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. 2023 Nov 3:10:1279613.
doi: 10.3389/fcvm.2023.1279613. eCollection 2023.

BMP2 gene transfer induces pericardial effusion and inflammatory response in the ischemic porcine myocardium

H H Pulkkinen  1 , A Kivistö-Rahnasto  1 , H Korpela  1 , M Heikkilä  1 , N Järveläinen  1 , S Siimes  1 , L Kilpeläinen  1 , N Laham-Karam  1 , S Ylä-Herttuala  1   2   3 , J P Laakkonen  1
Affiliations

BMP2 gene transfer induces pericardial effusion and inflammatory response in the ischemic porcine myocardium

H H Pulkkinen et al. Front Cardiovasc Med. .

Abstract

Pro-angiogenic gene therapy is being developed to treat coronary artery disease (CAD). We recently showed that bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor-A synergistically regulate endothelial cell sprouting in vitro. BMP2 was also shown to induce endocardial angiogenesis in neonatal mice post-myocardial infarction. In this study, we investigated the potential of BMP2 gene transfer to improve cardiomyocyte function and neovessel formation in a pig chronic myocardial infarction model. Ischemia was induced in domestic pigs by placing a bottleneck stent in the proximal part of the left anterior descending artery 14 days before gene transfer. Intramyocardial gene transfers with adenovirus vectors (1 × 1012 viral particles/pig) containing either human BMP2 (AdBMP2) or beta-galactosidase (AdLacZ) control gene were performed using a needle injection catheter. BMP2 transgene expression in the myocardium was detected with immunofluorescence staining in the gene transfer area 6 days after AdBMP2 administration. BMP2 gene transfer did not induce angiogenesis or cardiomyocyte proliferation in the ischemic pig myocardium as determined by the quantitations of CD31 or Ki-67 stainings, respectively. Accordingly, no changes in heart contractility were detected in left ventricular ejection fraction and strain measurements. However, BMP2 gene transfer induced pericardial effusion (AdBMP2: 9.41 ± 3.17 mm; AdLacZ: 3.07 ± 1.33 mm) that was measured by echocardiography. Furthermore, an increase in the number of immune cells and CD3+ T cells was found in the BMP2 gene transfer area. No changes were detected in the clinical chemistry analysis of pig serum or histology of the major organs, implicating that the gene transfer did not induce general toxicity, myocardial injury, or off-target effects. Finally, the levels of fibrosis and cardiomyocyte apoptosis detected by Sirius red or caspase 3 stainings, respectively, remained unaltered between the groups. Our results demonstrate that BMP2 gene transfer causes inflammatory changes and pericardial effusion in the adult ischemic myocardium, which thus does not support its therapeutic use in chronic CAD.

Keywords: BMP2; bone morphogenetic protein 2; coronary artery disease; gene therapy; inflammation; ischemic heart disease; myocardial ischemia; pig myocardium.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Study protocol for BMP2 gene transfer in ischemic pig myocardium. Functional measurements and blood sampling were done at all time points: on day −14 (ischemia operation), day 0 (gene transfer start), and day 6 (gene transfer end). Left ventricular ejection fraction (EF) and strain measurements were taken to evaluate the heart functionality. (A) Ischemia operation was performed 2 weeks before the gene transfer (day −14). A bottleneck stent was introduced to the left anterior descending artery (LAD) of the porcine hearts to limit perfusion and create myocardial ischemia. (B) Gene transfer started on day 0. The optimal gene transfer site (GT site) was detected using the NOGA mapping system. Injection catheters were used to inject AdBMP2 or AdLacZ vectors into the ischemic myocardium. (C,D) Six days after the gene transfer (day 6), the pigs were sacrificed, and samples were collected for post-operational measures. Myocardial samples were collected for RT-qPCR and histological analysis. Safety tissues were collected for histological analysis to determine the safety of the gene transfer. The size of pericardial effusion was measured to evaluate the safety of the GT on day 6. Image created with BioRender.com.
Figure 2
Figure 2
BMP2 transgene is successfully expressed in the pig myocardium after gene transfer. (A) Bottleneck stent in LAD caused ischemia and infarct of the anterior wall of the left ventricle and interventricular septum. Infarcted areas are marked with arrows. (B) Representative images of immunofluorescence staining of hBMP2 protein expression (BMP2 Ab, green) in the ischemic myocardium of AdLacZ- and AdBMP2-treated pigs at d6 after gene transfer (GT). Nuclei are stained with DAPI (blue). Scale bars, 100 µm. (C) Scoring of hBMP2 protein expression in AdLacZ- and AdBMP2-treated ischemic pig myocardium (five pigs/group, AdBMP2 = 16 samples, AdLacZ = 23 samples). hBMP2 protein was expressed in AdBMP2 (average score 2.53) but not in AdLacZ (average score 0.56)-treated hearts. Scores 0–3 were used, where 0 = absent, 1 = low, 2 = moderate, and 3 = high hBMP2 expression. The mean ± SD values are shown. The Mann–Whitney U test was used to determine the statistical significance. **p < 0.01. (D) Representative H&E images of the ischemic myocardium of AdLacZ- and AdBMP2-treated pigs at d6 after GT. Images from the infract area (*), border of the GT area (GT edge), GT area, and left ventricle posterior wall (negative control of GT) and ischemia are shown. Scale bars, 100 µm.
Figure 3
Figure 3
AdBMP2 induces pericardial effusion but has no effect on heart functionality. (A) Representative images of pericardial fluid at d6 after AdLacZ and AdBMP2 GT detected by transthoracic echocardiography. Only a physiological amount of pericardial fluid was seen in the AdLacZ group (arrow), whereas AdBMP2-treated animals had visually apparent pericardial fluid accumulation (arrowhead). (B) The size of the pericardial fluid effusion was determined in the end-diastole at d6 after GT. The effusion size was clinically relevant in the AdBMP2 group as it was 9.4 ± 3.2 mm. Compared to the AdLacZ group (n = 4), the size was significantly larger in the AdBMP2 group (n = 5). The mean ± SD values are shown. The Mann–Whitney U test was used to determine the statistical significance. *p < 0.05. (C) Ejection fraction (EF) under dobutamine-stress was measured at three time points: before ischemia operation (day −14), before GT (day 0), and after the GT (day 6). AdBMP2 GT did not alter EF from day 0 to day 6 (n = 3 or 6 pigs/time point). Mean ± SEM values are shown. The Mann–Whitney U test was used to determine the statistical significance. (D) Heart contractility was determined with strain analysis using the average global strain values from the myocardium taken at three time points: before ischemia operation (day −14), before GT (day 0), and after GT (day 6). Heart contractility did not change due to AdBMP2 (n = 5 pigs/time point) GT when compared with the AdLacZ group (n = 4–5 pigs/time point). Mean ± SEM values are shown. The Mann–Whitney U test was used to determine the statistical significance.
Figure 4
Figure 4
No off-target effects, myocardial injury, or toxicity were detected in AdBMP2-treated pigs. No elevations in (A) troponin I (TPI), (B) lactate dehydrogenase (LDH), (C) creatinine (CREA), (D) C-reactive protein (CRP), (E) alanine aminotransferase (ALAT), and (F) alkaline phosphatase (ALP) were detected. Blood values were measured at three time points: before ischemia operation (day −14), before GT (day 0), and after GT (day 6). Mean ± SEM values for each group are shown (n = 4–5 pigs/group).
Figure 5
Figure 5
BMP2 does not induce angiogenesis or affect cardiomyocyte apoptosis in the ischemic pig myocardium. (A) Representative images of CD31-stained pig myocardium indicate that no changes in the amount or size of the capillaries were detected in the AdBMP2-treated ischemic pig myocardium in comparison to AdLacZ control samples in 6 days GT. Scale bars: 100 µm. (B) The mean capillary area was determined from CD31-stained ischemic myocardial thin sections at day 6 after GT (n = 5 pigs/group). No statistically significant difference was detected between the groups. The mean ± SEM values are shown. The Mann–Whitney U test was used to determine the statistical significance. (C) Representative images of CC3-stained myocardial samples at day 6 after AdBMP2 and AdLacZ GT (n = 5 pigs/group, AdLacZ = 30 samples, AdBMP2 = 16 samples). Scale bars, 100 µm. Arrows point to CC3-positive cardiomyocyte nuclei and arrow heads to CC3-negative cardiomyocyte nuclei. (D) The number of CC3 expressing apoptotic cardiomyocytes was scored from whole slide myocardial thin sections at d6 after GT. AdBMP2 did not alter the number of CC3-positive cardiomyocytes in comparison to AdLacZ (n = 5 pigs/group). Scores 0–3 were used, where 0 = absent, 1 = low, 2 = moderate, and 3 = high amount of CC3-positive cardiomyocytes. The mean ± SD values are shown. The Mann–Whitney U test was used to determine the statistical significance.
Figure 6
Figure 6
AdBMP2 GT induces inflammation but has no effect on fibrosis. (A) Representative images of H&E-stained ischemic pig myocardium treated with AdBMP2 or AdLacZ at day 6 after GT. AdBMP2 GT induced infiltration of inflammatory cells. Scale bars, 100 µm. (B) The level of inflammation after the GT at d6 was evaluated by scoring the amount of infiltrated inflammatory cells from H&E-stained ischemic myocardial whole slide samples. AdBMP2 GT significantly increased the inflammatory cell infiltration in comparison to AdLacZ GT (n = 5 pigs/group, AdLacZ = 23 samples, AdBMP2 = 16 samples). Scores 0–3 were used, where 0 = no cells, 1 = low, 2 = moderate, and 3 = high number of inflammatory cells. The mean ± SD values are shown. The Mann–Whitney U test was used to determine the statistical significance. **p < 0.01. (C) Representative images of CD3+ T cells in the ischemic myocardial samples at d6 after AdBMP2 and AdLacZ GT. AdBMP2 increased the amount of CD3+ cells (arrows) in the myocardium in comparison to AdLacZ. Scale bars, 100 µm. (D) T-cell infiltration scoring from CD3-stained myocardial tissue sections. AdBMP2 GT significantly increased the number of CD3+ T cells in comparison to AdLacZ (n = 5 pigs/group, AdLacZ = 23 samples, AdBMP2 = 16 samples). The mean ± SD values are shown. The Mann–Whitney U test was used to determine the statistical significance. *p < 0.05. (E) Representative images of Sirius red–stained myocardial samples after AdBMP2 or AdLacZ GT at day 6. Scale bars, 100 µm. (F) The level of fibrosis was evaluated by scoring the amount of collagen from AdBMP2- or AdLacZ-treated myocardial samples stained with Sirius red (n = 5 pigs/group, AdLacZ = 23 samples, AdBMP2 = 16 samples). AdBMP2 did not induce fibrosis. Scores 0–3 were used, where 0 = physiological amount of collagen, 1 = low increase of collagen, 2 = moderate increase of collagen, and 3 = high increase of collagen. The mean ± SD values are shown. The Mann–Whitney U test was used to determine the statistical significance.
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