Stem cell therapy has emerged as an exciting new area of medicine and surgery. The plasticity of progenitor cells has resulted in positive remodeling and the regeneration of viable tissues in liver, brain, heart, and other organ systems. Among the many sources of adult stem cells, bone marrow-derived stem cells (BMSCs) have shown particular promise (1). Within BMSCs, bone marrow hematopoietic stem cells (HSCs) (2-4) and nonhematopoietic mesenchymal stem cells (MSCs) (5-12) have shown the ability for conferring protection and repair of damaged tissue. However, recent experimental studies questioning the engraftment and transdifferentiation of BMSCs (13), HSCs (14-16), and MSCs (17) have led to controversy regarding the mechanism behind these promising results. Indeed, we have previously demonstrated that BMSC differentiation is not required for cardioprotection; acute application of human BMSC into myocardium subjected to ischemia/reperfusion (I/R) improved functional recovery, decreased proinflammatory cytokine production, and decreased activation of proapoptotic caspases (18). No studies have tested whether murine BMSCs confer acute protection in an isolated perfused rat heart subjected to I/R injury.
Bone marrow-derived stem cells may instead mediate their beneficial effects via production of local factors. Dernbach and colleagues reported that circulating progenitor cells possess potent antioxidant properties (19, 20). In addition, Wang et al. (21) and others intimate that vascular endothelial growth factor (VEGF) may play a role in this BMSC paracrine-mediated cardioprotection. Vascular endothelial growth factor, a potent angiogenic stimulator, increases microvascular density and improves perfusion to ischemic regions. The transplanted adult progenitor cell, when faced with an inflammatory (hypoxic or infectious) environment, may release substances like VEGF to enhance its survival. Human adipose stem cells have been demonstrated to secrete VEGF in response to hypoxia and lipopolysaccharide (LPS) in vitro (22). Survival of HSC also depends on the secretion of VEGF (23). No study has demonstrated MSC production of VEGF in response to hypoxia and LPS.
Mesenchymal stem cells are a relatively underexplored population of BMSC, which may have advantages over the well-characterized HSC population. Ready availability from small aspirates of donor bone marrow, ease of expansion in an in vitro cell culture, simple isolation via plastic adherence, ability to evade rejection, and their multipotentiality for differentiation make MSCs ideal for clinical applications (1, 24). In contrast, isolation of adequate numbers of HSCs requires large volumes of marrow, and the cells are difficult to expand in culture (24).
Further studies of expanded BMSC populations indicate that in vitro expansion of BMSC may be limited. Bone marrow-derived stem cells senesce and lose their differentiation potential with increasing time in culture and passage (25). No studies have tested whether BMSCs lose their protective abilities with passage. No studies have determined whether BMSC activation, measured with production of growth factor, is affected by high passage. We hypothesize that murine MSCs are acutely cardioprotective against I/R injury in the isolated perfused rat heart and that high passage number has an adverse affect on MSC activation and MSC cardioprotection.
Normal male and female strain C57BL/6J mice (The Jackson Laboratory, Bar Harbor, Me) and normal (280-300 g, 9-10 weeks old) Sprague-Dawley rats (Harlan, Indianapolis, Ind) were fed a standard diet and acclimated in a quiet quarantine room for 1 week before the experiments. The animal protocol was reviewed and approved by the Indiana 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" (NIH publication No. 85-23, revised 1985).
Preparation of mouse bone marrow stromal cells
A single-step purification method using adhesion to cell culture plastic is used as previously described (26) with the following modifications: After killing 8-week old mice, mouse bone marrow stromal cells were collected from bilateral femurs and tibias by removing the epiphyses and flushing the shaft with complete media (Iscove modified Dulbecco medium [GIBCO Invitrogen, Carlsbad, Calif] and 10% fetal bovine serum [GIBCO Invitrogen]) using a syringe with a 23-G needle. Cells were disaggregated by vigorously pipetting several times. Cells were passed through a 30-μm nylon mesh to remove remaining clumps of tissue. Cells were washed by adding complete media, centrifuging for 5 min at 300 rpm at 24°C, and removing supernatant. The cell pellet was then resuspended and cultured in 75 cm2 culture flasks with complete media at 37°C. Mesenchymal stem cells were preferentially attached to the polystyrene surface; after 48 h, nonadherent cells in suspension were discarded. Fresh complete medium was added and replaced every 3 or 4 days thereafter. Mesenchymal stem cell cultures were maintained at 37°C in 5% CO2 in air. When the cultures reached 90% of confluence, the MSC culture was passaged; cells were recovered by the addition of a solution of 0.25% trypsi-EDTA (GIBCO Invitrogen) and replated in 75 cm2 culture flasks.
Experimental isolated heart groups
All isolated rat hearts were subjected to the same I/R protocol of 15-min equilibration period, 25 min of global index ischemia (37°C), and 40 min total reperfusion. Rats were divided into the following 4 experimental groups: (1) control hearts without intervention (n = 5); (2) hearts with passage 3 stem cell infusion (n = 3); (3) hearts with passage 5 stem cell infusion (n = 3); and (4) hearts with passage 10 stem cell infusion (n = 3). Bone marrow-derived stem cells were suspended in warm (37°C) oxygenated Krebs-Henseleit (KH) solution and infused (1 mL of 1 million cells) before global index ischemia.
Isolated heart preparation (Langendorff)
Hearts were isolated as previously described (27-29). Briefly, rats were anesthetized (sodium pentobarbital, 60 mg/kg intraperitoneally) and heparinized (500 U intraperitoneally), and hearts were rapidly excised via median sternotomy and placed in 4°C KH solution. The aorta was cannulated, and the heart was perfused in the isolated isovolumetric Langendorff mode (70 mmHg) with KH solution (in mmol/L: 11 dextrose, 110 NaCl, 1.2 CaCl2, 4.7 KCl, 20.8 NaHCO3, 1.18 KHPO4, and 1.17 MgSO4) at 37°C. The KH solution was bubbled with 95% O2/5% CO2 (Medipure) to achieve a PO2 of 450 to 460 mmHg, PCO2 of 39 to 41 mmHg, and pH of 7.39 to 7.41. Total ischemic time was less than 45 s. The perfusion buffer was continuously filtered through a 0.45-μm filter to remove particulates. A pulmonary arteriotomy and a left atrial resection were performed before insertion of a water-filled latex balloon through the left atrium into the left ventricle. The preload volume (balloon volume) was held constant during the entire experiment to allow continuous recording of the left ventricular developed pressure (LVDP). The balloon was adjusted to a mean left ventricular end-diastolic pressure of 8 mmHg (range, 6-10 mmHg) during the initial equilibration. Pacing wires were fixed to the right atrium and left ventricle, and hearts were paced at 6 Hz, 3 V, and 2 ms (approximately 350 beats per min) throughout perfusion. A 3-way stopcock above the aortic root was used to create global ischemia, during which the heart was placed in a 37°C degassed organ bath. Coronary flow was measured by collecting pulmonary artery effluent. Data were continuously recorded using a PowerLab 8 preamplifier/digitizer (AD Instruments Inc, Milford, Mass) and an Apple G4 PowerPC computer (Apple Computer Inc, Cupertino, Calif). The maximal positive and negative values of the first derivative of pressure (+dP/dt and −dP/dt) were calculated using PowerLab software. After reperfusion, the heart was removed from the apparatus, immediately sectioned, and snap frozen in liquid nitrogen.
Experimental cell culture groups
Passage 3 MSCs were plated in 12 well plates in a concentration of 1 × 106 cells per well per mL. Mesenchymal stem cells were divided into experimental groups (triplicate wells per group) and stressed with (1) increasing doses of LPS (0, 50, 100, and 200 ng/mL) and (2) hypoxia of 1 h. Mesenchymal stem cells from passages 3, 5, and 10 were also stressed with LPS 200 ng/mL. After 24 h (LPS and hypoxia) and 48 h (hypoxia) incubation, supernatants were harvested for VEGF-A enzyme-linked immunosorbent assay (ELISA). The experiment was repeated on 3 separate occasions (n = 6-11 wells per group).
Vascular endothelial growth factor enzyme-linked immunosorbent assay
Vascular endothelial growth factor release (specifically VEGF-A) by the MSC was determined with ELISA using a commercially available ELISA set (R&D Systems Inc, Minneapolis, Minn and BD Biosciences, San Diego, Calif). Enzyme-linked immunosorbent assay was performed according to the manufacturer's instructions. All samples and standards were measured in duplicate.
Presentation of data and statistical analysis
All reported values are mean ± SEM. Data were compared using 2-way analysis of variance with post hoc Bonferroni test or Student t test. A 2-tailed P < 0.05 was considered statistically significant.
Ischemia/reperfusion resulted in markedly decreased LVDP in all groups. Postischemic recovery of LVDP (expressed as percentage of preischemic function) was significantly higher (P < 0.05) in hearts with passage 3 MSCs (64.7% ± 10.2%) than control hearts (29.3% ± 2.2%; Fig. 1A). However, there was no significant difference in postischemic recovery of LVDP between hearts with passage 5 MSCs (38.4% ± 12.4%) and control hearts (Fig. 2A). Hearts with passage 10 MSCs conferred a decrease in postischemic recovery of LVDP (10.5% ± 0.7%) in comparison to controls (Fig. 3A).
Maximum positive and negative dP/dt were impaired at the start of reperfusion. Control hearts demonstrated more depression of +dP/dt and elevation of −dP/dt compared with hearts infused with passage 3 MSCs (Fig. 1, C and D). Hearts with passage 5 MSCs and without MSCs exhibited no difference in impairments of contractility and compliance (Fig. 2, C and D). Passage 10 MSCs conferred a decrease in contractility and compliance in comparison to controls (Fig. 3, C and D).
Mesenchymal stem cells activation
Hypoxia and LPS resulted in significant activation of passage 3 MSCs. One hour of hypoxia provoked significant VEGF production (% control) at 24-h incubation (120.0% ± 3.4% vs. 100.0% ± 1.6%) and 48-h incubation (112.2% ± 3.6% vs. 100.0% ± 0.8%), respectively, as shown in Figure 4A. Lipopolysaccharide also provoked significant VEGF production (% control) at 50 ng/mL (144.3% ± 2.9%), 100 ng/mL (143.5% ± 3.0%), and 200 ng/mL (145.3% ± 4.9%), respectively, in comparison to control (100.0% ± 1.4%) as shown in Figure 4B. In addition, LPS provoked significantly more VEGF production (pg/mL) in passage 3 (985.4 ± 30.4) versus passage 5 (677.4 ± 79.6) and passage 10 (415.8 ± 2.6) murine MSCs as shown in Figure 4C.
The results of this study are the first demonstration that (1) adult murine MSCs confer acute cardioprotection; (2) murine MSCs release significantly increased VEGF in response to LPS and hypoxia; and (3) MSC protection may depend on time in culture or passage.
Acute stem cell pretreatment represents a promising frontier for myocardial protection. Togel et al. found that MSC administration immediately after renal ischemia in rats conferred improved renal function, decreased proinflammatory cytokine production, and decreased apoptosis without MSC differentiation (17). In parallel, we previously demonstrated that human MSC administration immediately before cardiac ischemia improved functional recovery, decreased proinflammatory cytokine production, and decreased proapoptotic signaling (18). Indeed, the results of this study confirm the acute protective effects of stem cells; pretreatment with passage 3 murine MSCs significantly improved postischemic recovery of LVDP, contractility, and compliance. The acute use of MSCs in these studies precluded immediate stem cell differentiation as a cause of myocardial protection. Instead, MSCs may act as a stabilizing/protective "helper cell" during I/R. However, it remains unclear whether which acute mechanisms MSCs exert their myocardial protection after I/R.
Mesenchymal stem cells may mediate their acute protective effects via complex paracrine actions. Tang et al. determined that MSC implantation significantly increased VEGF expression and regional blood flow in ischemic hearts (30). Subsequently, Hiasa et al. determined that VEGF played an important role in the BMSC-induced myocardial protection from ischemia (31). In congruence with these findings, we found that MSC stimulation with both hypoxia and increasing doses of LPS resulted in significant release of VEGF. Vascular endothelial growth factor-A initiates its effects by binding with 2 distinct type II tyrosine kinase receptors, VEGF receptor (VEGFR) 1 and VEGFR2. Furumatsu et al. confirmed the expression of both VEGF-A and VEGFR in activated human MSCs (32). Hypoxia allows the stabilization of hypoxia-inducible factors that regulate both VEGF-A expression (33) and VEGFR1 upregulation (34). Vascular endothelial growth factor receptor 1 not only promotes stem cell survival but also stem cell recruitment and migration (35). Similarly, VEGFR2 is upregulated during hypoxia, and higher levels of circulating VEGFR2-expressing progenitor cells have been correlated with lower cardiovascular risk of death (36). Thus, it seems that the autocrine VEGF pathway by which stem cells promote their own survival (23) may also be associated with a paracrine VEGF pathway that confers protection to surrounding tissue. Determining whether BMSC release of VEGF confers protection to surrounding tissue via reduced apoptosis (22, 37, 38), decreased proinflammatory cytokines (39-41), or other mechanistic pathways (42-45) requires further investigation. Nevertheless, the organ protection seen in this and other studies after acute stem cell therapy suggests that MSCs mediate their beneficial effects not by transdifferentiation into target cells but perhaps instead by modulating local inflammation and stimulating endogenous repair mechanisms via the release of protective substances such as VEGF.
The potential of acute bone marrow stem cell-protective therapy is mitigated by observations that in vitro expansion seems to be limited by passage. Anatomically, bone marrow stem cells after extensive expansion in vitro undergo senescence and change both their shape and morphology (46). Functionally, recent studies indicate that cultured BMSCs progressively lose their proliferation potential with passaging or cell doubling (47, 48). Other investigations also determined that with time in culture, the differentiation potential of BMSCs was reduced, suggesting a preferential commitment of progenitors toward their target phenotype (49, 50). Interestingly, MSCs from old donors exhibited accelerated senescence evidenced by an increased number of senescence-associated beta-galactosidase cells as compared with MSCs harvested from young donors (51). However, this study represents the first investigation demonstrating that high passage adversely affects protection conferred. Hearts pretreated with passage 5 MSCs demonstrated no improvement in functional recovery, whereas passage 10 MSCs demonstrated worse myocardial function than controls. Moreover, this study demonstrated that cultured passage 5 MSCs released significantly lower levels of VEGF than passage 3 MSCs, and passage 10 MSCs produced significantly less VEGF than both passage 3 and passage 5 MSCs. However, it is unlikely that this decrease in VEGF production with increasing passage solely explains the discrepancy in cardioprotection conferred. Indeed, Ottino et al. determined that in low-passage-number endothelial progenitor cells, constitutive levels of VEGFR1 messenger RNA were detected, whereas at high passage numbers, no receptor expression was found by reverse transcriptase polymerase chain reaction (52). Whether this decrease in VEGFR1 with high passage can also be found in other stem cells remains unknown. Interestingly, others have found that stem cell expression of VEGFR2 increases with passage number, and that VEGFR2 may be associated with further differentiation (53). It also remains possible that our passage 10 MSCs may have undergone early differentiation; hearts treated with passage 10 MSCs may have been treated with progenitor cells that caused capillary trapping and subsequent decline in coronary flow and LVDP. Together, these factors may contribute in part to the diminished functional recovery in hearts treated with passage 10 MSCs. Thus, it seems that BMSCs do not replicate indefinitely in vitro and perhaps undergo senescence. The time in culture or passage number may have an adverse effect on local factor release, growth factor receptor upregulation, and subsequent organ protection. An ideal passage number may exist, which maximizes stem cell purity, potential, and protection conferred. This portends limited ex vivo expansion before possible therapeutic use.
These results demonstrate that murine MSCs are effective in attenuation of myocardial I/R injury. Planned ischemic events, such as those that occur during cardiac surgery, angioplasty, or transplantation, may allow an additional opportunity to observe the potential clinical benefit of adult stem cell pretreatment. However, the adverse effect of passage number highlights the challenges in maintaining undifferentiated MSCs. Mesenchymal stem cells are a novel potential agent for myocardial protection against I/R, but further investigations of its limitations are necessary to maximize this protection.
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