Angiogenesis in Chronic Wounds and the Role of Dehydrated Human Amnion/Chorion Membrane

Angiogenesis, the formation of new blood vessels from preexisting vasculature, represents a critical physiological process essential for proper wound healing. In chronic wounds, this process becomes significantly impaired, contributing to delayed healing and increased morbidity.

Recent advances in wound care have introduced dehydrated human amnion/chorion membrane (dHACM) as a promising therapeutic option for enhancing angiogenesis in non-healing wounds.

Here we explore the complex relationship between angiogenesis and wound healing, with special emphasis on its dysfunction in chronic wounds and how dHACM allografts offer potential solutions through their unique angiogenic properties.

The Fundamental Role of Angiogenesis in Wound Healing

Angiogenesis plays a crucial role in wound healing by forming new blood vessels from preexisting vessels that invade the wound clot and organize into a microvascular network throughout the granulation tissue. This dynamic process is highly regulated by signals from both serum and the surrounding extracellular matrix (ECM) environment1. In normal physiological wound healing, the microvasculature transitions from a state of homeostasis to an active state of new vessel formation following injury.

The disruption of the microvasculature due to injury leads to fluid accumulation, inflammation, and the development of hypoxia, which subsequently activates endothelial cells and initiates the angiogenic cascade7.

The angiogenic process during wound healing is characterized by highly orchestrated cell-cell and cell-extracellular matrix interactions. These interactions lead to the formation of new vascular structures from nearby intact blood vessels, ensuring adequate delivery of nutrients, oxygen, and regulatory factors necessary for tissue remodeling and regeneration3. 

During the proliferative phase of healing, the rapid and robust growth of capillaries creates a vascular bed containing many times more capillaries than normal tissue.

This temporary hypervascularization is a hallmark of normal wound healing, although over time, most of these newly formed capillaries regress, resulting in a final vascular density similar to that of normal skin8.

The Angiogenesis Cascade in Normal Wound Healing

Angiogenesis in wound healing occurs through a well-defined sequence of molecular and cellular events. The process begins with the release of angiogenic growth factors such as vascular endothelial growth factor (VEGF), placental growth factor (PlGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), angiogenin, and angiopoietin from damaged cells and platelets1. These factors lead to the rapid destabilization of local preexisting vessels and activation of local endothelial cells.

The angiogenesis cascade can be divided into several distinct steps. First, endothelial cell surfaces develop receptors to which angiogenic growth factors bind in preexisting venules. This growth factor-receptor binding activates signaling pathways within endothelial cells, leading to the release of proteolytic enzymes that dissolve the basement membrane of surrounding parent vessels1. Endothelial cells then proliferate and sprout outward through the basement membrane, migrating into the wound bed using integrins (αvβ3, αvβ5, and αvβ1) as cell surface adhesion molecules1.

As the process continues, matrix metalloproteinases (MMPs) dissolve the surrounding tissue matrix in the path of sprouting vessels, allowing vascular sprouts to form tubular channels that connect to create vascular loops. These vascular loops differentiate into afferent (arterial) and efferent (venous) limbs, and the new blood vessels mature by recruiting mural cells (smooth muscle cells and pericytes) to stabilize the vascular architecture. Finally, blood flow begins in the mature stable vessel1.

Temporal Phases of Angiogenesis in Wound Healing

The angiogenic process during wound healing can be further categorized into three temporal phases: initiation, amplification, and proliferation. In the initiation phase, basic fibroblast growth factor (bFGF) stored within intact cells and the extracellular matrix is released from damaged tissue. Bleeding and hemostasis in a wound also trigger angiogenesis, as thrombin upregulates cellular receptors for VEGF in the wound and promotes the release of gelatinase A (MMP-2), which facilitates the dissolution of basement membrane1.

During the amplification phase, macrophages and monocytes release numerous angiogenic factors, including PDGF, VEGF, Ang-1, TGF-α, bFGF, interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) into the wound bed. These factors synergize to enhance vascularization, while proteases that break down damaged tissue matrix further release matrix-bound angiogenic stimulators1.

The vascular proliferation phase is largely driven by hypoxia, which is an important stimulus for wound angiogenesis.

The hypoxic gradient between injured and healthy tissue triggers the expression of hypoxia-inducible factor-1 alpha (HIF-1α), which in turn promotes VEGF production17. VEGF, also known as vascular permeability factor, increases the permeability of capillaries, allowing the leakage of fibrinogen and fibronectin that are essential for the formation of the provisional extracellular matrix1.

Impaired Angiogenesis in Chronic Wounds

In chronic wounds, the normal healing processes, including angiogenesis, are significantly disrupted, resulting in delayed or inappropriate healing.

These wounds often occur in the presence of systemic diseases such as diabetes and atherosclerosis, which are accompanied by microvascular deficiencies2. The impairment of angiogenesis in chronic wounds leads to defective granulation tissue formation, which ultimately causes the failure of these wounds to progress through the proliferation phase of healing3.

Several cellular and molecular defects are associated with poor healing in chronic wounds. These include abnormalities in growth factor signaling, imbalances of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), and impaired recruitment of progenitor cells23. For instance, in chronic venous ulcers, researchers have observed increased proteolysis of VEGF-A as well as elevated levels of soluble VEGFR-1, which may neutralize VEGF-A activity and inhibit angiogenesis7.

The consequences of impaired angiogenesis in chronic wounds are profound. Without adequate blood vessel formation, the delivery of oxygen, nutrients, and growth factors to the wound bed is compromised, and the removal of waste products is impeded. This results in chronic tissue hypoxia and impaired micronutrient delivery, which further exacerbate tissue damage and inhibit healing1. Specific defects in angiogenesis have been identified in different types of chronic wounds, including diabetic ulcers, venous insufficiency ulcers, and ischemic ulcers1.

The Complex Relationship Between Inflammation and Angiogenesis

The relationship between inflammation and angiogenesis in wound healing is complex and bidirectional. The level of angiogenesis in wounds often correlates with the inflammatory response, largely because inflammatory cells produce an abundance of pro-angiogenic mediators8. However, recent research has challenged the traditional view that more angiogenesis is always better for wound healing.

Interestingly, wounds that heal exceptionally quickly and with less scar formation, such as those in fetal skin and oral mucosa, show a reduced angiogenic response but with more mature vessels that provide better oxygenation8. This suggests that the quality and organization of blood vessels may be more important than their quantity for optimal healing outcomes. Both the selective reduction of inflammation and the selective reduction of angiogenesis have now been proposed as potential strategies to improve scarring8.

Therapeutic Approaches to Enhance Angiogenesis in Chronic Wounds

Given the critical role of angiogenesis in wound healing and its impairment in chronic wounds, therapeutic strategies aimed at enhancing angiogenesis have emerged as promising approaches for treating non-healing wounds. These strategies include the use of angiogenic growth factors, gene therapy, cell-based therapies, and advanced wound care products such as dehydrated human amnion/chorion membrane (dHACM)57.

Growth factor-derived products have been applied in the treatment of neovascular deficiency in chronic wounds, with positive results3. Gene therapy, another promising strategy, can be classified into three main approaches: gene augmentation, gene silencing, and gene editing5. Despite the increasing number of encouraging results obtained using nucleic acids as active pharmaceutical ingredients of gene therapy, efficient delivery of nucleic acids to their site of action remains a key challenge5.

In recent years, human-derived placental tissues have emerged as effective treatments for chronic wounds due to their rich content of regenerative cytokines. Among these, dehydrated human amnion/chorion membrane (dHACM) allografts have shown particular promise in promoting angiogenesis and wound healing2.

dHACM: Composition and Angiogenic Properties

Dehydrated human amnion/chorion membrane (dHACM) is a tissue allograft derived from human placenta that has been processed using the PURION® Process to preserve the bioactivity of native amniotic growth factors and cytokines2. These allografts contain a large number of pro-angiogenic growth factors, including angiogenin, angiopoietin-2 (ANG-2), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), heparin binding epidermal growth factor (HB-EGF), hepatocyte growth factor (HGF), platelet-derived growth factor BB (PDGF-BB), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF)2.

What makes dHACM particularly effective is that these growth factors retain their biological activity even after the dehydration process2. This preservation of bioactivity enables dHACM to exert therapeutic actions both directly and indirectly by activating multiple signaling pathways that promote angiogenesis within healing wounds2.

The composition of dHACM differentiates it from other available wound healing products. While single growth factor therapies such as recombinant human PDGF (becaplermin) have shown modest efficacy in clinical trials, dHACM contains multiple angiogenic factors, specifically VEGF and bFGF, which are potent angiogenic cytokines that promote endothelial cell proliferation and migration2. dHACM also contains PlGF, which not only has direct angiogenic effects but acts synergistically with VEGF to stimulate wound angiogenesis2.

Mechanisms of dHACM in Promoting Wound Angiogenesis

The mechanisms by which dHACM promotes angiogenesis in wounds are multifaceted. First, dHACM directly stimulates angiogenesis through the angiogenic growth factors it contains2. When applied to a wound, these growth factors can bind to receptors on endothelial cells, activating signaling pathways that promote endothelial cell proliferation, migration, and tube formation.

Second, dHACM has been shown to stimulate human microvascular endothelial cells to increase production of a variety of angiogenic cytokines and growth factors2. This represents an amplification mechanism whereby dHACM not only provides its own angiogenic signals but also enhances the production of endogenous angiogenic factors by the cells within the wound environment. Specifically, soluble factors from dHACM have been shown to increase endogenous production of over 30 angiogenic factors by human microvascular endothelial cells, including granulocyte macrophage colony-stimulating factor (GM-CSF), angiogenin, transforming growth factor β3 (TGF-β3), and HB-EGF2.

Third, dHACM has been demonstrated to promote chemotactic migration of human endothelial cells in vitro26. In a standard transwell assay, human umbilical vein endothelial cells migrated toward pieces of dHACM tissue, suggesting that dHACM releases soluble factors that can recruit endothelial cells to enhance wound revascularization2.

Stem Cell Recruitment and Neovascularization

Another important mechanism by which dHACM promotes wound healing is through stem cell recruitment. Studies have shown that dHACM contains both stromal cell-derived factor 1 (SDF-1) and chemokine receptor type 4 (CXCR4), which are stem cell recruitment and homing factors4. In vitro studies have confirmed that dHACM can attract stem cells and stimulate the migration of mesenchymal stem cells, while in vivo studies have shown that stem cells home to sites of neovascularization, reflecting their role as endothelial progenitor cells4.

What distinguishes dHACM from cellular therapies is that instead of directly delivering cells with stemlike characteristics, dHACM releases factors that recruit endogenous stem cells, suggesting bona fide regenerative capability when used in wound management4. This ability to recruit the body’s own stem cells may explain the sustained benefits observed with dHACM treatment.

In Vivo Neovascularization and Long-Term Effects

In vivo studies have provided further evidence of dHACM’s angiogenic properties. Subcutaneous implantation of dHACM tissues in a murine model demonstrated a steady increase in vascularization through day 282. These implants progressed from being completely avascular following implantation to having a vascular density similar to both normal and healed skin within 4 weeks2. This dynamic vascular remodeling aligns with in vitro findings and is consistent with the time course observed in clinical trials2.

The prolonged pro-angiogenic effects from a single dHACM implant may offer practical benefits compared to advanced wound interventions that require more frequent applications2. The ability of dHACM to promote sustained angiogenesis over weeks rather than days could be particularly beneficial for chronic wounds, which often require extended periods to achieve complete healing.

Clinical Evidence for dHACM in Chronic Wound Healing

The clinical value of dHACM for treating non-healing wounds has been demonstrated in several studies. In a small, prospective, randomized clinical trial, Zelen et al. reported a significant increase in the healing rate of diabetic foot ulcers treated with dHACM compared to those treated with a standard therapeutic regimen2. Specifically, 77% and 92% of dHACM-treated wounds healed at weeks 4 and 6, respectively, compared to only 0% and 8% of controls24.

dHACM treatment has also shown efficacy in healing a variety of other wound types for which traditional therapies were ineffective, including venous leg ulcers, crush injuries, arterial insufficiency, immunological skin diseases, and snake bites2. Perhaps most notably, refractory wounds that healed after dHACM treatment were reported not to recur with long-term follow-up2, suggesting that dHACM promotes sustainable healing rather than temporary improvement.

These clinical results support the laboratory findings regarding dHACM’s angiogenic properties and suggest that the enhancement of angiogenesis may be a key mechanism by which dHACM promotes the healing of chronic wounds. The ability of dHACM to promote revascularization and tissue healing within poorly vascularized, non-healing wounds makes it a promising option for patients who have not responded to standard wound care protocols.

Conclusion

Angiogenesis plays a critical role in normal wound healing, and its impairment in chronic wounds contributes significantly to delayed or failed healing. The complex process of angiogenesis involves multiple growth factors, cell types, and signaling pathways that must be coordinated to achieve optimal wound vascularization. In chronic wounds, defects in angiogenesis lead to inadequate blood vessel formation, resulting in chronic hypoxia, impaired nutrient delivery, and compromised healing.

Dehydrated human amnion/chorion membrane (dHACM) represents an innovative approach to addressing impaired angiogenesis in chronic wounds. Through its content of multiple angiogenic growth factors with retained biological activity, dHACM directly stimulates angiogenesis while also promoting the amplification of angiogenic signaling through the induction of endogenous growth factor production. Moreover, dHACM’s ability to recruit endothelial cells and endogenous stem cells to the wound site further enhances its angiogenic potential.

The clinical success of dHACM in treating various types of chronic wounds substantiates its angiogenic properties and highlights the importance of targeting angiogenesis in wound healing therapies. As research in this field continues to advance, further understanding of the mechanisms by which dHACM and other angiogenic therapies promote wound healing may lead to even more effective treatments for patients suffering from chronic, non-healing wounds.

References

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