Postinfarct intramyocardial injection of mesenchymal stem cells pretreated with TGF-α improves acute myocardial function

Abstract

Stem cell-based therapies offer promising potential for myocardial infarction (MI), but endogenous molecules released in response to injury likely impair posttransplantation stem cell function. Stem cell-mediated cardioprotection occurs in part via paracrine effects, and transforming growth factor-α (TGF-α) has been shown to enhance paracrine function. However, it is unknown whether pretreating stem cells with TGF-α increases stem cell-mediated cardioprotection after acute MI. Mesenchymal stem cells (MSCs) were treated with TGF-α (250 ng/ml) for 24 h. Adult male Sprague-Dawley rat hearts were isolated and perfused using the Langendorff method. MI was induced by ligating the left anterior descending coronary artery. Postligation (30 min), vehicle or 1 × 10⁶ MSCs with or without pretreatment were injected in the infarct border zones, and the hearts were perfused for an additional 60 min. Left ventricular function was continuously measured, and infarct size was assessed with Evans blue dye and 2,3,5-triphenyltetrazolium chloride staining. Myocardial production of interleukin (IL)-1β and IL-6 and caspase 3 activation was also measured. Left ventricular function decreased significantly following coronary artery ligation but improved following injection of untreated MSCs and to a greater extent after injection of pretreated MSCs. In addition, the infarct area, myocardial caspase 3 activation, and IL-6 production were lowest in hearts injected with pretreated cells. Intramyocardial injection of TGF-α-pretreated MSCs after acute MI is associated with increased myocardial function and decreased myocardial injury. This strategy may be useful for optimizing the therapeutic efficacy of stem cells for the treatment of acute MI.

stem cell-based therapies offer promising means of improving myocardial function in the setting of acute and chronic myocardial ischemia. Stem cells may protect ischemic myocardium by homing to areas of injury (5); differentiating into terminal cell types, including endothelial cells (35); and by releasing a variety of growth factors and cytokines in a paracrine fashion, which in turn modulate local inflammation, promote angiogenesis, and improve cell survival (10, 15). Furthermore, it has been previously shown that mesenchymal stem cell (MSC) production of vascular endothelial growth factor (VEGF) may increase neoangiogenesis, limit infarct size, and improve postischemic cardiac function in experimental models of myocardial infarction (MI) (38).

To date, several randomized prospective clinical trials have demonstrated that delivery of autologous bone marrow-derived cells improves left ventricular function, infarct size, and left ventricular dimensions in the setting of acute or chronic myocardial ischemia (1). However, as was shown in the 18-mo follow-up of the Bone Marrow Transfer to Enhance ST-Elevation Infarct Regeneration (BOOST) trial (25), these benefits may have limited duration in part because of barriers to posttransplantation stem cell survival and function. As a result, strategies designed to augment MSC paracrine function have been employed in an attempt to improve their therapeutic efficacy (11). We have demonstrated that treating MSCs with transforming growth factor-α (TGF-α) can stimulate VEGF production in vitro (37). In addition, using a model of isolated heart perfusion, we observed that the intracoronary infusion of MSCs pretreated with TGF-α was associated with greater myocardial functional recovery after global ischemia-reperfusion when compared with infusion of untreated MSCs (12).

Cardiac surgical procedures present unique opportunities for highly specific, targeted injection of stem cells in areas of ischemic myocardium (32). Accordingly, we aimed to determine whether cells pretreated with TGF-α directly injected in the myocardium after acute MI would confer greater myocardial protection than untreated cells. We hypothesized that intramyocardial injection of MSCs pretreated with TGF-α would be associated with greater improvements in left ventricular function, coronary flow, infarct size, proinflammatory cytokine production, and apoptotic signaling. In addition, to isolate rat myocardial cytokine production, murine MSCs were used to minimize cross-detection of cytokines of myocardial or MSC origin.

MATERIALS AND METHODS

Animals.

Adult male 9- to 10-wk-old C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) and adult male Sprague-Dawley rats (280–300 g; Harlan, Indianapolis, IN) were fed a standard diet and acclimated in a quiet quarantine room for 1 wk before the experiments. The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Indiana University. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals [National Institutes of Health (NIH) publication No. 85Y23, revised 1996].

Preparation of mouse bone marrow mesenchymal cells.

A single-step purification method using adhesion to cell culture plastic was used as previously described (27) with the following modifications: mice were killed, and bone marrow cells were collected from bilateral femurs and tibias by removing the epiphyses and flushing the shafts with complete media [Iscove's Modified Dulbecco's Medium with 10% FBS (GIBCO Invitrogen, Carlsbad, CA)] using a syringe with a 23-gauge needle. Cells were washed by adding complete media, centrifuging for 5 min at 300 rpm at 24°C, and removing the supernatant. The cell pellet was then resuspended and cultured in 75-cm² culture flasks with complete media at 37°C, 90% humidity, and 5% CO₂ in air. MSCs preferentially attached to the polystyrene surface; after 48 h, nonadherent cells in suspension were discarded. Fresh complete medium was added and replaced every 3–4 days thereafter. After passage 3, cell surface marker expression was analyzed using flow cytometry. Cells were positive for Sca-1 and CD44 and negative for CD45, CD11b, and CD117 before and after TGF-α treatment (27, 33).

Experimental groups.

After three passages, 1.5 × 10⁶ MSCs were transferred to 75-cm² culture flasks in complete media with or without TGF-α (250 ng/ml) 24 h before infusion. The TGF-α dose was chosen based on previous work (37). Cells were recovered using a 0.25% trypsin-EDTA solution (GIBCO Invitrogen), centrifuged, and then resuspended in sterile PBS. A total volume of 200 μl containing 1 × 10⁶ cells was used for injection.

Isolated heart ischemia.

The method of isolated heart perfusion with regional ischemia has been previously described (8). Briefly, rats (n = 10/group) were anesthetized (60 mg/kg pentobarbital sodium ip) and heparinized (500 U ip). Hearts were rapidly excised via median sternotomy and placed in a 4°C modified Krebs-Henseleit solution (in mmol/l: 11 dextrose, 110 NaCl, 1.2 CaCl₂, 4.7 KCl, 20.8 NaHCO₃, 1.18 KHPO₄, and 1.17 MgSO₄). The aorta was cannulated, and the heart was perfused in an isovolumetric mode (70 mmHg) at 37°C and bubbled with 95% O₂. Total ischemic time was <45 s. A pulmonary arteriotomy was performed to facilitate coronary effluent collection. The left atrium was resected to allow insertion of a water-filled latex balloon in the left ventricle. The balloon was adjusted to a mean left ventricular end-diastolic pressure (EDP) of 8 mmHg (range, 6–10 mmHg) during equilibration. The preload volume was held constant during the entire experiment to allow continuous recording of the left ventricular developed pressure (LVDP). Pacing wires were fixed to the right atrium and left ventricle, and hearts were paced at 350 beats/min during equilibration and reperfusion.

Following a 10-min period of equilibration, the left anterior descending (LAD) artery was ligated using a 6–0 polypropylene suture ∼4 mm distal to its origin between the left atrium and conus arteriosus (Fig. 1A). For sham-operated animals, the suture was passed below the LAD through the myocardium and removed without tying. Visible cyanosis of the myocardial tissue distal to the ligature confirmed effective occlusion. After 30 min of occlusion, 1 × 10⁶ cells were injected in four sites along the border zone of the ischemic area. The hearts were perfused for an additional 60 min, and the coronary flow was measured by collecting effluent at regular intervals. Data were continuously recorded using a PowerLab 8 preamplifier/digitizer (AD Instruments, Milford, MA) and an Apple G4 PowerPC computer (Apple Computer, Cupertino, CA). The maximum positive and negative values of the first derivative of pressure (+dP/dt and −dP/dt, respectively) were calculated using PowerLab software.

Fig. 1.

A: representative diagram of the rat left-sided coronary vasculature and point of ligation of the left anterior descending (LAD) coronary artery. Shaded area represents area of ischemia, and the “X's” denote sites of injection. The left atrium is reflected superiorly. B: detection of labeled mesenchymal stem cells (MSCs) after myocardial injection. Cellular membranes were stained with DiI and nuclei with 4′,6-diamidino-2-phenylindole (DAPI). Cells appeared to remain localized within the myocardium (×100) and in close association with intramyocardial vessels (×200).

Myocardial staining and measurement of infarct size.

At the conclusion of the experimental run, a 1% solution of Evans blue dye was infused retrograde through the aortic cannula to demarcate the ischemic area-at-risk (17). After staining, the hearts (n = 3/group) were cut into serial sections, ∼2 mm thick, distal to the ligature and incubated in a 1% solution of 2,3,5-triphenyltetrazolium chloride for 30 min to determine infarct size. Sections were then fixed in 10% formalin for 1 h, and then three to four sections from each heart were scanned on both surfaces using a digital flatbed scanner. The infarct size was calculated as a percentage of left ventricular area using NIH image software (ImageJ, version 1.41o). Values from each section were averaged to determine a value for each heart.

Labeling and detection of injected cells.

To detect cell locations after injection, MSCs were colabeled with the membrane dye CM-DiI (Molecular Probes, Eugene, OR) and the nuclear dye 4′,6-diamidino-2-phenylindole (Invitrogen) at a concentration of 2 μg/ml cell suspension according to the manufacturers' instructions before cell collection (3). At the conclusion of the perfusion experiments, hearts (n = 2/group) were immediately sectioned transversely at the level of the ligation and cell injections. Tissue sections were fixed in 4% paraformaldehyde, embedded in HistoPrep medium (Thermo Fisher Scientific, Waltham, MA), and cut into 5-μM sections. With the use of a Nikon TE2000U inverted fluorescent microscope (Nikon, Melville, NY), cell fluorescence was assessed at ×100 and ×200 magnification. Images were captured using QCapture software (QImaging, Surrey, BC, Canada). Contrast adjustment was applied to the entire image in Adobe Photoshop (Adobe Systems, San Jose, CA) because of interface issues with QCapture and Photoshop per the journal ethics policy.

Enzyme-linked immunosorbent assays.

Rat myocardial interleukin (IL)-1β and IL-6 concentrations in myocardial tissue homogenates from the ischemic border zone and remote apical regions (n = 4/group) were measured using commercially available ELISA kits (R&D Systems, Minneapolis, MN). To evaluate potential cross-reactivity, murine IL-1β and IL-6 concentrations were also measured in the same samples using commercially available ELISA kits (BD Biosciences, San Jose, CA). All standards and samples were measured in duplicate.

Western blotting.

Western blot analysis was performed to measure intracellular caspase 3 activation in myocardial tissue from ischemic border zones. Myocardial homogenate protein extracts (10 μg/lane; n = 3/group) were electrophoresed on a 4–12% Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane. The membranes were blocked in a 5% nonfat milk solution for 1 h followed by incubation with primary antibodies against caspase 3 (Santa Cruz Biotechnology, Santa Cruz, CA) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Biodesign International, Saco, ME). After being washed, membranes were incubated in a 5% nonfat milk solution containing horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse immunoglobulin G secondary antibodies. Signal detection was performed using Supersignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). Caspase 3 band densities were normalized by the corresponding GAPDH signal.

Presentation of data and statistical analysis.

Reported values are means ± SE. Data were compared using one-way or repeated-measures ANOVA with post hoc Bonferroni correction. A P value <0.05 was considered statistically significant.

RESULTS

Evaluation of MSC transplantation in ischemic myocardium.

Injected MSCs remained localized within the myocardium (×100; Fig. 1B) and in close association with intramyocardial vessels (×200). MSCs within vessels were not observed.

TGF-α-pretreated MSCs improve myocardial function post-MI compared with untreated MSCs.

Following LAD ligation, intramyocardial injection of vehicle was associated with a nearly 50% reduction in LVDP compared with sham hearts (Fig. 2A). After injection of untreated MSCs, the LVDP improved to 67.26 ± 5.74 mmHg compared with 46.03 ± 5.19 mmHg for vehicle hearts (P < 0.05; Fig. 2B). However, the greatest recovery occurred after injection of MSCs pretreated with TGF-α (83.24 ± 4.38 mmHg). There were no significant changes in left ventricular EDP between any groups (Fig. 2, C and D). As with LVDP, the ±dP/dt showed improved recovery following MSC injection and to an even greater extent after injection with TGF-α-pretreated cells (Fig. 2, E-H).

Fig. 2.

Myocardial function of hearts injected with vehicle or MSCs with or without transforming growth factor-α (TGF-α) pretreatment following LAD ligation. Left ventricular developed pressure (LVDP) over time (A) and at end-reperfusion (B); end-diastolic pressure (EDP) over time (C) and at end-reperfusion (D); maximum positive value of the first derivative of pressure (+dP/dt) over time (E) and at end-reperfusion (F); maximum negative values of the first derivative of pressure (−dP/dt) over time (G) and at end-reperfusion (H). Results are means ± SE. P < 0.05, vehicle vs. sham (*), MSC vs. sham (#), MSC vs. vehicle (+), and MSC + TGF vs. vehicle (&).

Intramyocardial injection of MSCs improves coronary flow.

To assess the effect of cell treatments on coronary flow rates, coronary effluents were collected at regular intervals before and during ischemia. As seen in Fig. 3, vehicle injection after LAD occlusion was associated with a reduced coronary flow rate of 7.06 ± 0.54 ml/min compared with 10.5 ± 0.18 ml/min in sham-operated hearts (P < 0.05). In contrast, injection of MSCs with and without TGF-α pretreatment was associated with similar coronary flow rates (8.84 ± 1.17 and 8.59 ± 1.40 mmHg, respectively), which were greater than those of vehicle-injected hearts and equivalent to sham-ligated hearts.

Fig. 3.

Coronary flow rates over time. Injections of MSCs with or without TGF-α pretreatment were associated with improved coronary flow compared with vehicle injections. Results are means ± SE. P < 0.05, vehicle vs. sham (*), MSC vs. vehicle (+), MSC vs. sham (#), MSC + TGF vs. vehicle ($), and MSC + TGF vs. sham (@).

MSC injection reduces myocardial infarct size.

The left ventricular infarct size was normalized to the total left ventricular area in each heart. Intramyocardial injection of untreated MSCs was associated with a 20% decrease in infarct size compared with vehicle injection (38.3 ± 2.73% vs. 48.67 ± 1.63%, respectively) (Fig. 4, A-B). However, injection of TGF-α-pretreated cells was associated with the smallest infarct size (24.85 ± 1.25%) of any group. No infarction was observed in the apical region of hearts from any group (Fig. 4C).

Fig. 4.

Left ventricular infarct size following vehicle or stem cell injection. A: representative images of stained tissue sections through the level of left ventricular infarction. B: infarct size expressed as a percentage of left ventricular area. The left ventricular infarct size decreased following injection with MSCs and to a greater extent after injection with MSCs pretreated with TGF-α. C: representative sections from the apical region demonstrating no infarction in any group. Results are means ± SE. P < 0.05, MSC vs. vehicle (+) and MSC + TGF vs. MSC (‡).

Effect of MSC injection on myocardial IL-1β and IL-6 production.

Decreased myocardial IL-1β production was associated with injection of MSCs with or without TGF-α pretreatment (Fig. 5A). There was no difference between cell injection groups, however. In contrast, injection of TGF-α-pretreated but not untreated MSCs was associated with significantly less myocardial IL-6 production compared with vehicle-injected hearts (Fig. 5D). Both IL-1β and IL-6 were increased in the apical regions of hearts injected with vehicle, and there was no significant decrease in their tissue concentrations in hearts injected with either cell group (Fig. 5, B and E). Murine IL-1β and IL-6 levels (Fig. 5, C and F) were <1% and 10% of corresponding rat cytokine groups, respectively, indicating that the rat IL-1β and IL-6 concentrations were of rat myocardial origin, not murine MSC origin.

Fig. 5.

Myocardial concentration of interleukin (IL)-1β and IL-6 following LAD artery ligation. Intramyocardial injection of MSCs with or without TGF-α pretreatment was associated with decreased IL-1β production in the left ventricular ischemic border zone (A). In contrast, myocardial IL-6 production was significantly lower after injection of TGF-α-pretreated MSCs but not untreated cells (D). In the apical regions remote from the injection sites, IL-1β and IL-6 production (B and E) decreased to a lesser degree following injection of pretreated MSCs. Separate detection of murine IL-1β (C) and IL-6 (F) in the same samples revealed a low level of cross-reactivity. Results are means ± SE. P < 0.05, vehicle vs. sham (*), MSC vs. vehicle (+), and MSC vs. vehicle (&).

MSC injection decreases myocardial apoptotic signaling.

To assess ischemia-induced myocardial apoptotic signaling activation, caspase 3 levels were measured in the myocardial homogenates. Caspase 3 activation increased to the greatest degree in ischemic hearts injected with vehicle (Fig. 6). In addition, injection of TGF-α-pretreated but not untreated MSCs was associated with reduced caspase 3 activation compared with vehicle-injected hearts.

Fig. 6.

Myocardial caspase 3 activation following LAD ligation. Injection of TGF-α-pretreated MSCs but not untreated MSCs was associated with a decrease in caspase 3 activation compared with vehicle injection. Results are means ± SE. P < 0.05, vehicle vs. sham (*) and MSC + TGF vs. vehicle (&).

DISCUSSION

MSCs are a unique subpopulation of bone marrow-derived mononuclear cells whose multiple protective functions have gained particular interest in areas of myocardial regenerative therapy. These cells possess the ability to self-renew and differentiate into endothelial cells and cardiomyocytes (23, 31). In addition, MSCs secrete growth factors that in response to tissue injury act in a paracrine fashion to mitigate native cell apoptosis, increase neoangiogenesis, and modulate the local inflammatory response (10, 15). Moreover, allogeneic transfers of MSCs have exhibited the ability to inhibit local lymphocyte activation and evade immune recognition, properties that may make them suitable for off-the-shelf use (9).

While cell-based therapies for myocardial ischemia have shown early promise, poor posttransplantation survival and function are recognized barriers to the long-term success of these therapies (32). One potential strategy for overcoming these barriers is pretreating these cells with exogenous agents during ex vivo expansion before transplantation to maximize MSC paracrine function. One agent that has shown potential for augmenting MSC paracrine function and stem cell-mediated cardioprotection is TGF-α (12). TGF-α is a ligand of the epidermal growth factor (EGF) receptor, which is expressed by numerous cell types, including MSCs (28). Following EGF receptor activation, MSCs exhibit increased proliferation, migration, and survival (28, 34). We previously observed that TGF-α can directly stimulate MSC VEGF production, which is associated with improved myocardial functional recovery (12, 37). However, previous investigations of this pretreatment strategy used intracoronary stem cell delivery in the setting of global ischemia-reperfusion and not postinfarction intramyocardial injection, which may be more readily applied during cardiac surgical procedures. With the use of an ex vivo model of coronary artery ligation, the present study demonstrates that direct intramyocardial injections of TGF-α-pretreated cells were associated with superior improvement in left ventricular function as well as reduction in infarct size, inflammatory cytokine production, and apoptotic signaling compared with injections of untreated cells. Because these MSCs were washed of any media containing TGF-α before injection, these observed benefits likely resulted from an augmented MSC paracrine phenotype and not a direct effect of TGF-α.

We and others have reported the paracrine effects of stem cells on recipient tissues and cells (7, 10, 39). In particular, these effects include the ability of MSCs to modulate the local ischemia-induced inflammatory response. IL-1β is a proinflammatory cytokine produced in response to ischemia and other tissue injury (24). Within the heart, IL-1β contributes to ischemia-related myocardial dysfunction by altering intracellular calcium metabolism, upregulating nitric oxide synthase, and inducing expression of other cytokines and adhesion molecules (13, 16). In addition, increased myocardial production of IL-6 following ischemia has been shown to mediate cardiac dysfunction (26). Binding of IL-6 to its cell surface receptor decreases cardiomyocyte contractility and LV performance through regulation of calcium metabolism, and serum IL-6 levels have been shown to correlate with cardiovascular disease outcomes (6, 26). Our data suggest that MSCs mitigate the levels of these myocardial cytokines likely in association with the elevated paracrine factors we reported previously (12). However, because there was less reduction in IL-1β and IL-6 in the remote apical regions compared with the LV ischemic border zones following injection of pretreated cells, the protective benefits of the cell injections appear to be limited to areas around the injection sites and not the global myocardium. In addition, the observation that myocardial apoptosis was decreased following intramyocardial MSC treatment is consistent with previous evidence that stem cells may directly augment cardiomyocyte survival in the setting of myocardial ischemia (18). However, it remains unclear whether pretreated cells confer a longer duration of cytoprotection and modulation of inflammation beyond the acute phase of infarction.

Augmentation of regional tissue perfusion is another key protective property of MSCs that has been shown to improve myocardial perfusion as early as 2 days after infarction (29). However, the vasomodulatory effects of stem cell therapy immediately following MI are less well-defined. We observed that MSC delivery 30 min after the onset of ischemia improved coronary flow rates in association with improved myocardial function. The exact mechanisms for this observation remain unclear but may involve MSC paracrine function. In addition to promoting neoangiogenesis, VEGF is a potent stimulator of coronary microvessel dilatation (20). Activated MSCs may increase local tissue VEGF levels through direct production or by stimulating myocardial VEGF production in situ (14). Although TGF-α may induce VEGF secretion by MSCs, it is unclear whether this difference in VEGF expression translates into actual changes in coronary flow, since this parameter improved equally following injection of MSCs with or without TGF-α pretreatment in this study. Another possible mechanism of stem cell-mediated vasodilation may involve direct interaction between the stem and endothelial cells. During myocardial ischemia, coronary endothelial cells facilitate stem cell activation and homing to areas of injury (30). Further work is needed to elucidate these direct and indirect mechanisms of stem cell-mediated augmentation of coronary flow during acute ischemia.

In the present study, TGF-α was used solely as a stimulant of MSCs during ex vivo expansion and was not directly delivered to the ischemic myocardium in the cell suspensions. It, therefore, remains unknown whether TGF-α can exert cardioprotective effects alone. Activated endothelial cells, fibroblasts, and macrophages in the lung have been shown to produce TGF-α in response to oxidative stress (22, 36), but similar processes have not been assessed in the heart. Further work is needed to characterize this potential direct application of TGF-α during myocardial ischemia.

Limitations of this model of ex vivo coronary artery ligation include the inability to assess long-term neoangiogenesis and cardiac function in the setting of systemic physiological responses. In addition, we chose to deliver murine MSCs in rat hearts to facilitate measurement of rat myocardial cytokine production as opposed to potential MSC cytokine production by minimizing potential for cross-reactivity between cytokines of different tissue or cell origins. While this xenotransplantation may appear to be problematic, murine MSCs have been found to engraft into rat hearts following infarction and persist up to 4 wk following transplantation (21). Successful xenotransplantation has also been observed following transplantation of human MSCs in sheep or rat hearts (4, 19). This may in part be attributable to the ability of MSCs to evade (via decreased major histocompatibility class I expression) and even suppress local immunological responses (2, 9). Future experiments, however, may be conducted to evaluate success of this approach in a model of chronic myocardial ischemia. Despite these limitations, this model enables investigation of the effects of MSC injection in the acutely ischemic heart and provides a basis for future investigation of the effects of direct intramyocardial injection of TGF-α-pretreated stem cells during acute and chronic myocardial ischemia in vivo.

Perspectives and Significance

Although stem cell therapies for ischemic heart disease have shown early modest benefits for improving myocardial function, these improvements have been limited in duration and magnitude. Optimizing stem cell function during ex vivo expansion before transfer may be one method of improving overall clinical benefits of this application. Our results indicate that the intramyocardial injection of MSCs pretreating with TGF-α was associated with greater myocardial functional recovery during acute ischemia. Pretreating MSCs with TGF-α may be a rapid and effective means for enhancing stem cell function for the treatment of myocardial ischemia, and further work is needed to elucidate the benefits of this cell enhancement strategy in vivo.

GRANTS

This work was supported by the following National Institutes of Health (NIH) Grants: R01 GM-070628, R01 HL-085595, F32 HL-092718, F32 HL-092719, and F32 HL-093987 and the NIH Loan Repayment Program.

DISCLOSURES

The authors have no disclosures or conflicts of interest.