An Important Role of Blood and Lymphatic Vessels in Inflammation and Allergy

Angiogenesis and lymphangiogenesis, the growth of new vessels from preexisting ones, have received increasing interest due to their role in tumor growth and metastatic spread. However, vascular remodeling, associated with vascular hyperpermeability, is also a key feature of many chronic inflammatory diseases including asthma, atopic dermatitis, psoriasis, and rheumatoid arthritis. The major drivers of angiogenesis and lymphangiogenesis are vascular endothelial growth factor- (VEGF-)A and VEGF-C, activating specific VEGF receptors on the lymphatic and blood vascular endothelium. Recent experimental studies found potent anti-inflammatory responses after targeted inhibition of activated blood vessels in models of chronic inflammatory diseases. Importantly, our recent results indicate that specific activation of lymphatic vessels reduces both acute and chronic skin inflammation. Thus, antiangiogenic and prolymphangiogenic therapies might represent a new approach to treat chronic inflammatory disorders, including those due to chronic allergic inflammation.


Introduction
According to the World Allergy Organization, allergic disorders affect 30-40% of the world's population, and the prevalence is escalating to epidemic proportions. Much of the pathology of chronic allergic disorders such as atopic dermatitis and asthma is the long-term result of chronic allergic in�ammation at the site of allergen exposure [1]. us, to explore additional possibilities to treat chronic allergic disorders, it is of importance to understand the distinct pathomechanisms and properties of chronic in�ammation.
In�ammation in general is the response of tissues to harmful stimuli such as infectious agents, antigens, or physical and chemical damage. �esides the increased in�ammatory cell in�ltration into the in�amed tissue, it has become clear in the recent years that acute and chronic in�ammatory processes are associated with pronounced vascular remodeling. Angiogenesis and lymphangiogenesis, the growth of new blood vessels and of lymphatic vessels from preexisting ones, are involved in a number of physiological and pathological conditions such as wound healing, tumor growth, and metastatic spread [2][3][4][5]. Angiogenesis and lymphangiogenesis also occur in several chronic in�ammatory conditions, including rheumatoid arthritis, in�ammatory bowel disease, asthma, chronic airway in�ammation, atopic dermatitis, and psoriasis [6][7][8][9].
Even though blood and lymphatic vessels are key players in acute and chronic in�ammatory processes, and thus might serve as new therapeutic targets in in�ammatory and allergic diseases, there is currently no clinically approved treatment to speci�cally modulate the vasculature.

The Function of Blood Vessels and Lymphatic Vessels in Tissue Homeostasis
In vertebrates, there are two vascular systems: the cardiovascular and the lymphatic system. To exert their functions, both vascular systems build highly branched, tree-like tubular structures. In the cardiovascular system, the heart pumps the blood through arteries into smaller arterioles and into capillary beds. From there, the blood returns via venules and veins to the heart to proceed to the lungs for new oxygen loading. Under physiological conditions, the major functions of blood vessels include the supply of gases, �uid, nutrition, and signaling molecules to the tissues with the capillaries as the actual sites of exchange. At these sites, plasma leaks from the capillaries into the interstitium, driven by blood pressure and osmotic gradients. e lymphatic capillaries take up this protein-rich �uid, thereby maintaining not only tissue �uid homeostasis but also exerting immune surveillance. e lymphatic network is composed of blindbeginning thin-walled capillaries without pericyte coverage and with incomplete basal lamina as well as of collecting lymphatic vessels with a smooth muscle cell layer, a basement membrane, and valves, which prevent back �ow of lymph. e largest collecting lymphatic vessel, the thoracic duct, connects the lymphatic system with the cardiovascular system. In adults, physiological angiogenesis and lymphangiogenesis are uncommon. However, new lymphatic and blood vessels form during the female reproductive cycle, the hair cycle, and in healing wounds [10,11].

Anatomy of the Cutaneous and Pulmonary Vascular Systems
e epidermal layer of the skin is free of blood and lymphatic vessels. In the dermis, the blood vascular system is organized into a deep and a super�cial horizontal plexus with capillaries arising from the latter one [12,13]. e lymphatic vasculature also forms two plexuses in vicinity to the blood vessels. Branches from the super�cial lymphatic vessel plexus extend into the dermal papillae and descend into the larger lymphatic vessels in the lower dermis [14]. e bulk of blood microvascular vessels is located immediately below the epidermis, whereas the lymphatic vessels reside more distant to the epidermis [15]. Pulmonary blood vessels transport the low-oxygen blood from the heart to the lung, whereas the bronchial vessels supply the lung with nutrients and oxygen. e bronchial blood vessels arise from the aorta or intercostal arteries and enter the lung at the hilum. At the main stem bronchus, they branch and descend to the lower trachea and extrapulmonary airways. ey cover the whole lung from the bronchial tree to the terminal bronchioles, where the bronchial vessels anastomose with each other as well as with pulmonary vessels [16]. Studies of lymphatic vessels in normal human lung are rare, not only because for a long time lymphatic speci�c markers were not known, but also because in the lung the commonly used lymphatic marker lymphatic vessel endothelial hyaluronan receptor-(LYVE-)1 stains both lymphatic and blood vascular endothelial cells [17]. Recent studies using the speci�c lymphatic endothelium marker podoplanin showed that in human lung, lymphatic vessels extend beyond the respiratory bronchioles, accompanying intralobular arteries deep inside the lobule, the smallest unit of the lung [18,19]. In the murine trachea, the lymphatic network is highly ordered, and vessels are restricted to the mucosa located between the cartilage rings [8].

The Role of Blood and Lymphatic Vessels in �n�ammation
Both the blood and the lymphatic vascular system contribute to the body�s in�ammatory response. In acute in�ammation, blood vascular endothelial cells are activated by several in�ammatory mediators (e.g., vascular endothelial growth factor-(VEGF-)A, tumor necrosis factor (TNF-) , interleukin (IL)-6, and IL-1 ), leading to the typical signs of in�ammation, increased blood �ow as a conse�uence of vessel dilation and edema formation due to increased permeability of the blood vessels. Furthermore, the expression of adhesion molecules, such as intercellular adhesion molecule-(ICAM-)1, vascular cell adhesion molecule-(VCAM-)1, and E-selectin on activated blood vascular endothelial cells, enables the interaction between leukocytes and endothelium, a major event in the in�ammatory process [20,21]. In chronic in�ammation, the blood vasculature remains enlarged, hyperpermeable, and activated with high expression of adhesion molecules, leading to continuous extravasation of in�ammatory cells and �uid into the in�amed tissue. A number of in�ammatory conditions such as rheumatoid arthritis, in�ammatory bowel disease, asthma, atopic dermatitis, and psoriasis are characterized by pronounced angiogenesis [7,9,22,23].
Besides the blood vasculature, also the lymphatic vasculature plays an important role in in�ammation. Lymphatic vessels regulate the in�ammatory response by the transport of �uid, extravasated leukocytes, and antigen-presenting cells from the in�amed tissue to the lymph nodes and to other secondary lymphoid organs, thereby contributing to the decrease of in�ammation-induced edema and to the initiation of a speci�c immune response. e C-C chemokine receptor type (CCR) 7 expressed by dendritic cells is important for their migration into afferent lymphatic vessels which secrete the respective ligand chemokine (C-C motif) ligand (CCL) 21 [24]. Lymphangiogenesis occurs in several chronic in�ammatory conditions such as human psoriasis and mouse models of chronic skin in�ammation, chronic airway in�ammation, and rheumatoid arthritis [8,[25][26][27].

Mediators of Angiogenesis and Lymphangiogenesis in �n�ammation
In recent years, the understanding of in�ammatory angiogenic and lymphangiogenic processes such as endothelial cell growth, migration, and survival has increased, and a variety of involved mediators have been identi�ed. e most important molecule that controls in�ammation-driven angiogenesis is VEGF-A, a member of a family of angiogenic and lymphangiogenic drivers such as VEGF-C, VEGF-D, and placenta growth factor (PlGF) [6,28]. VEGF-A signals via its receptor tyrosine kinases VEGFR-1 and VEGFR-2 and thereby induces angiogenesis and lymphangiogenesis ( Figure 1). Expression of VEGF-A and VEGFR-2 is induced by cytokines such as TNF-, thereby linking angiogenesis with in�ammatory conditions [14,29]. VEGFR-1 is expressed on blood vessels, whereas VEGFR-2 is expressed Blood vascular endothelial cells Lymphatic endothelial cells Angiogenesis Lymphangiogenesis F 1: VEGF-binding properties and distinct VEGF receptor expression on lymphatic and blood vascular endothelium. VEGFs bind to the three VEGF receptor tyrosine kinases, leading to the formation of VEGFR dimers. Blood vascular endothelial cells express VEGFR-1 and VEGFR-2, whereas lymphatic endothelial cells express VEGFR-2 and VEGFR-3. VEGF-A-which binds both VEGFR-1 and VEGFR-2-can directly induce blood and lymphatic vascular remodeling. VEGF-C and -D bind VEGFR-3 and, aer proteolytic processing, also VEGFR-2, thus inducing angiogenesis and lymphangiogenesis.
on both blood and lymphatic vessels with high expression on tip cells [30]. Compared to VEGFR-2, VEGFR-1 has a higher affinity for VEGF-A but a lower kinase activity. VEGFR-1 has been reported to trap VEGF-A to prevent excess signaling via VEGFR-2 during embryogenesis, whereas in the adult the function of VEGFR-1 remains more elusive [31,32]. Several cytokines and chemokines such as IL-1, IL-8, IL-18, chemokine (C-X-C motif) ligand (CXCL) 3, and CXCL12 have also been reported to exert proangiogenic and lymphangiogenic activities (Table 1). In�ammatory lymphangiogenesis, as lymphangiogenesis in general, is mainly driven by VEGFR-2 and -3 signaling. us, VEGF-A-induced VEGFR-2 signaling plays not only a role in angiogenesis but also in lymphangiogenesis. VEGFR-3, which is expressed on lymphatic endothelial cells, binds VEGF-C and VEGF-D ( Figure 1). However, aer proteolytic cleavage, VEGF-C can also induce VEGFR-2 signaling [33][34][35]. VEGFR-2 and -3 signaling have been shown to be involved in in�ammatory lymphangiogenesis in mouse models of skin in�ammation and of airway in�ammation [8,36,37]. ese models will be discussed in more detail later on. e expression of the lymphangiogenic factor VEGF-C is induced in response to different proin�ammatory cytokines such as TNF-and IL-1 , most likely via the activation of the nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-B) pathway [38,39]. Furthermore, in�ammatory cells such as macrophages promote the formation of lymphatic vessels by secreting VEGF-C and VEGF-D [8,40]. TNF-might also contribute to in�ammatory airway lymphangiogenesis [39].

Blood and Lymphatic Vessels in Chronic ��in �n�ammation
Several skin diseases such as atopic dermatitis, contact dermatitis, UV damage, and psoriasis are associated with increased vascular remodeling ( Figure 2) [25,41,42]. In the lesional skin of atopic dermatitis and psoriasis, levels of the angiogenic growth factor VEGF-A are elevated [27,43,44], and in psoriasis patients, the plasma levels of VEGF-A correlate positively with the disease severity [45].
In recent years, a number of mouse models have been developed to study vascular remodeling in chronic skin in�ammation, for example, the epidermal speci�c JunB�C-Jun knockout mice [46], human psoriatic skin transplantation onto severe combined immunode�ciency-(SCID-)mice [47], and K14 (keratin14) VEGF-A transgenic mice [48] which have been developed in our laboratory. In these mice, murine VEGF-A164 is continuously expressed in epidermal keratinocytes under the control of the K14 promoter. Mice homozygous for this transgene develop a chronic cutaneous in�ammation at the age of approximately 5-6 months, which has most of the features of human psoriasis, namely epidermal hyperplasia and abnormal terminal differentiation of epidermal keratinocytes, typical leukocyte in�ltration including dermal CD4+ T cell and epidermal CD8+ T-cell accumulation as well as a pronounced increase in the number and size of blood and lymphatic vessels (Figure 3) [48]. In hemizygous K14-VEGF-A transgenic mice, the chronic skin in�ammation is inducible by applying the contact sensitizer oxazolone [25]. Several studies in these K14-VEGF-A transgenic mice by our laboratory and others have validated these mice as a valuable model for chronic cutaneous in�ammation with a relevant involvement of vascular remodeling. In this model, the small molecular VEGFR inhibitor NVP-�A�2881 showed strong anti-in�ammatory actions with a reduction in in�ammation-induced angiogenesis and lymphangiogenesis [49]. Furthermore, treatment with the spe-ci�c anti-VEGFR-2 antibody DC101 inhibited skin in�ammation, in�ammatory cell in�ltration, and angiogenesis [37], indicating that angiogenesis plays an important role for disease maintenance and progression. e importance of the lymphatic vasculature in in�ammation was also studied in these mice. Surprisingly, blockade of VEGFR-3 signaling increased the severity of skin in�ammation. Conversely, speci�c activation of the lymphatic vasculature by intracutaneous injections of recombinant VEGF-C156S, a speci�c ligand of VEGFR-3, reduced chronic skin in�ammation [37]. Taken together, hemizygous K14-VEGF-A tg mice represent a reliable model to study in�ammation-induced vascular remodeling in the skin and to test the potential effectiveness of new anti-in�ammatory drugs. However, psoriasis is a human-speci�c disease that is not naturally observed in animals. us, different genetic and xenotransplant mouse models have been developed to mimic some features of human psoriasis [50]. Considering the substantial differences between mouse and human skin, however, most of these models do not fully recapitulate all characteristics of the human disease. erefore, for speci�c pathogenetic studies, the evaluation of different psoriasis mouse models might be advantageous. In this regard, it has been recently reported that topical treatment with Imiquimod, a toll-like receptor 7 and 8 agonist, triggers psoriasis-like skin lesions in humans and in mice, with an involvement of the IL23/IL17 axis, known to play a crucial role in human psoriasis [51].

Blood Vessels in Asthma
Asthma is a chronic in�ammatory disease of the airways that is characterized by airway hyperresponsiveness, episodic air�ow limitations, and a decline in lung function. ese symptoms are caused by chronic in�ammation and airway remodeling, including increased thickness of the lamina reticularis [52,53], smooth muscle hyperplasia/hypertrophy, and increased vascularity [54,55] in small and large airways [56]. In the 1960s, Dunill demonstrated for the �rst time the involvement of blood vessels in asthma by showing swollen bronchial mucosa with dilated and congested capillaries in lung samples of subjects who died of acute asthmatic attacks [57]. Since then, increased airway vascularity was not only found in severe but also in mild asthmatic cases, and it is nowadays a well established �nding that the number of blood vessels and the tissue area covered by blood vessels are increased in asthmatic patients compared to healthy subjects (reviewed in [55]). In mild asthma cases, the increased submucosal vascularity in the inner area of the medium airway might contribute to air�ow limitation, as indicated by the inverse correlation of vascularity and forced expiratory volume, a measure for airway obstruction [56]. VEGF-A, the major driver of angiogenesis, was also increased in sputum samples of patients with mild asthma compared to healthy controls [58]. VEGF-A and VEGFR-1 mRNA levels were increased in lung biopsy specimens from patients with mild-to-moderate asthma compared to healthy controls. e reported colocalization of VEGF-A with CD68, major basic protein and Chymase-positive cells suggests that macrophages, eosinophils, and mast cells are a major source of VEGF-A in the lung [59,60], which thus may contribute to angiogenesis in asthma. Conversely, it has been shown-in different in vitro and in vivo settings-that eosinophils, mast cells, and macrophages are also in�uenced by angiogenic factors. Due to their expression of VEGFR-1, these cells are able to migrate towards VEGF-A gradients [61][62][63][64][65]. erefore, increased VEGF-A levels at the site of in�ammation contribute to the recruitment of different in�ammatory cells which themselves can secrete proin�ammatory mediators.
McDonald and colleagues established the Mycoplasma pulmonis infection model of chronic airway in�ammation as a valuable tool for investigation of chronic in�ammatory airway diseases in mice. is model shows several-though not all-characteristic features of asthma such as in�ammatory cell in�ux, angiogenesis, mucosal edema, epithelial changes, �brosis, and bronchial hyperreactivity [8,66]. Shortly aer M. pulmonis infection, mucosal blood vessels enlarge by endothelial cell proliferation, and angiogenesis reaches a plateau at 14 days aer infection [8]. Surprisingly, VEGF receptor blocking studies showed that this pathological angiogenesis might not be driven by VEGF-A [8]. However, blocking of TNF-signaling by an anti-TNF-antibody dramatically reduced blood vessel remodeling 14 days aer M. pulmonis infection, suggesting that TNF-signaling is involved in this angiogenic process [39].

Lymphatic Vessels in Chronic Airway Disease
Edema formation results when the amount of leakage from the blood vessels exceeds the capacity of lymphatic vessels for drainage. Such edemas are a cardinal sign of chronic in�ammation, and indeed increased microvascular permeability as well as edema are features of asthma [67]. However, knowledge of lymphatic involvement in edema formation in asthma is remarkably sparse. Only recently, it was shown that lymphangiogenesis increased with advanced stages of idiopathic pulmonary �brosis, a chronic lung disease of unknown etiology with an insidious onset, leading to ventilatory restriction and respiratory failure [68].
In the M. pulmonis driven mouse model of chronic airway in�ammation, there is also a dramatic remodeling of lymphatic vessels [8]. Robust lymphangiogenesis is most abundant on the surface facing the cartilage rings that grow towards the overlaying airway epithelium. Interestingly, aer 4 weeks of treatment with antibiotics, lymphatic sprouting was completely inhibited, but lymphatic vessels regressed only partially, compared to the almost complete regression of blood vessels [8]. VEGF-C, VEGF-D, and TNF-are major drivers of this remodeling. Blockade of VEGFR-3 signaling via soluble VEGFR-3-Fc or by an anti-VEGFR-3 antibody almost completely prevented lymphangiogenesis in tracheas of infected mice [8]. In contrast, adenoviral overexpression of VEGF-C in the murine trachea leads to enhanced lymphatic �lopodia formation and to sprouts similar to those seen in the M. pulmonis infected mice. Immuno�uorescence stainings showed that mucosal in�ammatory cells, in particular F4/80� macrophages, are a major source of VEGF-C in this model. Reduced lymphangiogenesis aer infection with M. pulmonis was also observed aer inhibition of TNF signaling by a blocking antibody and in TNFR-1 ko mice [39].

Conclusions and Outlook
ere is clear evidence that in humans, vascular remodeling occurs in many chronic in�ammatory disorders. Even though different anti-in�ammatory drugs are on the market, there is no speci�c therapy that interferes with the pathological vascular changes that occur during in�ammation. Angiogenesis and lymphangiogenesis are tightly linked to chronic in�ammation, and targeting the blood vessels and lymphatic vessels has been shown to be an effective strategy in different experimental mouse models of chronic in�ammation. One has to keep in mind, however, that in most conditions the vascular activation likely represents a downstream event that maintains the in�ammatory process, but not the pathogenetic cause of the respective disease, which oen has remained unknown. Nonetheless, antiangiogenic and prolymphangiogenic therapies might represent new approaches to treat chronic in�ammatory disorders, including those due to chronic allergic in�ammation.

Authors' Contribution
S. Zgraggen and A.M. Ochsenbein equally contributed to this work.