Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction

Abstract

In a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) results in the expansion and directed differentiation of endogenous heart progenitors in a mouse myocardial infarction model. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with DNA vectors and temporal control with VEGF inhibitors revealed the greatly increased efficacy of pulse-like delivery of VEGF-A. Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control progenitor cell fate and thereby enhance long-term organ repair.

Figure 1. Highly efficient, transient gene transfer in vivo using modRNA

a-b. Luciferase DNA or modRNA was delivered by myocardial injection. Protein expression was assayed by bioluminescence. c. Time course of luciferase activity after injection of luciferase DNA or modRNA (100 μg) or vehicle only (control) per heart. d. Cre DNA or modRNA, delivered by myocardial injection, catalyzed cardiac recombination, detected by X-gal staining (blue) for Cre-activated expression of LacZ from R26^fsLacZ. e-g. Indicated doses of Cre DNA were injected intramyocardially into R26^fsLacZ hearts. DNA plasmid inefficiently transfected cells expressing endothelial, cardiomyocyte, and smooth muscle lineage markers. Lines labeled i, ii, and iii indicate planes of section in panel f. Bar = 400 μm (e) or 50 μm (f-g). h-j. Indicated doses of Cre modRNA were injected intramyocardially into R26^fsLacZ hearts. modRNA efficiently transfected cells expressing endothelial, cardiomyocyte, and smooth muscle lineage markers. Lines labeled i, ii, and iii indicate planes of section in panel i. Bar = 400 μm (h) or 50 μm (i-j). k. Summary of transfection efficiency of single 100 μg DNA or modRNA injection into cardiac muscle (left ventricle). l. Kinetics of VEGFA modRNA expression in cardiac cells cultured in vitro. * For a-l, n=3, Representative of 2 independent experiments.

Figure 2. VEGF-A modRNA enhanced formation of functional, non-leaky vessels

a. VEGF-A modRNA, injected into the infarct region at the time experimental myocardial infarction (myocardial infarction), increased vascular density in the peri-infarct region. Seven days after myocardial infarction, the vascular plexus was highlighted by Microfil followed by imaging of cleared hearts. The indicated treatments were injected within the region demarcated by the dashed lines. b. VEGF-A modRNA reduced scar area and TUNEL⁺ cells and increased capillary density at 1 week and 4 weeks after myocardial infarction. Capillary density and TUNEL⁺ fraction were measured in infarct border zone (left ventricle). Masson's trichrome was used to evaluate scar area (see also Supplementary Figure 4). n=3. c. Beneficial activity of VEGF-A modRNA required KDR signaling. Mice were treated with myocardial infarction and VEGF-A modRNA. KDR inhibitors SU5614 or PTK787, administered from one day before myocardial infarction to tissue collection at 7 days after myocardial infarction, blocked beneficial effect of VEGF-A modRNA. d. Experimental design to assess functional angiogenesis. VEGF-A modRNA was injected into the myocardium at the time of LAD ligation. After one week, isolectin B4 and FITC-dextran beads (70 kDa) were injected into the tail vein to assess connection to the systemic vasculature and vascular permeability, respectively. e. Vessels formed under the influence of VEGF-A DNA, but not VEGF-A modRNA, or VEGF-A DNA with Avastin, neutralizing VEGF-A antibodies (given IP twice a week) were permeable to FITC-dextran (yellow arrows). Scale bar = 50 μm. f. Density of luminal structures and leaky vessels quantification for different treatments. g. Macroscopic myocardial edema in VEGF-A DNA (yellow frame), but not VEGF-A modRNA, treated hearts. Scale bar = 5 mm. *For a-g, n=3, Representative of 2 independent experiments.

Figure 3. VEGF-A modRNA improved outcome in a murine myocardial infarction model

a. Short term survival curve after myocardial infarction and the indicated treatments. VEGF-A modRNA, DNA, or vehicle were injected into the infarct region at the time of LAD ligation. Avastin was injected twice weekly starting on post-myocardial infarction day 3. P-values were calculated using the Mantel-Cox log-rank test. b. Long term survival curve after myocardial infarction and VEGF-A modRNA or control treatments. VEGF-A modRNA improved survival at one year compared to control treatment. P-value was calculated as in (a). c. MRI assessment of left ventricular systolic function. Images show left ventricular chamber (outlined in red) in diastole and systole. d. Left ventricular systolic function (ejection fraction) was better preserved 21 days after LAD ligation in the VEGF-A modRNA group compared to control. P-value was calculated using paired-test. Sham control, n=3, control or VEGF-A modRNA group, n=5.

Figure 4. VEGF-A modRNA reduced scar area and apoptosis and increased capillary density and WT1⁺ cells proliferation after myocardial infarction in a KDR-dependent manner

a. Marker gene analysis showed that VEGF-A modRNA dramatically upregulated Wt1 expression. qRT-PCR was performed on peri-infarct tissue 3 days post-myocardial infarction. Expression was calculated relative to vehicle-treated heart (dashed line). b. Quantitation of Wt1⁺ cells in the infarct border zone (left ventricale) of immunostained heart sections. VEGF-A modRNA increased frequency of WT1⁺ cells at 1 week and 4 weeks after myocardial infarction but not after sham treatment. c. Increase in Wt1⁺ cells in myocardial infarction + VEGF-A modRNA-treated hearts required signaling through KDR. Samples were analyzed as in (b), 1 week after myocardial infarction. d. FACS-based quantitation of Wt1+ cells after myocardial infarction and control or VEGFA modRNA treatment. WT1⁺ epicardial progenitors were isolated from dissociated Wt1^GFPCre heart by GFP FACS sorting. VEGF-A modRNA treatment increased the frequency of GFP⁺ (Wt1-expressing) cells 1 week after myocardial infarction. Red numbers within the region of interest indicate the fraction of cells that were GFP⁺ (WT1⁺). e. KDR expression on WT1⁺ epicardial progenitors was measured by FACS. Dissociated Wt1^GFPCre/+ hearts were stained for KDR, then analyzed by FACS. The histogram shows KDR immunostaining intensity on WT1⁺ epicardial progenitors (GFP⁺). myocardial infarction and VEGF-A modRNA treatment increased KDR mean fluorescence intensity (MFI) on these progenitors. f. VEGF-A protein increased, and KDR antagonists reduced proliferation of FACS-purified WT1⁺ epicardial progenitors. Cell number was measured using an automated cell counter at days 4, 8 and 14 of cell culture. *For a-f, n=3, Representative of 2 independent experiments.

Figure 5. VEGF-A modRNA induced WT1⁺ epicardial progenitor proliferation and shifted differentiation towards the endothelial lineage

a. VEGF-A modRNA increased endothelial marker gene expression in FACS-purified GFP⁺ cells after myocardial infarction. Gene expression, determined by qRT-PCR, was calculated relative to GFP⁺ cells isolated from control-treated, post-myocardial infarction hearts. n=3 Representative of 2 independent experiments. b. Experimental design of clonal assays to assess VEGF-A modulation of WT1⁺ epicardial cell fate decisions. Individual FACS-purified cardiac WT1⁺ cells were deposited in 96 well dishes, clonally expanded, and assessed for differentiation to the indicated lineages by qRT-PCR. One VEGF-A-naïve, VE-Cadherin negative clone was subcloned and the individual subclones were further tested in the clonal differentiation assay. c. VEGF-A modRNA promoted Wt1⁺ epicardial progenitor differentiation towards the endothelial lineage. Each row represents an individual clone, and each column indicates the relative qRT-PCR-measured expression of the indicated lineage marker. The precentage of clones with detectable expression of each lineage marker is indicated at the bottom of each column. d. VEGF-A modRNA effect on Wt1⁺ epicardial progenitor differentiation was recapitulated in the subclonal assay. This confirmed multipotency of WT1⁺ epicardial cells, and made polyclonal contamination highly unlikely.

Figure 6. VEGF-A modRNA promoted differentiation of EPDCs towards the cardiovascular lineage in vivo

a. Genetic lineage tracing was used to follow the fate of EPDCs after myocardial infarction with VEGF-A or Luc modRNA treatment. Tamoxifen treatment of Wt1^CreERT2/+::R26^mTmG mice prior to myocardial infarction irreversibly labeled epicardial cells and their descendants with GFP. b. FACS analysis of dissociated hearts 1 week after myocardial infarction indicated that VEGF-A modRNA increased the frequency of EPDCs expressing the endothelial marker PECAM1 (indicated in red). c. VEGF-A modRNA increased endothelial and cardiomyocyte marker gene expression in FACS-sorted EPDCs one week after myocardial infarction. Expression of each marker was measured by qRT-PCR and displayed relative to expression in control-treated EPDCs. d. Immunofluorescent analysis of EPDC fate by Wt1^CreERT2 genetic lineage tracing. Expression of smooth muscle (smMHC), endothelial (PECAM1), and cardiomyocyte (TNNI3) markers by GFP⁺ EPDCs was assessed by immunostaining and confocal microscopy. Bar = 30 μm. e. Quantitation of d. A minimum of 2000 EPDCs were analyzed in each post-myocardial infarction heart treated with Luc modRNA (n=2) or VEGF-A modRNA (n=5) from 2 independent experiments. The graph shows the percentage of GFP⁺ EPDCs that co-expressed the indicated lineage marker. f. Cre modRNA gel-mediated tracing of epicardial cell fate was used to follow the fate of EPDCs after myocardial infarction with VEGF-A or Luc modRNA treatment. Cre modRNA gel, applied to R26^mTmG mice 2 weeks prior to myocardial infarction, irreversibly labeled epicardial cells and their descendants with GFP. g. Cre modRNA gel selectively labeled epicardial cells with GFP in R26^mTmG mice. Note that labeled cells were restricted to the epicardium in controls (Sham or myocardial infarction and Luc modRNA treatment). However in myocardial infarction hearts injected with VEGF-A modRNA, labeled cells were found both in the epicardial layer and within the myocardium, and differentiated into myocytes (yellow asterisk) and non-myocytes (white arrowheads). Bar = 50 μm. h. Immunofluorescent analysis of EPDC fate with Cre modRNA gel lineage tracing. Bar = 30 μm. i. Quantitation of h. A minimum of 2000 Cre-gel labeled cells were analyzed in each post-myocardial infarction heart treated with Luc modRNA (n=3) or VEGF-A modRNA (n=4) from 2 independent experiments. The graph shows the percentage of GFP⁺ EPDCs that co-expressed the indicated lineage marker.

Figure 7. Suggested model for the role of VEGF-A modRNA on EPDCs differentiation in vivo

Schematic summary of results. In the native state, myocardial infarction stimulates amplification of WT1⁺ EPDCs, which remain confined to the epicardial layer. VEGF-A modRNA in the context of myocardial infarction augments amplification of WT1⁺ EPDCs, increases their mobilization into the myocardial layer, and enhances their differentiation towards the endothelial lineage.