Therapeutic vascularization in regenerative medicine

Abstract

Therapeutic angiogenesis, that is, the generation of new vessels by delivery of specific factors, is required both for rapid vascularization of tissue-engineered constructs and to treat ischemic conditions. Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis. However, uncontrolled expression can lead to aberrant vascular growth and vascular tumors (angiomas). Major challenges to fully exploit VEGF potency for therapy include the need to precisely control in vivo distribution of growth factor dose and duration of expression. In fact, the therapeutic window of VEGF delivery depends on its amount in the microenvironment around each producing cell rather than on the total dose, since VEGF remains tightly bound to extracellular matrix (ECM). On the other hand, short-term expression of less than about 4 weeks leads to unstable vessels, which promptly regress following cessation of the angiogenic stimulus. Here, we will briefly overview some key aspects of the biology of VEGF and angiogenesis and discuss their therapeutic implications with a particular focus on approaches using gene therapy, genetically modified progenitors, and ECM engineering with recombinant factors. Lastly, we will present recent insights into the mechanisms that regulate vessel stabilization and the switch between normal and aberrant vascular growth after VEGF delivery, to identify novel molecular targets that may improve both safety and efficacy of therapeutic angiogenesis.

Keywords: extracellular matrix; genetic therapy; ischemia; neovascularization; tissue engineering; vascular endothelial growth factor.

Figure 1

Sprouting and intussusception: two alternative modes of angiogenesis. Schematic representation of the processes generating new vascular structures by sprouting (A–C) or by intussusception (vascular splitting; D–I). J–M, Immunofluorescence images of vessels undergoing intussusception after vascular endothelial growth factor delivery in murine skeletal muscle, stained for endomucin (endothelial cells, green), laminin (basal lamina, red), and with DAPI (nuclei, blue). Circumferentially enlarged vessels displayed: (a) no degradation of the basement membrane; (b) intraluminal filopodia‐like protrusions from the endothelial layer (white arrowheads in high‐magnification panels K and M); and (c) mature intraluminal tissue pillars (white arrows in high‐magnification panel M). * represents vascular lumen; scale bars = 20 μm in all panels

Figure 2

Functional outcomes of vascular endothelial growth factor (VEGF) dose distribution in tissue. A, Heterogeneous dose distributions (eg, by gene therapy vectors) lead to hotspots of excessive expression that remains localized in the microenvironment around producing cells (red spots, upper right panel) and lead to toxic effects. Reducing the total dose does not completely avoid toxic hotspots even if therapeutic levels are achieved in some areas (blue spots, upper middle panel), until the total dose is so low that mostly ineffective levels are achieved (gray spots, upper left panel). B, Homogeneous distribution of the total dose allows therapeutic levels (blue spots, upper middle panel) to be achieved and to harness the therapeutic window of VEGF delivery

Figure 3

Fluorescence‐activated cell sorting (FACS)‐purification of genetically modified progenitor populations expressing homogeneous vascular endothelial growth factor (VEGF) levels. A, Retroviral vector carrying a bicistronic cassette, in which the sequence of VEGF is linked to that of the membrane‐bound reporter CD8 through an Internal Ribosome Entry Site (IRES) sequence. B, Progenitors of interest are transduced with this retroviral vector. C, After integration in the cell chromatin, the IRES sequence enables cotranslation of both proteins from the same mRNA molecule. Therefore, the amount of CD8 on the cell membrane reflects that of secreted VEGF regardless of their absolute level of expression. D, Transduced cells in the primary population express heterogeneous VEGF levels depending on their viral copy number and the transcriptional activity of the chromatin integration sites. E, These can be FACS‐sorted into homogeneous subpopulations stably expressing specific VEGF levels based on the intensity of membrane CD8 staining

Figure 4

Strategies for matrix decoration with engineered recombinant factors. A, Fibrin matrices can be decorated with engineered growth factors to mimic extracellular matrix (ECM) functions. Taking advantage of the coagulation cascade, an octapeptide substrate of the TransGlutaminase factor XIII (TG hook) can be fused to growth factors (GFs), enabling their covalent crosslinking to fibrin. Specific domains of ECM proteins (eg, fibronectin) can also be incorporated through a TG hook to exploit their natural affinity for different GFs. B, Endogenous ECM can be decorated with therapeutic GFs engineered to exhibit super‐affinity to a broad range of ECM components