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CALL FOR PAPERS
Regulation and Function of Stem Cells in the Cardiovascular System
1Cardiovascular Division, Department of Medicine, University of Minnesota, Minneapolis, Minnesota; and 2Department of Biochemistry, University at Buffalo, Buffalo, New York
Submitted 12 August 2005 ; accepted in final form 11 December 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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mesenchymal stem cells; left ventricular hypertrophy; metabolism; vascular endothelial growth factor; vessels; perfusion
Both experimental and clinical evidence indicates that cellular transplantation into the LV myocardium improves the LV contractile performance of failing hearts (3, 18, 20, 21, 25, 28). The underlying mechanisms of this improvement remain unclear. Transplanted cells may regenerate myocytes and new vessels (21, 30). They also release cytokines such as vascular endothelial growth factor (VEGF) that can exert a trophic effect on host cardiac cells (11, 26, 29). Thus VEGF is a multifunctional growth factor that not only promotes neovascularization but also regulates cell proliferation, migration, and survival. In skeletal muscle, VEGF also has beneficial effects on oxidative respiration that are independent of the increased neovascularization (33).
Cell transplantation into diseased (usually ischemic or infarcted) hearts typically delivers cells into or adjacent to a poorly perfused segment by using intramyocardial injection. These regions are often partially necrotic and infiltrated by macrophages and other immune-responsive cells. Under these conditions, the majority of transplanted cells die, although LV function improves (31). An alternative method of stem cell delivery via coronary artery catheter in normal dogs resulted in significant occlusion of microvessels by the stem cells that resulted in microinfarctions (34). In contrast, the cardiac venous system has robust collateral vessels, and regional coronary venous occlusion does not induce myocardial injury. Hence, coronary venous retroperfusion is being evaluated as a technique for virally based gene therapy and cell delivery to myocardium (4).
The aims of the present study were 1) to evaluate the efficacy of coronary venous delivery of autologous bone marrow mesenchymal stem cells (MSCs) into pressure-overloaded LV myocardium and 2) to determine the myocardial structural and functional consequences of autologous MSCs transplantation on the progression of LVH after ascending aortic banding. We hypothesized that transplantation of both unmodified MSCs and VEGF-overexpressing MSCs (VEGF-MSCs) would attenuate LVH and improve myocardial perfusion, contractile function, and bioenergetic characteristics. We further hypothesized that improvement would be greatest in hearts receiving VEGF-MSCs.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-galactosidase reporter gene lac-Z was purchased from the University of Iowa Gene Transfer Vector Core (1). Swine VEGF165 expression vector was kindly provided by Dr. J. Canty (State University of New York at Buffalo). Isolation of Swine MSCs
MSCs from bone marrow were isolated by gradient density centrifugation (21, 27). Bone marrow was aspirated from the sternum of healthy Yorkshire pigs into a syringe containing 6,000 U heparin and diluted with Dulbecco's PBS in a ratio of 1:1. The marrow sample was carefully layered onto the Ficoll-Paque-1077 (Sigma) in a 50-ml conical tube and centrifuged at 400 g for 30 min at room temperature. The mononuclear cells were collected from the interface, washed with 2–3 vol of Dulbecco's PBS, and collected by centrifugation at 1,000 rpm. The cells were resuspended and seeded at a density of 200,000 cells/cm2 in T-75 flask coated with 10 ng/ml fibronectin and cultured in medium consisting of 60% low-glucose Dulbecco's modified Eagle's medium (GIBCO BRL), 40% MCDB-201 (Sigma), 1 x insulin transferin selenium, 1 x linoleic acid bovine serum albumin, 0.05 µM dexamethasone (Sigma), 0.1 mM ascorbic acid 2-phosphate, 2% FCS, 10 ng/ml PDGF, 10 ng/ml EGF, 10 U/ml penicillin, and 100 U/ml streptomycin. After 3 days, nonadherent cells were removed by replacing the medium. The attached cells grew and developed colonies in 
5–7 days. After 10 days, the primary cultures of MSCs reached nearly 90% of confluence; cells were subcultured by incubation with trypsin. The first passage cells were plated at 4,000–5,000 cells/cm2 and further cultured 2 days for the transduction with VEGF or AdRsvlac-Z.
Cell Phenotype and Differentiation Potential Phenotype
CD44, CD45, CD90, myosin heavy chain (MHC) class I, MHC class II, SWC3A, and SLA-DR were detected by flow cytometry. MSCs (0.5–1 x 106) were placed in 100 µl BSA/PBS solution for each phenotype test and incubated with 2 µg primary mouse monoclonal antibodies (MAbs) against pig CD44, CD45, CD90, MHC class I, MHC class II, SWC3A, and SLA-DR for 40 min at 4°C. The second polyclonal antibody IgG against mouse, FITC conjugated (1 µg/tube) was added and incubated at 4°C for an additional 30 min in a dark room. Mouse IgG (2 µg) instead of primary MAbs was added to 0.5–1 x 106 cells for a negative control.
Adenoviral Transduction
VEGF was subcloned to the shuttle vector pacAd5CMVK-Np. Viruses were prepared and titrated by the Gene Transfer Vector Core Laboratory at University of Iowa. Adenovirus infections were performed 24 h after plating. Cells were incubated for 3 h at 37°C with 0.5 ml of serum-free culture medium containing the virus at the appropriate concentration and then re-fed with fresh 2% serum medium. Viral concentrations used for transduction of the nuclear -galactosidase reporter gene lac-Z were described previously (21).
Persistence of Adenoviral Transduction in In Vitro MSCs
It has been frequently reported that, in in vivo studies of myocardium transfected with adenovirus vectors, expression of the transduced gene peaks at 7 days after transduction and then rapidly declines (e.g., see Ref. 22). Possible mechanisms of the persistent presence of lac-Z and VEGF gene expressions observed at 4 wk after transplantation were examined in two in vitro experiments. In the first experiment, swine MSCs expressing the transduced reporter gene lac-Z were cultured under either low-density (1,000 cells/cm2) or high-density conditions (i.e., to confluence). This experiment was designed to examine whether transgene expression persisted if MSCs proliferation was limited. Culture media were changed every 2 days. In the second experiment, swine MSCs expressing swine VEGF165 DNA adenovirus were studied under the culture conditions described above. VEGF mRNA expression was quantified by quantitative real-time RT-PCR (QRT-PCR).
Effects of VEGF-MSCs on HL-1 Cell Apoptosis
HL-1 myocytes (from Claycomb Laboratory, University of Louisiana) were plated onto fibronectin-gelatin-coated plates or flasks and cultured in Claycomb medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1 mM norepinephrine, and 2 mM L-glutamine as previously described (7, 37). For the conditioned medium experiment, swine MSCs were transfected with 100 plaque-forming units (pfu)/cell of VEGF adenovirus and nuclear lac-Z adenovirus. Six hours after infection, cells were washed three times and placed in a MSC culture medium as previously described (20). The conditioned medium was harvested 48 h after culture. As a control, a portion of the medium was incubated for 48 h without MSCs. HL-1 cells were cultured in the conditioned medium for 24 h, during which time they were exposed to 2% oxygen (hypoxia) to induce apoptosis. For the coculture experiment, HL-1 cells were plated in 12-well plates at a total density of 5 x 105 (1:30 ratio of MSCs to HL-1 cells) in half MSC medium and half Claycomb medium. Before being cocultured, HL-1 cells were labeled with Vybrant CFDA SE cell tracer kit (Molecular Probes). Labeled HL-1 cells were extensively washed and cocultured with MSCs- or VEGF-modified MSCs. The cell cocultures were then incubated for 24 h at 2% oxygen (hypoxia) or 21% oxygen (normoxia). Apoptosis was assessed by staining with Hoechst 33342 (H33342 [GenBank] ) dye and then quantifying the percentage of apoptotic nuclei (300 cells counted per sample) in the CFDA-labeled subset by identifying cells with H33342 [GenBank] staining (35, 36).
Induction of LVH and Cell Transplantation
Severe concentric LVH was produced in swine as previously described in detail (38). Briefly, Yorkshire pigs (45 days old) were anesthetized with pentobarbital sodium (25–30 mg/kg iv), intubated, and mechanically ventilated. A right thoracotomy was performed. While LV and distal aortic pressures were simultaneously measured, the band was tightened until a 55- to 60-mmHg peak systolic pressure gradient was achieved across the narrowing. A silicone elastomer catheter (1.0 mm ID) was inserted into the interventricular cardiac vein. The vein was ligated proximal to the point of catheter entry, and 30 million autologous VEGF-MSCs or MSCs were slowly injected through the catheter. The catheter was then removed, and the entry site was repaired. The chest was closed in layers, and the animal was allowed to recover. LVH developed progressively because the area of aortic constriction remained fixed in the face of normal body growth. Approximately 4 wk after banding, the animals were returned to the laboratory for MRI, magnetic resonance spectroscopic (MRS), and hemodynamic measurements.
Magnetic Resonance Imaging Studies
All MRI studies were performed on a Siemens Medical Vision System operating at 1.5 T 3 days before the final MRS and physiological study. The animals were sedated with ketamine (20 mg/kg im), anesthetized with pentobarbital sodium (30 mg/kg, iv), intubated, and ventilated with a respirator. Animals were placed on their left side in an 18-cm diameter Helmholtz coil. Imaging sequences were gated to the ECG while respiratory gating was achieved by triggering the ventilator to the cardiac cycle between data acquisitions. A detailed account of the imaging and analysis methodologies has been reported previously (23, 41). LV systolic wall thickening fraction (ST%) was measured in the anterior wall by using the following equation: ST% = 100% x (ls – ld)/ld, where ls is LV thicknesssystole (in mm) and ld is LV thicknessdiastole (in mm).
31P and 1H NMR Spectroscopic Techniques
Measurements were performed in a 40-cm bore, 4.7-T magnet interfaced with a SISCO (Spectroscopy Imaging Systems, Fremont, CA) console. Radio frequency transmission and signal detection were performed with a 25-mm diameter coil sutured to the epicardium. The LV pressure signal was used to gate NMR data acquisition to the cardiac cycle while respiratory gating was achieved by triggering the ventilator to the cardiac cycle between data acquisitions (19, 40). 31P and 1H NMR frequencies were 81 and 200.1 MHz, respectively. Spectra were recorded in late diastole with a pulse repetition time of 6–7 s. This repetition time allowed full relaxation for ATP and inorganic phosphate (Pi) resonances and 95% relaxation for the phosphocreatine (PCr) resonance (19, 23). PCr resonance intensities were corrected for this minor saturation; the correction factor was determined for each heart from two spectra recorded consecutively without transmural differentiation, one with 15 s repetition time to allow full relaxation and the other with the 6 s repetition time used in all the other measurements. Detailed descriptions of 1H and 31P spectroscopic methods have been published previously (19, 23, 40).
31P resonance intensities were quantified by using integration routines provided by the SISCO software. ATP-
resonance was used for ATP determination. Because data were acquired with the transmitter frequency positioned between the ATP- and PCr resonance, off-resonance effects on these peaks were virtually nonexistent. The numerical values for PCr and ATP in each voxel were expressed as ratios of PCr/ATP. Pi levels were measured as changes from baseline values (
Pi) by using integrals obtained in the region covering the Pi resonance. 1H deoxymyoglobin resonances were only detectable during occlusion of the coronary artery supplying the myocardium under the NMR coil. Hence, there was no evidence of myoglobin desaturation under basal state or HCW conditions as previously reported (23).
Myocardial Blood Flow
Myocardial blood flow was measured by using 15-µm-diameter microspheres labeled with gamma-emitting radionuclides (141Ce, 51Cr, 95Nb, 85Sr, or 46Sc) as described previously (9, 41).
Experimental Preparation for MRS Study
Animals were anesthetized with pentobarbital sodium (30 mg/kg iv), intubated, and ventilated with a respirator with supplemental oxygen. Arterial blood gases were maintained within the physiological range by adjustments of the respirator settings and oxygen flow. A heparin-filled polyvinyl chloride catheter (3.0 mm OD) was introduced into the right femoral artery and advanced into the ascending aorta. A sternotomy was performed and the heart suspended in a pericardial cradle. A second heparin-filled catheter was introduced into the LV through the apical dimple and secured with a purse-string suture. A similar catheter was placed into the left atrium through the atrial appendage. A 25-mm-diameter NMR surface coil was sutured onto the LV anterior wall. The pericardial cradle was then released, and the heart was returned to its normal position. The surface coil leads were connected to a balanced-tuned circuit external and perpendicular to the thoracotomy incision. The animals were then placed in a Lucite cradle and positioned within the magnet.
Experimental Protocol
Hemodynamic measurements and 31P NMRS spectra were first obtained under basal conditions. Midway through the 10-min NMR spectroscopic acquisition period, a microsphere injection was performed for determination of myocardial blood flow. Arterial blood gases were measured every 10 min, and the respirator was adjusted to maintain the normal physiological PO2, PCO2, and pH. After baseline data were obtained, dobutamine and dopamine were infused simultaneously (each 20 µg·kg–1·min–1 iv) to induce a high cardiac workstate (HCW). After allowing 10 min to achieve a steady state, all measurements were repeated.
Cell Engraftment Rate Determination
After the terminal MRS study (4 wk after cell transplantation), every heart was cross-sectioned into 8 to 10 rings. Odd-numbered rings were used for histological studies, and even-numbered rings were snap frozen for QRT-PCR. For histological analysis, every ring was divided into 10 to 12 pieces. After X-gal staining, tissues were embedded in Tissue-Tek OCT compound (Fisher Scientific) and frozen in a liquid nitrogen-cooled isopentane. Frozen tissue sections (10-µm thick) were sectioned on a cryostat. Total cell nuclei were stained with 4',6'-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). The engraftment cell number was analyzed by X-gal and DAPI double-positive nuclei in every 10th serial sections.
RNA Isolation and cDNA Preparation
Snap-frozen LV specimens were pulverized in liquid nitrogen. Total RNA was isolated by using RNeasy columns with RNase-free DNase treatment. Total RNA (1 µg) was used for reverse transcription reactions with oligo (dT)18 as a primer. Primer sequences and reaction parameters are depicted in Table 1.
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Changes in mRNA levels under different experimental conditions were compared by QRT-PCR analysis with the use of the Light Cycler Thermocycler (Roche Diagnostics) as previously described (36).
5-Bromo-2'-deoxyuridine Analysis
The functional activity of VEGF secreted by the transduced MSCs was assessed via a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay (Boehringer Mannheim, Tokyo, Japan) of endothelial cells cultured in conditioned media harvested from VEGF-MSCs.
Immunohistochemistry and immunofluorescenses
Tissue samples were chilled in cold 2-methylbutane for 1 h, embedded in Tissue-Tek OCT (Fisher Scientific), and sectioned into 10-µm slices with the use of a cryostat. Immunohistochemistry and immunofluorescence staining was performed as previously described (35, 36). Mouse anti-human von Willebrand factor (vWF) antibody and fluorescence-labeled secondary antibodies (Molecular Probes) were used.
Data Analysis
One-way ANOVA identified the presence of significant differences in group means. Scheffé's multiple comparison test evaluated the significance of pairwise differences between group means. Differences were considered statistically significant at a value of P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Autologous VEGF-MSCs. MSCs isolated from adult swine bone marrow were positive for CD44, CD90, SWC3A, and human leukocyte antigen (HLA) class I and were negative for CD45 and HLA class II; these findings are in agreement with a previous report (8). Adenoviral transductions of MSCs with swine VEGF165 DNA and the lac-Z DNA control were 90% efficient. QRT-PCR detected both endogenous and exogenous porcine VEGF mRNA. VEGF-MSCs transduced at 10 pfu/cell and 100 pfu/cell expressed VEGF mRNA at levels, respectively, 10 and 30 times greater than the endogenous VEGF mRNA levels in lac-Z-MSCs (Fig. 1A). Immunohistochemical evaluation confirmed the significantly increased expression of VEGF in transduced MSCs (Fig. 1B).
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36 h, X-gal staining had almost disappeared within 7 days (data not shown). In contrast, under high-density culture conditions, during which MSC proliferation was inhibited, the X-gal staining remained unchanged for 4 wk (data not shown). Similarly, in the low-density culture group, VEGF mRNA expression was almost absent at 7 days (data not shown), whereas in the high-density culture, VEGF mRNA expression was 14-fold and fivefold greater than that of similarly cultured nontransduced MSCs at 2 wk and 4 wk after transduction, respectively (Fig. 1C). These data demonstrate that, under in vitro conditions, the increased -galactosidase and VEGF mRNA expressions induced by adenovirus transduction can be maintained for at least 4 wk when MSC proliferation is limited. We assessed the functionality of VEGF secreted by VEGF-MSCs in two ways. The first employed a BrdU incorporation assay by using human umbilical endothelial cells (HUVECs) cultured in the conditioned media harvested from VEGF-MSCs (Fig. 2A). HUVECs were cultured in normal MSCs medium for 24 h followed by 24 h in serum-free medium and then incubated in the conditioned medium obtained from either VEGF or lac-Z transduced MSCs (with 10 µl of BrdU solution added) for 12 h (stock solution was 1 mM BrdU in Dulbecco's PBS). A negative control was established by using 10 µl of plain PBS. BrdU labeling was positive in 35 ± 4% of the HUVECs cultured in VEGF-MSCs conditioned media but essentially absent in HUVECs cultured in lac-Z-MSCs conditioned media (Fig. 2A), suggesting that functional VEGF was secreted by the transduced MSCs. The lack of response of HUVECSs to media conditioned by lac-Z-MSCs also suggests that the quantities of endogenous VEGF or other growth factors that might be secreted by lac-Z-MSCs under these conditions were below the threshold of detection of this bioassay.
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In Vivo Studies
Anatomic data.
Nine of the 19 subjects in the untreated LVH group developed ascites (100–1,000 ml), and these animals had increased LV end-diastolic pressure values. Taken together, these data indicated the presence of biventricular decompensation. Therefore, we divided the untreated LVH group into two subgroups based on the presence or absence of ascites [LVH and congestive heart failture (CHF), respectively]. Ascites was absent in both MSCs-treated groups of banded animals. The LV weight-to-body weight ratio (LVW/BW, in g/kg) increased by 50% in the untreated LVH group without ascites as well as in both LVH groups that received MSCs (P < 0.05; Table 2). In contrast, LVW/BW was increased by 118% in CHF hearts (P < 0.05 vs. all other groups; Table 2). Concordantly, the ratio of right ventricular weight to BW was increased in all banded groups; however, this increase was only significant in the CHF group (P < 0.05 vs. all other groups; Table 2). Hence, both MSCs and VEGF-MSCs prevented the development of the severe LVH and decompensation that developed in 49% of the untreated group of pressure-overloaded hearts.
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Myocardial blood flow data. The regional myocardial blood flow data are summarized in Table 4. In both the anterior and posterior walls, basal state blood flows were moderately (but significantly; P < 0.05) higher in the VEGF-MSCs group than the other groups (Table 4). At HCW, myocardial blood flow rose substantially in all groups (P < 0.05; Table 4). This increase was significantly higher in the VEGF-MSC group (P < 0.05).
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40% more energy (as reflected in the RPP data shown in Table 4) than achieved by the group with the next highest RPP. Myocardial inorganic phosphate levels, expressed as Pi/PCr (zero at baseline because of the inability to detect intracellular Pi), rose significantly (P < 0.05) during HCW in all but the LVH + VEGF-MSCs group (N = 0.26 ± 09; LVH = 0.23 ± 0.08; CHF = 0.50 ± 0.04; LVH + MSCs = 0.17 ± 0.08; LVH + VEGF-MSCs = 0.04 ± 0.05). It should be noted that Pi released from the hydrolysis of PCr (as reflected by the fall in the PCr/ATP ratio) was the source of the increased cytosolic Pi during HCW. Transmurally differentiated spectra obtained at baseline and during HCW indicated that the observed PCr/ATP reductions were most prominent in the subendocardial (data not shown). These data indicate that VEGF-MSCs transplantation improves the bioenergetic response to HCW in pressure-overloaded myocardium even though it does not significantly attenuate the basal state reduction of PCr/ATP.
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-galactosidase and cardiac-specific protein (TnT, TnI, MHC, and phospholamban, suggesting cardiomyocytes differentiation), the number of these cells is too small to have direct contribution to the LV contractile function. In addition, there is a possibility that these cells are derived from fusion of transplanted stem cells with native myocytes (2, 24). MSCs promote neovascularization. Figure 4A illustrates the robust neovascularization promoted by engrafted VEGF-MSCs. Immunofluorescence staining for vWF indicated significant angiogenesis in VEGF-MSCs-treated hearts, with more vWF-expressing capillaries in LVH + VEGF-MSCs transplanted hearts compared with normal, LVH, and LVH + MSCs-transplanted hearts. The mean number of vWF-positive capillaries per high-power field was 42 ± 5 in the VEGF-MSCs-treated LVH group compared with 32 ± 2 for the normal group, 27 ± 3 for the untreated LVH groups, and 34 ± 2 for MSCs-treated group (n = 6 pigs; P < 0.01; VEGF-MSCs vs. other groups). Additionally, quantitative RT-PCR revealed a significant increase in vWF mRNA expression in the VEGF-MSCs-treated LVH hearts compared with normal, untreated LVH, and MSCs-treated LVH hearts (n = 5 pigs; anterior wall; P < 0.01; Fig. 4B). In the group transplanted with non-VEGF-transduced MSCs, vWF mRNA was also significantly increased compared with the untreated LVH group. VEGF mRNA expression in VEGF-MSCs-treated hearts was also significantly elevated when compared to normal, untreated LVH, and non-VEGF-transduced MSCs-treated hearts (n = 5 pigs; P < 0.001; Fig. 4C).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Stem Cell Delivery via the Coronary Venous System
Passage of infused cells through the walls of microvessels and their consequent infiltration into tissues occurs at the site of postcapillary venules. Because the heart possesses rich postcapillary venous collaterals, we assumed that autologous MSCs delivered via a coronary venous catheter would flow retrogradely into these small venules and hypothesized that obstruction of outflow of the infused vein would result in an increase of the dwell time of MSCs and facilitate engraftment. The histological data indicate that MSCs became engrafted in the whole heart (anterior wall > posterior wall) without the development of areas of micronecrosis that have been reported after coronary artery infusion of MSCs in some (34) but not all studies. The current findings are consistent with previous studies of the efficacy of adenoviral gene delivery by coronary venous retrotransfusion in which the efficacy and transmural homogeneity of transduction were both far superior to those achieved by coronary artery administration (4). Many recent advances in coronary vein catheterization have been made by clinical cardiac electrophysiologists (6). Hence, it is likely that the coronary venous approach for cell delivery can be adapted for percutaneous catheter-based MSCs administration.
VEGF-MSCs Engraftment Promotes Neovascularization and Increases Myocardial Blood Flow
The experimental data demonstrate that, at 4 wk after transplantation, tissue VEGF mRNA levels are 3.3 times greater in LVH hearts receiving VEGF-MSCs than in untreated LVH and normal hearts. Furthermore, this change was accompanied by enhanced angiogenesis in hearts with engrafted VEGF-MSCs as evidenced by a significantly increased myocardial capillary density in that group. Although a very small number of MSCs-derived endothelial cells were detected in the VEGF-MSCs-transplanted LVH hearts (data not shown), their small numbers preclude any significant contribution to the increase in capillary density. Both basal and HCW myocardial blood flows were significantly higher in both the anterior and posterior LV walls of VEGF-MSCs-treated hearts than in other groups. Although the comparatively high myocardial blood flow values in the HCW VEGF-MSCs group can probably be explained by the very high RPP values present in that group, the explanation is not valid for basal state measurements in which RPP values in the LVH groups were comparable. Therefore, the basal state myocardial blood flow measurements indicate that the aforedescribed neovascularization as a structural basis has a functional correlate.
In hypertrophied nonfailing and failing hearts, increased systolic wall stress of the LV would be expected to increase myocardial energy demands while oxygen delivery might be limited by an impaired coronary flow reserve (10, 15). These and other considerations have led to the notion that a chronic imbalance of the energy supply-demand relationship might contribute to impaired contractile performance and altered bioenergetic characteristics of the hypertrophied and failing hearts (16). Consistent with this view, concentric LVH secondary to ascending aortic banding is associated with vascular rarefaction that is most prominent in the subendocardial layer of the LV wall (5, 32). This factor undoubtedly contributes to the physiological finding that the subendocardial layer in hearts with LVH is most vulnerable to ischemia during tachycardia (13, 40). In this regard, it is interesting to note that the VEGF-MSCs-induced increase of basal state myocardial blood flow is most prominent in mid-myocardium and the subendocardial layer layers of the LV (Table 4).
MSCs Transplatation Increases Myocardial Contractile Reserve
In previous studies (12, 23, 39), we employed catecholamine infusion to determine maximal inducible contractile performance and M
O2. In the present study, maximal catecholamine-induced elevation of RPP was used to determine the in vivo CR of the experimental groups. The RPP of LVH + VEGF-MSCs hearts during catecholamine stimulation was 40% greater than that of any of the other banded groups and reflected the higher LV systolic pressure present in that group. Impressively, CR of VEGF-MSCs group was comparable to that of normal hearts (165%) despite the markedly higher afterload present in the grafted group. Moreover, the higher CR present in the VEGF-MSCs group occurred despite the presence of LVH of severity comparable to that present in both the untreated compensated LVH group and the LVH group treated with nontransduced MSCs. The systolic thickening fraction data also demonstrate an increase of catecholamine-induced contractile performance in hearts that received MSCs and VEGF-MSCs transplantation (P < 0.05; Table 4). Hence, both hemodynamic and MRI wall thickening data indicate that VEGF-MSCs transplantation at the time of induction of pressure overload is associated with preservation of myocardial contractile capacity despite the development of moderate LVH.
MSCs Transplantation Improves Myocardial Bioenergetics
In normal dogs, myocardial HEP levels and PCr/ATP do not change over moderate ranges of workload (RPP up to 30–35,000 mmHg·beats·min–1) produced by pacing and catecholamine infusion (17, 39). In contrast, at higher cardiac workloads (RPP > 35,000–40,000), we and others observed that myocardial PCr levels fell and calculated ADP levels rose (39). These data suggest that when the rate of ATP expenditure rises sufficiently (the specific RPP value is species dependent), then cytosolic free ADP levels must increase to maintain the steady-state rate of mitochondrial ATP synthesis (17, 23, 39). We have also reported that reductions of basal state PCr/ATP were linearly correlated with the severity of LV chamber hypertrophy and dysfunction in pressure-overloaded hearts (38). This abnormality was most severe in hearts showing evidence of decompensation (12). The data from the present study are consistent with the earlier data because both untreated and MSCs-transplanted LVH groups showed reduced basal state PCr/ATP levels, and these reductions were most severe in the CHF group. Also, consistent with previous observations, during HCW, PCr/ATP fell significantly in normal hearts and untreated LVH hearts and also in MSCs-treated LVH hearts. Notably, the HCW-associated fall in PCr/ATP was not present in the VEGF-MSCs group despite the much higher RPP value achieved by that group. Although it has been shown that the HCW-associated fall of PCr/ATP in normal and hypertrophied myocardium is not the result of decreased tissue oxygenation (12, 23, and current data), the actual mechanisms of the PCr/ATP response to HCW remain unclear. Therefore, it is not known how VEGF-MSCs engraftment abrogates this response. Similarly, the reason why baseline PCr/ATP levels in VEGF-MSCs-treated hearts were reduced despite the abrogation of HCW-associated reductions also remains to be established. Importantly, because the ATP consumption rate is tightly coupled to MO2 (which is linearly related to RPP), the extremely high RPP observed during HCW underscores the fact that the mitochondrial ATP synthetic reserve of the VEGF-MSCs group is at least comparable to that of the normal group.
How Does VEGF-MSCs Engraftment Protect the Pressure-Overloaded LV?
There are at least several potential mechanisms of protection: 1) The notion that increased VEGF levels present in cell transplanted hearts and the consequent increase of vascularity prevent the emergence of the blood flow supply-demand imbalance associated with (untreated) progressive LVH is supported, but not proven, by the current data. 2) The possibility that the engrafted cells make a direct and significant structural contribution to the hypertrophied heart via transdifferentiation to endothelial cells and cardiomyocytes is unlikely because the number of engrafted cells is modest and relatively few of these differentiate to endothelial cells and cardiomyocytes. 3) Another possibility is that the paracrine effects of VEGF secreted by engrafted MSCs on cardiomyocytes directly contribute to the marked attenuation of the deleterious consequences of LV pressure overload. Both the presence of VEGF receptors (VEGFR) on cardiomyocytes and the fact that receptor binding of VFGF activates intracellular signaling pathways within cardiomyocytes are well established (11, 14, 26, 29). This view is supported by our in vitro observations that hypoxia-induced apoptosis in HL-1 cardiac cells is markedly attenuated by the addition of culture medium conditioned by VEGF-MSCs and that this protection is eliminated by the concordant addition of anti-VEGF antibodies. Therefore, the beneficial effects of increased myocardial VEGF levels in pressure-overloaded myocardium may result from activation of protein kinases such as PI-3K
that, in turn, activate downstream antiapoptotic and physiological hypertrophy signaling pathways (8, 14). It is also possible that cardiomyocyte VEGFR stimulation limits the activation of pathological hypertrophy signaling pathways resulting from stimulation of Gq protein-coupled receptors. Taken together, these data suggest that the beneficial effects of VEGF-MSCs transplantation may result from both 1) VEGF-induced neovascularization and 2) the paracrine effects of VEGF on antiapoptotic and hypertrophic signaling pathways in the overloaded cardiomyocytes.
Potential Limitations
Although myocardium containing engrafted VEGF-MSCs has a significantly elevated level of VEGF mRNA, the specific cells that overexpress VEGF mRNA have not been defined in this study. Because mRNA overexpression after in vivo transfection of the heart with adenovirus vectors is often of relatively short duration (22), it is possible that the major source of myocardial VEGF mRNA expression at 4 wk after transplantation is located in the new blood vessels induced by VEGF secreted by MSCs during the early posttransplant period. However, the fact that the engrafted MSCs still express the transduced lac-Z reporter gene product 4 wk after transplantation is strong evidence that they also continue to express VEGF mRNA. This view is consistent with our in vitro observations that VEGF mRNA overexpression can persist for at least 4 wk in transduced but nonproliferating MSCs; these in vitro data raise the possibility that limited proliferation of engrafted cells is the basis for continued VEGF overexpression by these cells. However, additional study will be needed to explain the failure of immune system clearance of the adenovirus vector in the transplanted MSCs given that the adenovirus vector does not persist in cardiomyocytes after in vivo myocyte transfection. It could be speculated that the lack of immune system exposure to the adenovirus vector (because it was already incorporated in the MSCs at the time of transplant) could also have prolonged expression of the transduced genes in engrafted MSCs. Last, although the mechanisms of the beneficial responses to MSCs transplantation (including the effects of treatment on antiapoptotic and other signaling pathways in the overloaded cardiomyocytes) await future study, it should be noted that the main purpose of this study was to determine whether MSCs transplantation would be beneficial to pressure-overloaded myocardium undergoing hypertrophy, a question not previously addressed to our knowledge. The current data have answered this question affirmatively and provide the basis for future studies of the molecular mechanisms of VEGF-MSCs protection.
In conclusion, in this swine model of concentric LVH, coronary vein infusion of autologous VEGF-overexpressing autologous MSCs at the time of aortic banding 1) resulted in significant engraftment of MSCs; 2) attenuated hypertrophy and prevented progression to LV decompensation; 3) induced myocardial neovascularization and increased myocardial blood flow; 4) abrogated the HCW-associated fall in PCr/ATP seen in all other experimental groups; and 5) preserved LV contractile function despite the development of moderate LVH. In contrast, nontransplanted, nonfailing LVH hearts with moderate hypertrophy (as well as CHF hearts) had decreased contractile function. Hence, VEGF-MSCs transplantation ameliorates many of the maladaptive changes that occur in myocardium undergoing pressure overload-induced hypertrophy. The molecular mechanisms underlying these beneficial effects remain to be elucidated.
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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.
* Xiaohong Wang and Qingsong Hu contributed equally to this work. 
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  J. Feygin, A. Mansoor, P. Eckman, C. Swingen, and J. Zhang Functional and bioenergetic modulations in the infarct border zone following autologous mesenchymal stem cell transplantation Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1772 - H1780. [Abstract] [Full Text] [PDF]
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