| Background: Myocardial infarction (MI) remains the most common cause of cardiac morbidity and mortality in the developed world. Acute myocardial ischemia causes rapid death of cardiomyocytes and vasculature, leading to left ventricular (LV) remodeling, including progressive fibrotic myocardium replacement, LV dilation, and heart failure. In the last decade, stem cell therapy has been shown to be a promising method for treating MI, and mesenchymal stem cells (MSCs) have been considered a good option because of their unique biological attributes. When introduced to an infarcted heart, MSCs prevent deleterious remodeling and improve recovery. Increasing evidence demonstrates that the beneficial effect of MSCs is significantly mediated through incompletely understood indirect paracrine actions, supplying multiple therapeutic growth factors and cytokines regulatory of the ongoing MI pathological processes. In recent studies, pigment epithelium-derived factor (PEDF), a member of the serpin protease inhibitor family lacking protease inhibitory activity, was identified as a major MSC-secreted protein. PEDF is a multifunctional, pleiotropic protein with antiangiogenic, antioxidant, anti-inflammatory, antitumorigenic, and neuroprotective properties, with cell-type dependent biological function. Thus, PEDF might be contributive to MSC paracrine actions regulating ongoing MI pathologic processes, but has not been investigated.Most clinical studies have used autologous stem cells for MI treatment. Patients suffering MI typically are of advanced age. However, the impact of donor age upon MSCs therapeutic efficacy remains uncertain. Clarification of this issue and underlying mechanisms are of utmost importance, as their elucidation will give insight regarding MSC genetic modification with age and any potential reversibility. In the present study, MSCs from older mice secreted significantly greater PEDF levels compared to young mice. We hypothesized that PEDF plays a critical role in MSC paracrine functioning during the pathological processes associated with MI. Furthermore, age-related PEDF expression may critically influence MSC therapeutic efficacy in MI treatment.Objective: Despite the promising potential of utilizing MSCs for the treatment of myocardial infarction MI, MSCs derived from older donors are less efficacious than those from younger donors. The underlying mechanisms contributing to this effect are not completely understood. Here, we determine how age-related expression of pigment epithelium-derived factor (PEDF) in MSCs affects their therapeutic efficacy for MI.Methods: (1)Laboratory animals, Isolation, culture, and characterization of MSCs: MSCs were isolated and expanded from young (8-week-old) and older (18-month-old) male C57BL/6 mice via standard protocols.13 Third-passage cells were used for experiments. To validate the enriched MSCs population, flow cytometry characterized the surface antigens, and in vitro differentiation of cultured MSCs was performed. (2)Detection of PEDF expression in vitro: RT-PCR analysis was performed to detecte PEDF mRNA level. PEDF concentrations released by MSCs were measured via mouse PEDF ELISA kit. (3)Adenoviral vectors and MSCs transduction: Human type 5 adenoviral vectors expressing human-PEDF (Ad.PEDF) from a cytomegalovirus (CMV) immediate early promoter were generated, and the E1 and E3 regions of the viral genome were replaced with a reporter gene (GFP). Control vectors (Ad.Null) not expressing PEDF were constructed and produced concomitantly. In addition, the Ad.shPEDF vector, in which a short hairpin RNA (shRNA) was driven by the U6 promoter, and GFP was driven by an independent CMV promoter, which resulted in dramatically attenuated PEDF levels, was generated for further experiments. Ad.shctrl (Ad-Scramble) vector served as a control. MSCs were incubated with adenoviruses at infection multiplicity of 3000 for 2 hours. Transduction efficiency was analyzed 24 hours after transduction by flow cytometry and inverted microscopy. (4)Model of myocardial infarction and MSCs transplantation: Eight week-old male C57BL/6 mice were subjected to small left thoracotomy, temporary cardio-exteriorization, and placement of 6.0 silk suture below origin of the left anterior descending coronary artery (LCA). The heart was replaced immediately into the thoracic cavity, with thoracic air evacuation to avoid pneumothorax. 4.0×106 MSCs/mouse were tail-vein injected 0.5-1 hour post MI. Control animals underwent LCA ligation with only saline injection. (5)Detection of MSCs recruitment and PEDF expression in vivo: To investigate MSC recruitment, alternate sets of 8μm thick serial vertical sections of the infarcted heart were mounted on gelatin-coated slides for fluorescent microscope examination, and images were analyzed by Image-Pro Plus. Infarct region PEDF expression was identified by immunofluorescence. Sections were sequentially incubated with goat anti-human PEDF primary antibodies or rat anti-mouse PEDF primary antibodies and Texas Red-conjugated secondary antibody. Furthermore, PEDF levels were also determined by a human or mouse PEDF ELISA kit. (6)Analysis of cellular profile in the MI area: On day 7 post-MI, hearts were fixed and sectioned. Slides were incubated with primary antibodies for CD31,αSMA, vimentin, Cardiac Troponin I, and F4/80. After PBS rinsing, slides were incubated overnight with biotinylated goat anti-mouse IgG or donkey anti-rat IgG antibodies, further incubated with Texas red-conjugated streptavidin in PBS. Cell nuclei were stained with 4′, 6′-diamino-2-phenylindole. (7)Measurement of LV fibrosis and LV function: Left ventricle cross sections (4μm thick) at the midpapillary muscle level were Trichrome-Masson stained. For LV functional analysis, mice were anesthetized by intraperitoneal 1% sodium pentobarbital injection (50mg/kg body weight) before a micro-catheter was inserted into the LV via the right carotid artery. LV function and electrocardiogram data were recorded using a Biopac Data Acquisition System. (8)Co-culture of cardiac fibroblasts with MSCs for proliferation and migration assays: Young MSCs, old MSCs, Y&Ad.Null, Y&Ad.PEDF, O&Ad.shctrl, or O&Ad.shPEDF cells were plated (1.5×105 cells/cm2) in a Transwell insert with 0.4μm pores, while neonatal mouse cardiac fibroblasts (CFs) were plated as control. The insert was then placed into 24-well plates, where CFs were plated (2×10~5 cells/cm~2). CF proliferation was evaluated 48 hours after co-culture under hypoxia using MTT assay. CF migration assay was carried out using a Transwell insert of 8μm pore size. Chemotaxis was induced by MSCs in the lower compartment. CFs were serum-starved overnight, and 5×10~4 cells were seeded in the upper compartment. Cells were allowed to migrate for 8 hours under hypoxic conditions. The filters were stained with crystal violet for microscopy. A blinded, single person counted the number of cells that migrated to the lower filter surface. (9)The direct effects of PEDF on cardiac fibroblasts: Cardiac fibroblasts were stimulated for 8 hours with 0-200 ng/ml recombinant human (rh) PEDF under hypoxia, and the MTT assay was performed. Cell cycle distribution was detected by a Calibur flow cytometer. For western blotting analysis, 50μg total proteins from each cell extract was resolved with SDS-PAGE and transferred onto a polyvinylidence difluoride membrane. The membrane was then incubated sequentially with primary anti-cyclin D1, anti-p27, or anti-glyceraldehyde-3-phosphate dehydrogenase and horseradish peroxidase-conjugated secondary antibodies; bands were detected with enhanced chemiluminescence. The CF migration assay was carried out using a Transwell insert as described above, except that rh-PEDF was used for chemotaxis in the lower compartment. AnnexinV-FITC apoptosis detection kit determined apoptotic cell percentage, and flow cytometry analysis was performed. Moreover, CFs were stained with Hoechst 33258. Apoptotic CFs were identified on the basis of their nuclear morphology (presence of condensed chromatin and fragmentation). The nuclei from 5 random fields were counted by a single blinded person, and the percentage of apoptotic nuclei relative to total nuclei was calculated. (10)Statistical analyses: Data were expressed as mean±standard error of the mean (SEM). Multiple group comparisons were evaluated via one-way ANOVA followed by least significant difference (LSD)-t test for post hoc analysis. Comparisons between two independent groups were analyzed using the Student t test. Analyses were performed using SPSS software. P values less than 0.05 were considered to be statistically significant and tests were performed two-sided.Results: (1) MSCs from older donors express and secrete more PEDF than younger donors: After 3 passages, MSC pluripotency was confirmed by capacity to differentiate into adipogenic and osteogenic lineages in vitro. By flow cytometry analysis, cultured MSCs expressed CD29, CD44, CD73, CD90 and CD105, and were devoid of hematopoietic markers CD11b, CD34 and CD45. Notably, RT-PCR and ELISA revealed that, in comparison to young MSCs, significantly greater PEDF expression was found in older MSCs under both normoxic and hypoxic conditions. (2)Adenoviral vector transduced MSCs recruit to the infarct region, and regulate PEDF levels: We used adenoviral vectors carrying human-PEDF and or short hairpin RNA (shRNA) targeting mouse-PEDF to overexpress PEDF in young MSCs or knock down PEDF expression in older MSCs respectively. All adenoviral vectors were designed to carry a GFP reporter. The transduction efficiency of these adenoviral vectors was analyzed. Utilizing our mouse MI model, we intravenously injected either: saline, young MSCs, old MSCs, Y&Ad.Null, Y&Ad.PEDF, O&Ad.shctrl, or O&Ad.shPEDF. On Day 1 after MI, a large number of GFP positive cells were found dispersed in the infarct region. Furthermore, no significant differences were observed in the green fluorescent intensity of infarcted hearts among Y&Ad.Null, Y&Ad.PEDF, O&Ad.shctrl, and O&Ad.shPEDF groups, indicating similar numbers of MSCs dispersal in the infarct region among these groups. Subsequently, we performed immunofluorescent staining and ELISA to identify the ability of genetically modified MSCs in the infarct region to regulate local PEDF levels. Greater PEDF levels were found in the infarct region after administration of MSCs of older compared to young origin. Adenoviral vectors were used to overexpress human-PEDF or block mouse-PEDF expression in young or old MSCs in vivo respectively; thereby leading to PEDF level significantly changes in the infarct region. (3)Decreased efficacy of old MSCs for MI treatment is associated with increased PEDF expression: In order to study whether different PEDF levels could affect the efficacy of MSCs for MI, we performed gross cardiac morphology and function analyses. Compared to saline treatment, both young and old MSCs significantly attenuated fibrosis post MI, but to greater extent after young MSC treatment. Compared to saline treatment, administration of young MSCs after MI markedly improved LV functional parameters. Compared to young MSC treatment, administration of old MSCs after MI increased the ratio of the pre-ejection period/left ventricular ejection time and LV end-diastolic pressure, and decreased maximum LV pressure, as well as the +dp/dt and–dp/dt, all data suggestive of decreased therapeutic ability of old MSCs compared to young MSCs. Importantly, compared with Y&Ad.Null cells, Y&Ad.PEDF not only significantly increased LV fibrosis, but also impaired LV function. In contrast, O&Ad.shPEDF decreased the LV fibrosis area, and ameliorated LV function compared with old MSCs transduced with Ad.shctrl, supporting the notion that impaired therapeutic efficacy of old MSCs is associated with increased PEDF secretion. (4)MSCs regulate the cellular profile in the MI area via paracrine factor PEDF: To investigate in the underlying mechanism contributing to age-diminished efficacy against MI injury, we analyzed the cellular profile of the infarct region after administration of saline, young MSCs, or old MSCs. In infarcted myocardial sample regions seven days after MI, confocal microscopy images demonstrated significantly increased density of vascular endothelial cells (ECs and CD31-positive), vascular smooth muscle cells (VSMCs andαSMA-positive), and macrophages (F4/80-positive) in mice having received young or old MSCs compared to saline. In contrast, fibroblast (vimentin-positive) density was significantly lower in mice receiving young or old MSCs compared to saline. The infarct area of hearts subjected to old MSCs contained significantly fewer ECs, VSMCs, and macrophages, but more fibroblasts compared to those subjected to young MSCs. To investigate whether the differential cellular profile between young and old MSCs was caused by variant PEDF level expression, we assessed the resultant cellular profile following administration of genetically modified MSCs. Interestingly, we observed only a small proportion of ECs, fibroblasts, VSMCs, and macrophages in the infarct region of hearts receiving CD31-, vimentin-,αSMA-, or F4/80- cells (each with GFP double positivity, confirmatory of viral infectivity). Importantly, PEDF overexpression in young MSCs (Y&Ad.PEDF group vs Y&Ad.Null group) not only decreased the total number of ECs, VSMCs, and macrophages (total CD31- orαSMA- or F4/80 positive cells), but also the number of MSC-derived ECs, VSMCs, and macrophages in the infarct region, whereas both fibroblast (all vimentin positive cells) and MSC-derived fibroblast populations were increased. Furthermore, knocking down PEDF expression in old MSCs (O&Ad.shPEDF group vs O&Ad.shctrl group) increased the total number of ECs, VSMCs, and macrophages, as well as MSC-derived ECs, VSMCs, and macrophages in the infarct region, whereas both fibroblast population and MSC-derived fibroblast density was decreased. These results suggest that MSCs can alter the cellular profile within a MI area through PEDF paracrine factor, and furthermore, this cellular profile alteration can be influenced by the age-specific type PEDF expression present. (5)PEDF secreted by MSCs stimulates cardiac fibroblast proliferation and migration in dose dependent manner: To provide further evidence that MSC-secreted PEDF can affect fibroblast populations in the infarct region, we performed a Transwell co-culture experiment with CFs and MSCs. We demonstrated that CF proliferation was slower when they were cultured with young MSCs than with old MSCs. Furthermore, this inhibitory effect was markedly weaker when CFs were co-cultured with Y&Ad.PEDF group. Moreover, CF proliferation was much slower when they were cultured with old MSCs after PEDF expression had been knocked down. In fact, our data showed that PEDF protein alone stimulated CF proliferation under hypoxic conditions in dose-dependent fashion. PEDF (200 ng/ml) significantly increased the number of CFs in S phase, up-regulated cyclin D1 expression, and down-regulated P27. However, 200 ng/ml PEDF did not have a pronounced effect on CF apoptosis during hypoxia. Additionally, we demonstrated that either old MSCs with naturally high PEDF expression, or young MSCs with overexpressed PEDF, attracted more CFs in the Transwell system. In contrast, knocking down PEDF expression in old MSCs attenuated CF chemotaxis. Furthermore, by using PEDF protein to substitute MSCs in the lower compartments of the Transwell system, PEDF alone was consistently shown to be an important chemoattractant of the MSC secretome.Conclusions: (1)This is the first report that PEDF is a paracrine factor serving an important role in MSCs'therapeutic effect for treatment of MI. Thus, our results strongly support the notion that paracrine effects play an important role in reparative processes after MSC administration in the ischemic heart. We show for the first time that PEDF is one of the most important factors involved in MSC paracrine function. (2)An additional novel finding of our study is the significantly increased PEDF expression in MSCs with age. Notably, the impaired therapeutic ability of old MSCs is predominantly caused by increased PEDF secretion, a heretofore new underlying mechanism of age-related decrease in MSC therapeutic efficacy. (3)Finally, the knock down of PEDF expression in old MSCs significantly improves therapeutic efficacy against MI, illustrative of exciting potential clinical applications. |
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