Chimeric Adeno-Associated Virus-Mediated Cardiovascular Reprogramming for Ischemic Heart Disease.

Here, we demonstrated chimeric adeno-associated virus (chimeric AAV), AAV-DJ-mediated cardiovascular reprogramming strategy to generate new cardiomyocytes and limit collagen deposition in cardiac fibroblasts by inducing synergism of chimeric AAV-expressing Gata4, Mef2c, Tbx5 (AAV-GMT)-mediated heart reprogramming and chimeric AAV-expressing thymosin β4 (AAV-Tβ4)-mediated heart regeneration. AAV-GMT promoted a gradual increase in expression of cardiac-specific genes, including Actc1, Gja1, Myh6, Ryr2, and cTnT, with a gradual decrease in expression of a fibrosis-specific gene, procollagen type I and here AAV-Tβ4 help to induce GMT expression, providing a chimeric AAV-mediated therapeutic cell reprogramming strategy for ischemic heart diseases.

Introductuon

Cardiovascular disease is caused by reduced or blocked blood supply to the heart muscle, which is usually associated with damages to the arteries, leading to the heart failure. It is the primary silent killer, constituting the major cause of worldwide morbidity and mortality. There is as yet no effective treatment because postnatal cardiomyocytes (CMs) have little regenerative capacity, indicating that cardiac regeneration and/or angiogenesis strategies should be developed for treatment.[1] Owing to the advent of stem cell technologies, we now have several options for cardiac regeneration.[2] The first approach is to supply functional CMs directly to the ischemic site; however, there are issues with this approach, such as cell delivery, integration, rejection, and cellular maturation,[1,3] as well as obtaining enough number of pure CMs. Other approaches include the use of proximal lineage cells, such as cardiac progenitor cells[4,5] or stimulation of resident stem cells surrounding ischemic sites in the heart by inductive signals.[6] However, there are many barriers for obtaining the expected therapeutic efficiency, such as obtaining enough functional stem cells to generate functional CMs and promote tissue regeneration and overcoming fibrosis. The recent transdifferentiation technology of reprogramming fibroblasts into CMs[7] is a very attractive method because it provides a fast route for generating functional CMs to replace cardiac fibroblasts (CFs) residing in the ischemic area. However, the efficiency of the approach[7,8] is very low (∼5% cardiac gene+/reporter gene+ cells after 1 week). Efforts have been made to improve the efficiency by addition or modification of transcription factors and the use of miRNAs and/or small molecules; however, the efficiency still needs to be improved for further application.[9−12]

In vivo reprogramming, compared with in vitro reprogramming, can be assisted by the native environment, coupled with neighboring cells (“the niches”), to promote cell survival and maturation, thereby showing higher efficiency.[8,9,13−15] However, these promising and advanced methods are dependent on retroviral or lentiviral systems, and are thus not yet feasible for further clinical application for safety reasons. For clinical translation of an in vivo cardiac reprogramming strategy, finding an alternative biomaterial with gene delivery function that is safe and efficient and can deliver the defined cardiogenic transcription factors, Gata4, Mef2c, and Tbx5 (GMT), into the ischemic sites is therefore a major prerequisite.[16−18] Many researchers have utilized nanosized natural[19,20] or synthetic materials,[21,22] protein or peptide nanospheres,[23,24] and polymersomes[25,26] and virus-based vehicles[27−31] to transfer some genetic information/function into target cells.

Cardiotropic vector, such as adeno-associated virus (AAV), capable of sustained expression of therapeutic proteins[32,33] can thus be considered as designed viral nanoparticles (VNPs) for gene therapy to promote myocardial protection and rescue. VNPs can provide controllable design and engineering and batch-to-batch consistency, compared to synthetic or natural protein preparation. The AAV is a nonpathogenic VNP with a linear single-stranded DNA genome that contains inverted terminal repeats at each AAV genome terminus for expressing eukaryotic functional gene.[34,35] Herein, we investigated the use of AAV, a safe and effective gene delivery system because these viruses do not integrate unexpectedly into the host genome and are not as immunogenic as adenovirus.[36,37] AAVs infect a relatively broad range of cells efficiently, whereas other viruses have a limited range of host cell types they can infect.[38,39] The safety and usage of AAV for clinical settings was approved by the European Medicine Agency in 2012.[40] The merits of using AAV for our study include that it can transduce dividing and nondividing cells, as well as provide nonpathogenic, efficient transduction, persistent gene expression, and low immunogenicity.

In addition, a clinical benefit can be expected from adjuvant neovascularization therapies (angiogenesis/arteriogenesis) in ischemic diseases of the heart or peripheral muscle. To increase the effect of the AAV-transgene system for in vivo cardiac reprogramming, therefore, we adopted the approach of stimulating blood vessel formation by providing angiogenic growth factors that are suitable for the treatment of vascular insufficiencies.[41−43] Thymosin β4 (Tβ4) is an actin-sequestering protein to rearrange cytoskletal proteins and thus participates in cell motility. It stimulates endothelial cell migration, adhesion, and tubule formation involved in angiogenesis[44] and can induce the differentiation of epicardial progenitor cells into endothelial cells.[45,46] It also attenuates the inflammatory response, protects from apoptosis,[47,48] and enhances the survival, proliferation, and migration of cardiac cells,[49] thereby demonstrating its potential for cardiac repair.[50] Tβ4 also serves as an antifibrotic, anti-inflammatory, and angiogenic protein to protect and facilitate regeneration of injured or damaged tissues.[40,44−48,51] Its antifibrotic effects on cardiac fibroblasts (CFs) in vitro and in vivo showed a cardioprotective effect,[52,53] which is from the inhibition of collagen synthesis was mediated by its specific receptor.[54] Its angiogenic and antifibrotic effects are associated with the normalization of organ function.[46] Importantly, AAV-Tβ4 has recently been described as a powerful tool to promote micro- and macrovessel growth in murine and porcine species.[55] Herein, we used AAV-expressing thymosin β4 (AAV-Tβ4), together with AAV-GMT, as a combined treatment to promote ischemic heart repair in an acute myocardial ischemia model. The direct intramyocardial injection of retroviral vectors expressing GMT with pretreatment of thymosin β4 peptide has previously been introduced.[13] Similarly, the chimeric AAV-DJ capsid was previously used to deliver GMT to mouse embryonic fibroblast (MEFs) in vitro.[18] However, to our knowledge this is the first report of using chimeric AAV-DJ to deliver GMT + Tβ4 to post-infarct mouse hearts for a highly efficient in vivo cardiac reprogramming strategy with clinically applicable potency. We proposed that this clinically available AAV-mediated system can be used for cardiovascular regeneration, by achieving synergism of GMT-mediated heart repair and effects of Tβ4, an angiogenic and cardioprotective peptide, to improve the system efficacy (Figure ).

Figure 1

Schematic of heart recovery in combination therapy using chimeric AAV-GMT and AAV-Tβ4. AAV-mediated cardiac repair and regeneration by achieving GMT-based cardiac conversion and Tβ4-based angiogenic niche regeneration. Chimeric AAV-mediated niches induced by GMT and Tβ4 can work successfully to improve heart function. This AAV-based synergism can be easily achieved and has promise as the next therapeutic strategy for cardiovascular disease.

Results and Discussion

We used AAV-DJ[56,57] to produce capsid and rep proteins and AAV-Helper to aid the assembly of AAV-transgenes and produce the required AAV-transgene viruses. AAV-DJ was engineered using DNA shuffling technology, which created a chimeric capsid, resulting in chimeric AAV with broad-range tropism and with higher efficiency for fibroblasts. Transgene cassettes (AAV-Gata4, -Mef2c, -Tbx5, -Tβ4, and -GFP) driven by the cytomegalovirus promoter were constructed and produced using AAV-DJ and Helper (Figure a). AAV-GFP was coinfected to allow the assessment of virus transduction. Functionality was further confirmed by transduction of 293T, MEFs, and mouse cardiac fibroblast (MCFs) at a dose of 1 × 108 genome copies per 25 T flask (Figure b). 293 T showed over 80% transduction efficiency (GFP+ cells/total cells) 3 days after transduction. Of note, AAV-mediated transgene expression gradually increased in MEFs and MCFs, which reached ∼60–80% at 1 week after transduction and lasted for 4 weeks. Over 80% MCF transduction efficiency was observed at 28 days (Figure c).

Figure 2

Production of AAV-Gata4, Mef2c, Tbx5, thymosin β4, and GFP by using AAV-DJ and helper. (a) Construction of AAV-transgene cassette, (b) long-last expressions of transgenes in mouse fibroblasts (MEF: mouse embryonic fibroblast, MCF: mouse cardiac fibroblast), (c) gradual increase of AAV-transduction efficiency was observed in MCF.

To investigate how well GMT and Tβ4 in an AAV system work for heart reprogramming and/or heart regeneration, four experimental groups were used and AAV-GFP was coinfected to allow the assessment of virus transduction: AAV-GFP (no GMT or Tβ4; group 1; GFP group), AAV-GFP + AAV-GMT (group 2; GMT group), AAV-GFP + AAV-Tβ4 (group 3; Tβ4 group), and AAV-GFP + AAV-GMT + AAV-Tβ4 (group 4; GMTTβ4 group). Primary MCFs cultured in Dulbecco’s modified eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) were transduced with the AAV-transgene cassettes, and the cardiac gene expression was examined in transduced cells in each group. The dose of each vector was held constant between groups. Immunofluorescent staining of cTnT and vimentin (VIM; fibroblast maker) showed that the virus-infected (GFP-expressing) cells in the GMT, Tβ4, or GMTTβ4 group gradually increased the expression of cTnT and decreased the expression of VIM, whereas the GFP group (AAV-GFP only) showed no cTnT expression and high VIM expression (Figure S1). Quantitative RT-PCR showed that the expression of the cardiomyocyte-specific genes, Actc1 (cardiac α-actin), Gja1 (gap junction α1-protein; connexin 43), Myh6 (α-myosin heavy chain), Ryr2 (ryanodine receptor 2), and cTnT (cardiac troponin T; cardiomyocyte marker), gradually increased in a time-dependent manner (Figure S2a). The gradual increase in GMT expression in AAV-Tβ4-transduced cells (Figure S2b) showed that Tβ4 may somehow contribute positively to GMT activity.

Correlation between GFP expression (AAV-transgene transduced cells) and cardiac-specific proteins expression, cTnT and Sarcomeric α actinin (αSA), as for cardiomyocyte conversion, was then examined (Figure ). The white regions in the cells show GFP expression, whereas co-localized regions of cTnT expression with GFP are shown in green (Figure a, up), which is opposite in terms of co-localized regions of VIM expression with GFP (Figure b, bottom). Transduction efficiencies (GFP+ cells/total cells) at day 28 were ∼80 and ∼60% in MCFs and MEFs, respectively. The cTnT-expressing cells were counted and expressed as a percentage of GFP-expressing cells (cTnT+ cells/GFP+ cells), with 0, 55 ± 7, 13 ± 11, and 67 ± 24% in MCFs and 0, 20 ± 1, 23 ± 4, and 57 ± 15% in MEFs, for groups 1–4 (Figure b). cTnT expression was highest in the GMTTβ4 group (Figure b). αSA staining also showed that the GMTTβ4 group had the highest αSA staining (Figure c). Interestingly, the relative RNA expression of Col1a2, a fibrosis-specific gene, was highest only in the GFP-only group (Figure d).

Figure 3

Correlation of AAV-transduction (GFP expression) and cTnT/αSA expression. (a) Co-localization with GFP regions were shown in green. (b) Percent of cTnT+/GFP+ cells in MCF and MEF. (MCF: mouse cardiac fibroblast, MEF: mouse embryonic fibroblast, scale bar = 20 μm) (c) Sarcomeric α-actinin (αSA) expression in each group. (d) Relative expression of fibrosis-specific gene, Col1a2, which was decreased in other three groups than AAV-GFP-only group.

Next, in vivo efficacy of AAV-transgene transfer into the mouse myocardial infarction (MI) model was examined. Mouse MI model was mimicked by left anterior descending (LAD) ligation. Mice were randomly divided into four groups (AAV-GFP only, GFP; AAV-GMT, GMT; AAV-Tβ4, Tβ4; and AAV-GMT plus Tβ4, GMTTβ4). After ligation, each group of mice received an intramyocardial injection of their specified AAV-transgenes at a dose of 1 × 109 genome copies per transgene, keeping the dose of vector bearing each transgene constant between groups. To check whether AAV-GMT, AAV-Tβ4, and AAV-GMTTβ4 promote cardiac repair may be via in vivo reprogramming. Improved heart repair can be observed first by Masson’s trichrome and hematoxylin and eosin (H&E) staining (Figure a, up). The GMT, Tβ4, and GMTTβ4 groups showed significant improvement with a decreased fibrotic area and increased wall thickness, compared to the GFP group (Figure a, bottom). Importantly, the GMTTβ4 group showed significantly enhanced improvement compared to the other groups.

Figure 4

Improved heart repair was observed by AAV-transgene treatment. (a) Histological assessment of heart sections from animals with MTc (Masson’s trichrome) and H&E (hematoxylin and eosin) staining (up). Infarct size (left) and infarct wall thickness (right) (bottom). Infarct size was presented as a percentage of the left ventricular free wall circumferential length, and infarct wall thickness was presented as a percentage of the thickness of septal wall (data are represented as mean ± standard deviation (SD), n = 5, *p < 0.01). (b) CD31 and GFP expression and PCNA and caspase-3 expression (up). Enhanced vessel growth and survival were observed in the combined treatment group (down) (data are represented as mean ± SD, n = 3, *p < 0.01, **p < 0.05 vs the GFP group, scale bar = 20 μm).

Improved heart structure by AAV-GMTTβ4 may be attained by in vivo cardiac reprogramming, first by GMT and then remodeling of injured tissues by Tβ4.[13,40,44−48,51] To investigate the antifibrotic effect and further assess the in vivo cardiac reprogramming, αSA (Actn2), compared to collagen type I (Col1a2), expression was analyzed in each group. Immunostaining of αSA and collagen type I (COI) (Figure S3a, up) was performed. The percent ratio of the COI-stained area versus the αSA-stained area was lowest in the GMTTβ4 group and lower in the GMT and Tβ4 groups compared to the GFP group, in the order GMTTβ4 < GMT < Tβ4 < GFP. The αSA ratio was reversed and highest in the GMTTβ4 group (GMTTβ4 > GMT > Tβ4 > GFP; Figure S3a, bottom). This correlation between the reduced fibrotic area (COI expression) and increased cardiac area (αSA expression) suggests that the fibrotic area is converted into cardiac tissue (meaning in vivo cardiac reprogramming). cTnT-expressing cells among GFP+ cells were also found in the AAV-transgene-treated groups (Figure S3b, up). Co-localized regions of the GFP-expressing area (the virus-infected region) with cTnT expression are examined. The average percentage of coexpressing cells (cTnT+/GFP+) was determined to be ∼2% in GFP, ∼22% in GMT, ∼7% in Tβ4, and ∼32% in GMTTβ4 groups (Figure S3b, bottom). From these results, we conclude that GMTTβ4 is the most effective combination for cardiac reprogramming.

Improved heart repair by Tβ4 co-treatment may be also related to its angiogenic effects. When we investigated CD31 expression with GFP in AAV-transgene-transduced cardiac tissue (Figure b up; CD31 in red, GFP in green), enhanced blood vessel formation was found, which may enhance regeneration/survival and reduce cell death (Figure b up; PCNA for proliferating cells in red, caspase-3 for dead cells in green). Enhanced vessel formation seems to be primarily induced by Tβ4 and was highest in the co-treatment group (Figure b, bottom). This was also associated with heart regeneration/survival (PCNA+ cells in Figure b, bottom). Reduced numbers of caspase-3-expressing cells may be related to cardiac repair/reprogramming. The co-treated group showed the lowest number of caspase-3-expressing cells, followed by the GMT group (caspase+ cells, Figure b, bottom).

AAVs are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, nonpathogenic nature, and low immunogenicity. They have been approved for the clinical treatment of lipoprotein lipase deficiency in Europe.[21,45,46] In addition, many engineered AAV variants, with novel and biomedically valuable cell tropism, are now available.[35,46] In this study, AAV-DJ was used to construct chimeric AAV-Gata4, -Mef2c, -Tbx5, and -Tβ4. Gradual and long-lasting (even after 28 days) expression in cardiac fibroblasts was confirmed. A gradual increase in cardiac-specific gene expression, with a gradual decrease in fibrosis-specific gene expression, correlated with GMT transduction and was contributed to by Tβ4. This is in accordance with a previous report that Tβ4 plays a role in inducing epicardial progenitor-derived de novo cardiomyocytes and neovascularization,[27,30] in addition to its original role in endothelial cell stimulation. It is likely that Tβ4 stimulates the niche resident stem cells in cardiac tissues. It is likely that Tβ4 also stimulates stem cells residing in the cardiac niche, consistent with the cTnT-stimulating effect of Tβ4 on MCF and MEF cells in vitro, as shown in (Figure b).

Improved heart repair was in accordance with these findings. The αSA to COI ratio and in vivo transduction efficiency showed that heart repair is primarily induced by cardiac reprograming with GMT but that Tβ4 co-treatment is the most effective. As expected, the Tβ4-induced niche may promote neovascularization, as Tβ4 groups had higher capillary/vascular density, with more proliferating cells. Lower caspase-3+ cell numbers in the GMT group may be because of GMT-mediated cardiac reprogramming (attenuated fibrosis). The percentage of cTnT+/GFP+ cells was used in this study as a metric of the efficiency of transdifferentiation to a more cardiac-like phenotype, but this metric is built on the assumption that every GFP+ cell was equally transduced by up to three separate AAV particles bearing different transgenes. This assumption may be appropriate in regions close to the injection site where the proportion of GFP+/total cells may approach 100%, but will break down with distance from the injection site as the multiplicity of infection (moi) declines. In contrast to previous reports using retroviral vectors,[13] immunostains for α-SA in the current study failed to detect the striations characteristic of sarcomeres in GFP+ cells and none of the GFP+/Actn2+ cells in this study resemble the shape of elongated cardiomyocytes. Therefore, the functional consequence of AAV-mediated GMT transduction remains to be studied. In all cases, the co-treatment group showed the highest efficacy in both heart reprogramming and regeneration.

In conclusion, AAV-mediated niches induced by GMT and Tβ4 work successfully in terms of heart repair. This AAV-based synergism can be easily achieved and has promise as the next therapeutic strategy for cardiovascular disease in the near future.

Experimental Section

Reagents

Dulbecco’s modified eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (Thermo Scientific Inc., Waltham, MA). pAAV-MCS, pAAV-GFP, pAAV-DJ, and pAAV-Helper were obtained from Cell Biolab Inc. (San Diego, CA). Anti-CD31, cTnT, and PCNA antibodies were obtained from Abcam (Cambridge, MA). Anti-caspase-3 and vimentin were obtained from Cell Signaling Technology Inc. (Beverly, MA). Antisarcomeric α-actinin was from Sigma-Aldrich (St. Louis, MO). AlexaFluor 488, 594, and 647 secondary antibodies were from Invitrogen (Warszawa, Poland). All other reagents and chemicals, unless otherwise stated, were purchased from Sigma.

Cell Culture

Human embryonic kidney-293T cells were grown in high-glucose DMEM containing 10% FBS, penicillin (100 U/mL), and streptomycin (10 μg/mL). Cells were kept under standard conditions: 37 °C, 5% CO2, and humidified atmosphere. Primary MEFs were isolated from BALB/C mouse embryos (12.5–13.5 days post-coitum).[47] Primary MCFs were isolated from 2 days to 3 weeks old C57BL/6 mice. Hearts were removed, washed in cold PBS, chopped, and digested with collagen type-II solution, as previously described.[13] A single-cell suspension was obtained by gentle trituration and passed through a 40 μm cell strainer (Nunc, Thermo Scientific Inc.). For primary MCF culture, the excised mouse hearts were minced into small pieces of less than 1 mm3, digested with 0.03 unit of Liberase (Roche, Indianapolis, IN), and cultured for 10 days in F12/DMEM/15% FBS media on gelatin-coated dishes. Migrated fibroblasts were harvested and filtered with 40 μm cell strainers (BD Bioscience, San Jose, CA) to avoid contamination with heart tissue fragments. After 4–24 h of infection, the medium was replaced with DMEM/M199/10% FBS medium and changed every 2–3 days.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

qRT-PCR was conducted using total RNA with the SYBR Green Master Mix kit (Roche) and individual primer sets (Table S1) for Actc1 (actin, α cardiac muscle 1), Myh6 (cardiac muscle myosin; myosin heavy chain 6), Ryr2 (ryanodine receptor 2), Gja1 (connexin 43; gap junction α1-protein), cTnT (cardiac Troponin T), Col1a2 (collagen 1a2), and BAT (β-actin) by using a LightCycler 96 machine (Roche). mRNA levels were normalized to BAT.

Construction of Chimeric AAV

The pAAV plasmids containing GFP, Gata4, Mef2c, Tbx5, or Tβ4 were constructed using multicloning sites and standard protocols. The pAAV vectors, along with pAAV-DJ and pAAV-Helper, were transfected into 293T cells using Lipofectamine2000 (Invitrogen) to generate functional cAAVs. Cells were harvested and lysed by freeze-thaw cycling after 72 h.

AAV Purification and Genome Copy Number Determination

AAVs were purified using an AAV purification kit (Cell Biolab). The AAV genome copy number was determined using the SYBR Green Master Mix kit (Roche) and quantitative PCR (qPCR) in a LightCycler 96 Real-Time PCR machine (Roche). The same sets of primers that were used for AAV titer determination were used for quantifying AAV genome copy (gc) numbers.

LAD Ligation and In Vivo AAV Administration

Five mice per group were subjected to MI by ligation of the LAD coronary artery, as described previously.[48] Immediately after LAD ligation, each group of mice received an intramyocardial injection of AAV-transgene (1 × 109 genome copies) in a total volume of 30 μL at three different border sites of ligated area. The mice were euthanized at 28 days post-MI, and structural assessment of their hearts was performed. All five mice from each group were included in the final tissue analysis by immunohistochemistry.

Immunohistochemistry

The excised hearts of the sacrificed mice were retrogradely perfused with PBS to wash the coronary vasculature and LV and fixed with 4% paraformaldehyde overnight at 4 °C. Each tissue sample was embedded in paraffin. Sections (2 μm) stained with hematoxylin and eosin and Masson’s trichrome were used to calculate fibrosis size and wall thickness using Image Pro version 4.5 (Media Cybernetics, Bethesda, MD). The sections were subjected to immunofluorescence staining using standard protocols. Primary antibodies against GFP (abcam ab13970), cTnT, Vimentin (BD Bioscience BD550513), CD31 (abcam ab28364), collagen type I (abcam ab21286), and αSA (Sigma A2172) were used to investigate AAV transduction (GFP), fibroblasts (Vimentin), vascular regions (CD31), fibrosis (COI), or cardiogenic regions (cTnT or αSA). Primary antibodies against proliferating cell nuclear antigen (PCNA; Abcam) and Caspase-3 (Santa Cruz Biotechnology) were used for examining proliferation and cell death in ischemic tissues. Sections were counterstained with DAPI (Vector Laboratories, Burlingame, CA) and examined using a FluoView 1000 confocal microscope (Olympus, Tokyo, Japan).

Quantitative Morphometry

Histological assessment was performed using Image J software (NIH, Bethesda). Infarct area was recognized by Masson’s trichrome staining. Infarct size was measured with a percentage of the left ventricular free wall circumferential length and infarct wall thickness with a percentage of the thickness of septal wall. GFP and cardiac protein expressing cell numbers were counted with counterstaining with DAPI. Co-localized regions of both protein expressions were determined by Image J software. Five mice in each group were used to analyze the morphology. Two–three numbers of microscope slides/mouse and three numbers of fields analyzed per mouse were used to get the analyzed data. Images in border sites of ligated area were taken.

Statistical Analyses

Data are presented as mean ± SD (where appropriate) and were considered to differ significantly if the p value was less than 0.05.