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1β1-impact on inflammation
* Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, ETH Zurich, Switzerland; and
Shiseido Life Science Research Center, Yokohama, Japan
1Correspondence: Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, Wolfgang-Pauli-Str. 10, HCI H303, CH-8093 Zurich, Switzerland. E-mail: michael.detmar{at}pharma.ethz.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1β1 isoform of soluble guanylate cyclase (sGC), that vascular endothelial growth factor-A potently induces sGC1β1, and that nitric oxide (NO) -induced LEC proliferation, migration, and cGMP production in LECs are specifically dependent on sGC1β1. Moreover, the specific sGC inhibitor NS-2028 completely prevents ultraviolet B-irradiation-induced lymphatic vessel enlargement, edema formation, and skin inflammation in vivo. These findings identify a crucial role of the NO/sGC1β1/cGMP pathway in modulating lymphatic vessel function. The blockade of sGC1β1 signaling might serve as a novel therapeutic strategy for inhibiting lymphangiogenesis and inflammation, in addition to its effects on the blood vasculature.—Kajiya, K., Huggenberger, R., Drinnenberg, I., Ma, B., and Detmar, M. Nitric oxide mediates lymphatic vessel activation via soluble guanylate cyclase 1β1-impact on inflammation.
Key Words: lymphangiogenesis • vascular endothelial growth factor-A • UVB • endothelium • angiogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. There is increasing evidence that lymphatic vessels also actively participate in acute and chronic inflammation. The chronic inflammatory skin disease psoriasis is characterized by pronounced cutaneous lymphatic hyperplasia, and chronic skin inflammation in mice is also associated with lymphatic endothelial cell (LEC) proliferation and lymphatic hyperplasia (3)
. Furthermore, kidney transplant rejection is frequently accompanied by lymphangiogenesis (4)
, and LEC-derived chemokines such as CCL21 might promote the inflammatory process (5)
. Lymphangiogenesis has also been observed in experimental models of chronic airway inflammation (6)
. Recently, we found that acute skin inflammation and edema formation induced by ultraviolet B (UVB) irradiation are associated with hyperpermeable, leaky lymphatic vessels that are functionally impaired (7)
. UVB irradiation of the skin also increases expression of vascular endothelial growth factor (VEGF)-A, and systemic blockade of VEGF-A leads to diminished UVB-induced lymphatic vessel abnormalities and skin inflammation in mice (7)
. These findings indicate that VEGF-A-mediated impairment of lymphatic vessel function promotes edema formation and inflammation. However, the mechanisms and functional consequences of lymphangiogenesis under inflammatory conditions are largely unknown.
Nitric oxide (NO) is a diatomic free radical molecule that is synthesized by a family of enzymes known as nitric oxide synthases (NOS). There are three isoforms of NOS: the calcium-dependent endothelial NOS (eNOS), neuronal NOS (nNOS), and a calcium-independent inducible NOS (iNOS) (8)
. NO regulates a number of signaling processes; in the vascular system, NO effects include vasodilation and increased vascular permeability (9)
. Soluble guanylate cyclase (sGC) is the only known physiological receptor for NO. On binding to NO, the activity of sGC is increased up to 400-fold, thereby promoting the conversion of GTP to cGMP and pyrophosphate. Synthesized cGMP regulates various effector proteins, including protein kinases, phosphodiesterases, and ion channels (10)
. The sGC is a heme-containing heterodimeric protein consisting of 73- to 82-kDa alpha and 70-kDa beta-subunits (11)
. Two isotypes of human sGC have been identified (12)
. The originally identified human 3 and β3 subunits have been renamed 1 and β1 (13)
, and sGC1β1 is mainly expressed in human heart and lung. The sGC 2/β2 isotype has been shown to localize to synaptic membranes in the brain (14)
. The sGCs have important roles in smooth muscle contractility, platelet reactivity, as well as in NO-induced hypotension in septic shock (15)
. Moreover, the sGCs play a major role in the mediation of normal and pathological blood vascular angiogenesis (16)
, and inhibition of sGC inhibits neovascularization in the chicken chorioallantoic membrane assay (17)
.
Recent studies have shown that NO is produced and released by lymphatic endothelial cells, possibly regulating lymphatic permeability and flow (18)
and that stimulation of iNOS activity in tumors is correlated with expression of the lymphangiogenic growth factor VEGF-C (19)
. However, little is known about the direct contributions of NO or of distinct sGC isoforms toward the control of normal and pathological lymphatic vessel function.
Because our previous gene array-based transcriptional profiling studies of cultured human LECs vs. blood vascular endothelial cells (BVECs) revealed highly increased expression levels of sGC subunits 1 and β1 in LECs, compared with BVECs, we investigated the importance of the NO/sGC system for LEC function in vitro and in vivo. Here, we show that NO-induced LEC proliferation and migration are sGC dependent and that NO-induced cGMP production in LECs is specifically dependent on sGC1β1. Importantly, the specific sGC inhibitor NS-2028 completely prevented ultraviolet B (UVB) irradiation-induced lymphatic vessel enlargement and skin inflammation and edema formation. These findings identify a crucial role of the NO/sGC1β1/cGMP pathway in mediating lymphatic function. The blockade of NO/cGMP signaling might therefore serve as a novel therapeutic strategy for inhibiting lymphangiogenesis and inflammation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 21)
. The lineage-specific differentiation was confirmed by real-time RT-PCR for the lymphatic vascular markers Prox1, LYVE-1, and podoplanin, and for the blood vascular endothelial markers VEGF receptor-1 and VEGF-C, as well as by immunostains for CD31, Prox1, and podoplanin as described (20
, 21)
. Cells were cultured in endothelial basal medium (Cambrex; Verviers, Belgium) supplemented with 20% fetal bovine serum (FBS; Life Technologies, Paisley, UK), antibiotics, 2 mM L-glutamine, 10 µg/ml hydrocortisone, and 0.025 mg/ml N-6,2-O-dibutyryl-adenosine 3',5'-cyclic monophosphate (all from Fluka; Buchs, Switzerland) for up to 11 passages.
Quantitative real-time RT-PCR
Total cellular RNA was isolated from confluent BVEC and LEC cultures following five rounds of passage using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). After exposure to RQ1 RNase-free-DNase (Promega, Madison, WI, USA), the mRNA expression of vascular lineage-specific genes and of sGC1, β1 and eNOS was investigated in triplicate by quantitative real-time TaqMan RT-PCR using the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and predesigned probes and primers (Applied Biosystems assay IDs Hs00181365_m1, Hs00168325_m1, Hs00167166_m1). Each reaction was multiplexed with β-actin primers (forward 5'-TCACCGAGCGCGGCT-3', reverse 5'-TAATGTCACGCACGATTTCCC-3') and probe (5'-JOE-CAGCTTCACCACCACGGCCGAG -TAMRA-3') as an internal control. Triplicate cultures of LECs were also incubated with 20 ng/ml of human recombinant VEGF-A165 (R&D Systems, Minneapolis, MN, USA) for 1, 4, 8, or 24 h (control cells were incubated in media alone), followed by RNA extraction and real-time TaqMan RT-PCR analysis of sGC1 and sGCβ1 expression.
Immunoblotting
Immunoblot analyses of sGC1, sGCβ1, and eNOS were performed as described previously (21)
. Confluent cultures of BVECs and LECs were homogenized in lysis buffer, and protein concentrations were determined using the BCA-Kit (Pierce Biotechnology, Rockford, IL, USA). Equal amounts of lysates (100 µg protein) were immunoblotted with a rabbit polyclonal antibody against sGC1 (Sigma, Saint Louis, MO, USA), sGCβ1 (Cayman Chemical, Ann Arbor, MI, USA), or eNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Specific binding was detected by the enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA). Equal loading was confirmed with an antibody against β-actin (Sigma).
Immunostains
Immunofluorescence analysis was performed on 6-µm cryostat sections of human neonatal foreskins using polyclonal antibodies against LYVE-1 (kindly provided by Dr. David Jackson, Oxford, UK), sGC1, sGCβ1, eNOS (as described above), or with mouse monoclonal antibodies against human podoplanin (22)
(clone D2–40; Signet Laboratories, Dedham, MA) or CD34 (BD Pharmingen, San Jose, CA, USA), and corresponding secondary antibodies labeled with AlexaFluor488 or AlexaFluor594 (Molecular Probes; Eugene, OR, USA). Immunohistochemical analysis was performed on 5 µm AMEX-fixed mouse skin sections as described (23)
using antibodies against the macrophage monocyte marker MOMA-2 (BMA Biomedicals AG, Augst, Switzerland) or LYVE-1. Double immunofluorescence stains for Ki67 (rat anti-mouse Ki67; Dako, Baar, Switzerland) and LYVE-1 were performed to visualize proliferating lymphatic endothelial cells. Routine hematoxylin-and-eosin staining was also performed. Sections were examined by a Nikon E-600 microscope (Nikon; Melville, NY, USA), and images were captured with a SPOT digital camera (Diagnostic Instruments; Sterling Heights, MI). Computer-assisted morphometric analyses of representative LYVE-1-stained sections were performed as described (3)
.
Proliferation, migration, and tube formation assays
LECs (1.5x103) or BVECs were seeded into fibronectin-coated 96-well plates as described (21)
. For proliferation studies, quinduplicate wells were exposed to various concentrations (0 or 0.1–10 µM) of SNAP (S-nitroso-N-acetyl-D,L-penicillamine) (Cayman Chemical) in EBM containing 2% FBS. In additional assays, LECs were incubated with 10 µM SNAP in the presence or absence of the specific guanylate cyclase inhibitor NS-2028 (100 nM, Cayman Chemical). After 72 h, cells were incubated with 5-methylumbelliferylheptanoate, as described previously (21)
. The fluorescence intensity, proportional to the number of viable cells, was measured using a Spectra Max GEMINI EM (Bucher Biotech AG, Basel, Switzerland). Haptotatic cell migration was studied as described (21)
, using 24-well FluoroBlok inserts of 8-µm pore size (Falcon, Franklin Lakes, NJ). LECs (100 µl; 4x105 cells/ml) in serum-free EBM containing 0.2% delipidized BSA were seeded into the upper chambers and were incubated for 3 h at 37°C in the presence of SNAP (0.1–10 µM). In additional migration studies, cells were preincubated with 100 nM NS-2028 for 30 min and were then seeded into the upper chambers in the presence of SNAP (10 µM) with or without 100 nM NS-2028. After 3 h incubation at 37°C, cells on the underside of the inserts were stained with Calcein-AM (Molecular Probes), and the fluorescence intensity, proportional to the number of viable cells, was measured using a Spectra Max GEMINI EM as described (21)
. Tube formation assays were performed as described (21)
. Confluent LECs were overlaid with 0.5 ml of a neutralized isotonic bovine dermal collagen type I solution (Vitrogen, Palo Alto, CA) with or without SNAP (1 or 10 µM). After 6 h, cells were fixed with 4% paraformaldehyde for 30 min at 4°C. Representative images were captured, and the total length of tube-like structures per area was measured using the IP-LAB software as described (21)
. All studies were performed in triplicate. Statistical analyses were performed using the unpaired Student’s t test.
siRNA transfection and cGMP immunoassay
After trypsinization, LECs (5x105) were resuspended in 100 µl of basic nucleofactor solution. Cells were transfected by electroporation (Nucleofactor II, Amaxa Biosystems), using 1.6 µg of siRNA containing the following double-stranded oligonucleotides: GC1: CCUUGUACAUAUAUCAGAUtt and GGCACCCUUAAAGAUUUUUtt; and GC2: CGAUACAGCAGACUCUCAAtt and GCUAUGCUCUGAUGUUUCAtt. Control siRNA (Silencer negative control #1 siRNA; Ambion, Cambridgeshire, UK) comprising a 19-bp scrambled sequence with 3'dT overhangs was used as a control; the sequence has no significant sequence homology to any known gene sequence. At 72 h after transfection, cells were used for RNA purification or for cGMP assays. Efficient knockdown of mRNA expression was confirmed by TaqMan real-time RT-PCR for sGC1 and sGC2 (Applied Biosystems assay ID for sGC2 was Hs00181365_m1). For cGMP enzyme immunoassays, 1 x 104 siRNA-transfected LECs were seeded into individual wells of a 96-well plate and were incubated with 2% FBS-containing medium. After 24 h, cells were incubated with several concentrations of SNAP (1–100 µM) for 15 min. The cellular cGMP concentrations were measured using an EIA immunoassay kit (Amersham Biosciences; Freiburg, Germany).
UVB irradiation
A total of 15 female albino HR-1 hairless mice (Hoshino Laboratory Animal, Tokyo, Japan) that were 8 wk old were exposed to a single dose of 200 mJ/cm2 of UVB irradiation using 10 Toshiba FL-20 SE fluorescent lamps that deliver energy in the UVB (280–340 nm) wavelength range with a maximum energy at a wavelength of 305 nm, as described previously (24)
. One day before UVB irradiation, the right ears were given daily topical applications of 1 mM SNAP, 10 µM of NS-2028, or 1 mM of SNAP together with 100 µl of 10 µM NS-2028 in a 50% ethanol solution until day 3 following UVB irradiation (5 total applications of test materials). Only the vehicle was applied to the left ears of all mice (controls). The thickness of the ears was measured every day. On day 4 following UVB irradiation, mouse ears were collected and fixed in cold acetone (AMeX procedure) (23)
and were processed for histological analysis of paraffin sections. All animal studies were approved by the Shiseido Life Science Research Center Committee on Research Animal Care.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1β1 by cultured LECs compared with BVECs
, 21)
. These studies revealed that among several other genes, soluble sGC 1 was expressed at much higher levels (a mean 14.9±3.48-fold increase) in LECs compared with BVECs. Increased expression of sGC β1 (2.09±1.5-fold) and of eNOS (13.0±1.8-fold) was also observed in LECs, whereas expression levels of iNOS, sGC2, and sGCβ2 were below detectable levels. These findings were confirmed by quantitative TaqMan real-time RT-PCR, revealing a >140-fold increase of sGC1, a >30-fold increase of sGCβ1 (Fig. 1
A), and a three-fold increase of eNOS expression in LECs, compared with BVECs (Fig. 1B
). Immunoblot analyses demonstrated that the protein expression levels of sGC1, sGCβ1, and eNOS were also increased in LECs (Fig. 1C
). Expression of sGC1 and sGCβ1 mRNA increased following exposure of LECs to 20 ng/ml of VEGF-A, with a more than 6-fold increase in sGC1 expression after 4 h (Fig. 1D
).
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sGC1, sGCβ1, and eNOS are expressed by lymphatic vessels in situ
We performed differential immunofluorescence analyses of lymphatic vessels in frozen sections of human neonatal foreskin samples. Lymphatic vessels were specifically detected by the anti-human podoplanin antibody D2–40 (22)
and by an antibody against the lymphatic-specific hyaluronan receptor LYVE-1 (25)
. The majority of D2–40-positive lymphatic vessels also showed immunoreactivity for sGC1 (Fig. 2
A–C) and sGCβ1 (Fig. 2D-F
). As expected, both sGC1 and sGCβ1 were expressed by epidermal keratinocytes (data not shown) and by CD34-positive dermal blood vessels (Fig. 2K-P
). Lymphatic vessels also expressed eNOS (Fig. 2G-I
).
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NO-induced LEC proliferation and migration are dependent on GC
To further characterize the effects of NO on LEC functions, LECs were exposed to the NO donor SNAP. We found that SNAP induced LEC proliferation in a dose-dependent manner, with a minimal effective concentration of 1 µM (P<0.001; Fig. 3
A). SNAP-induced proliferation was significantly blocked in the presence of the sGC inhibitor NS-2028 (P<0.05), whereas NS-2028 itself did not have any effect on untreated LECs (Fig. 3A
). SNAP promoted the proliferation of human blood microvascular endothelial cells with a minimal effective concentration of 10 µM (P<0.001; Fig. 3B
). SNAP also promoted haptotactic migration of LECs, as potently as the established (lymph)angiogenic factor VEGF-A, with a minimal effective concentration of 10 µM (P<0.001; Fig. 3C
). The addition of NS-2028 strongly inhibited the promigratory effect of SNAP (P<0.001). At a minimal effective concentration of 1 µM, SNAP induced in vitro cord formation in confluent LEC cultures covered with a type I collagen gel by the 6-hour time point (P<0.001; Fig. 3D, E
).
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NO-induced cGMP production in LECs is dependent on sGC1
Binding of NO to GC results in the catalysis of GTP to cGMP (26)
. To investigate which subtype of sGC is responsible for mediating the effects of NO in LECs, we studied NO-mediated cGMP production after specific, siRNA-mediated knockdown of sGC1 or sGC2. Quantitative real-time RT-PCR confirmed a >85% knockdown of sGC1 expression, but no significant change in the level of sGC2 expression, by 48 h after transfection with sGC1-specific siRNA, compared with control cells. The sGC2-specific siRNA reduced expression of sGC2 by >90% but did not affect sGC1 levels (Fig. 4
A, B). The control siRNA-transfected and sGC2 siRNA-transfected LECs increased cGMP production following SNAP exposure (a minimal effective concentration of 1 µM) (Fig. 4C, E
). However, induction of cGMP production by SNAP was completely prevented in LECs transfected with sGC1-siRNA (Fig. 4D
).
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UVB-induced edema formation and skin inflammation are promoted by the NO donor SNAP and are inhibited by the GC inhibitor NS-2028
We have previously shown that UVB irradiation of the skin up-regulates expression of VEGF-A and is associated with edema, inflammation, and lymphatic vessel enlargement and leakiness (27)
. Moreover, systemic blockade of VEGF-A reduced the UVB-induced lymphatic vessel abnormalities and the level of skin inflammation in mice, indicating that VEGF-mediated impairment of lymphatic vessel function promotes UVB-induced inflammation (7)
. Because VEGF-A induces the expression of sGC1β1 in LECs, we performed in vivo investigations of whether NO regulates lymphatic function and whether inhibition of GC activity could reduce UVB-induced skin damage. To this end, HR-1 hairless mice were exposed to a single dose of 200 mJ/cm2 of UVB irradiation. Beginning one day before irradiation, the right ears of the mice were given daily topical applications of the NO donor SNAP, the guanylate cyclase inhibitor NS-2028, SNAP in combination with NS-2028, or vehicle alone. Ear thickness was measured each day to determine the level of skin inflammation and edema formation.
Skin inflammation and edema formation were clearly detectable in control mice 2 days after UVB irradiation, with a maximal ear swelling on day 3 (Fig. 5
A). Mice that were exposed to SNAP had significant increases in ear swelling (P<0.01 on day 2, P<0.05 on days 3 and 4), whereas ear swelling was significantly reduced in mice exposed to NS-2028 (P<0.01 on day 2, P<0.05 days 3 and 4) (Fig. 5A
). Combined exposure to SNAP and NS-2028 reduced the SNAP-induced augmentation of the UVB response (P<0.01 on days 2 and 3; P<0.05 on day 4).
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Histological analysis of samples taken from ears of mice 4 days after UVB irradiation revealed increased edema formation in the dermis of mice exposed to SNAP (Fig. 5C
), compared with control mice (Fig. 5B
). In contrast, edema formation was decreased in mice exposed to NS-2028 (Fig. 5D
), and SNAP-induced edema formation was strongly reduced in the presence of NS-2028 (Fig. 5E
). Immunohistochemical analysis of samples for the macrophage/monocyte marker MOMA2 demonstrated inflammatory cell accumulation in the dermis of the control mice (Fig. 5F
), whereas fewer MOMA-2-positive cells were found in samples from NS-2028 mice (Fig. 5H
). SNAP exposure increased the number of infiltrating macrophages (Fig. 5G
), whereas NS-2028 blocked macrophage infiltration (Fig. 5I
).
The effects of GC inhibition on the number and size of cutaneous lymphatic vessels were evaluated by immunohistochemistry using an antibody against the lymphatic specific hyaluronan-receptor LYVE-1. In agreement with previous studies, we found enlargement of lymphatic vessels in UVB-irradiated mice that were given only vehicle (controls; Fig. 6
A, E). There was a pronounced enlargement of lymphatic vessels in the dermis of SNAP-treated mice (Fig. 6B, F
), compared with control mice (Fig. 6A, E
), whereas NS-2028 reduced the enlargement of lymphatic vessels (Fig. 6C, G
). NS-2028 also inhibited the SNAP-induced enlargement of lymphatic vessels (Fig. 6D, H
). Computer-assisted morphometric analyses of LYVE-1-stained sections confirmed these findings and revealed a significant increase of the average size of lymphatic vessels (730.7 µm2±34.9 µm2; P < 0.05) and of the area occupied by lymphatic vessels (2.18%±0.52%; P<0.05) after SNAP exposure (Fig. 6I, J
). Conversely, the mean size of lymphatic vessels was significantly decreased following exposure to NS-2028 (350.1 µm2±19.5 µm2; P<0.05; Fig. 6J
). The densities of lymphatic vessels were comparable between all groups (Fig. 6G
). Differential immunofluorescence stains for the proliferation marker Ki67 and for LYVE-1 revealed proliferating lymphatic endothelial cells in SNAP-treated skin (Fig. 6M
) but not in vehicle-treated controls (Fig. 6L
). Together, these findings reveal an important role of the NO/sGC1β1/cGMP pathway in mediating lymphatic vessel function in inflammation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1β1 as an important mediator of lymphatic vessel function. We found that sGC1 is more highly expressed by cultured LECs than by BVECs, that VEGF-A potently induces LEC expression of sGC1, that NO-induced LEC proliferation and migration depend on sGC activity, and that sGC1 is the only receptor that mediates the effects of nitric oxide on cGMP production in LECs. Furthermore, NO promotes lymphatic vessel dilation and edema in vivo, and UVB-induced lymphatic vessel dilation and skin inflammation are potently blocked following sGC inhibition. Blockade of sGC might therefore serve as a new strategy to improve lymphatic drainage and to inhibit inflammation.
There has been a recent surge of interest in the lymphatic vascular system, mainly because of its emerging role in tumor metastasis to the lymph nodes (28
, 29)
. The quest to identify lymphatic-specific growth factors and differentiation markers has been hampered, however, by the lack of reliable markers to distinguish between the lymphatic and the blood vascular endothelial cell lineage. The recent identification of several lymphatic specific genes, including Prox1, LYVE-1, and podoplanin, has cleared the path for molecular investigation into lymphatic lineage-specific differentiation and function and also for the reliable isolation and comparison of LECs and BVECs. Thus, we and others have been able to perform comparative transcriptional profiling of these cells and to identify novel lymphatic lineage signature genes (20
, 30
, 31)
. Because our comparison of several primary cell lines revealed that sGC1 is one of the most strongly LEC-specific genes and because of the established importance of the NO pathway in blood vascular physiology and pathology (9)
, we investigated key molecular players in the NO/cGMP pathway and their functional relevance in LECs.
We found that in addition to sGC1, sGCβ1 and eNOS (but not sGC2, sGCβ2, or iNOS) were more highly expressed by LECs than by BVECs, as determined by quantitative real-time RT-PCR and immunoblot analyses. There are two isoforms of sGC, sGC1β1, and sGC2β2 (32)
. Our results indicate that sGC1β1 is the only functional NO receptor in LECs, because specific knockdown of sGC1 completely inhibited the cGMP production induced by the NO donor SNAP in these cells, whereas SNAP-induced cGMP production was not affected by knockdown of sGC2. Moreover, LEC proliferation and migration were increased following incubation with SNAP, and this NO-induced effect could be completely abolished by the GC inhibitor NS-2028. These findings indicate that NO promotes LEC proliferation and migration via the sGC1β1 and cGMP pathway, rather than through the cGMP-independent pathways reported to mediate the effects of VEGF-E on human umbilical vein endothelial cell migration (33)
.
NO has potent vasoactive effects. In arteries, activation of eNOS increases diffusion of NO to the underlying vascular smooth muscle cells, where it stimulates sGC to produce more cGMP, leading to actin disassembly and vasodilation. In contrast, lymphatic capillaries are not ensheathed by pericytes or smooth muscle cells but consist of a single layer of lymphatic endothelial cells (reviewed in ref. 2
) In the presence of increased interstitial fluid pressure, usually caused by increased leakage from blood vessels, lymphatic capillaries are thought to be passively "pulled" open by anchoring filaments that directly connect the lymphatic endothelial cells with elastic fibers in the extracellular matrix—possibly facilitating fluid drainage via the lymphatic vascular system. However, our findings and previous studies indicate that lymphatic vessel dilation might also be actively induced by mediators that act directly on LECs (18)
. Because we found that sGC1β1 is selectively expressed by cultured LECs (compared with BVECs) and by lymphatic capillaries in the skin and because NO exerts direct effects on LEC migration and tube formation in vitro, it is tempting to speculate that activation of the NO pathway might also directly target LECs to induce lymphatic capillary dilation in vivo. In addition, NO could negatively affect the pumping activity of larger, collecting lymphatic vessels, contributing further to lymphatic capillary dilation via lymphatic stasis (34)
. In future studies, it will be of interest to investigate the effects of NO on the cytoskeletal rearrangement of LECs and the interaction between anchoring filaments and the extracellular matrix.
Edema is a cardinal feature of inflammatory diseases and results when the amount of fluid leakage from inflamed blood vessels exceeds the capacity of lymphatic vessels for drainage (6)
. Whereas abundant research efforts have characterized the molecular control of blood vascular permeability, little is known about the mechanisms that regulate lymphatic vessel function in inflammation. We have previously shown that lymphatic vessels have an important role in UVB-induced edema and skin inflammation and that UVB irradiation leads to enlarged and leaky lymphatic vessels (7
, 27)
. Importantly, the UVB effect on lymphatic vessels was mediated by VEGF-A because systemic inhibition of VEGF-A prevented UVB-induced edema and lymphatic enlargement, whereas overexpression promoted edema formation and lymphatic dysfunction (7)
. Enhanced blood vessel leakiness leads to increased lymph formation, which might contribute to the observed enlargement of lymphatic vessels. However, in previous experiments in PlGF-transgenic mice and in thrombospondin-2–deficient mice—that were characterized by enhanced blood vascular leakiness in the skin—we did not detect enlargement of lymphatic vessels (3)
. Moreover, the colocalization of Ki67-positive nuclei and LYVE-1-positive lymphatic endothelial cells that we observed in samples obtained from SNAP-treated skin, indicates that active lymphatic endothelial cell proliferation contributes to the observed enlargement of lymphatic vessels.
Our present study identifies the NO/sGC1β1/cGMP pathway as an important mediator of VEGF-A’s effects on lymphatic vessel function in inflammation. VEGF-A promotes this pathway by at least two distinct mechanisms, since VEGF-A treatment of LECs, in addition to increasing sGC1β1 expression, also increased expression of iNOS and thereby NO production (data not shown). Blockade of sGC by NS-2028 completely prevented UVB-induced lymphatic enlargement and edema formation, indicating that VEGF-A’s effects are dependent on cGMP production. In addition, inhibitory effects on the leakiness of blood vessels might contribute to the effects of NS-2028 in vivo. Overall, our findings indicate that inhibition of this pathway in lymphatic endothelium might represent an additional strategy to slowing or preventing inflammation. Because high levels of eNOS activity have been linked to tumor progression (35)
, it will be of interest to investigate whether the NO/sGC1β1/cGMP axis is also involved in mediating tumor-associated lymphangiogenesis and metastasis to the lymph nodes.
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ACKNOWLEDGMENTS |
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Received for publication April 23, 2007. Accepted for publication August 9, 2007.
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3-fold in LECs (B). Results are expressed as mean value ± the SD from 3 independent matched pairs of primary LECs and BVECs. C) Immunoblot analyses confirmed higher levels of sGC
)2 days after UVB irradiation, with a maximal ear swelling observed on day 3. Swelling was significantly increased in mice whose ears were exposed to SNAP (
) (P<0.01 on day 2, P<0.05 on days 3 and 4), whereas swelling was significantly reduced in ears exposed to NS-2028 (triangle symbol). Combined exposure to SNAP and NS-2028 (NS) (•) strongly reduced the SNAP-induced augmentation of the UVB response. Ear swelling is expressed as the increase (
) in thickness (µm) compared with pre-UVB exposure values. Data are expressed as mean ± SE (n=5). *P < 0.05; **P < 0.01. B–E) Histological analysis of ear samples collected on day 4 after UVB irradiation revealed increased edema formation in the dermis of SNAP-treated mice (C), compared with control mice (exposed to vehicle, B). In contrast, edema formation was decreased in ears exposed to NS-2028 (D), and SNAP-induced edema formation was reduced in the presence of NS-2028 (E). B–E) hematoxylin-eosin stains. F–I) Immunohistochemical stains for the macrophage/monocyte marker MOMA2 (brown) demonstrated inflammatory cell accumulation in the dermis of control mice (vehicle, F), whereas fewer MOMA-2–positive cells were found in samples of ears exposed to NS-2028 (H). SNAP increased macrophage infiltration in ear tissues (G), and this increase was blocked in the presence of NS-2028 (I). Scale bar = 100 µm.