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ANGIOGENESIS, A KEY STAGE IN TISSUE REGENERATION, IS MODULATED BY PLATELETS.
Sarah BERNDT PhD1, Gilles CARPENTIER2, Axel TOLLANCE PhD1,3, Antoine TURZI1
1 Regen Lab SA, 1052 Le Mont-sur-Lausanne, Switzerland.
2 Gly-CRRET Research Unit 4397, Université Paris-Est Créteil, Créteil, France.
3 Department of Orthopedic Surgery, Geneva University Hospitals & Faculty of Medicine, Geneva, Switzerland .
Summary
Angiogenesis in vivo and in vitroin physiological and pathological processes, is a multifactorial process. It involves a plethora of signaling molecules and pathways, cells in dynamic dialogue and countless cytokines and growth factors to generate a functional and stable vascular system. This vascularization, however, is one of the prerequisites for tissue regeneration, as it ensures a continuous supply of nutrients and oxygen. These considerations found their way into modern tissue engineering when the vital importance of a vascular network was increasingly studied.
The future of regenerative approaches will inevitably combine the field-proven strategies of tissue engineering with modern biomolecular techniques in the scientific environment of stem cells and gene therapy.
The delicate balance between mechanical properties, tissue support and angiogenic stimulation will be the focus of regenerative medicine research for years to come.
In this work, we focused on the potential use of platelet-derived preparations (platelet-rich plasma (PRP), PRP combined with hyaluronic acid (PRP-HA) and platelet lysates (PL)) for controlled angiostimulation.
Keywords: angiogenesis, platelets, regeneration.
Figure 1.
Mechanisms regulating angiogenesis by platelets. In budding angiogenesis (BA), nutrient deprivation, hypoxia and pro-angiogenic factors prompt endothelial cell (EC) activation and vascular growth, where tip cells guide highly proliferative stem cells.
Circulating platelets can contribute to AB by (1) preserving vascular integrity by binding either to exposed extracellular matrix (ECM) or to ECs.
Activated platelets can also (2) release angiogenic molecules and platelet microparticles (PMPs) at proximity of ECs (adapted from Roweth and Battinelli, 2024 [5]).
INTRODUCTION: ANGIOGENESIS, KEY STAGE IN TISSUE REGENERATION
Angiogenesis, the process by which new blood vessels form from existing ones, plays a crucial role in tissue regeneration. Indeed, when tissue is damaged or needs to grow, adequate vascularization is essential to provide the nutrients, oxygen and growth factors required for tissue repair or growth [1].
In the context of tissue regeneration, angiogenesis can be stimulated in a number of ways, including the use of growth factors, cell therapies or biomimetic materials. By promoting the formation of new blood vessels, the vascularization of damaged tissue is improved, accelerating the healing process and promoting more complete tissue regeneration [2].
Understanding and manipulating angiogenesis in the context of tissue regeneration opens the way to many promising medical applications, notably in the treatment of wounds.
vascular disease and tissue degeneration [3]. By optimizing this complex biological process, we can significantly improve clinical outcomes and promote patient health and well-being.
With regard to angiogenesis, platelets play a crucial role by releasing various growth factors and cytokines that promote the formation of new blood vessels from existing ones.
These include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet growth factor (PDGF) and other pro-angiogenic molecules [4].
When tissue damage occurs, platelets are activated and recruited to the site of injury. They then release these growth factors, stimulating the proliferation and migration of endothelial cells, thus promoting the formation of new blood vessels for tissue repair and healing. In addition, platelets can interact with other cells involved in angiogenesis, such as endothelial cells and immune cells, to regulate the process in a coordinated fashion.
Understanding the role of platelets in angiogenesis is crucial for developing new therapeutic strategies aimed at modulating this process in various pathological contexts, such as cardiovascular disease, cancer and tissue regeneration.
Extensive research is underway to elucidate the underlying mechanisms and exploit the therapeutic potential of platelet-derived products in modulating angiogenesis [5].
3D CULTURE MODELS IN VITRO ARE INVALUABLE TOOLS FOR STUDYING ANGIOGENESIS, AS THEY ENABLE US TO MORE FAITHFULLY RECREATE THE THREE-DIMENSIONAL ENVIRONMENT FOUND.
The main advantages of these models for studying angiogenesis are as follows:
█ Realistic representation of the cellular environment
3D cultures enable cells to develop in a three-dimensional environment closer to the one they encounter. in vivoThis is due to the different cell types involved and their interaction with the surrounding matrix. This promotes more physiological cell-cell and cell-matrix interaction and more faithful gene expression, which can lead to more relevant results.
█ Precise control of experimental conditions
3D models offer the possibility of more precise control over experimental conditions, such as culture medium composition, growth factor concentration and substrate rigidity. This enables researchers to better understand the underlying mechanisms of angiogenesis and identify the factors that regulate it.
2D cell culture.
BENEFITS
- - Easy handling and imaging
- -Often lower cost
- - Greater availability of cell lines
- - More suitable for certain types of cell analysis, such as cell proliferation or cytotoxicity assays
ADVANTAGES
- - Inability to faithfully reproduce the 3D environment of tissue in vivo
- - Limitations in the representation of cell interaction and cell morphology
- -Less representative of in vivo physiological phenomena
- -May lead to less predictive results for therapy efficacy
3D cell culture
BENEFITS
- - More faithful reproduction of in vivo tissue architecture
- - Better representation of cellular interactions and morphology
- - Enables the study of complex biological phenomena such as angiogenesis and metastasis
- -Can provide more predictive results for drug or therapy evaluation
ADVANTAGES
- -Increased complexity of cell lines adapted to 3D models
- -Potentially higher costs
- -Less availability of cell lines suitable for 3D models
- -Increased risk of non-standardization and reproducibility of results
- - Time-consuming
█ Study of temporal dynamics
3D models enable us to follow the evolution of angiogenesis over time, which is crucial for understanding the different stages of the process, such as blood vessel formation, maturation and remodeling. It also enables us to study how different cell types interact and coordinate their activities during angiogenesis.
█ Testing drugs and therapies
3D crops in vitro can be used to assess the efficacy of drugs and therapies targeting angiogenesis, such as angiogenesis inhibitors used in cancer treatment, or pro-angiogenic agents used to promote tissue regeneration. These models enable faster, more cost-effective preclinical testing, while reducing the need for animal models.x.
THE FIBRIN ANGIOGENESIS BEAD (FBA) MODEL IS A 3D ANGIOGENESIS TECHNIQUE. IN VITRO (Figure 3) [7]
In this model, polymer beads or microspheres are coated with endothelial cells, the cells that line blood vessels.
Figure 3. Steps in angiogenesis analysis with fibrin gel beads.
- The first day consists in attaching the endothelial cells to a microcarrier (bead).
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The second day involves incorporating the coated beads into a fibrin clot composed of growth factors and the compound of interest, and seeding a layer of fibroblasts onto the gel surface.
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Finally, after 48 h incubation at 37°C, the beads are imaged on day 4 (adapted from Clavane et al. 2022) using a high-throughput automated microscope.
These beads are then incorporated into a fibrin gel, a protein present in the blood that forms a clot when activated. Once the beads have been incorporated into the fibrin gel, a layer of fibroblasts is deposited on top of the fibrin gels to secrete the growth factors required for spontaneous angiogenesis.
A culture medium is added in which the compounds of interest to be tested are diluted: in our case, platelet-based preparations: PRP (RegenPRP), PRP-HA (Cellular Matrix) and platelet lysates (Platelet Max).
Over time, endothelial cells proliferate under the influence of various stimuli provided by fibroblasts and growth factors from platelet-based preparations.
They then migrate out of the beads to form true capillary tubes containing lumens, replicating the angiogenesis process observed step by step. in vivo.
In a Nature© publication (Carpentier et al, Scientific Report 2020) [8In order to increase the statistical power of the quantifications performed (Figure 4).
This model coupled with this new quantification offers a practical and reproducible means of studying the underlying mechanisms of angiogenesis and assessing the efficacy of different therapeutic agents, such as anti-angiogenic drugs or pro-angiogenic agents.
What's more, it makes it easy to visualize morphological and functional changes in the blood vessels formed, in response to the growth factors released by platelets.
It is an invaluable tool for vascular biology research and for the development of new therapies in regenerative medicine and vascular pathophysiology [...9].
IN OUR STUDY IN VITROWE EVALUATED AND COMPARED THE CAPACITY OF THREE PLATELET-DERIVED PREPARATIONS
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Platelet-rich plasma (PRP),
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PRP with hyaluronic acid (PRP-HA)
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Platelet lysates (PL) at different concentrations (5-40 %)
For their biological effects on human umbilical vein endothelial cells (HUVEC) in :
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Their metabolism,
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Their viability,
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Their senescence,
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Secretion of angiogenic factors,
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and 2D angiogenic capacities (endothelial tube formation test or EFTA),
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and in 3D (fibrin bead assay or FBA) [10] (Figure 5).
PRP, PRP-HA and PL induce different angiogenic responses.
We have shown that although all three preparations are derived from platelets, they contain diverse mixtures of growth factors that trigger different stages of the angiogenic process spatially and temporally.
PLs are powerful stimulators of endothelial proliferation, but in an unorchestrated way, hindering proper endothelial tube formation.
Figure 4. Development of a new method for morphometric analysis of angiogenesis in the fibrin bead model based on "vascular tree" detection. From [8].
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A A. Image of the initial sample showing the detected sphere. B. Enhanced strong gradients and background suppression. C. Medium" threshold. D. Final binary segmentation. E. Binary segmentation skeleton. F. Final skeleton after cleaning the inside of the sphere. Scale bar: 200 μm.
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B. Vector object detection in skeletonized trees. A.Ends which are red dots surrounded by yellow (inset 1). B. Detection of Branches (green) and Segments (magenta). Inset 2 shows an artificial branch (cyan) (because it's too small) which will be removed by the program. Inset 3 shows the fusion of two close junctions into a single one (blue border). C. Representation of the final analysis, including the anchor junctions (purple, inset 4), which intersect the sphere boundary (red).
Figure 5. Platelet-derived products modulate angiogenesis differently in the 3D fibrin bead assay.
A. Human EC 3D cultures (HUVEC) were treated with platelet-derived products (PRP, PRP-HA and PL) at different concentrations (5-40 %) for 4 days. Representative images of a massive increase in angiogenesis (PRP 20, PRP-HA 20) or mild endothelial proliferation from EC-coated beads (PL20) compared with control conditions (control and heparin) on day 4. Scale bar: 150 μm.
B. Quantification of morphometric parameters of the capillary network was performed by a computerized method on images taken on day 4. Representative parameters measured were total length, total branch length, number of tips and number of anchor junctions per sphere. Graphs are representative of three independent experiments. One hundred spheres were quantified for each experimental condition. Data from Berndt et al. Biomedicines. 2021 [10].
PRP and PRP-HA containing live platelets induce the best angiogenic response in the complex 3D FBA model, which recapitulates the entire angiogenic process.
PRP and PRP-HA may also exhibit anti-aging properties on HUVECs, as endothelial senescence is diminished [10].
Hyaluronic acid, as a natural biodegradable hydrogel, promotes the controlled release of PRP growth factors.
Secretome profiling explains biological activities by differential secretion of potent angiogenic factors when platelets are cultured alone or in the presence of HUVECs.
IN CONCLUSION
PRP alone or combined with hyaluronic acid is a reservoir of growth factors that promotes angiogenesis for cellular tissue engineering applications. in vitro.
Moreover, the results are of great interest for clinical applications where platelet-derived preparations are used for angio-regenerative therapy. in situ to accelerate wound healing and tissue repair.
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