Abstract
Angiogenesis is essential for wound healing, and angiogenesis impairment can result in chronic ulcers. Studies have shown that the sympathetic nervous system has an important role in angiogenesis. In recent years, researchers have focused on the roles of sympathetic nerves in tumor angiogenesis. In fact, sympathetic nerves can affect angiogenesis in the wound healing of soft tissues, and may have a similar mechanism of action as that seen in tumorigenesis. Sympathetic nerves act primarily through interactions between the neurotransmitters released from nerve endings and receptors present in target organs. Among this, activation or inhibition of adrenergic receptors (mainly β-adrenergic receptors) influence formation of new blood vessels considerably. As sympathetic nerves locate near pericytes in microvessel, go along the capillaries and there are adrenergic receptors present in endothelial cells and pericytes, sympathetic nerves may participate in angiogenesis by influencing the endothelial cells and pericytes of new capillaries. Studying the roles of sympathetic nerves on the angiogenesis of wound healing can contribute to understanding the mechanisms of tissue repair, tissue regeneration, and tumorigenesis, thereby providing new therapeutic perspectives.
Introduction
Angiogenesis is a complicated and multistage process. It plays an important part in physiologic processes (e.g., embryogenesis, wound healing, reproduction) and pathologic processes (e.g., atherosclerosis, age-related macular degeneration, autoimmune diseases, cancer) [10, 24]. During the formation of new vessels, endothelial cells (ECs) proliferate, migrate and subsequently form tube-like structures [42]. This process is followed by pericytes recruitment to ECs, insertion into the basement membrane of capillaries, and encircling of ECs. Role of pericytes has been shown to be critical for the development of new capillaries into mature vessels with normal structures and functions. It has been reported that pericytes and ECs determine and regulate the stability of vessel walls in unison [33].
Recent studies have shown that the noradrenergic system has an important role in retinal angiogenesis [26]. Persistent activation of the sympathetic nervous system (SNS) has been demonstrated to promote angiogenesis and tumor growth in mouse models of ovarian carcinoma [27]. Furthermore, simultaneous blockade of alpha and beta adrenergic receptors (α-ARs and β-ARs, respectively) has been shown to delay wound healing in rats with full-thickness skin defects on the backs [35]. In guinea-pigs with third-degree burn wounds on bilateral thighs, the number of capillaries in wounds is detected to decrease, and peak regeneration of capillaries in granulation tissue was shown to be postponed after chemical sympathectomy [40]. Eventually, blood vessels in vivo and in vitro are damaged in β2-AR knockout mice [7]. The studies outlined above suggest that the SNS has an important role in angiogenesis.
It can be said that tumorigenesis has similar biologic processes to wound healing: inflammation, cell proliferation/differentiation, angiogenesis, and tissue molding. Of these parameters, angiogenesis is very important because it provides oxygen and nutrients for wound healing and tumor growth. A study on human breast-carcinoma xenografts in mice shows that connective tissue cells promote tumor growth by secreting a protein involved in angiogenesis during wound healing [29]. This mechanism can be applicable to all types of carcinomas [13].
Thus, focusing on the regulation of angiogenesis by the SNS may be important for exploring the mechanisms of wound healing in soft tissues, chronic ulcers, and tumors.
Classification and distribution of sympathetic nerve fibers in the vascular system
The SNS is an important part of the autonomic nervous system. Sympathetic nerves comprise pre-ganglionic and post-ganglionic fibers. The former are cholinergic fibers that release acetylcholine, which activates M and N receptors. Post-ganglionic fibers (adrenergic fibers) primarily secrete norepinephrine(NE), as well as small quantities of neuropeptide Y(NPY) and dopamine(DA), to activate α-ARs and β-ARs. Nevertheless, post-ganglionic fibers (which are found in sweat glands and skeletal muscle) release acetylcholine. Recently, research teams have started to focus on the roles of neurotransmitters of post-ganglionic fibers and α-ARs/β-ARs in angiogenesis.
Blood vessels in the skin microcirculation are innervated abundantly by sympathetic nerves [23]. In the upper dermis of the hind paw skin of rats with chronic constriction injured right sciatic nerve [52] and lower lip dermal of rats with damaged mental nerve [39], sympathetic nerves sprout to form a mesh-like pattern around blood vessels. In the vasa recta capillaries of Sprague– Dawley rats kidneys, sympathetic nerves run along the capillaries in close proximity to pericytes, and dominate regulation of vessel diameter by pericytes [9]. Sympathetic nerves have also been found near the pericytes of choroidal microvessels of rats, and pericytes are under the control of them [41]. However, in humans, it is not known if sympathetic nerves locate along capillaries in the skin and form a mesh-like pattern around capillaries.
It has been reported that ECs of brain capillaries in rats are closely associated with adrenergic fibers [25], and functional adrenergic receptors have been detected in bovine retinal microvascular pericytes [56]. Retinal [26] and dermal [28] microvascular ECs in humans, as well as murine retinal capillary nets [26] have been shown to express β-ARs. Furthermore, β-ARs coupled to adenylate cyclase have been observed in the brain capillaries of rats [25]. Alpha-1D-ARs have been detected in human umbilical vein endothelial cells (HUVECs) [48], and α2-ARs have been documented in the uterine artery endothelial cells (UAECs) of ewes [17]. However, it has not been reported if α-ARs are present in capillaries, how they affect the function of capillaries, and how they are involved in angiogenesis. Sympathetic nerves may play an important part in angiogenesis primarily via the major structures of capillaries: ECs and pericytes.
Sympathetic nerves and angiogenesis
Sympathetic neurotransmitters and angiogenesis
It has been demonstrated that NE is involved in the growth and development of tumors [49]. NE promotes angiogenesis in tumors in restrained mice with ovarian cancer by increasing expression of vascular endothelial growth factor (VEGF) [27]. It also increases the angiogenesis and invasion potential of tumors through upregulation of VEGF expression in nasopharyngeal cancer, ovarian cancer, melanoma, and oral cancer [46]. This phenomenon has been attributed mainly to reduced generation of hypoxia-inducible factor-1α, during which α1-ARs and β-ARs have been found to have a significant role and the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/Akt/p70S6K pathway has been shown to be involved in [31]. Besides VEGF, NE increases the angiogenesis and invasion capacity of tumors by upregulation of expression of interleukin (IL)-8 and IL-6 in human melanoma cell lines [51]. The mechanism detected in the effects of NE seems to be activation of β-ARs in tumor cells leads to a significant increase in the synthesis and release of the angiogenic factors IL-8, IL-6 and VEGF [47].
In normal conditions, NE has complicated biologic effects. Mediated by β2-ARs and β3-ARs, NE promotes the proliferation of ewes UAECs derived from pregnant(P-UAECs) [17]. Acting via the β-AR/cAMP/PKA pathway, NE induces HUVEC proliferation by provoking expression of VEGF mRNA in HUVECs, and this effect may be related to NE-induced activation of α1-ARs coupled with extracellular regulated protein kinases [43]. However, in the cardiac ECs of neonatal rats, NE has been found to cause apoptosis via activation of β-ARs (β2-ARs > β1-ARs), the caspase-2 pathway after down-regulation of Bcl-2 expression [11] and a reactive oxygen species-dependent c-Jun N-terminal kinase pathway [12]. Thus, the opposing effects of NE on ECs from different sources in vitro merit attention. Furthermore, NE increases VEGF expression in mice, leading to an increased density of capillaries in ischemic gastrocnemius muscle [16]. It has been shown that angiogenesis in wounds increases in NE-depleted mice compared with mice with intact NE function, which could be attributed to NE-stimulated apoptosis of ECs [32]. In a mouse model of hind-limb ischemia, NE stimulates the mobilization of endothelial progenitor cells (EPCs) [16]. Therefore, the role of NE in angiogenesis may differ if conditions change.
In addition to the main neurotransmitter (NE), sympathetic nerves also release other neurotransmitters, primarily DA(an inhibitory catecholamine) [6], and NPY(a potent angiogenic factor in vivo and in vitro), which activates several stages of angiogenesis [57]. One study has suggested that HUVECs possess NPY and its receptors. HUVECs are not only the origin of the autocrine NPYsystem but also the site of NPY action [59]. NPY induces HUVEC adhesion to the extracellular matrix, as well as the proliferation, migration, and formation capillary-like tubes on Matrigeltrademark or collagen [57]. In ischemic tissues, depending on endothelial nitric oxide synthase(eNOS) activation and the VEGF pathway [21], NPY up-regulates its expression and that of its receptors probably via its Y2 receptors, which leads to angiogenesis [57]. In an angiogenic assay in mice, NPY is considered to be as powerful as VEGF or basic fibroblast growth factor (bFGF) in terms of angiogenesis [59].
Shome et al. [44] have found that the potent anti-angiogenic effect of DA is mediated primarily by its D2 receptors. They have also found that, through the induction of angiogenesis in wound sites, specific antagonist of the DA D2 receptor markedly accelerates wound healing in mice with full-thickness skin defects on their back. As an inhibitory catecholamine, DA has the opposite effect to that of NE with regard to tumor angiogenesis. Some studies have shown that DA, by acting specifically through its D2 receptors, suppresses tumor growth in vitro and in vivo by inhibiting the effects of VEGF/vascular permeability factor of bone marrow-derived endothelial progenitor cells (BM-EPCs) coupled with tumor ECs [4]. DA also inhibits mobilization of BM-EPCs, and DA levels have been shown to decrease in stressed mice with ovarian cancer. Recently, several studies in mice have suggested that DA substitution neutralizes the acceleration of NE on tumor growth by inhibiting tumor angiogenesis [27]. In gastric cancer, endogenous levels of DA have been found to be significantly lower than those of surrounding normal tissues, suggesting that DA is a endogenous “tumor blocker” [5]. Cancer development necessitates considerable angiogenesis, so DA has been shown to be a new inhibitor of tumors because of its anti-angiogenic function [8]. In addition, during DA treatment in restrained mice with ovarian cancer, focal adhesion kinase is activated after cAMP levels increase or PKA is activated, resulting in increased pericyte coverage of tumor ECs and acceleration of maturation of blood vessels [27]. It has been shown that exogenous application of DA changes the morphology and function of blood vessels by influencing tumor ECs and pericytes. A mechanism of action has been postulated for this phenomenon whereby, mediated directly by DA D2 receptors on the surface of tumor ECs and pericytes, DA upregulates expression of Krüppel-like factor-2 in tumor ECs and angiopoietin-1 in pericytes, leading to increased stability of blood vessels with abnormal structure and function in tumors [6]. Above all, DA is an appealing molecule to develop ways to enhance the efficacy of therapies for diseases associated with abnormal blood vessels and tumors [27].
As a hormone secreted by the adrenal medulla, epinephrine(E) acts via α-ARs and β-ARs. It is under the control of the sympathoadrenal medullary axis. E has the same pro-angiogenesis effects and mechanisms as NE in tumors [47]. Acting through β3-ARs, E promotes the migration of bovine aortic endothelial cells. During this process, the Rac1-PKA-Akt pathway makes a significant contribution [18]. Mediated by β-ARs instead of α-ARs, E increases the proliferation of P-UAECs [17] (Table 1).
In conclusion, as an integral part of homeostasis, the SNS secretes substances that have opposing effects upon angiogenesis (Fig. 1).
Sympathetic adrenergic receptors and angiogenesis
We have demonstrated that β2-ARs are important regulators in wound healing. Multiple models in vivo and in vitro have suggested that β2-ARs can modulate angiogenesis in the skin and soft tissues. For instance, activated β2-ARs expressed on skin ECs have been found to reduce vascular permeability and enhance angiogenesis in hind-limb ischemic tissues. β2-AR activation induced by stress has also been shown to increase tumor angiogenesis (though the new vessels are disorganized and immature). Acting through a cAMP-dependent pathway, β2-AR agonists increase VEGF (released by macrophages) levels, thereby leading to angiogenesis [32]. In addition, it has been reported that β2-ARs partly reverse the damaged angiogenesis in animal models of cardiovascular disease (e.g., spontaneously hypertensive rats) and that β2-AR overexpression increases VEGF levels in EC culture supernatants [7]. Pathologic angiogenesis, a key change observed in several ischemic/hypoxic retinal diseases, is dependent (at least in part) on β2-AR activity. In mice with hypoxia-inducible retinopathy, β2-AR blockade decreases levels of pro-angiogenic factors as well as pathologic angiogenesis in the retina, resulting in effective modulation of retinal angiogenesis [26]. Conversely, β-AR activation (primarily β2-AR) can induce apoptosis of cardiac ECs in neonatal rats in vitro. Studies have shown that β2-AR activation in vivo reduces angiogenesis in wounds and, in turn, wound vascularization increases in mice after β2-AR blockade. Thus, β2-ARs can regulate angiogenesis, but whether β2-ARs increase or decrease angiogenesis after activation is not known [32]. Actions of β2-ARs on angiogenesis are dependent upon several factors.
It has been shown that β3-AR activation on human retinal ECs induces their proliferation and migration [45]. The non-selective β-AR agonist isoproterenol has been found to inhibit the migration and tube formation of human dermal microvascular endothelial cells (HDMECs) by reducing secretion of pro-angiogenic FGF-2 from HDMECs. After isoproterenol treatment, angiogenesis in wound sites has been shown to decrease in mice with full-thickness skin defects on the back [28]. However, several studies have suggested that isoproterenol stimulates proliferation of infant hemangioma endothelial cells (IHECs) [14, 26, 29]. In animal models of lung cancer and breast cancer, tumor vascularization increases significantly after β-AR activation and, in turn, the density of blood vessel decreases. Furthermore, upon activation of β-ARs in multiple types of tumor cells, the synthesis and release of the angiogenic factors VEGF, IL-8 and IL-6 increase significantly, causing accelerated tumor growth [47]. Conversely, β-AR blocked by propranolol suppresses the proliferation/migration of HUVECs, as well as the tube formation of HDMECs and human brain microvascular endothelial cells, thereby leading to impaired angiogenesis [19]. In IHECs, propranolol increases apoptosis and inhibits cell proliferation in a similar manner [15]. Apart from downregulation of VEGF expression, propranolol suppresses angiogenesis of hemangiomas by inhibition of bFGF expression [55]. Propranolol is also thought to inhibit proliferation of infant hemangioma(IH) pericytes and normal pericytes from the placentas and retinas of human placenta and retina in vitro [20], so has been used in IH treatment. Romana et al. showed that administration of propranolol (p.o.) increases blood-vessel density in the granulation tissue of rats with normal blood glucose [38] or streptozotocin-induced diabetes mellitus (DM) rats [37] with full-thickness skin defects on their back, although outcomes of wound healing are contradictory (propranolol delays wound healing in normal-blood-glucose rats but facilitates wound healing in streptozotocin-induced DM rats) merely because of the different blood glucose levels. Pro-angiogenic effects of propranolol in vivo may be attributed to an increased number and increased migration of mast cells(MCs; the histamine and heparin released from MCs promote the growth and migration of ECs) [38] and synthesis of nitric oxide(nitric oxide can increase VEGF secretion and MC number) [37]. In a study in severely burned rats(third-degree, 10% of total body surface area), propranolol(6 mg/kg, p.o.) has found to increase secretion of matrix metalloproteinase-2, cell proliferation, collagen deposition, myofibroblast density, and re-epithelization in wounds, but capillary density decreases in the experimental group compared with the control group [36]. Therefore, the roles of propranolol on angiogenesis are complicated. Possibly, in tumors, β-AR activation induces angiogenesis and inhibition suppresses angiogenesis. In wound healing, β-AR inhibits angiogenesis after activation, and promotes it indirectly after inhibition, except if an β-AR antagonist is given at a low dose via the oral route. However, in vitro, non-selective activation and inhibition of β-ARs in normal ECs reduce angiogenesis. Thus, the effects of β-ARs upon angiogenesis differ as conditions change. Hence, the influences of clinical conditions on drug effects must be considered carefully (Table 2).
In a proliferation assay of ewe P-UAECs, activation of β2-ARs and β3-ARs promote cell division, whereas α-ARs doesn’t have a role [17]. It has been reported that blockade of only α-ARs has no effects on blood-vessel density and healing in cutaneous wounds of rats [38]. Though phenylepinephrine, a nonvasoconstrictive α-AR agonist, has been shown to augment the proliferation and migration of HUVECs, and capillary generation [47]. Hence, sympathetic nerves may have a role in angiogenesis mainly through β-ARs, whereas α-ARs are less important.
In conclusion, regulation of angiogenesis by the SNS is complicated. It is influenced by several factors: species, in vitro/in vivo conditions, and blood glucose levels.
Sympathetic nerves and pericytes, basement membrane and EPCs
Previously, studies on angiogenesis focused on ECs but, in recent years, attention has been shifted to pericytes and EPCs because both have important roles during angiogenesis. It has been shown that sympathectomy in the eye reduces expression of the steady-state mRNA of platelet-derived growth factor-BB (PDGF-BB) in rats, causing a decrease in number of pericytes because PDGF regulates the growth, survival and activities of pericytes [50]. Lee et al. [20] have found that propranolol inhibits the proliferation of normal pericytes. In β1-AR-deficient mice, significantly increased death of retinal pericytes has been documented, as well as simultaneous increases in expression of tumor necrosis factor-α and caspase-3 [30]. Pericytes are essential for angiogenesis, and the VEGF synthesized and secreted by pericytes can promote angiogenesis [3]. Therefore, sympathetic denervation or functional changes can influence angiogenesis by inducing pericyte survival or death. In addition, sympathetic nerves also regulate the constituents of the basement membrane, so sympathetic denervation can lead to a thickened basement membrane. One study shows that after sympathectomy of the eye in rats, the thickness of the basement membrane of capillaries in layers of ganglia in the retina increases [50]. Finally, EPCs are considered to be important resources of post-natal angiogenesis. Acting throughα-AR, β-AR and Akt/eNOS pathways, sympathetic nerves regulate the mobilization and pro-angiogenic ability of EPCs. With augmentation of sympathetic activities and NE secretion during wound healing, EPC mobilization increases, as does the number of EPCs in the peripheral circulation, spleen and BM [16].
Sympathetic nerves and vascular smooth muscle cells (VSMCs)
In the wound sites of soft tissues, there are also growing, larger vessels containing VSMCs, which have critical roles in angiogenesis through communication with neighboring ECs [22]. Thus, any changes in VSMCs levels by sympathetic nerve/sympathetic neurotransmitters/sympathetic adrenergic receptors can affect angiogenesis.
α1-ARs have been reported to be present in VSMCs, mediating cell constriction [1]. Rat aortic SMCs express all three α2-AR subtypes (α2A, α2B, α2C). It has been shown that α2-AR activation is involved in VSMCs contraction. Mediated by nitric oxide released from ECs, however, α2-AR activation promotes VSMCs relaxation. In addition, α2-AR activation of rat aortic SMCs stimulates the activity of mitogen-activated protein kinase and cell migration, markedly reduces the intensity of F-actin labeling, but do not promote cell proliferation [34]. NPY is a strong mitogen for VSMCs. Mediated by Y1 and Y2 receptors, NPY promotes VSMC growth over a wide range of concentrations in a bimodal fashion, probably contributing to capillary angiogenesis and collateral formation [58]. Furthermore, the D4 receptor is expressed in VSMCs, and its activation inhibits insulin-mediated and angiotensin II-mediated proliferation and migration of VSMCs. Meanwhile, activation of D1-like or D3 receptors also inhibits insulin-mediated proliferation of VSMCs [54].
Conclusion
Recently, researchers have become increasingly interested in the roles of sympathetic nerves. Sympathetic nerves run along capillaries in close proximity to pericytes [9, 52], and adrenergic receptors are present in ECs [17, 48] and pericytes. In addition, denervation [40] and abnormalities [7, 35] of the SNS affect angiogenesis. Therefore, sympathetic nerves may have important roles in angiogenesis mainly through the major structures present in capillaries: ECs and pericytes. However, studies on sympathetic nerves, endothelial cells/pericytes and angiogenesis at the present are not comprehensive enough, which will probably be a research direction of angiogenesis in the future. As an integral part of homeostasis, the SNS secretes substances with opposing effects on the regulation of angiogenesis: NE, E, DA, and NPY. They interact with receptors present in ECs, pericytes and other target cells. Furthermore, the different states of ARs in vivo or in vitro can lead to different outcomes of angiogenesis. Above all, regulation of angiogenesis by the SNS is extremely complex and influenced by several factors: species, in vivo/in vitro, animal model, and injury conditions.
Angiogenesis is essential for wound healing. New vessels deliver oxygen and nutrients to damaged tissues and facilitate debris removal. This process increases the metabolic demands associated with healing. As a consequence, angiogenesis impairment can result in chronic wounds. Subjects with DM exhibit different degrees of angiogenesis impairment that can cause healing disorders(e.g., ulcers). Among this, sympathetic nervous damages and secondary vascular dysfunctions are worth attention. Studies have shown that dysfunction [2] and decreased activities [49] of sympathetic nerves can be detected in the early stages of DM. Furthermore, early dysfunction of the SNS has been reported to participate in the development of diabetic foot ulcers [23]. Summing up, diabetic sympathetic damages may result in angiogenesis impairment in diabetic patients, thus paly an important role in the occurence of diabetic chronic wounds, but the reliability and mechanisms need further study.
Conflicts of interst
The authors declare that there is no conflict of interests regarding the publication of this article.
Footnotes
Acknowledgments
This paper was supported by grants from the Guangzhou City Science and Technology Project (2012J4100044), the National Natural Science Foundation of China (81171812 and 81272105), and the National Basic Science and Development Program (973 Program, 2012CB518105).