Angiogenesis and scar formation in healing wounds : Current Opinion in Rheumatology

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MEDICAL PHYSIOLOGY AND RHEUMATIC DISEASES: Edited by John Varga

Angiogenesis and scar formation in healing wounds

DiPietro, Luisa A.

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Current Opinion in Rheumatology 25(1):p 87-91, January 2013. | DOI: 10.1097/BOR.0b013e32835b13b6
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Abstract

Purpose of review 

One well described feature of wound healing is the ingrowth of new capillaries or angiogenesis. At its peak, the capillary content in healing wounds may reach three or more times that of normal uninjured tissue. This new vasculature is required to restore oxygenation and allow the growth of new tissue to fill the wound space. This review examines the assumption that a capillary content in excess of normal density is essential for adequate healing.

Recent findings 

The regulation of wound angiogenesis has been demonstrated to involve both proangiogenic and antiangiogenic stimuli, with the level of capillary growth reliant upon both sets of factors. Several studies now show that normal skin wounds heal adequately even when the angiogenic response is artificially reduced. In normal skin, a reduction of capillary growth to a level consistent with normal tissue does not affect wound closure and may even lead to highly favorable long term healing outcomes.

Summary 

The angiogenic response in normal wounds may exceed what is needed for optimal repair.

INTRODUCTION

The presence of a functional vasculature is essential to the viability of nearly all multicellular tissues. The importance of a capillary bed to tissue survivability is highlighted when tissue injury repair occurs. Angiogenesis, or the process by which new blood vessels are formed, is a crucial component of wound healing, and the normally healing wound demonstrates robust capillary growth followed by controlled capillary regression. The process and regulation of wound angiogenesis have been studied intensively. Nevertheless, several critical questions about the functionality of the wound vasculature and the influence of capillary growth on the repair process remain unanswered.

THE HEALING WOUND AS A MODEL OF REGULATED ANGIOGENESIS

In healing wounds, an initial vigorous angiogenic response results in a vessel density that far exceeds that of normal uninjured tissue [1]. During the resolution phase of healing, the majority of the new vessels disappear, and the vessel bed is pruned back to normal vascular density. The regression process is highly regulated and involves the selective apoptosis of many of the newly formed capillaries.

The onset of angiogenesis is positively regulated by soluble factors, most notably vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-2, platelet derived growth factor (PDGF), and members of the TGF-β family; other factors such as cardiac ankyrin repeat protein (CARP) also play important roles [2–5]. Although the proangiogenic factors within healing wounds are well described and many, the regulation of vascular pruning is not as well understood. The onset of vascular regression coincides with sharp decreases in proangiogenic stimuli, and one previously accepted concept was that the simple decrease in proangiogenic signal led to the loss of capillaries. However, experiments that have attempted to provide a sustained level of proangiogenic stimuli to wounds demonstrate that a continued presence of high levels of proangiogenic factors cannot alone effectively maintain high levels of wound vascularity [6]. Thus, the regression of the vascular bed in wounds is thought to involve an active antiangiogenic environment, rather than passive process. The initiation of vascular regression in the wound is likely to be influenced by changes in the extracellular matrix that occur during the resolution of healing. In addition, several specific factors that are known to be antiangiogenic, including thrombospondin, interferon gamma-induced protein 10 (IP-10)/CXC motif chemokine 10 (CXCL10), and Sprouty-2, have now been implicated as mediators of vascular regression in wounds [7,8▪,9]. More recently, esophageal cancer-related gene-4 (Ecrg4) has also been described as a factor that is critical to wound resolution [10▪]. Overall, then, the net level of capillaries derives from a complex set of signals that modulate over the time course of repair.

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Box 1:
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ANGIOGENESIS IN WOUNDS: WHAT IS AN ADEQUATE AMOUNT?

One widely accepted view is that wound healing requires a robust and vigorous angiogenic response [11,12]. In support of this concept, there is little argument that the cellular proliferation, migration, and metabolic activities that are needed to knit tissues back together generate a demand for oxygen and nutrients. Inadequate angiogenesis has been implicated in the pathology of the chronic and poorly healing wounds that occur in persons with venous stasis disease, in diabetic individuals, and in the aged [13–16]. These nonhealing wounds may exhibit an angiogenic response that is deficient or nonfunctional. In contrast, the amplified angiogenesis that is seen in normally healing tissue may be excessive. This somewhat unorthodox notion is supported by at least five separate studies. These studies have demonstrated that under specific conditions that reduce (but do not eliminate) the angiogenic response, full thickness wounds in normal animals heal completely adequately. The conditions include the blockade of capillary growth using antibodies to VEGF, treatment with a variety of antiangiogenic agents, and a blockade of integrin signaling [17–22].

The concept that a reduced but functional angiogenic response might satisfy the demands of the healing wound is also supported by studies of wounds that heal exceptionally well. Oral mucosal wounds, a location known to heal very rapidly, exhibit a less robust angiogenic response than skin wounds [23]. Other studies have shown that non-scar forming wounds produced on the second-trimester fetus exhibit a lesser angiogenic response [24].

Although the concept that wounds can heal well with a reduced angiogenesis may seem heretical, the findings might be explained by studies of vascular function in wounds. Several studies now suggest that many of the capillaries that are formed in early healing wounds are not highly functional. A carefully performed and elegant study by Bluff et al.[25] demonstrates that most of the new vasculature that is histologically apparent in the healing wound is not effectively perfused. Moreover, the vasculature that is produced under situations of high proangiogenic pressure, such as that found in solid malignancies, has been shown to be tortuous, leaky, and is often highly ineffective in delivering adequate blood flow [26,27]. At least a few reports suggest that many of the capillaries formed in the early healing wound are immature and highly permeable [28]. Together, these studies suggest that the typical pattern of wound angiogenesis includes an initial dense bed of capillaries that are not highly functional. Therapeutics that partially block this capillary growth might yield an abridged yet functional vasculature that provides excellent perfusion. Moreover, a reduction in the initial burst of capillary growth would both reduce edema and lessen the need for vascular regression.

THE LINK BETWEEN EXCESS CAPILLARY GROWTH AND SCAR FORMATION

Several recent studies suggest a link between the surplus capillary growth seen in healing wounds and scar formation. Neutralization of VEGF via antibody treatment has been used to effect an approximately 50% reduction in peak wound vascularity in adult skin wounds. These treated wounds close normally and also showed a significant reduction in wound scar width [24]. More recent studies have suggested an association of robust capillary growth with the development of keloids and peritoneal adhesions [29▪,30]. Moreover, a recent comparison of capillary content in human normotrophic and hypertrophic scars demonstrated that hypertrophic scar formation is associated with higher levels of angiogenesis [31▪]. This evidence has led to the suggested use of antiangiogenic therapy to reduce scar formation [24,32].

Although multiple studies now suggest that a reduction in angiogenesis has no effect on wound closure, and might in fact lead to improved wound resolution, conflicting findings do exist on this topic. In some studies, specific doses and types of antiangiogenic treatments have been shown to slow wound healing. One possible explanation for such discrepancies is that some antiangiogenic mediators have effects well beyond the angiogenic response [33,34]. For example, the antiangiogenic agent SU5416, an agent that inhibits the receptor tyrosine kinases that are critical for VEGF signaling, both inhibits angiogenesis and delays wound healing [34]. The delay in healing, however, was shown to be mediated by an SU5416 inhibition of TGF-β1 activation, an effect outside of its direct antiangiogenic activity. Of clinical relevance is the known association of the use of antiangiogenic therapies for patients with malignancies with poor healing outcomes. While this might be considered presumptive evidence for a net negative influence of antiangiogenic treatments on human wound healing, patients with malignancies most often receive combinatorial therapies that might create an underlying condition of impaired healing. In humans, the effect of reducing angiogenesis in wounds of normally healing individuals without additional comorbidities is unknown.

HOW MIGHT THE ANGIOGENIC RESPONSE INFLUENCE HEALING OUTCOMES?

The observed link between robust angiogenesis and a fibrotic outcome in wounds suggests multiple possible mechanisms, some of which are direct and some of which are indirect (Fig. 1). One obvious direct possibility is that proangiogenic factors not only stimulate capillary growth, but also simultaneously act to encourage a fibrotic phenotype in fibroblasts and other cells. Indeed, the majority of soluble mediators, including most proangiogenic factors, have a multitude of potential activities. In line with this, the impact of proangiogenic factors can directly extend to fibroblasts, as many proangiogenic factors, such as VEGF, adenosine, members of the TGF family, and others, can bind to receptors on fibroblasts and influence the function [35,36]. In this case, blockade of the proangiogenic factor might also directly block the effect on fibroblasts. This concept has been recently demonstrated in a study [37▪] that investigated the effect of the blockade of the adenosine A2A receptors in wounds. A2A receptors are found on both endothelial cells and fibroblasts; adenosine mediates both angiogenesis and fibroblast fibrotic activity via this receptor. In an in-vivo wound healing model, the blockade of A2A receptors caused decreased angiogenesis in tandem with decreased scar formation. The concept of dual direct effects, on both endothelial cells and fibroblasts, seems plausible for certain mediators. However, an argument against this fairly simple explanation is the timing of production of many of the critical proangiogenic factors that are found in wounds. The highest level of proangiogenic factors in wounds is seen in the early proliferative phase; levels of most of the known proangiogenic factors are quite low by the time wound resolution and collagen refinement begin.

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FIGURE 1:
Points of intersection of the wound angiogenic response and fibroblast function in healing wounds. White boxes indicate items that may influence both processes via direct effects. Gray boxes are elements that may result from a robust angiogenic response and then influence fibroblast function. Intersecting elements are shown at the approximate time sequence at which they might act. Arrows indicate the direction of influence.

A second possible mechanism by which a large burst of capillary growth might influence healing outcomes is edema. As discussed above, rapidly growing capillaries may be highly permeable, and the resulting edema is known to negatively impact healing [38,39]. The influence of edema on downstream outcomes of healing has not been well considered experimentally. Nevertheless, levels of extracellular fluid and tissue tension on fibroblasts might be hypothesized to affect fibroblast responses as wound resolution occurs.

In healing wounds, vessel regression requires endothelial cell apoptosis, and the regression process generates a substantial apoptotic load. Apoptotic endothelial cells may themselves influence wound resolution in several ways. Although long considered benign, apoptotic cells have been connected to fibrotic outcomes in several pathologic states, including chronic transplant vasculopathy, cardiac failure, and pulmonary fibrosis [40–45]. Of direct relevance to resolving wounds, conditioned media derived from apoptotic endothelial cells has been shown to influence fibroblasts, increasing focal adhesions and levels of alpha-smooth muscle actin [46]. This fibrotic effect has been suggested to derive from the release of connective tissue growth factor (CTGF), a factor previously implicated in fibrosis. Beyond the direct release of factors, the removal of apoptotic cells could also modulate tissue repair. Phagocytosis of apoptotic endothelial cells by macrophages or other cell types may cause these cells to release profibrotic mediators [47▪]. Additional investigations of the interactions between the regressing apoptotic endothelium and fibrotic outcomes in wounds will be needed to sort this out.

Several additional mechanisms might be part of the explanation for how robust angiogenesis influences scar formation. High levels of angiogenesis frequently occur in tandem with, and indeed often as a result of, acute inflammation. Many proinflammatory mediators are both profibrotic and proangiogenic. In this context then, the intersection of high levels of inflammation, angiogenesis, and scar formation is associative, not causative. In opposition to this concept, though, at least one study [24] suggests that a simple artificial reduction of wound angiogenesis, absent a reduction in inflammation, will lead to diminished scar. Nevertheless, a rich and vast literature supports the idea that inflammation and inflammatory mediators support fibrotic outcomes, suggesting that further exploration of the inflammation–angiogenesis–scar linkage is needed.

CONCLUSION

There is little argument that normal skin wound healing requires the ingrowth of capillaries to restore adequate tissue oxygenation and provide nutrient support. This review has highlighted some recent studies to suggest that the robust angiogenesis seen in normal wounds can be pared down quite significantly without inhibiting wound closure. Additional findings support the idea that angiogenesis in skin wounds is often excessive and might be linked to poor healing outcomes, including scars. Additional studies will be needed to more fully understand the intersection of angiogenesis, wound healing, and scar formation.

Acknowledgements

None.

Conflicts of interest

There are no conflicts of interest.

Research in the author's laboratory is supported by the National Institute of General Medical Sciences of the National Institutes of Health under award numbers R01-GM50875 and P20-GM078426. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • ▪ of special interest
  • ▪▪ of outstanding interest

Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 152).

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Keywords:

angiogenesis; apoptosis; fibrosis; scars; wound healing

© 2013 Lippincott Williams & Wilkins, Inc.