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 ± 3.17â mm; AdLacZ: 3.07 ± 1.33â 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.
ABSTRACT
Three-dimensional image analyses are required to improve the understanding of the regulation of blood vessel formation and heterogeneity. Currently, quantitation of 3D endothelial structures or vessel branches is often based on 2D projections of the images losing their volumetric information. Here, we developed SproutAngio, a Python-based open-source tool, for fully automated 3D segmentation and analysis of endothelial lumen space and sprout morphology. To test the SproutAngio, we produced a publicly available in vitro fibrin bead assay dataset with a gradually increasing VEGF-A concentration ( https://doi.org/10.5281/zenodo.7240927 ). We demonstrate that our automated segmentation and sprout morphology analysis, including sprout number, length, and nuclei number, outperform the widely used ImageJ plugin. We also show that SproutAngio allows a more detailed and automated analysis of the mouse retinal vasculature in comparison to the commonly used radial expansion measurement. In addition, we provide two novel methods for automated analysis of endothelial lumen space: (1) width measurement from tip, stalk and root segments of the sprouts and (2) paired nuclei distance analysis. We show that these automated methods provided important additional information on the endothelial cell organization in the sprouts. The pipelines and source code of SproutAngio are publicly available ( https://doi.org/10.5281/zenodo.7381732 ).
Subject(s)
Endothelial Cells , Neovascularization, Physiologic , Mice , Animals , Neovascularization, Physiologic/physiology , Endothelium , Cardiovascular Physiological Phenomena , InformaticsABSTRACT
The BMP/TGFß-Smad, Notch and VEGF signaling guides formation of endothelial tip and stalk cells. However, the crosstalk of bone morphogenetic proteins (BMPs) and vascular endothelial growth factor receptor 2 (VEGFR2) signaling has remained largely unknown. We demonstrate that BMP family members regulate VEGFR2 and Notch signaling, and act via TAZ-Hippo signaling pathway. BMPs were found to be regulated after VEGF gene transfer in C57/Bl6 mice and in a porcine myocardial ischemia model. BMPs 2/4/6 were identified as endothelium-specific targets of VEGF. BMP2 modulated VEGF-mediated endothelial sprouting via Delta like Canonical Notch Ligand 4 (DLL4). BMP6 modulated VEGF signaling by regulating VEGFR2 expression and acted via Hippo signaling effector TAZ, known to regulate cell survival/proliferation, and to be dysregulated in cancer. In a matrigel plug assay in nude mice BMP6 was further demonstrated to induce angiogenesis. BMP6 is the first member of BMP family found to directly regulate both Hippo signaling and neovessel formation. It may thus serve as a target in pro/anti-angiogenic therapies.
