After hindlimb ischemia operation, gene therapy was performed by a single injection of 50 μg of plasmid DNA. Function and tissue integrity of mice treated with SN improved because of increased blood perfusion, as measured by LDPI. SN gene therapy using a very low concentration of SN (5 μg) was not effective compared to GFP, whereas an intermediate dose (25 μg) showed complete recovery of perfusion after 4 weeks. This observation underlines the finding that unlike standard pharmacological therapies, gene-based delivery systems do not have a classic dose response. There is often a dose at which an effect is observed, whereas at lower doses, no effect is observed (with no intermediate response).24
As shown previously in the cornea model15 and in a stroke model of ischemia,18 SN increased density of capillaries, but vessels surrounded by α-SMA–positive cells representing arterioles and arteries were also increased. Recruitment of pericytes/SMCs is mediated by PDGF-BB secreted by stimulated ECs.7 We show in this work that SN induces SMC chemotaxis and association of SMCs with ECs in vitro (Online Figure VI). Although SN induced upregulation of PDGF-BB in ECs and SMCs, we found that SN-induced effects were not blocked by a PDGF-BB–neutralizing Ab, indicating direct, PDGF-BB–independent action of SN on SMCs and ECs (Online Figure VI). SN also upregulated b-FGF in ECs, but VEGF, another classic angiogenic cytokine, was not upregulated by SN in HUVECs. In this context, it might be interesting that VEGF was shown recently to inhibit vessel maturation and pericyte function when applied in combination with PDGF by inhibition of PDGF signaling.29
The NO pathway is important for angiogenesis, as shown by impaired stromal cell–derived factor–induced angiogenesis by inhibition of NO synthase, by stimulation of this pathway by a variety of angiogenic cytokines, and by induction of angiogenesis by NO donators and gene therapy with eNOS.22,30–33 We, therefore, investigated whether SN is capable of stimulating this pathway and found that SN induced stimulation of eNOS in HUVECs and that SN induced EC migration and in vitro angiogenesis, as well as that in vitro vasculogenesis was blocked by the NO inhibitor L-NMMA. Furthermore, inhibition of NO by L-NAME also inhibited therapeutic angiogenesis in vivo as shown by inhibition of SN gene therapy–mediated effect on blood perfusion (measured by LDPI) and on angiogenesis, arteriogenesis, and vasculogenesis (as shown by capillary, arteriole and EPC density).
We demonstrated in a previous publication that SN induced mobilization and incorporation of EPCs to sites of angiogenesis in vivo and exerted chemotactic and antiapoptotic effects on these cells in vitro.16 These effects might also play a role for SN-mediated EPC recruitment after SN gene therapy in hindlimb ischemia. In this work, we show by FACS analysis that SN-induced homing of EPCs to ischemic hindlimbs, but no significant mobilization was observed, probably because of lack of increased systemic SN levels after SN gene therapy (Online Figures II and IV). Further potential mechanisms, especially of SN-induced EPC homing to ischemic tissues, must be elucidated in future studies. Also perivascular cells,34 such as monocytic cells, might play a role in SN-induced vasculogenesis (Online Figure V).
In summary, this study demonstrates that intramuscular SN gene therapy induces therapeutic angiogenesis, arteriogenesis, and vasculogenesis, leading to improved function and tissue integrity in the mouse hindlimb ischemia model. SN-induced effects are dependent on the NO system, and SN upregulated other angiogenic cytokines, such as b-FGF or PDGF-B. Several angiogenic cytokines are under investigation in clinical trials for use in therapeutic angiogenesis.35 It remains to be determined in further toxicological studies whether SN gene therapy might also be applied in patients experiencing critical limb ischemia without options for standard revascularization.
We thank the Biooptics Core Facility of the Medical University Innsbruck for use of the confocal microscope.
Sources of Funding
This work was supported by Oesterreichische Nationalbank grant 10189 (to R.K.), Joseph Skoda Award of the Austrian Society of Internal Medicine (to P.S.), and Austrian Science Fund grant P 21021-B05 (to R.K.).
Original received April 20, 2009; revision received September 1, 2009; accepted September 17, 2009.
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