The process of angiogenesis, which is defined as the growth of blood vessels from preexisting vessels through migration and proliferation of endothelial cells, is pivotal in wound healing.
For multicellular organism to grow beyond size of 100 to 200 pm, they must recruit new blood vessels by angiogenesis. The formation of new capillaries is characterized by stimulation of endothelial cells from their quiescent state, migration and proliferation, tube formation, stabilization and differentiation. Hoying summarized the cellular events of angiogenesis into four stages. Stage I: The breakdown of the basement membrane and the formation of a new blood vessel sprout. Stage II: Extension of the sprouting vessels through the migration and proliferation of endothelial cells. Stage III: The new vessel sprout begins to reform its basement membrane and recruit pericytes. Stage IV: Vessel stabilization and maturation begins with the recruitment of smooth muscle cells and the differentiation of mesenchymal cells. The rate of angiogenesis ranges from 158 µm/day to 300 µm/day. It is faster than the rate of bone apposition, which is 110 µm/day, in the first 4 weeks of bone regeneration.
Even though angiogenesis is a part of organ development, more attention has been focused on its roles in wound healing process. The phenomenon of angiogenesis has long been familiar to surgeons and pathologists dealing with inflammation, trauma and tumors.
During wound healing, angiogenesis occurs as a part of the physiologic response to injury. In humans, as early as day 4 after wound injury, angiogenesis begins and is the main component (60%) of granulation tissue, whose formation is an important process of wound healing. During this stage, under the control of growth factors including Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF), Platelet-Derived Growth Factor (PDGF) and Transforming Growth Factor Beta (TGF-β), endothelial cells and fibroblasts enter the wound, followed by capillary formation and then collagen formation.
In addition to endothelial cell proliferation, migration of endothelial cells is a key event during angiogenesis. D’Amore and Thompson reported that during the process of angiogenesis, migration of endothelial cell migration is more critical than proliferation and considered it is a principle confirmed in studies showing that angiogenesis is promoted by growth factors that primarily or exclusively affect migration. Endothelial cell proliferation provides a supply of new cells for newly developing vasculature. The proliferation of endothelial cells is thought to only occur at the leading tips of angiogenic vessels just behind the migratory cells.
To summarize, an endothelial cell that has been activated to participate in angiogenesis begins to migrate following activation. Following migration, signals of proliferation will result in additional cells for the developing new vessel. Once the new vessel has been formed, a new basement membrane must be established to maintain a quiescent endothelium.
Regulation of Angiogenesis by Growth Factors
Angiogenesis is under the control of growth factors, including VEGF, PDGF, FGF and TGF-β. The regulation of angiogenesis by growth factors is a complex orchestration of molecular signals, many of which have only recently been discovered. A brief introduction of the sources and functions of the growth factors that are important in angiogenesis follows.
PDGF is produced by platelets and endothelial cells and stimulates smooth muscle cells to divide and produce FGF and VEGF. It serves also as a chemoattractant for pericytes. It is reported that pericytes, which are pluripotential cells surrounding blood vessels, have osteogenic potential.
VEGF is produced by macrophages and smooth muscle cells. It induces endothelial cells to relax their cell-to-cell connections, resulting in hyperpermeability of the vessel and produce matrix-degrading proteins that allow for cell migration. It is reported that the human fracture hematoma, present after injury, has potent angiogenic activity that appears to be predominately due to VEGF. Inhibition of VEGF activity disrupts repair of femoral fractures and cortical bone defects in mice. VEGF is also hypothesized as the major agent by which angiogenesis and osteogenesis are tightly coupled during bone repair.
FGFs are heparin-binding proteins and are released when the matrix is disrupted during injury. FGF functions as a mitogen for many cell types including endothelial cells, chondrocytes, fibroblasts and smooth muscle cells. It regulates cell proliferation, migration and differentiation as well as musculoskeletal development. The exact mechanism by which FGF stimulates bone repair remains uncertain, but it is reported that FGF induces angiogenesis and stimulates mitogenesis of mesenchymal cells and osteoblasts, which might be mediated and modulated by TGF-β.
TGF-P is stored and circulated as an inactive peptide associated with a latency associated peptide, which when removed, results in action of TGF-β. It is involved in regulating cell differentiation and proliferation. It is released by degranulating platelets in the hematoma and by extracellular matrix at the site of damage. TGF-β could stimulate recruitment and proliferation of mesenchymal cells (stem cells, chondroblasts, and osteoprogenitors), and might affect inflammation and angiogenesis. Saadeh et al reported that TGF-β induces VEGF ex
Extracellular Matrix (ECM) in Angiogenesis
The previously mentioned functions of soluble growth factors justify their importance in the regulation of angiogenesis. In addition, insoluble regulators of angiogenesis, i.e. extracellular matrix proteins and matrix components, may also demonstrate efficacy in the stimulation of angiogenesis. The angiogenic signals for endothelial cells to migrate need to be accompanied with an extracellular matrix interaction.
Angiogenesis is a dynamic process of wound healing as fibrin clot is replaced by blood vessel-rich granulation tissue and then is subsequently replaced by collagenous scar in soft-tissue wound healing or by new bone tissue in bone regeneration. Migration of endothelial cells and development of new capillary tubule structure are dependent not only on the cells and cytokines present, but also the production and organization of ECM components in both granulation tissue and endothelial basement membrane. The ECM consists of proteinaceous and nonproteinaceous molecules that provide the tissue-specific extracellular architecture to which cells attach.
ECM is critical for normal vessel growth and maintenance, by acting as both scaffold support, through which endothelial cells may migrate, and reservoir and modulator for growth factors to mediate intercellular signals. These processes are mediated by transmembrane protein named integrins. These classes of integrins allow for communication between the cell and the outside environment and provide a mechanism by which a cell can migrate through the ECM environment.