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Cellular Plasticity in the Cardiovascular System
1Department of Physiology, Cardiovascular Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112; and 2Department of Anesthesiology, Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
Submitted 12 February 2004 ; accepted in final form 31 March 2004
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ABSTRACT |
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angiogenesis; coronary circulation; therapeutic angiogenesis
The enormous potential of gene therapy to stimulate angiogenesis and collateral growth in the heart has yet to be realized. Several cytokines have been identified as putative agents mediating collateral growth and angiogenesis, including VEGF, basic fibroblast growth factor (bFGF), and platelet-derived growth factor (5, 7, 13, 24). Several of these factors have been used to treat patients with coronary or peripheral vascular disease. For example, VEGF is a potent angiogenic factor; it appears to be linked to collateral growth and is used in clinical trials applying various strategies, including viral vectors, naked DNA, or purified recombinant proteins (6, 10). Another clinical trial used bFGF to treat patients with IHD (25). Although animal experiments using gene therapy to stimulate angiogenesis have generally succeeded, the above-mentioned trials, and many others, for therapeutic coronary angiogenesis have generally failed. Although there are likely myriad reasons for failure, an obvious explanation is poor transfection efficiency.
In addition to poor transfection efficiency, there are other challenges for gene therapy, including directed or organ-specific transfection and nonspecific effects of the transfection, e.g., inflammation. Typically, gene therapy in the heart has been based on direct injection of plasmid or viral vectors into the myocardium. The main problem arising from injection at many sites is that the transfection is not uniform. Moreover, a sizable portion of the vector enters the bloodstream and, thus, transfects many other organ systems. To solve these problems, we propose that genes can be uniformly transfected into the myocardium via intracoronary administration of autologous cells. We believe that vascular smooth muscle cells (VSMC) are the ideal carrier, because they are easily obtained (and thus serve as an suitable autologous line), rapidly divide, and are efficiently transfected in culture. VSMC administered by intracoronary injection are expected to lodge mainly at the precapillary portions, because they are larger in diameter than capillaries [their estimated diameter from cell volume (2) is 14–18 µm] and, thus, serve to deliver a gene in an organ-specific manner. A further rationale for using cell-based gene therapy was shown by Yau et al. (32), who found that VEGF gene transfer by direct injection of cells into infarcted myocardium improved left ventricle perfusion. Thus cell-based gene delivery has potential as an organ-specific, angiogenic gene therapy.
Another important aspect of VSMC as carriers for genes pertains to their limited plasticity. There are indications that certain undifferentiated cells can evolve into VSMC (11, 12), but the general consensus is that, on expression of the VSMC phenotype (contractile or synthetic), they cannot revert to an undifferentiated cell (9, 20). This quality of limited plasticity can be advantageous if the intent is to use VSMC as a device for gene transfer; thus the differentiation or dedifferentiation of the cells is not a concern.
We previously studied coronary collateral growth using a repetitive coronary occlusion model and found that a repetitive occlusion protocol (2 min of occlusion every hour, 8 times/day, for 21 days) results in collateral-dependent flow equal in magnitude to that in the normal zone (21, 30). This increase in collateral flow was very rapid, and, within 3 wk, flow in the collateral zone equaled that in the normal zone. In the present study, to evaluate the angiogenic effect of this therapeutic strategy, we adapted the occlusion protocol so that collaterals would develop more slowly. For this reason, we implemented a protocol using four occlusions per day for 21 days and evaluated whether collateral development was accelerated after administration of VSMC transfected with VEGF.
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METHODS |
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Procedures. Mongrel dogs of either gender weighing 25–30 kg were used for these studies.
In an initial surgical procedure, a small piece of jugular vein wall was excised from anesthetized dogs to harvest autologous VSMC. Dogs were allowed to recover for 
3 wk. VSMC were isolated from the excised veins and established in culture by standard procedures (19). The cells were grown in DMEM containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.5 µg/ml amphotericin B. Confirmation that the cells were primarily VSMC was done by inspection of cell morphology. On the basis of these analyses and our experience with immunocytochemical staining for smooth muscle myosin and calponin, we believe that the cultures were 98% VSMC. Cultures of VSMC were passed as needed and split at 1:4 ratios.
In a second surgical procedure, dogs were anesthetized with propofol (50 mg/kg iv) and maintained with isoflurane (1.5–2.0%) in 100% oxygen via positive-pressure ventilation with a cuffed endotracheal tube. Arterial blood pH, PO2, and PCO2 were monitored at selected intervals with a blood gas analyzer (model 313, Instrumentation Laboratories). A left thoracotomy was performed under sterile conditions, and heparin-filled catheters were implanted in the thoracic aorta and right atrial appendage for measurement of arterial pressure and fluid administration, respectively. The heart was suspended in a pericardial cradle, and 1.5- to 2.0-cm sections of the proximal left anterior descending coronary artery (LAD; immediately distal to the 1st diagonal branch) were isolated. A balloon cuff vascular occluder (In Vivo Metric) was placed around the LAD for production of brief coronary occlusion. A heparin-filled catheter was positioned in the left atrial appendage for measurement of left atrial pressure and administration of radioactive microspheres for measurement of myocardial blood flow. An intramyocardial multiport catheter (0.8 mm OD, 0.4 mm ID, intramyocardial portion is Teflon catheter) was inserted into the LAD perfusion territory for sampling of myocardial interstitial fluid (MIF). All catheters and leads were secured, tunneled subcutaneously, and exteriorized between the scapulae via several small incisions. After the intracoronary cell transplantation, the chest wall was closed in layers, and the pneumothorax was evacuated by a chest tube. Each dog was fitted with a jacket to prevent damage to the instruments and catheters that were housed in an aluminum box within the jacket pocket. After surgery, each dog was treated with procaine penicillin G (25,000 U/kg im), gentamicin (4.5 mg/kg im), and the analgesic buprenorphine (0.02 mg/kg im). Animals were trained to stand quietly in a restraining sling.
MIF. MIF was collected from the LAD region each morning before subsequent experimentation. Isotonic saline (4 ml) was flushed into the catheter as 4 ml of aspirate were withdrawn. After collection, the sample was immediately placed on ice, aliquoted, frozen, and stored at –80°C until analysis.
Regional myocardial perfusion. Carbonized plastic microspheres (15 ± 2 µm diameter; NEN, Boston, MA) labeled with 141Ce, 103Ru, 51Cr, or 95Nb were used to measure regional myocardial perfusion, as described previously (30). Transmural tissue samples obtained from normal coronary artery perfusion territory (left circumflex coronary artery region) and the ischemic zone (LAD region) were selected for mapping of myocardial blood flow at the conclusion of each experiment.
Construction of expression vectors. pSEAP2-Control (Clontech, Palo Alto, CA) containing the secreted alkaline phosphatase (SEAP) gene was used to ascertain in vivo transfection efficacy. MIF SEAP expression was assayed with an SEAP chemiluminescence detection kit (Clontech).
pIRES2-EGFP (Clontech), which has an enhanced green fluorescence protein (EGFP) gene, was used as a control vector. A VEGF expression vector (pVEGF/EGFP) was constructed by cloning human VEGF-165 into the EcoRI site of pIRES2-EGFP. This plasmid has an internal ribosomal entry site (IRES) that allows two proteins to be expressed, so this vector coexpressed VEGF and EGFP in mammalian cells. DNA was transfected into the VSMC (P5) on the day before transplantation with LipofectAMINE PLUS Reagent (Life Technologies) according to the manufacturer's instructions.
Cell transplantation. Immediately before transplantation, the transfected cells were trypsinized, dispersed in PBS, and washed three times, with centrifugation for 5 min at 1,500 rpm. The supernatant was decanted, and the cells were resuspended in 5–16 ml of heparinized plasma from a host dog at 1–3 x 106 cells/ml (a total of 5 x 105 cells/kg). After filtration with a cell strainer (70-µm mesh), the cell suspension was injected into the LAD over 2 min through a 25-gauge needle temporarily placed into the proximal LAD. This intracoronary injection occurred at the second surgery when instruments were implanted.
In vivo experimental protocol. In all experiments, coronary collateral growth was evaluated in dogs treated with intracoronary injection of autologous VSMC containing plasmids expressing reporter genes (EGFP and SEAP) and/or VEGF. After the second surgery, dogs were allowed to recover for 7–10 days before commencement of the repetitive occlusion protocol. After recovery, 2-min occlusions were performed once every 2 h, 4 times a day, for up to 21 days. In preliminary experiments to ascertain the effectiveness of the transfection protocol, we administered VSMC that were transfected with pSEAP2-Control (n = 7). After completion of these experiments, we proceeded with the study of two groups: 1) a sham group of dogs (n = 6) was subjected to repetitive occlusions, but not cell transplantation, and 2) a control group of dogs (n = 4) received cells transfected with a control vector (pIRES2-EGFP) and repetitive occlusions. Cells, transfected with pVEGF/EGFP, were administered to the experimental group (VEGF group receiving repetitive occlusions, n = 7). Hemodynamics were monitored daily. MIF samples were collected from the perfusion territory of the LAD daily. We measured coronary collateral blood flow by injecting radioactive microspheres at day 0 (before initiation of the repetitive occlusion protocol to obtain native collateral flow) and at days 7, 14, and 21.
RT-PCR. To evaluate the distribution of transplanted VSMC, RT-PCR was employed for detection of EGFP mRNA. At the conclusion of the protocol, heart, lung, liver, kidney, and spleen tissues were excised and cleaned of adherent fat in 4°C phosphate-buffered saline. These tissues were flash frozen in liquid nitrogen and stored at –80°C until analysis. Total RNA was isolated using TRIzol (Life Technologies), and RT-PCR was performed. The sequence of EGFP sense primer was 5'-TGAACCGCATCGAGCTGAAGGG-3' and that of antisense primer was 5'-TCCAGCAGGACCATGTGATCGC-3'. The sequence of S15 sense control primer was 5'-TTCCGCAAGTTCACCTACC-3' and that of antisense primer was 5'-CGGGCCGGCCATGCTTTACG-3'.
ELISA. VEGF levels in MIF were quantified by ELISA (R&D) according to the manufacturer's directions. The results were compared with a standard curve constructed using dilutions of recombinant human VEGF (each assay carried out in duplicate for each MIF sample). The absorbance was measured at 450 nm using a microplate reader.
EGFP fluorescence. Frozen heart tissues were embedded in OCT compound (VWR Scientific products), and 10-µm-thick sections were sliced. EGFP fluorescence was observed by fluorescence microscopy (Eclipse TE2000-S, Nikon), and the images were captured with a charge coupled device camera (Photometrics Cool SNAP, Roper Scientific).
Statistical analyses. Values are means ± SE. Changes in parameters between groups and over time were compared by two-way ANOVA for repeated measurements. If significant differences were observed, the post hoc Bonferroni-Dunn test was employed to detect specific differences between the groups and across time. The level of significance was accepted at P < 0.05.
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RESULTS |
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EGFP mRNA expression was detected only in the heart, and not in other tissues (Fig. 1A). EGFP fluorescence was detected primarily along microvessels (Fig. 1B). In the VEGF group, VEGF levels in MIF were significantly elevated from the control group receiving EGFP plasmid only and consistent with values measured at early time points of a protocol when collateral development is stimulated (21). Although the levels progressively declined during the 21-day protocol, the values remained higher than those from the control or sham animals at all time points (Fig. 2).
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25% to a peak of >80%. Generally, the transfection efficiencies were 60–70%. Collateral blood flow. On day 0, collateral-dependent flow was similar in all groups. In the VEGF group, collateral-dependent flow was significantly increased at day 7 (0.56 ± 0.17 ml·min–1·g–1) and was higher at all successive time points until day 21 compared with control or sham animals (Fig. 3A). However, collateral-dependent flow did not reconstitute levels equivalent to normal zone blood flow (collateral flow was 69% of normal zone flow at day 21; Fig. 3B).
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DISCUSSION |
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Methodological considerations. Several kinds of cells have been used as vector cells for cell-based angiogenic gene therapy (3, 14, 28, 32). One advantage to using autologous cells is the elimination of the problem associated with allotypic differences. Autologous cells do not appear to elicit inflammatory reactions, which may potentially lead to graft rejection. Although autologous stem cells are proposed donor cell candidates for cell therapy (26), these cells have the capacity to differentiate into unexpected cell types with unforeseen ramifications (17). It is reasonable to assume that modification of these stem cells with new genes would further increase the risk for cell changes. So, terminally differentiated cells or certain specific progenitor cells will be more suitable for cell-based gene therapy.
Some cells, e.g., skeletal muscle myocytes, when transplanted into the heart, can increase arrythmogenic risk (22). Direct cell injection into the myocardium would seem more likely than intracoronary administration to produce arrhythmias because of myocardial injury and heterotopic cell transplantation. Even with intracoronary administration, it is possible that some types of cells will migrate out of the vascular system by "homing" to an area of chemoattraction. Intracoronary administration of VSMC has the potential to avoid these problems, because VSMC are among the major components of the vascular wall, and on the basis of our images, VSMC localize around the vessel. This is important, because they will, therefore, not affect the impulse-conducting system. Another facet of using VSMC to carry genes is that the cells can be harvested from a small piece of vascular tissue (and thus be autologous and eliminate immune reactions), and they are easily amplified in culture. VSMC grow robustly in culture and are efficiently transfected with plasmids (16). Moreover, the ability to migrate is important, because the initial injection of 5 x 105 cells/kg produces some coronary embolization; yet within 48 h the cells have migrated from the lumen to the interstitium. Campbell et al. (3) also reported that VSMC injected into the jugular vein were found within small arterioles in the lung, and the majority of these cells transmigrated through the endothelial layer within 24 h. On the basis of the reasons cited above, VSMC have the potential to be an ideal vector cell to carry genes for potential therapies.
We opine that it is the lack of plasticity in VSMC that makes them an ideal carrier for genes. Although it could be argued that the phenotype of the VSMC could shift toward a synthetic cell, which could accelerate the process of intimal hyperplasia, this was not our observation; we observed the VSMC taking up residence on the outside of small microvessels. Because nitric oxide confers a quiescent phenotype (31) and VEGF stimulates endothelial production of nitric oxide (15), we speculate that the transfection of VEGF would also serve to prevent the development of the synthetic phenotype.
We traced EGFP gene transfected into VSMC by RT-PCR and imaging techniques. We detected EGFP only in the heart, and the protein was visualized around microvessels, implying that VSMC migrated out of the small vessels and became resident in the proximity of the blood vessel. We speculate that this feature of VSMC gene transfer makes VSMC ideal for therapeutic angiogenesis applications, because the secreted products are localized to the area of blood vessels. Thus not only are the cells targeted to an organ, but they naturally target to the vasculature, which makes them ideal for therapeutic angiogenesis.
We believe that gene transfer via autologous VSMC is cardiac specific and homogeneous. There is a potential risk for side effects when cell-based organ-specific delivery of genes encoding secretory proteins is utilized, because secreted protein will enter the bloodstream. It has been proposed that gene delivery into another organ, such as the liver, would be expected to produce effects in the heart (18). In this aspect, VSMC seem better than endothelial progenitor cells, which are also proposed as vehicle cells for gene therapy (14), because endothelial progenitor cells can flow through capillaries if they do not home to a tissue site (1).
Suzuki et al. (27) reported that 1 x 106 skeletal myoblasts were successfully grafted into the rat heart through intracoronary infusion, but 1 x 107 cells caused a large myocardial infarction. In the dog heart, however, we estimated that 1 x 106 cells/kg was the maximum limit for cell injection into a major coronary artery without deleterious effects. This estimation was based on our previous microsphere study in which up to 5 x 106 25-µm microspheres were administered by intracoronary infusion without evidence of dysfunction or infarction (4). In the present study, we applied 5 x 105 cells/kg VSMC without occurrence of infarction, arrythmias, or sustained alterations in hemodynamics (pressures and heart rate). Moreover, administration of cells expressing EGFP did not induce collateral development or VEGF expression. Ninomiya et al. (23) injected fibroblasts (5 x 106) transfected with bFGF into the coronary circulation in pigs. These investigators reported that this treatment improved function and collateral blood flow in a model of chronic myocardial ischemia.
Collateral development. Collateral-dependent flow was the measured index of collateral development. Collateral flow was significantly increased within 7 days in the VEGF group from the beginning of the repetitive occlusion protocol. In the final 2 wk of the protocol, collateral flow never reached the level attained in the eight-occlusion group. This could be due to 1) a decrease of VEGF expression that was observed in the transfection group and/or 2) the requirement that VEGF work in concert with other factors to produce optimal coronary collateral growth.
We previously found that a repetitive 2-min coronary occlusion protocol of eight occlusions per day for 21 days results in collateral-dependent flow equal in magnitude to that in the normal zone (21, 30). In the present study, we implemented a protocol using four occlusions per day for 21 days. This very weak ischemic stimulation with a four-occlusion protocol did not promote collateral development in the absence of VEGF: no collateral growth in sham or control EGFP groups. In contrast, coronary collateral flow increased in the VEGF group. We initiated the repetitive occlusion protocol 7–10 days after cell transplantation. Although MIF VEGF level was significantly elevated (132 pg/ml at day –3) in the VEGF group in this preocclusion protocol period, the collateral-dependent flow was not increased at the beginning of the occlusion protocol and rapidly increased during the first 7 days of the protocol. This implies that ischemia is a prerequisite for the actions of VEGF to exert its effects on collateral growth. Taken together, ischemic stimulation with four occlusions per day is not sufficient to produce collateral growth, but when VEGF is introduced via autologous VSMC, collateral growth occurred.
Our results of accelerated collateral growth are even more dramatic than those in the literature using gene therapy (32) or therapy with mesenchymal stem cells (29). Specifically, we found that, within 1 wk, collateral flows were 50% of that in the normal zone. This is roughly a doubling of the rate of collateral development observed when we stimulated growth using a canine model with eight occlusions per day (21, 30).
In conclusion, cardiac-specific gene delivery was achieved using intracoronary administration of autologous VSMC transfected with the VEGF gene. This protocol effectively promotes collateral development and may provide an effective means by which the full potential of angiogenic gene therapy may be realized.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1B-adrenoceptor gene expression in aortic and coronary smooth muscle is influenced by cyclical stretch. In Vitro Cell Dev Biol Anim 31: 595–600, 1995.
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P < 0.05 vs. sham and control.