Catecholamines in the regulation of angiogenesis in cutaneous wound healing

Abstract

Angiogenesis involves the formation of new blood vessels from preexisting ones, and it is an essential step during cutaneous wound healing, which supports cells at the wound site with nutrition and oxygen. Impaired angiogenesis in the wound tissues results in delayed wound closure and healing. Among the regulators of angiogenesis, the role of catecholamines (epinephrine, norepinephrine, and dopamine) is of interest due to their diverse roles in the process of wound healing. While both norepinephrine and epinephrine mostly inhibit the angiogenic process in cutaneous wounds, dopamine, the other member of the catecholamine family, has interesting and contradictory roles in the regulation of angiogenesis in the wound beds, depending on the type of dopamine receptor involved. The stimulation of dopamine D2 receptors negatively regulates the angiogenic process in normal dermal wounds and thereby delays healing, whereas the stimulation of dopamine D1 receptors promotes angiogenesis and expedites healing in diabetic wounds. Importantly, catecholamines also play important roles in other pathological conditions, and specific agonists and antagonists of catecholamines are available for the treatment of some disorders. Therefore, such drugs may be utilized for the management of angiogenesis to promote the healing of dermal wounds. This review provides a broad overview of the angiogenic process during cutaneous wound healing and the regulatory roles played by catecholamines during the process.

INTRODUCTION

Cutaneous wound healing (tissue repair and regeneration) is an essential physiological process required for the restoration of the integrity of skin after any trauma (acute injury, surgery, or other factors that cause the breakdown of intact skin) to maintain physiological homeostasis.^1,2 It is a highly orchestrated complex process involving large numbers of cell types and factors. Classically, the process involves four phases: hemostasis, inflammation, proliferation, and remodeling. Successful wound healing relies upon rapid hemostasis to limit blood loss, appropriate inflammation, differentiation, proliferation, and successful migration of mesenchymal cells to the wound area, sufficient neovessel formation or angiogenesis, prompt re-growth of epithelial tissue over the wound area, as well as proper collagen synthesis and alignment in the regenerating tissue.^3,4All these processes must occur in the right sequence at a specific time and for an appropriate duration.^4,5 Any variation in the processes can result in impaired tissue repair and deficient healing of the wound, resulting in the development of chronic wounds. Non- healing wounds present a challenging clinical problem—they constitute a significant source of morbidity as well as substantial societal and economic burden.^{6, 7}

Among all the processes, neovascularization or angiogenesis, which is the formation of new blood vessels from the preexisting vasculature, represents an essential component of optimal wound healing due to its primary impact from the beginning of the process (after skin injury) until the end of the remodeling phase.^8,9 In wound healing, new capillaries first appear in the wound bed 3–5 days after injury along with granulation, and the creation of a provisional matrix is initiated. The new capillaries rapidly grow into the wound and produce an abundant network of new blood vessels to support cells at the wound site with nutrition and oxygen. Inadequate ability to reestablish functional blood supply to the injury site can cause chronic non-healing wounds.¹⁰ Several endogenous growth factors and hormones¹¹ regulate the process of angiogenesis in cutaneous wound healing. Therefore, identifying and validating these endogenous factors that regulate angiogenesis in the wound bed is of immense interest for the development of potential therapeutic targets.^12,13

Catecholamines (CA) have definitive roles in the regulation of angiogenesis in diseases;^14–24 however, their roles in wound healing are highly underappreciated. This review provides the readers with a broad overview of the roles played by the three different CA in the angiogenic process during cutaneous wound healing. Furthermore, it discusses the potential mechanisms through which they influence the wound physiology.

ANGIOGENESIS AND WOUND HEALING

The normal vasculature is in a state of quiescence, which is maintained by a balance between basal levels of pro-angiogenic factors, such as vascular endothelial growth factor-A (VEGF-A) and fibroblast growth factor, and anti-angiogenic factors, such as angiopoietin1 (Ang1) and pigment epithelium-derived factor.^10,25–27 An injury disrupts this homeostasis, leading to a low oxygen tension/hypoxic state, which is an important activator of the endothelial cells (EC).²⁸ In the initial stages of wound healing, leukocytes and neutrophils are recruited to the site of injury.²⁹ Thereafter in the proliferative stage, macrophages, neovessels, and loose connective tissue form the granulation tissue.^30,31Active angiogenesis is the hallmark of the proliferative stage and involves the growth of neovessels that are characterized by their immaturity, permeability, and redundancy.^8–10,32 Angiogenic response in this phase takes place along with fibroblast migration, proliferation, and collagen synthesis. After an injury has occurred, the microvascular EC lining the inner surface of blood vessels is activated by hypoxia, which activates hypoxia-inducible factor- 1 (HIF-1) and consequently stimulates the process of angiogenesis by upregulating target pro-angiogenic genes such as VEGF-A.^26,33 Pro-angiogenic factors are also released by monocytes, platelets, and fibroblasts during angiogenesis, and VEGF-A is the most prominent cytokine that regulates this process. In addition to promoting angiogenesis, VEGF-A also increases vascular permeability and contributes to wound edema.^33,34 Other pro-angiogenic factors associated with wounds include fibroblast growth factor −2, platelet-derived growth factor (PDGF), and members of the transforming growth factor beta (TGF-β) family.^34–37 The activated EC then severs their interactions with the neighboring EC, degrades the basement membrane, and digests their surrounding extracellular matrix (ECM) components by secreting matrix metalloproteinases (MMP).³⁸ Activated EC along with other cells, like fibroblasts, platelets, smooth muscle cells, and monocytes/macrophages, secrete important proangiogenic cytokines such as VEGF-A, PDGF, and TGF-β. These further help the EC to invade and migrate through the ECM, proliferate, and reestablish cell-cell contacts, thereby forming new capillaries.³⁸ In wound healing, the neovessels grow into the wound at a rapid rate, forming a network of newly formed blood vessels that exceed the numbers of vessels in the normal skin by many folds. However, the new vessels formed are highly disorganized and poorly perfused.^34,39Once immature granulation tissue and microvasculature fill up the wound bed, resolution factors trigger vascular maturation and remodeling.³⁹ During this phase, the new capillaries are remodeled along with the connective tissue and epithelium; they gain normal vascular permeability, blood flow, and are pruned to exhibit normal vascular branching.³⁹ Pericytes, a type of mural cells, help in this wound maturation phase by stabilizing the microvascular capillaries and the vascular basement membrane.^{40, 41} At the end of this phase, the granulation tissue is remodeled due to the reabsorption of excess blood vessels and the disappearance of fibroblasts, and ultimately, a scar composed primarily of dense collagen along with some intermittent, widely scattered fibrocytes and blood vessels are formed.^34,39The role of angiogenesis in the different phases of wound healing are represented in Figure 1.

FIGURE 1:

Schematic diagram showing that angiogenesis is an important step during the proliferative phase of dermal wound healing (Created with BioRender.com). VEGF-A = Vascular Endothelial Growth Factor-A, FGF = fibroblast growth factor, PDGF = platelet-derived growth factor, TGFβ = transforming growth factor beta

DIABETIC WOUNDS AND ANGIOGENESIS

Diabetic wound healing and angiogenesis present a global challenge clinically as well as in research settings. As evident in both clinical and experimental diabetes, the disease affects all stages of wound healing, resulting in an impaired healing capacity.^39,42,43 Disruptions in hemostasis and inflammation, matrix deposition, and angiogenesis in the wound bed are associated with non-healing cutaneous diabetic wounds. Furthermore, cutaneous wounds in diabetics show a dysfunctional inflammatory response, microbial invasion, epithelial breakdown, and impaired immune function along with an abnormal expression of chemokines. The underlying factor associated with inadequate wound healing in diabetes is impaired vascular response.⁴²

During normal wound healing, angiogenesis depends upon an intricate balance between pro- and anti-angiogenic factors, which maintains the balance between vessel growth promotion, vessel maturation, and quiescence.^4,8,10 However, the scenario changes during diabetes, where a decrease in the pro-angiogenic stimulus is noted.^39,42 In diabetes, macrophages, one of the major sources of VEGF-A and other pro-angiogenic mediators, as well as fibroblasts undergo phenotypic changes and have altered functions.⁴⁴ The levels of VEGF-A are deficient in experimental and clinical diabetic wounds. Besides, diabetes also alters the levels of anti-angiogenic factors, which leads to reduced angiogenesis and delayed wound healing.^{39, 43}

Furthermore, vascular integrity is disturbed in diabetes as a result of elevated levels of systemic glucose, which adversely affects EC and makes them dysfunctional; consequently, this causes several microvascular and macrovascular complications.^39,45 Thus, wound healing under diabetic conditions exhibits decreased vascularity and capillary density. These processes, in turn, disrupt tissue regeneration and restoration of a functional vascular system, which leads to delayed wound closure.^39,43 The angiopoietin 1/angiopoietin 2/TEK tyrosine kinase (Ang1/Ang2/Tie2) pathway also plays a role in the defective angiogenic process seen in diabetic wounds. The ratio of Ang1 to Ang2 is decreased in diabetes, which results in the disruption of vessel maturation.^{42, 46, 47}

CATECHOLAMINES IN THE SKIN

Dopamine (DA), norepinephrine (NE)/noradrenaline, and epinephrine (E)/adrenaline are the naturally occurring CA derived from amino acid tyrosine.^48,49 They play important roles as neurotransmitters in the brain or sympathetic nervous system, or as neurohormones produced by the adrenal medulla.^48,49 Neurons, as well as non-neuronal cells of different tissues, including the skin^50–52, produce DA, NE, and E.

CA are the principal autonomic neurotransmitters in the skin, as 25–50 % of sympathetic nerve terminals target skin effectors, innervate blood vessels, sweat glands, and hair follicles, and emerge as nerve fibers in the dermal and epidermal layers of the skin.⁵³ The skin is also capable of synthesizing CA, their degrading enzymes, and high-affinity receptors, which have been identified in nerve fibers, keratinocytes, and melanocytes.⁵⁰

NE and E act through α (α1 and α2) and β (β1 and β2) adrenergic receptors in target cells.^12,50 These receptors are part of the classic seven-transmembrane G-protein-coupled receptor (GPCR) family. α1 (α1a, α1b, and α1d) adrenoceptors (AR) couple to phospholipase C via Gqα, leading to the formation of diacylglycerol and inositol trisphosphate and increased intracellular calcium level, while α2 (α2a, α2b, and α2c) AR couple to Giα, causing the downregulation of adenylate cyclase and consequently the inhibition of intracellular cyclic AMP (cAMP).⁵⁰ β-AR activates adenylate cyclase to increase intracellular cAMP via Gsα.⁵⁰ α- and β-AR are present on epidermal and dermal cells and are also expressed in dermal blood vessels,⁵⁴ while β2- and α1-AR are expressed mostly on keratinocytes.^50,52,55

DA acts on specific receptors present in target cells belonging to the GPCR family, which are divided into D1 (D1 and D5) and D2 (D2, D3, and D4) classes of receptors. Activated D1 class of receptors increases Gs-dependent intracellular cAMP, whereas the D2 class activates Gi proteins and inhibits intracellular cAMP. In addition, D2-like receptors via Gβγ subunits can inhibit N- and L-type calcium channels leading to the activation of G protein-regulated inwardly rectifying potassium channels.^50,56 Keratinocytes express D2-like receptors; D4 receptors are mostly found in the uppermost part of the epidermis, whereas D2 receptors occur in the basal layer of the epidermis.⁵⁰

CA plays vital roles in cutaneous homeostasis and inflammation.^50,57 Furthermore, it plays diverse roles in cutaneous wound healing by either positively or negatively affecting the process, which in turn either speeds up or delays wound closure.

CATECHOLAMINES, ANGIOGENESIS AND CUTANEOUS WOUNDS

Epinephrine and norepinephrine

The roles of E and NE in the process of wound healing have been demonstrated by studies that have mainly shown the effect of stress on the healing of wounds, as both acute and chronic stress enhance circulating and tissue CA in the body due to the activation of the sympathoadrenal system.^57,58 The results of these studies indicate that stress has adverse effects on the cutaneous wound restoration process and that the process of wound healing is impaired in stressed animals due to the high levels of CA, which leads to the activation of AR.^57,58 Both E and NE act through AR,^48,49 and hence, in this review, we will discuss the roles of NE and E along with AR modulation.

In general, it has been shown that the blockade or inhibition of β2-AR promoted angiogenesis in wound bed by increasing proangiogenic growth factors and therefore expedited the healing of wounds, which confirmed the deleterious effect of these receptors on the overall wound-healing process.^59,60,61 However, β2-AR is expressed in all types of cells in the skin-like epithelial cells, macrophages, and EC that take part in the wound repair process; therefore, their roles in the wound-healing process are multifaceted.^62,63 In particular, β2-AR activation in EC suppressed the angiogenic processes in the wound bed by inhibiting factors such as collagen III deposition, myofibroblasts density, and re-epithelialization, and by prolonging the overall healing time, which indicates that β2-AR blockade may improve angiogenesis and expedite the healing of these wounds.⁶³ A study using murine excisional skin wound models showed that the stimulation of β-AR in EC caused delayed wound healing through the inhibition of EC functions such as mobilization, proliferation, and tube formation capabilities of dermal EC, which are crucial for the process of angiogenesis.⁶² The study demonstrated that the activation of β-AR resulted in the inhibition of secretion of proangiogenic growth factors, which included fibroblast growth factor 2 from human dermal microvascular endothelial cells (HDMEC) and VEGF from keratinocytes. The findings further showed that in isolated HDMEC, the stimulation of β-AR caused impaired angiogenic response via a cAMP-dependent but PKA-independent pathway.⁶²

Although high levels of systemic CA in chronically stressed mice have been implicated with impaired and delayed wound healing via activation of β-AR, interestingly, a significant increase in the number of blood vessels has been reported in these stressed mice compared to non-stressed control animals. This increase in the blood vessels has been attributed to stress or CA induced vasoconstriction, which resulted in increased tissue hypoxia, i.e., reduced blood flow and oxygen availability to the skin and other extremities^64,65 and therefore more angiogenesis in the wound bed. The infusion of exogenous E (level achieved in acute stress) also reduced blood perfusion in tissues, an essential part of tissue regeneration and healing.⁶⁶ Treatment with propranolol, a β adrenergic blocker, abrogated the systemic effects of CA by activating MMP‐2 and MMP‐9 and increasing collagen turnover, along with less vascularized and more organized collagenous granulation tissue.^59,61

NE, like E in most of the cases, was shown to affect the overall wound-healing process negatively.^67,68 The role of NE in wound bed has been demonstrated in studies that either use pharmacological blockade of α or β receptors or use global knockout (of these receptors) mice to study the effect of these receptors on the overall wound-healing process. NE-depleted mice showed increased wound angiogenesis⁶⁹ and skin wounds were more vascularized in β-AR antagonist-treated rats.⁷⁰ However, the effect of NE on isolated EC is contradictory and mostly context-dependent.⁶⁹ Different studies have indicated that NE could directly stimulate proliferation and hypertrophy of large vessel-derived EC as well as promote apoptotic cell death in cardiac-derived EC.^71,72

In addition to β-AR, the other AR, that is, α1/α2 AR, also play substantial roles in wound angiogenesis and wound restoration processes. α2A/α2C receptor-knockout animals showed a high level of CA that is commonly observed in stressed individuals and animals suffering from chronic inflammatory conditions.^68,73 The deletion of the α2A/α2C‐AR significantly promoted cutaneous wound healing and wound closure by increasing angiogenesis, TGF‐β 1/2/3 expression, keratinocyte migration, collagen deposition, and myofibroblastic differentiation.⁶⁸

In summary, the role of E and NE in wound angiogenesis needs more in-depth investigation. From available reports till date it is difficult to single out the effects of these two CA on angiogenesis during wound healing due to the presence of AR not only on EC but also on other cell types such as keratinocytes, fibroblasts, macrophages that are present in wound beds and take part in the wound healing response. The main effects of AR activation and inhibition on the angiogenic process of wound healing have been summarized in Table 1.

Table 1:

Effects of AR activation and inhibition on the angiogenic process of wound healing

Receptor activation/ inhibition	Effect on angiogenesis and wound healing	Reference
β2-AR activation	inhibition of mobilization, proliferation, and tube formation capabilities of dermal EC; delayed wound healing	62
β2-AR activation	inhibition of secretion of fibroblast growth factor 2 from human dermal microvascular endothelial cells (HDMEC), and VEGF from keratinocytes; delayed wound healing	62
β–AR inhibition	stimulates MMP‐2 and MMP‐9 and promotes collagen turnover and less vascularized and more organized collagenous granulation tissue; faster wound healing	59
α1/α2 AR deletion	increases angiogenesis, TGF‐β 1/2/3 expression, keratinocyte migration, collagen deposition, and myofibroblastic differentiation; faster wound healing	68

Dopamine

In addition to its essential role in the central nervous system (CNS), DA also plays important regulatory roles in different peripheral organs, including the skin.^50,56,74,75 The nerve endings present in the skin synthesize DA, and keratinocytes, the most abundant cell type in the epidermis, and also express enzymes that synthesize and metabolize DA.⁵⁰ Different cell types in the skin, such as the keratinocytes, also express different DA receptors.⁵⁰ Our group has reported that EC express D2 receptors and dermal fibroblasts express D1 receptors.^76,77 Differential expression of the D2 and D4 receptors has been demonstrated in the different epidermal layers^50,74 of the skin. D2 receptors on the epidermal keratinocytes play a critical role in cutaneous barrier homeostasis.⁷⁴

The regulatory role of DA in VEGF-A-mediated angiogenesis, via its D2 receptors present in the EC and endothelial progenitor cells, was reported by our laboratory and confirmed by other research groups.^{16,18, 20,78} In the context of wound healing, we have identified that DA plays either a negative or a positive role depending on whether the wound is a normal cutaneous wound or a diabetic wound.^76,77 Our group has reported that DA D1 receptor activation in dermal fibroblasts could promote angiogenesis and shorten healing time both in type 1 (streptozotocin-induced) and type 2 (db/db) diabetic mice. DA D1 receptor activation stimulated the production of VEGF-A by the cells through the protein kinase A pathway and the phosphorylation of cAMP response element-binding protein.⁷⁷

In contrast, reports from our laboratory have also indicated that DA can negatively regulate the angiogenic process in normal dermal wounds via the D2 receptors. DA through its D2 receptors downregulated VEGF induced expressions of homeobox transcription factor (HoxD3) and its target genes α5 and β1 integrins,⁷⁶ which play critical roles in wound angiogenesis and thereby slowed down the healing process. Treatment with specific D2 receptor antagonist, eticlopride, could significantly accelerate the process of healing by inducing angiogenesis through the upregulation of HoxD3 and its target, α5β1 integrin.⁷⁶ Our studies have thus demonstrated that DA plays opposing roles in normal and diabetic cutaneous wounds, which involves the differential activation of D1 or D2 receptors.^76,77

Several studies have indicated that angiogenesis can also be induced by mesenchymal stem cells (MSCs) that differentiate into EC and promote neoangiogenesis.^79–82 During the process of wound healing, circulating MSCs migrate to the wound site by chemotaxis and subsequently get incorporated into neovessels and are transdifferentiated into EC.^83,84 Furthermore, these incorporating MSCs release different proangiogenic factors, such as VEGF-A, which further promote neovessel formation and healing.^82,85,86 A report from our group indicated that the concentration of DA in the synaptic clefts negatively regulated the mobilization of MSCs into the wound sites. DA acting through its D2 receptors present on MSCs inhibited VEGF-A-induced phosphorylation of Akt, which in turn suppressed the polymerization dynamics of the actin cytoskeleton. This decreased actin polymerization subsequently inhibited the migration of MSCs and inhibited angiogenesis.⁸⁶ DA mediated regulation of angiogenesis during cutaneous wound healing has been summarized in Figure 2.

FIGURE 2:

Schematic diagram of dopamine mediated regulation of angiogenesis during wound healing (Created with BioRender.com). VEGF= Vascular Endothelial Growth Factor, pAKT= phopho-AKT, cAMP=cyclic adenosine 3′,5′-monophosphate, HoXD3=Homeobox D3, D1 receptor= Dopamine D1 receptor, D2 receptor = Dopamine D2 receptor

CONCLUSION AND FUTURE DIRECTIONS

Few reports have suggested that wound closure can proceed normally even with decreased angiogenesis, which may occur with antiangiogenic agents or blockade of signaling pathways,^34,87,88 implying that angiogenesis might not solely be responsible for the regulation of the process. However, several other reports have suggested that angiogenesis plays critical roles in the cutaneous wound healing process by supporting cellular proliferation, migration, and other metabolic activities. Therefore, approaches that can promote angiogenesis during wound healing may be considered as possible therapeutic strategies to improve wound healing. CA neurotransmitters or neurohormones are important regulators of physiological angiogenesis.^{14–17,19, 22–24,75–78} NE and E inhibit the process of angiogenesis and thereby retard the healing process.^59–63 On the contrary, DA inhibits angiogenesis during post-ischemic healing by suppressing angiotensin receptor type 1 expression in EC and either promotes or inhibits cutaneous wound angiogenesis, depending on whether the wound is diabetic or normal, through the activation of DA D1 or D2 receptors, respectively.^{14–21,75–77}

Furthermore, one of the early events in granulation tissue formation during wound healing is angiogenesis.^8,9 To date, there are only a few studies that have looked into the role of CA in angiogenesis during wound healing. Therefore, it is necessary to conduct such studies to fill in the significant gaps in the knowledge about the exact roles of CA in wound healing. These studies might help to identify new therapeutic approaches to manage this critical step of the wound healing response. Lastly, it is of utmost importance that the mechanism of cutaneous wound healing is well-understood to develop strategies to overcome impaired wound healing. Results from clinical trials have confirmed that DA D2 receptor agonists can inhibit VEGF-A-mediated angiogenesis in diseases.⁷⁸ Besides, both β blockers and DA agonists are used clinically for different disorders, and they are considered to have established safety profiles.^78,89 Therefore, clinical trials may be conducted with these compounds towards the development of therapeutic agents for cutaneous wounds.

ABBREVIATIONS

CA

catecholamines

VEGF-A

vascular endothelial growth factor-A

Ang1

angiopoietin1

EC

endothelial cells

HIF-1

hypoxia-inducible factor-1

PDGF

platelet-derived growth factor

TGF-β

transforming growth factor beta

ECM

extracellular matrix

MMP

metalloproteinases

Ang2

angiopoietin 2

Tie2

TEK tyrosinekinase

DA

Dopamine

Norepinephrine

norepinephrine

E

epinephrine

GPCR

G-protein-coupled receptor

AR

adrenoceptors

cAMP

cyclic AMP