Therapeutic angiogenesis aims at restoring blood flow to ischemic tissues by the generation of new vessels. Our research focuses on the basic principles governing the growth of blood vessels and translating these concepts into novel regenerative medicine approaches to: 1) treat ischemic diseases (coronary and peripheral artery diseases), and 2) improve the rapid vascularization of tissue engineered grafts.
The projects, at the interface between fundamental research and clinical translation, rely on the close interaction between basic scientists and clinical surgeons. Key approaches are the use of precursor cells genetically engineered to express controlled levels or combinations of angiogenic factors, in order to provide both vascular growth and tissue regeneration and combining the specific advantages of cell and gene therapy, as well as controlled release of recombinant proteins by smart biomaterials.
1. Cellular and molecular mechanisms of VEGF-induced angiogenesis
Vascular endothelial growth factor (VEGF) is the master regulator of vascular growth, but its uncontrolled expression can cause aberrant angiogenesis and angioma-like vascular tumors (Ozawa & Banfi 2004). Our understanding of angiogenesis is mostly based on developmental models, in which new vessels sprout to vascularize tissues. However, we found that VEGF delivery to muscle (the therapeutic target tissue) at the doses needed for functional benefit, induces angiogenesis without sprouting, but rather by circumferential enlargement of vessels, followed by longitudinal splitting (“intussusception”; Gianni-Barrera 2013). The mechanisms regulating intussusceptive angiogenesis are essentially unknown and likely to differ from those of sprouting.
Taking advantage of highly controlled cell-based and biomaterial-based factor delivery platforms we developed, we are pursuing a systematic investigation of the mechanisms that regulate the vascular switch between normal and aberrant angiogenesis in vivo under clinically relevant conditions, as well the stabilization and persistence of newly induced vessels (Groppa 2015). These studies include the analysis of the stage-specific mRNA and miRNA ex vivo vascular transcriptomes and non-invasive in vivo imaging, in order to identify novel and more specific molecular targets for therapeutic angiogenesis approaches.
Groppa E et al. VEGF dose regulates vascular stabilization through Semaphorin3A and the Neuropilin-1+ monocyte/TGF-b1 paracrine axis. EMBO Mol Med, 2015. Pubmed.
Gianni-Barrera R et al. VEGF over-expression in skeletal muscle induces angiogenesis by intussusception rather than sprouting. Angiogenesis, 2013. Pubmed.
Ozawa CR and Banfi A et al. Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and aberrant angiogenesis. J Clin Invest, 2004. Pubmed.
- Roche Translational Medicine Hub Innovation Fund Grant: Unraveling the VEGF-induced angiogenic switch by in vivo vascular transcriptome analysis.
2. Controlled Factor Delivery
We are developing novel methods to deliver the VEGF gene alone or in combination with modulating factors to increase its safety and efficacy in vivo, using transduced progenitors, gene therapy vectors and controlled release of recombinant proteins by smart biomaterials.
The transition between normal and aberrant angiogenesis depends on the VEGF amount in the microenvironment around each producing cell rather than on the total dose, since VEGF remains tightly localized in the extracellular matrix (Ozawa & Banfi 2004). In order to translate this biological concept into a clinically applicable approach, we developed a high-throughput FACS-based technology to rapidly purify transduced progenitors expressing specific VEGF levels (Misteli 2010).
On the other hand, to avoid the need for genetic modification and improve clinical applicability, in collaboration with Jeffrey Hubbell (University of Chicago, USA) we developed a state-of-the-art biomaterial platform based on fibrin hydrogels that enables independent control of the dose and duration of release of matrix-bound growth factors, by which we could identify a 500-fold range of VEGF concentrations inducing only physiological capillary networks, which were long-term stable and therapeutically effective in ischemic wounds (Sacchi 2014).
Martino MM et al. Extracellular matrix and growth factor engineering for controlled angiogenesis in regenerative medicine. Front Bioeng Biotechnol, 2015. Pubmed.
Sacchi V et al. Long-lasting fibrin matrices ensure stable and functional angiogenesis by highly tunable, sustained delivery of recombinant VEGF164. Proc Natl Acad Sci USA, 2014. Pubmed.
Misteli H et al. High-throughput FACS purification of transduced progenitors expressing defined VEGF levels induces controlled angiogenesis in vivo. Stem Cells, 2010. Pubmed.
- European Union, H2020 Project (646075): ELASTISLET: Tailored elastin-like recombinamers as advanced systems for cell therapies in diabetes mellitus: a synthetic biology approach towards a bioeffective and immunoisolated biosimilar islet/cell niche.
3. Cardiac revascularization
Ischemic heart disease is one of the most frequent causes of death worldwide and there is a large unmet clinical need for approaches to: 1) restore flow to ischemic myocardium; and 2) provide functional support to the damaged myocardium by external apposition of tissue-engineered contractile patches.
We could previously show that controlled VEGF expression by FACS-purified progenitor populations could induce effective vascularization both inside and outside of thick, engineered cardiac patches (Marsano 2013; Boccardo & Gaudiello 2016), as well as therapeutic angiogenesis in ischemic myocardium (Melly 2012 and 2017). Currently we are aiming at obtaining robust, normal and long-lasting vascular growth in the myocardium by controlled delivery of specific doses and combinations of recombinant factors through an optimized fibrin-based platform we developed, thereby dispensing with the safety concerns attached to the genetic modification of progenitors and improving clinical applicability. These projects are carried out in collaboration with the Cardiac Surgery of Basel University Hospital (Prof. Friedrich Eckstein and PD Dr. Anna Marsano) and the Cardiac Surgery of the Catholic University of Louvain/Namur (Prof. Benoit Rondelet and Dr. Ludovic Melly).
Melly LF et al. Myocardial infarction stabilization by cell-based expression of controlled VEGF levels. J Cell Mol Med, 2017. In press.
Boccardo S and Gaudiello E et al. Engineered mesenchymal cell-based patches as controlled VEGF delivery systems to induce extrinsic angiogenesis. Acta Biomaterialia, 2016. Pubmed.
Marsano A et al. The effect of controlled expression of VEGF by transduced myoblasts in a cardiac patch on vascularization in a mouse model of myocardial infarction. Biomaterials, 2013. Pubmed.
Melly LF and Marsano A et al. Controlled angiogenesis in the heart by cell-based expression of specific VEGF levels. Hum Gene Ther Methods, 2012. Pubmed.
4. Accelerated healing of ischemic diabetic wounds
Chronic foot ulcers in diabetic patients cause severe morbidity and are characterized by poorly vascularized and ischemic tissue, although blood flow in large vessels is normal or only mildly impaired. Taking advantage of the unique tools we previously developed, we will investigate the potential for controlled co-delivery of VEGF and PDGF-BB proteins from a fibrin-based optimized platform to restore functional flow and accelerate healing in a chronic wound model in diabetic mice. This project is carried out in collaboration with the Vascular Surgery of Basel University Hospital (Prof. Lorenz Gürke and PD Dr. Thomas Wolff).
Gianni-Barrera R et al. Long-term safety and stability of angiogenesis induced by balanced single-vector co-expression of PDGF-BB and VEGF164 in skeletal muscle. Sci Rep, 2016. Pubmed.
Mujagic E et al. Induction of aberrant vascular growth, but not of normal angiogenesis, by cell-based expression of different doses of human and mouse VEGF is species-dependent. Hum Gene Ther Methods, 2013. Pubmed.
Wolff T et al. FACS-purified myoblasts producing controlled VEGF levels induce safe and stable angiogenesis in chronic hind limb ischemia. J Cell Mol Med, 2012. Pubmed.
Banfi A et al. Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB. FASEB J, 2012. Pubmed.
- European Union, FP7 Marie Curie Initial Training Network (ITN; CP-IP 317304): AngioMatTrain: Development of biomaterial-based delivery systems for ischemic conditions – An integrated pan-European approach.
Successful generation of tissue-engineered bone substitutes requires: 1) robust osteogenic differentiation of implanted progenitors, and 2) rapid vascularization of the graft in vivo. We will develop an engineered fibrin-based matrix, decorated with an optimal combination and duration of signaling molecules, to both drive robust bone formation by adipose- and bone marrow-derived mesenchymal progenitors and promote graft vascularization in an intraoperative approach (i.e. without intervening in vitro culture of progenitors). In particular we will investigate the potential of signaling pathways that we identified in previous projects and that play a dual role in both processes, in order to couple angiogenesis and osteogenesis at a molecular level. This project is carried out in collaboration with the Plastic and Reconstructive Surgery of Basel University Hospital (Prof. Dirk J. Schäfer and Dr. Maximilian Burger).
Grosso A et al. It takes two to tango – Coupling of angiogenesis and osteogenesis for bone regeneration. Front Bioeng Biotechnol, 2017. Pubmed.
Helmrich U et al. Osteogenic graft vascularization and bone resorption by VEGF-expressing human mesenchymal progenitors. Biomaterials, 2013. Pubmed.
Di Maggio N et al. FGF-2 maintains a niche-dependent population of self-renewing highly potent non-adherent mesenchymal progenitors through FGFR2c. Stem Cells, 2012. Pubmed.