Vasoactive intestinal peptide (VIP) is a neuropeptide that exerts various vascular and cardioprotective functions and regulates immune function and inflammatory response at multiple levels. However, its role in inflammatory cardiovascular disorders is largely unknown. Myocarditis and atherosclerosis are two inflammatory and autoimmune cardiovascular diseases that cause important adverse circulatory events. In this study, we investigate the therapeutic effects of VIP in various well-established preclinical models of experimental autoimmune myocarditis and atherosclerosis. Intraperitoneal injection of VIP during the effector phase of experimental autoimmune myocarditis in susceptible BALB/c mice significantly reduced its prevalence, ameliorated signs of heart hypertrophy and injury, attenuated myocardial inflammatory infiltration, and avoided subsequent profibrotic cardiac remodeling. This effect was accompanied by a reduction of Th17-driven cardiomyogenic responses in peripheral lymphoid organs and in the levels of myocardial autoantibodies. In contrast, acute and chronic atherosclerosis was induced in apolipoprotein E–deficient mice fed a hyperlipidemic diet and subjected to partial carotid ligation. Systemic VIP treatment reduced the number and size of atherosclerotic plaques in carotid, aorta, and sinus in hypercholesterolemic mice. VIP reduced Th1-driven inflammatory responses and increased regulatory T cells in atherosclerotic arteries and their draining lymph nodes. VIP also regulated cholesterol efflux in macrophages and reduced the formation of foam cells and their presence in atherosclerotic plaques. Finally, VIP inhibited proliferation and migration of smooth muscle cells and neointima formation in a mouse model of complete carotid ligation. These findings encourage further studies aimed to assess whether VIP can be used as a pharmaceutical agent to treat heart inflammation and atherosclerosis.
This article is featured in In This Issue, p.3665
Vasoactive intestinal peptide (VIP) is a 28-aa neuropeptide with a broad distribution in the body and with diverse cardiovascular effects that include vasodilatation of the cerebral and coronary artery circulations and improvement in myocardial contractile performance (1, 2). Exogenous VIP regulates blood flow in many organs and increases heart rate and myocardial contractility. Endogenous VIP seems to exert cardioprotective actions because myocardial VIP depletion is related to the development of cardiomyopathy and myocardial fibrosis, and low VIP circulating levels are directly associated with adverse prognosis in acute myocardial infarction and chronic heart failure (2–6). In contrast, VIP exerts a regulatory role in the homeostasis of the immune system (7). VIP is a potent anti-inflammatory factor that affects both innate and adaptive immunity and many studies with experimental preclinical models have demonstrated the efficiency of VIP in the treatment of inflammatory and autoimmune disorders, including sepsis, rheumatoid arthritis, type I diabetes, Sjogren’s disease, inflammatory bowel disease, experimental autoimmune encephalomyelitis, and experimental autoimmune uveitis (7–14). Various studies demonstrate that the therapeutic actions of VIP in experimental autoimmunity are mainly exerted through the coordinated regulation of the balance between self-reactive T cells (mainly Th1 and Th17 cells) and regulatory T (Treg) cells and their products, the modulation of dendritic cell (DC) function, and the deactivation of inflammatory macrophages (7–18). Evidence also indicates that these immunoregulatory mechanisms could be exerted in patients with autoimmune disorders (19).
There is increasing evidence that autoimmunity plays an important role in the pathogenesis of inflammatory cardiovascular diseases (CVDs) such as atherosclerosis, myocarditis, and dilated cardiomyopathy (DCM) (20). Atherosclerosis is a pathological condition that underlies several important adverse vascular events including ischemic cardiomyopathy, stroke, and peripheral arterial disease. It is responsible for most of the cardiovascular morbidity and mortality in the Western world today. .In the past decade, atherosclerosis has come to be recognized as active and inflammatory rather than simply a passive process of lipid accumulation in arterial intima or a reparative event after endothelial injury. Now, atherosclerosis is generally considered as an intramural chronic inflammation resulting from an inadequate regulation of adaptive immune responses to interactions between modified lipoproteins, infiltrating macrophages and lymphocytes, and the normal cellular elements of the arterial wall such as smooth muscle cells (SMC) (20–22). The advanced human atherosclerotic plaque contains effector-memory activated T cells, mostly exhibiting a Th1 cell–associated cytokine secretion pattern, including IFN-γ and TNF-α, and atherosclerotic patients display low-density lipoprotein–specific autoantibodies in circulation (22). Moreover, several lines of evidence in experimental murine models demonstrated proatherogenic effects of Th1 cells and their cytokines, and that Treg cells could counterbalance them (21, 22).
Myocarditis and subsequent DCM are major causes of heart failure in young patients. This condition is characterized by infiltration of inflammatory cells into the myocardium with consequent loss of myocytes and development of fibrosis. Beside genetic susceptibility, myocarditis is induced by a variety of exogenous agents, including toxins, viruses, bacteria, and parasites (20, 23–25). Moreover, myocardial injury and exposure to cardiac self-antigens can induce postinfectious autoimmune responses, which play a role in the pathogenesis of myocarditis and DCM (20, 24, 26). A recent study demonstrated that persistent heart failure was associated with high percentages of Th17 cells and IL-17–promoting cytokines, and the myocarditis/DCM phenotype included a decrease in Treg cells, which may contribute to disease severity (26). Many affected DCM patients develop heart Ag–specific autoantibody responses, and clinical trials showed that immunosuppressive treatments resulted in being beneficial mainly for affected patients showing chronic inflammatory heart disease and absence of viral particles in the heart (27). Moreover, studies in rodent models have revealed that myocarditis can be induced by immunization with cardiac myosin or myosin-derived peptides in adjuvant or by adoptive transfer of cardiac myosin-activated CD4+ T cells (24, 25, 28).
Despite its well-described cardioprotective and immunoregulatory actions in various experimental models, the effects of VIP in CVDs that course with exacerbated inflammation and autoimmunity are largely unknown. The aim of this study is to investigate the potential therapeutic effect of VIP in various murine models of myocarditis and atherosclerosis, namely experimental autoimmune myocarditis (EAM) induced by immunization with a fragment of cardiac myosin in susceptible BALB/c mice, in a model of accelerated atherosclerosis induced in apolipoprotein E–deficient (apoE−/−) mice subjected to partial carotid ligation and fed a high-fat Western diet and in a chronic model of atherosclerosis induced in hypercholesterolemic apoE−/− mice. We found a significant protective effect of VIP in all these models. As previously described in other autoimmune models, we observed that VIP exerted its therapeutic action in experimental myocarditis and atherosclerosis by regulating the balance between Th1 or Th17 cells and Treg cells, which is crucial for the progression of both diseases. Moreover, we observed for the first time, to our knowledge, that VIP impairs critical pathological events of the atherosclerotic process, other than T cell responses, including circulating cholesterol, generation of foam cells and neointima formation, and vascular remodeling.
Materials and Methods
Animals and ethical statement
Male BALB/c (7–8 wk old), female C57BL/6 apoE−/− (6 wk old), and male FVB/NJ (7 wk old) mice were obtained from Charles River and housed in a temperature- and humidity-controlled environment (22 ± 1°C, 60–70% humidity) in individual cages (10 mice per cage, with wood shaving bedding and nesting material) with a 12 h light/dark cycle (lights on at 7:00 am) and tap water ad libitum. The experimental protocols of this study conform to European Union Directive 2010/63 and followed the ethical guidelines for investigations with experimental animals approved by the Ethics Review Committee for Animal Experimentation of Spanish Council of Scientific Research.
Experimental model of autoimmune myocarditis
We immunized susceptible BALB/c mice (presenting Ags in a H2d context) s.c. with 100 μg murine heart muscle–specific peptide derived from MyHC-α (MyHC614–629, Ac-RSLKLMATLFSTYASADR-OH, in which two arginines were included at both ends to increase its solubility; purity >93%; AnaSpec) emulsified 1:1 in CFA (Difco) on days 0 and 7, as previously described (25). At day 7, animals were randomly distributed in different experimental groups: 1) mice that received i.p. PBS (control group) three times/week starting at day 7; 2) mice that received i.p. VIP (1 nmol, which corresponds to 3.3 μg of mouse VIP; from Bachem) three times per week starting at day 7; 3) mice that received i.p. VIP at 1 nmol starting at day 11; or 4) mice that received i.p. VIP at 1 nmol starting at day 15 (a total of six injections in all cases). The dose of VIP used in this study was selected in base of results obtained in previous experiments performed in other autoimmune and vascular models (7). On day 21 after EAM induction, we sacrificed mice by carbon dioxide inhalation, collected blood by cardiac puncture, perfused the animals with cold PBS, and removed heart, spleen, and cranial mediastinal lymph nodes. We isolated serum from blood to determine the levels of the cardiac damage biomarker brain natriuretic peptide (BNP), and inflammatory cytokines and MyHC-specific Abs were determined as described below. Hearts were weighed, macroscopically scored, and processed for histopathological analysis and for protein isolation (25, 29). H&E-stained heart sections were scored blindly by two independent investigators according to a semiquantitative scale of inflammatory infiltrates (0, no focal inflammatory infiltrates; 1, up to 5% of the cross-sectional area of the heart section; 2, 6–20%; 3, 21–50%; 4, >50% of a cross-section involved). Moreover, images were acquired in a blinded fashion using an Olympus microscope and the area of myocardium and surrounding tissue affected by myocarditis (consisting of inflammatory cells and myocardial necrosis) relative to the entire area was determined by a computer-assisted analyzer (Image J software, NIH) as previously described (29). Values for three ventricular regions were averaged for each heart, and the mean percentage of the affected area for each group was calculated. In addition, the presence of inflammatory infiltrates was confirmed by flow cytometric analysis of mononuclear cells isolated from the heart as described below. Sections of hearts isolated 21 and 50 d after EAM induction were also stained with PicroSirius Red to evaluate collagen deposits and signs of fibrosis, and the area ratio (affected/entire area as a percentage) was automatically quantified with the Fibrosis HR software (ImageSP) (29). Cell suspensions of cardiac draining lymph nodes (DLNs) and spleens were isolated 14 or 21 d after EAM induction and used for flow cytometric analysis and to study self-reactive responses as described below.
Experimental model of acute atherosclerosis
To induce atherosclerosis in hyperlipidemic mice, we subjected female C57BL/6 apoE−/− mice to partial ligation of left carotid artery. Because female apoE−/− mice develop markedly increased atherosclerotic lesions compared with male apoE−/− mice (30), we used mice with this sex to evaluate the effects of VIP in all our atherosclerosis studies. Anesthesia was induced by i.p. injection of a xylazine (10 mg/kg) and ketamine (80 mg/kg) mixture. A ventral midline incision (5 mm) was made in the neck, and three of four caudal branches of left carotid artery (external carotid, internal carotid, and occipital artery) were ligated with 6-0 silk suture, leaving the superior thyroid artery intact. One day after surgery, animals were fed a high-fat Western diet (38% fat, 0.15% cholesterol, diet U8958, SAFE) and received i.p. PBS (control) or mouse VIP (1 nmol) every 2 d for a period of 3 wk. Mice were sacrificed with carbon dioxide 14 or 21 d after surgery. We monitored plasma cholesterol (Accuntrend Plus Kit; Roche) every week in blood samples collected from the tail. Some animals were subsequently perfused with cold PBS and with 4% paraformaldehyde (PFA)/0.1 M phosphate buffer pH 7.4 (buffered PFA), and the ligated and contralateral unligated arteries were isolated for morphometric analysis. In the other animals, ligated and unligated carotids were isolated to determine protein expression, to analyze inflammatory infiltration, and for ex vivo artery culture. Moreover, we isolated the ipsilateral and contralateral deep cervical lymph nodes (used as DLNs of the carotid artery), spleen, and mesenteric lymph nodes (used as non-DLN controls) for flow cytometric analysis as described below. For morphometric analysis, the isolated carotid arteries were fixed in 4% buffered PFA for 6 h, and then subjected to cryopreservation in 30% sucrose/0.1 M phosphate buffer at 4°C, embedding in OCT compound and freezing. We obtained 20 cryosections (6 μm thick) at the artery segment, compressing from 0.5 to 1.5 mm proximal to carotid bifurcation. We measured the area and volume occupied by atherosclerotic lesion and by arterial medial layer in five H&E-stained carotid sections and in five Oil Red O–stained carotid sections in a blinded manner using a Zeiss microscope and ImageJ software. Furthermore, to determine the number and phenotype of infiltrating cells in ligated and unligated carotids (isolated at day 21 postligation), a single-cell suspension was obtained by digestion of the arteries as described below.
To determine the effect of VIP on the production of cytokines by atherosclerotic carotids, segments of ligated and unligated carotid arteries isolated from untreated atherosclerotic mice at day 21 were ex vivo–cultured in complete DMEM (DMEM supplemented with 100 U/ml penicillin/streptomycin, 2 mM l-glutamine, and 10% FCS) in the presence or absence of VIP (100 nM) and stimulated with PMA (25 ng/ml) plus ionomycin (0.5 μg/ml) in 24-well plates at 37°C and 5% CO2. After 24 h, we determined the cytokine contents in culture supernatants by using specific sandwich ELISAs (BD Pharmingen). Moreover, total proteins were obtained from ligated and unligated carotid arteries isolated from untreated and VIP-treated atherosclerotic mice at day 21 as previously described (31), and the content of cytokines in protein extracts was determined by using specific sandwich ELISAs (BD Pharmingen).
Experimental model of chronic atherosclerosis
To induce chronic atherosclerosis, we fed female apoE−/− mice (6 wk old) with a high-fat Western diet for 16 wk. Animals received i.p. PBS (control) or mouse VIP (1 nmol) three times/week during 15 wk, starting 1 wk after initiation of Western diet. We monitored plasma cholesterol every 4 wk in blood samples collected from the tail. When indicated, naive female C57BL/6 mice (22 wk old) that were fed a normal chow diet were used as reference. Ten or sixteen weeks after initiating a Western diet, mice were sacrificed with carbon dioxide, and spleen, DLN heart, aortic arch and aorta (deep cervical, cranial mediastinal, lumbar aortic, and medial iliac lymph nodes), and mesenteric lymph nodes (used as non-DLN controls) were collected for flow cytometric analysis. In some animals, artery segments containing heart aortic sinus and aortic arch were dissected at week 16 and processed for determination of RNA expression or digested for flow cytometric characterization of inflammatory cell infiltration as described below. Moreover, the descending aorta comprising from proximal ascending thoracic aorta to iliac bifurcation was microdissected in situ at week 16, and the atherosclerotic plaques were quantified after Sudan IV staining as described below. Some animals were sacrificed at week 16, perfused with cold PBS and 4% buffered PFA, and the segments containing heart aortic sinus and aortic arch were isolated, fixed in 4% buffered PFA for 6 h, cryopreserved in sucrose, embedded in OCT compound, cryosectioned, stained with Oil Red O, and processed for morphometry analysis as described below.
Sudan IV and Oil Red O staining
Descending aorta was fixed in 4% buffered PFA, depleted of adventitial fat and stained with 5% Sudan IV (dissolved in 50% acetone/35% ethanol) for 15 min, destained with 80% ethanol for 5 min, extensively washed in tap water for 1 h, pinned flat onto a white rubber board, opened longitudinally using a dissecting microscope, and photographed. Total aortic area and Sudan IV–positive lesion area were quantified in the images using ImageJ software.
Transversal and/or longitudinal cryosections (8 μm thick) of carotid artery, aortic sinus, and aortic arch were sequentially obtained and processed for H&E staining or for Oil Red O staining. For Oil Red O staining, sections were rinsed with 60% isopropanol for 5 min, stained with 0.5% Oil Red O/60% isopropanol (20°C, 10 min), destained with 60% isopropanol for 2 min, and extensively washed with distiller water. Finally, nuclei were counterstained with hematoxylin. Images of stained sections were acquired in a Zeiss microscope, and the area occupied by the plaques in arteries and heart aortic sinus was measured in a blinded fashion using ImageJ software.
Experimental model of arterial neointima formation
To induce SMC hyperplasia in vivo, we subjected male FVB/NJ mice (anesthetized as above) to permanent complete ligation of the left common carotid artery near its bifurcation as previously described (32). FVB/NJ mice are genetically susceptible to suffer marked vascular remodeling upon artery ligation (33). Animals received i.p. PBS (control) or VIP (1 nmol) every 2 d starting 1 d after artery ligation. The ligated and contralateral unligated arteries were isolated 4 wk after ligation and processed for morphometric analysis. We used five cryosections (6 μm thick) at 0.5–1.5 mm proximal to carotid bifurcation stained with H&E to measure areas of intima and media in a blinded manner using ImageJ software.
Measurement of autoreactive response in EAM
Single-cell suspensions (106 cells/ml) from DLNs and spleens were obtained 21 d postimmunization and were stimulated in complete RPMI medium (RPMI 1640 supplemented with 100 U/ml penicillin/streptomycin, 2 mM l-glutamine, 50 μM 2-ME, and 10% FCS) with MyHC614–629 (10 μM). To evaluate polyclonal stimulation, cells were cultured with 1 μg/ml anti-CD3 Ab or with 2.5 μg/ml Con A (Sigma). After 72 h of culture, we evaluated cell proliferation by an addition of 2.5 μCi/ml [3H]thymidine during the last 8 h of culture and determination of cpm. After 48 h of culture, we measured the contents of cytokines and chemokines in culture supernatants by specific sandwich ELISAs (BD Pharmingen). To determine whether VIP affects the autoreactive response in vitro, spleen and DLN cells isolated at day 21 from mice with EAM were stimulated with MyHC614–629 (10 μM) in the absence or presence of VIP (10 nM).
Measurement of levels of BNP, VIP, cytokines, and anti-MyHC Abs in serum and cardiac and carotid protein extracts
BNP was determined by using a competitive ELISA (RayBio), VIP was determined by competitive ELISA (Phoenix Pharmaceuticals), and the cytokines IFN-γ, IL-17, IL-6, and TNF-α were measured by using specific sandwich ELISAs (BD Pharmingen), following the manufacturer’s recommendations (31). To determine anti-MyHC IgG Abs, ELISA 96-well plates were coated overnight at 4°C with 2 μg/ml MyHC614–629 in PBS. After washing with PBS containing 0.05% Tween 20, nonspecific binding was blocked with PBS containing 10% FBS for 2 h at room temperature. After washing three times, serum samples diluted 1/50 were added and incubated for 2 h at room temperature. After four washes, biotin-conjugated goat anti-mouse IgG, IgG1, or IgG2a Abs (Jackson ImmunoResearch Laboratories) were added and incubated at room temperature for 1 h, followed by six washes and incubation with avidin-peroxidase for 30 min. After eight washes, plates were developed using 2,2′-azino-di-(3-ethylbenzothiazoline-6-sulphonic acid) as substrate for peroxidase, and the OD was measured using a microplate reader.
Isolation of heart- and artery-infiltrating cells
Hearts were harvested from EAM mice and placed in a 37°C water bath. The aorta was cannulated with a 22-gauge needle, and hearts were perfused at a constant flow of 1.1 ml/min at 37°C for 3 min with a calcium-free bicarbonate-based perfusion buffer containing 120 mM NaCl, 5.4 mM KCl, 1.2 mM NaH2PO4, 20 mM NaHCO3, 5.6 mM glucose, 5 mM taurine, 1.6 mM MgCl2, and 10 mM 2,3-butanedione monoxime, as previously described (34). The hearts were perfused for an additional 7 min with a perfusion buffer supplemented with 0.9 mg/ml collagenase type D (Roche) and 0.5 mg/ml protease type XIV (Sigma). The hearts were then placed into a Petri dish containing chilled 1% FBS and 0.05% sodium azide in PBS and manually dispersed into a single-cell suspension using razor blades. Single-cell suspensions were sequentially filtered through 70 and 40 μm cell strainers, suspended in PBS containing 3% FBS, and used for flow cytometric analysis.
We obtained single-cell suspensions from atherosclerotic arteries as previously described (35). Briefly, carotids and aortas were cut into small pieces in a Petri dish to improve tissue digestion and then incubated at 37°C for 1 h in a mixture of 450 U/ml collagenase I, 125 U/ml collagenase XI, 60 U/ml DNase I, and 60 U/ml hyaluronidase I (all from Sigma, diluted in PBS). Cell suspensions were filtered through 70 μm cell strainers and used for flow cytometric analysis.
Flow cytometric analysis
For analysis of cells infiltrating the heart, carotid, and aorta, single-cell suspensions were isolated 21 d after EAM induction or carotid ligation or 16 wk after initiating high-fat diet (see above) and were incubated with anti-2.4G2 Ab (Mouse BD Fc Block, 1:100, 4°C, 10 min) to avoid nonspecific binding to Fc-receptors and with 7-Aminoactinomycin D (1:100; Calbiochem) to exclude dead cells, washed in PBS/0.1% BSA. Cells were surface-stained with allophycocyanin-conjugated anti-CD4, FITC-conjugated anti-CD11b, and PE-conjugated anti-CD45 mAbs (each at 4–5 μg/ml, 30 min, 4°C; BD Biosciences) and were analyzed in a FACSCalibur flow cytometer (BD Biosciences). Data were acquired until at least 100,000 events were collected from a live gate using forward and side scatter plots and 7-Aminoactinomycin D staining.
To determine the number of cells expressing cytokines, infiltrating inflammatory cells were isolated from hearts of mice with EAM at day 21, from ligated and unligated carotids at day 21 after ligation, and from aortic sinus and aortic arch from apoE−/− mice at week 16 after initiating a high-fat diet as well as from spleen, DLN, and non-DLN cells isolated at days 14 and 21 after EAM induction or from apoE−/− mice at days 14 and 21 after carotid ligation or at weeks 10 and 16 after initiating a high-fat diet that was activated with 25 ng/ml PMA for 14 h in the presence of monensin (1.33 μM) for the last 6 h. Cells were incubated with anti-2.4G2 Ab plus 7-Aminoactinomycin D, washed in PBS/0.1% BSA, and were then stained with allophycocyanin-conjugated anti-CD4 mAb as described above. After extensive washing, cells were fixed and/or permeabilized with Cytofix/Cytoperm solutions (BD Biosciences), stained with PE-conjugated anti–IL-17 and FITC-conjugated anti–IFN-γ mAbs (2 μg/ml, 30 min, 4°C; BD Pharmingen), and analyzed in a FACSCalibur flow cytometer.
For Foxp3 staining, carotid and aortic cell isolates and DLN cells were isolated from mice 14 and 21 d after carotid ligation or 10 and 16 wk after initiating a high-lipid diet or from mice 14 and 21 d after EAM induction and incubated with FITC-conjugated anti-CD25 and allophycocyanin-conjugated anti-CD4 mAbs (5 μg/ml; BD Biosciences) for 1 h at 4°C. After extensive washing, cells were fixed and/or permeabilized (eBioscience), stained with PE-conjugated anti-Foxp3 Ab (5 μg/ml; eBioscience) for 30 min at 4°C, and analyzed in a FACSCalibur flow cytometer. In all cases, we used isotype-matched Abs (BD Biosciences) as controls.
Determination of gene expression by real-time PCR
Total RNA was isolated from aortic arches following the manufacturer’s protocol (TriPure; Roche). Precipitated RNA was treated with DNase 1 (Sigma-Aldrich) before reverse transcription (RevertAid First Strand cDNA Synthesis Kit; Thermo Fisher Scientific). SYBR Green quantitative PCR (SensiFast SYBR No-ROX mix; Bioline) was performed on the Bio-Rad CFX using the following conditions: 95°C for 5 min followed by 35 cycles at 95°C for 30 s, annealing (see temperature below for each gene) for 30 s, and extension at 72°C for 30 s. Primer sequences and temperature of annealing were as follows: TNF-α (5′-GCGACGTGGAACTGGCAGAAGAG-3′ [forward], 5′-TGAGAGGGAGGCCATTTGGGAAC-3′ [reverse], annealing at 68°C), IFN-γ (5′-ACACTGCATCTTGGCTTTGC-3′ [forward], 5′-TTGCTGATGGCCTGATTGTC-3′ [reverse], annealing at 58°C), CD68 (5′-CCATCCTTCACGATGACACCT-3′ [forward], 5′-GGCAGGGTTATGAGTGACAGTT-3′ [reverse], annealing at 60°C), IL17 (5′-CTGTGTCTCTGATGCTGTTG-3′ [forward], 5′-ATGTGGTGGTCCAGCTTTC-3′ [reverse], annealing at 60°C), ABAC1 (5′-GGACTTGCCTTGTTCCGAGAG-3′ [forward], 5′-GCTGCCACATAACTGATAGCGA-3′ [reverse], annealing at 64°C), peroxisome proliferator–activated receptor (PPAR)-γ (5′-GCCCTTTGGTGACTTTATGGA-3′ [forward], 5′-GCAGCAGGTTGTGTTGGATG-3′ [reverse], annealing at 60°C). The expression of each gene was normalized against the expression of the housekeeping gene GAPDH (5′-AACTTTGGCATTGTGGAAGG-3′ [forward], 5′-ACACATTGGGGGTAGGAACA-3′ [reverse]) in every PCR.
Determination of lipid accumulation and cholesterol efflux in macrophages
Macrophages were generated by differentiating bone marrow cell precursors isolated from untreated C57BL/6 mice (sacrificed by carbon dioxide) and then incubated in complete DMEM in the presence of macrophage-colony stimulating factor (20 ng/ml; PeproTech) for 6–8 d. To determine the accumulation of lipid droplets, macrophages (5 × 105) were seeded in coverslips (8 h, 37°C, in complete DMEM) and incubated in DMEM supplemented with 100 U/ml penicillin and streptomycin, 2 mM l-glutamine, and 2% FCS in the presence of oxidized low-density lipoprotein (oxLDL;50 μg/ml; KALEN Biomedical) and mouse VIP (100 nM). After 24 h, cells were fixed in 4% buffered PFA (10 min, 22°C), and intracellular neutral lipids were stained with Oil Red O (0.15%, 15 min, 22°C). After extensive washing in water, macrophages were counterstained with hematoxylin and observed in a Zeiss microscope. Images were analyzed with ImageJ software to quantify the content of intracellular lipid droplets per cell and determine the percentage of foam cells.
To determine the cholesterol efflux, macrophages (105) were cultured in 96-well plates with complete DMEM. After 16 h, cells were incubated in DMEM-BSA (DMEM supplemented with 100 U/ml penicillin and streptomycin, 2 mM l-glutamine, and 0.2% fatty acid-free BSA) in the presence of oxLDL (50 μg/ml) and [3H]cholesterol (0.5 μCi/ml; PerkinElmer) with or without mouse VIP (100 nM) for 10 h. Cells were then washed with DMEM-BSA and incubated again with ApoAI (10 μg/ml; Sigma) with or without VIP (100 nM). After 10 h, culture supernatants were collected and cells lysed in NaOH (0.1 M, 5 h, 22°C). The cpm in cell lysates and supernatants were quantified in a MicroBeta TriLux Counter, and the ApoAI-mediated efflux of cholesterol was determined by using the following formula: % efflux = (cpm supernatant/cpm supernatants + lysates) × 100.
Western blot analysis
Mouse macrophages differentiated from bone marrow were cultured in complete DMEM in the absence or presence of oxLDL (50 μg/ml) and mouse VIP (100 nM). After 16 h, cells were lysed by incubation with lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% SDS, and 10 μg/ml of a mixture of proteinase inhibitors) for 30 min on ice. Proteins extracts (20 μg/lane) were separated on 7.5% SDS-polyacrylamide gels and blotted onto polyvinylidene difluoride membranes (Millipore) using a semidry system. Membranes were blocked with TBS–Tween 20/5% nonfat dry milk for 1 h at room temperature and subsequently probed overnight at 4°C with primary rabbit anti-mouse CD36 or ATP-binding cassette A1 (ABCA1) Abs (at 1/1000; Novus Biologicals), rabbit anti-mouse PPAR-γ or SRB1 Abs (at 1/500; Cell Signaling), or rabbit anti-mouse GAPDH Ab (at 1/5000; R&D Systems). Immunodetection was performed by incubation with a peroxidase-conjugated anti-rabbit Ab (at 1:5000, 2 h, 20°C; DakoCytomation) and was developed with ECL detection system (ECL plus; Amersham). Protein expression was represented as a percentage of expression in densitometry units relative to GAPDH.
SMC proliferation and migration assays
We evaluated proliferation of human aortic SMC (hAoSMC; from Clonetics, Verviers, Belgium) by measuring the incorporation of [3H]thymidine. hAoSMCs (104 per well) were cultured for 12 h in SmGM-2 medium (Clonetics) in 96-well plates and serum starved for 24 h in SmBM medium (Clonetics) before stimulation with platelet-derived growth factor (PDGF; 10 ng/ml) in the absence or presence of different concentrations of VIP. We used hAoSMCs incubated with medium alone as unstimulated controls. We added 1 μCi [3H]thymidine per well for the last 12 h of the 24 h culture and determined the incorporation of [3H]thymidine on a MicroBeta TriLux Counter.
To measure hAoSMC migration, cells expanded in SmGM-2 medium were trypsin-detached and cultured in SmBM medium containing 0.1% BSA at 104 cells per well in the upper chamber of a 48-well migration system with 8 μm pore size polycarbonate filters (Neuro Probe, Gaithersburg, MD) coated with 100 μg/ml collagen type I and 10 μg/ml vitronectin. We added PDGF (10 ng/ml) as a chemoattractant in the lower chamber and VIP to the cells immediately before placing them in the upper chamber. We allowed cell migration for 6 h (37°C, 5% CO2), removed the nonmigrated cells with a cell scraper, fixed the filter bottom face with cold methanol, stained the cells with DAPI, and counted migrated cells with a Nikon fluorescence microscope.
All data are expressed as mean ± SEM. We analyzed data for statistical differences using unpaired two-tailed Student t test to compare two groups or one-way ANOVA followed by Fisher’s protected least significant difference test to compare multiple groups. To compare the severity scores of myocarditis between two groups, the Mann–Whitney U test was used. A p value < 0.05 was considered to be statistically significant.
VIP treatment reduces heart inflammation in animals with acute EAM
In this study, we used a well-established murine model of autoimmune myocarditis that mirrors important aspects of human inflammatory DCM and is widely used for assaying new therapeutic strategies against the inflammatory chronic phase of the disease, in which autoimmunity is the prevailing cause for ongoing disease (24, 25). In this model, signs of inflammation in the heart and autoantibodies against cardiac myosin in sera are only evident during the progression phase of the disease, between days 10 and 21 postimmunization (24, 25). During the late phase, following day 21 postimmunization, myocardial inflammation declines and fibrosis gradually replaces it (24, 25). As expected, mice immunized with cardiac myosin peptide and sacrificed at day 21 postimmunization showed the severe signs of myocarditis characterized by marked enlargement of heart (Fig. 1A) and areas of pericardial calcification (data not shown). Myocarditis was accompanied by an increase in serum BNP (Fig. 1A), a biomarker of cardiac dysfunction (36). The hypertrophy of the heart correlated with the presence of severe and disseminated inflammatory infiltration of leukocytes in pericardium and myocardium and of focal areas of pericardial myocyte necrosis (Fig. 1B). Persistent myocardial inflammation and associated tissue injury induced profibrotic remodeling, which is revealed initially by the presence of incipient disseminated epicardial Sirius Red–positive areas at day 21 and of later extended fibrotic areas in myocardium at day 50 postimmunization (Fig. 1C). Systemic treatment with VIP during the progression phase of EAM (from day 7 to day 19 postimmunization) significantly reduced the disease severity, assessed by improved signs of heart hypertrophy and reduction of serum BNP levels (Fig. 1A) and of myocardial inflammatory infiltration (percentage of infiltrated area: 21.2 ± 2.4 in untreated EAM, 2.6 ± 0.8 in VIP-treated EAM; p < 0.001) (Fig. 1B). Inhibition of heart inflammation by VIP markedly reduced the subsequent appearance of myocardial collagen deposits (Fig. 1C). Moreover, injection of VIP reduced disease prevalence (percentage of animals with histological scores >0) from 90 to 45% at the end of the study (Fig. 1B). We still observed a significant protective effect when the initiation of treatment with VIP was delayed to day 11, but not to day 15 (a late time point at the progression phase of the disease) (Fig. 1A). A flow cytometric analysis of the heart infiltrates revealed that treatment with VIP reduced the presence in myocardium of inflammatory CD45+ leukocytes, CD4+ lymphocytes, and CD11b+ cells (mostly monocytes) (Fig. 2A).
VIP modulates cardiomyogenic T cell responses in animals with EAM
We next investigated the mechanisms underlying the decrease in severity of EAM following treatment with VIP. In myocarditis, progression of the autoimmune response involves the development of autoreactive CD4 T cells in peripheral lymphoid organs, their entry into the myocardium, and subsequent recruitment of inflammatory cells (24, 26). Moreover, Abs directed against cardiac myosin play a critical role in the development of EAM and its progression to DCM (20, 24, 26, 28). Analysis of intracellular cytokine staining of the myocardial infiltrating CD4+ cells revealed that the injection of VIP reduced the numbers of IL-17+ cells (Th17) and IFN-γ+ cells (Th1) in mice with EAM (Fig. 2A). Moreover, treatment with VIP diminished the heart and serum amounts of TNF-α, IL-6, and IL-17 (Fig. 2B, 2C), which are cytokines that were previously described to be mechanistically linked to myocardial inflammation and heart failure (20, 23, 24, 26). Importantly, treatment with VIP attenuated the increase in levels of MyHC-specific IgGs, particularly IgG2a and IgG1 autoantibodies, observed in mice with EAM (Fig. 3).
We next studied whether the effect found for VIP in EAM was exerted by modulating T cell–driven cardiomyogenic responses in peripheral lymphoid organs. Treatment with VIP of mice with EAM impaired the capacity of spleen and DLN T cells (isolated at the peak of the disease) to proliferate and produce IL-17, IFN-γ, IL-2, and CXCL-10 in a recall response to the cardiomyogenic Ag MyHC (Fig. 4A). Importantly, VIP treatment did not suppress the capacity of T cells to respond adequately to polyclonal stimulation with Con A or anti-CD3 Abs (Supplemental Fig. 2). Intracellular cytokine determination showed that treatment with VIP significantly reduced the increase in the number of Th17 cells observed in DLNs of EAM mice, whereas it did not affect the number of IFN-γ–producing Th1 cells (Fig. 4B). Moreover, animals treated with VIP showed a significant increased presence of CD25+Foxp3+ Treg cells in the CD4+ cell population of DLNs isolated during the effector phase of the disease (at day 14), but not isolated at day 21 after EAM induction (Fig. 4B). These results suggest that VIP could downregulate the activation of cardiomyogenic T cell responses in peripheral lymphoid organs by modulating the balance between Th17 and Treg cells. Finally, we found that VIP suppressed in vitro cell proliferation and cytokine production by MyHC-stimulated T cells isolated from DLNs (Fig. 4C) and spleens (data not shown) of animals with EAM, suggesting that VIP could exert a direct effect on cardiomyogenic T cells and/or APCs.
VIP ameliorates atherosclerosis in partially ligated carotid artery of hyperlipidemic mice
We next investigated the potential therapeutic effect of VIP in two models of atherosclerosis in genetically susceptible apoE−/− mice fed a high-fat Western diet. In the first acute model, we ligated three of the four caudal branches of the left common carotid artery and then fed animals with a high-fat diet for 21 d. This partial ligation causes disturbed blood flow with low and oscillatory shear stress and accelerated atherosclerosis in the carotid artery of hyperlipidemic mice (37). In comparison with contralateral unligated artery, ligated carotid developed severe atherosclerotic plaques that progressively occupied the vascular lumen (Fig. 5A, 5B). The plaques were characterized by the predominant presence of lipid-loaded cells stained with Oil Red O, which probably corresponded to foam cells (Fig. 5A). Flow cytometric analysis of single-cell isolates of ligated carotids showed a high presence of CD45+ leukocytes, including CD11b+ macrophages and CD4+ T lymphocytes (Fig. 5C). Systemic administration of VIP, three times per week, starting 1 d after artery ligation, resulted in a strong decrease in the formation of atherosclerotic plaques in the ligated carotid and a reduced presence of macrophages and CD4 T cells in the plaque (Fig. 5A–C). Moreover, VIP impaired the increase in the carotid medial layer area observed in untreated atherosclerotic mice, which occurs as a consequence of vascular remodeling induced by artery ligation and atherosclerosis (Fig. 5B).
VIP impairs Th1/Th17-driven inflammatory responses in atherosclerotic mice
As expected, the hyperlipidemic diet increased the serum levels of cholesterol in apoE−/− mice, and although a slight decrease in cholesterol concentration was observed in VIP-treated mice at day 21 postligation (Supplemental Fig. 3A), these levels are probably still high enough to support any role in the therapeutic action of VIP in atherosclerosis. Because atherogenic T cells, mainly Th1 cells, play critical roles in development and progression of the atherosclerotic plaque (20–22), we evaluated the effect of VIP treatment in the presence of these pathogenic cells in plaques and in lymphoid organs. Injection of VIP reduced the number of IFN-γ–producing CD4 Th1 cells infiltrating the ligated carotids of apoE−/− mice fed a hyperlipidemic diet (Fig. 5D) and strongly downregulated the expression of inflammatory cytokines that are linked to development of atherosclerosis (20) in the artery (Fig. 5E). Moreover, the addition of VIP to ex vivo cultures of ligated carotid arteries diminished the production of TNF-α and IFN-γ (Fig. 5F), suggesting that VIP could also exert a local effect at the vascular level in atherosclerotic plaques.
Moreover, we observed that the number of total cells and of IFN-γ– and IL-17–producing CD4 cells in DLNs of the ligated carotid increased with respect to the corresponding DLNs isolated in the contralateral side of the same animal (DLNs of the unligated artery) or DLNs of naive mice (Fig. 6A, 6B) or with respect to spleen or mesenteric lymph nodes (non-DLNs) of apoE−/− mice fed a Western diet for 21 d (data not shown), supporting that a major immune response occurs in DLNs of ligated carotids. Treatment with VIP decreased the number of total cells in DLNs, especially of both Th1 and Th17 cells (Fig. 6A, 6B). In contrast, the percentage of Foxp3+ Treg cells in the CD4 population of DLNs and in the ligated carotid artery significantly increased with VIP administration (Fig. 6C).
VIP decreases the formation of plaques in a model of chronic atherosclerosis
Next, we tried to confirm the protective effects of VIP in a second model of chronic atherosclerosis in apoE−/− mice fed a high-lipid Western diet for 16 wk. Intraperitoneal injection of VIP, three times per week, starting 1 wk after initiation of diet, reduced the number and size of atherosclerotic lesions in descending aorta, aortic sinus, and aortic arch (Fig. 7A–C). Remarkably, 50% VIP-treated mice did not show evidence of plaque formation in the descending aorta at the end of treatment. Again, VIP administration slightly reduced circulating cholesterol in apoE−/− mice fed a high-lipid Western diet (Supplemental Fig. 3B), but because cholesterol levels were still high (above 600 mg/dl), this effect probably does not play a major role in the antiatherosclerotic action of VIP. As expected, aortic arches isolated from apoE−/− mice fed a high-lipid diet showed high mRNA expression of IFN-γ and the inflammatory cytokine TNF-α, many CD11b+ macrophages (also confirmed by CD68 gene expression) and IFN-γ–producing CD4+ T cells, and a marginal presence of IL-17–producing CD4+ T cells (Fig. 7D, 7E). Treatment with VIP significantly reduced all these atherogenic inflammatory mediators (Fig. 7D, 7E). Moreover, the VIP injection decreased the number and percentages of Th1 and Th17 cell populations in aortic DLNs (Fig. 8A) and spleen (data not shown). At the same time, apoE−/− mice treated with VIP showed elevated percentages of Foxp3+ Treg cells in aorta artery cell isolates and in cardiac and aortic DLNs in comparison with untreated mice (Fig. 8B). These findings suggest that downregulation of the atherogenic T cell–mediated responses at the vessels and lymphoid organs during the effector phase of the disease could play a role in the protective effect of VIP in atherosclerosis.
VIP reduces the formation of foam cells
Beside self-reactive T cells, accumulation of macrophages in the arterial intima layer plays a key role in the early events of atherogenesis. These macrophages accumulate cholesterol ester and oxidized derivatives through increased uptake of oxLDL. This accumulation causes the macrophages to become atherogenic foam cells and induces an inflammatory response. Therefore, we tested whether VIP regulated lipid accumulation in macrophages exposed to oxLDL. The presence of oxLDL in macrophage cultures significantly increased the percentage of foam cells, and VIP decreased it (Fig. 9A). Cholesterol accumulation in macrophages depends on the following three independent events: oxLDL uptake, intracellular balance between free cholesterol and cholesterol ester, and cholesterol efflux to extracellular acceptors such as apoE or apoAI. Treatment of oxLDL-activated macrophages with VIP decreased the expression of the scavenger receptor CD36 but not of SRB1, both involved in the uptake of oxLDL (Fig. 9B). In macrophages, VIP signals mainly by activating the cAMP-protein kinase A (PKA) pathway (reviewed in Ref. 7), and agents that elevate cAMP are potent inducers of cholesterol efflux (38, 39). As expected, forskolin (a cAMP-induced agent) and VIP enhanced cholesterol efflux by oxLDL-activated macrophages (Fig. 9C). Because this effect was observed in the presence of apoAI in the culture, the membrane transporter ABCA1 emerged as a potential mediator of the effect of VIP in cholesterol efflux. Indeed, VIP significantly augmented the expression of ABCA1 in oxLDL-activated macrophages (Fig. 9B). In agreement with this observation, we found that VIP also increased the expression of PPAR-γ, an inducer of ABCA1 expression (40), in macrophages exposed to oxLDL (Fig. 9B). Interestingly, treatment with VIP increased the expression of ABCA1 and PPAR-γ relative to the macrophagic marker CD68 in aortas isolated from atherogenic apoE−/− mice (Fig. 9D), suggesting that the effect observed for VIP in foam cells in vitro could be also exerted in vivo.
VIP inhibits proliferation and migration of SMCs and neointima formation
Proliferation and migration of SMCs are key steps for the progression of atherosclerosis. In response to vascular injury, the medial SMCs proliferate and migrate into the intima, where they proliferate and secrete abundant extracellular matrix to form the neointima (21). Therefore, we assayed the effect of VIP in the proliferation and migration of human aortic SMCs activated with PDGF, a potent mitogen and driver of migration for vascular SMCs (21). As previously reported for other SMC types and stimuli (41), we confirmed that VIP impaired the proliferative and migratory responses of aortic SMCs to PDGF in vitro (Supplemental Fig. 4). Therefore, we tested for the first time, to our knowledge, the effect of VIP on an established model of neointima hyperplasia induced by complete ligation of the carotid artery in susceptible FVB/NJ mice fed a standard diet (in the absence of a atherogenic milieu) (32). In this model of blood flow cessation, after an early phase of inflammatory cell recruitment, medial SMCs rapidly proliferate and migrate toward the lumen, leading to extensive neointima formation after 4 wk (Fig. 10). Systemic injection of VIP strongly reduced neointima formation in the ligated artery (Fig. 10). Therefore, through this effect on neointima formation, VIP could limit the outward vascular remodeling observed during the progression of atherosclerosis.
VIP is a neuropeptide that has previously emerged as an anti-inflammatory factor that regulates self-reactive responses in various experimental models of autoimmune disorders (7, 9–14). In this study, we provide evidence for the first time, to our knowledge, that VIP could be considered a protective therapeutic agent for CVDs that are caused by exacerbated inflammatory and autoimmune responses. Using two well-characterized mouse models of acute autoimmune myocarditis and atherosclerosis, we demonstrate that VIP attenuated heart hypertrophy, myocardial inflammation and injury, and ameliorated atherosclerotic plaque formation. Our data indicate that the effects of VIP in acute EAM and atherosclerosis are mainly exerted during the effector phase of both diseases, in which innate and adaptive immune responses play pivotal roles. Thus, the beneficial effects of VIP in EAM and atherosclerosis were associated with inhibition of production of autoantibodies and inflammatory cytokines and/or with the impairment of infiltration of inflammatory and T cells into the myocardium and the developing atherosclerotic lesion. This effect is mostly exerted by regulating the cardiomyogenic and atherogenic sensitization in the peripheral immune compartment. Our findings suggest that treatment with VIP could impair the activation and/or expansion of tissue-specific self-reactive Th17 and Th1 cell clones. Although the role played by Th1 and Th17 cells in EAM and atherosclerosis, respectively, remains controversial, numerous studies in human and animals models largely demonstrate that Th17 cells and IL-17 are critically involved in the generation of cardiomyogenic T cell responses and the establishment of myocardial inflammation (23, 24, 26, 42–47) and that Th1 cells and IFN-γ enhance development of atherosclerotic lesions and contribute to lesion rupture through various pathological events (20–22, 48–51). Treatment with VIP diminished the presence of Th1 and Th17 cells and their derived cytokines in inflamed myocardium and in aorta- and carotid-bearing atherosclerotic plaques. This effect could be mainly mediated at the peripheral level in lymphoid organs because treatment with VIP reduced the number of Th1 and Th17 cells in lymph nodes that drain atherosclerotic aorta and carotid arteries and inflamed heart.
Beside this effect of VIP at the peripheral lymphoid organ level and the subsequent impairment of inflammatory infiltration, our data also indicate that even when they reach the arterial wall, atherogenic T cells and infiltrating macrophages could be locally deactivated by VIP. Indeed, numerous studies identified VIP as a potent regulator of macrophage function that regulates the secretion of a plethora of inflammatory mediators by activated macrophages, including inflammatory cytokines, chemokines, and enzymes (7). Many of the macrophage-derived inflammatory mediators that are regulated by VIP have been previously involved in myocardial and arterial injury (20–26). In fact, the current study demonstrated that VIP is able to reduce the inflammatory milieu in atherosclerotic arteries and inflamed heart. Moreover, we observed for the first time, to our knowledge, that VIP impaired the formation of foam cells in response to the atherogenic factor oxLDL in vitro. This could be also occurring in vivo because mice treated with VIP showed less lipid-loaded macrophages in the atherosclerotic lesion area. This finding is important from a pathological point of view because accumulation of foam cells in the intima layer is critical in the progression of atheromatous plaque (20–22). This effect is directly mediated by an increase in the efflux of cholesterol from macrophages, and partially by a decrease in oxLDL uptake, because VIP significantly decreased the expression of CD36, a scavenger receptor involved in the uptake of cholesterol, and increased the expression of ABCA1, a membrane transporter critically involved in promoting cholesterol efflux from macrophages to extracellular acceptors (38, 39). Moreover, VIP increased PPAR-γ expression, a transcription factor that upregulates ABCA1 in macrophages (40). Interestingly, aortas isolated from VIP-treated atherosclerotic mice showed increased expression of ABCA1 and PPAR-γ, which supports a link between these cholesterol efflux-inducing factors and the reduced numbers of aortic foam cells observed in animals treated with VIP.
Whether VIP regulates activation and/or expansion of cardiomyogenic and atherogenic T cell clones acting directly on T cells or indirectly on APCs is still unknown. Previous data support actions of VIP on both T cells and DCs. Thus, VIP induces cell cycle arrest and inhibits the secretion of IL-2 and IFN-γ in clonally activated T cells through a mechanism that depends on cAMP production (52, 53), supporting a direct effect on T lymphocyte activation. At the same time, VIP blocks the differentiation of Th1 cells indirectly by regulating DC functions through various nonexcluding mechanisms, such as inhibition of IL-12 production and differential regulation of costimulatory molecules and of chemokines (54–56). Moreover, VIP generates a tolerogenic DC phenotype in vitro and in vivo, which is characterized by low expression of costimulatory molecules and high production of IL-10 and its capacity to impair Th1 and Th17 activation and expansion (17, 18, 57). These tolerogenic DCs induced by VIP also generated a population of IL-10–producing T cells with suppressive and/or regulatory functions on other T cells (57). Interestingly, it has been reported that IL-10–producing T cells that are also regulated by DCs are able to protect against EAM (58). Beside an indirect generation of Treg cells through tolerogenic DCs, VIP is able to directly induce a population of FOXP+ Treg cells with suppressive functions through a mechanism that involves cell cycle arrest and induction of CTLA-4 expression (52, 53, 59). Several studies have associated the effect of VIP in autoimmunity to the generation of Treg cells (15, 16, 60, 61). In this study, we also observed significant changes induced by VIP in this cell population in arteries and DLNs of atherosclerotic and EAM mice, at least at early disease stages, promoting beneficial Treg/Th1 and Treg/Th17 balances during the progression of both diseases. It was reported in experimental models that peripheral activation and/or generation of Treg cells and their recruitment to atherosclerotic plaque and myocardium limits the progression of the lesion and myocardial inflammation (20–24, 26, 42). Moreover, it is tentative to speculate that the induction of Treg cells by VIP could be also related to the inhibition of cardiomyogenic Th17 cell responses. Although an in vitro study shows controversial results about the effect of VIP on the differentiation of Th17 cells (62), several in vivo studies in experimental type I diabetes and rheumatoid arthritis clearly demonstrated that VIP impairs the generation and activation of this T cell population, which is in agreement with our results in CVDs (9, 60). The inhibitory effect of VIP in Th17 cell population could be partially related with the decrease in antimyosin IgG Abs that we observed in animals with EAM treated with VIP, which is a process with clinical significance (63). Because B cells mostly lack VIP receptors (7, 64), the effects of this neuropeptide on the production of autoantibodies must be explained by its action on Th1 and Th17 cells and their role in B cell differentiation and IgG class switch recombination (65, 66).
Although our data indicate that VIP could improve myocarditis clinical signs during the effector phase of disease by impairing cardiomyogenic T cell responses in the periphery and that the reduction of cardiac fibrosis that we observed after VIP treatment probably is a consequence of the reduction of acute myocardial inflammation, especially of Th17 cells, we cannot rule out its local effects in the myocardium. We found that delayed administration of VIP to animals with established inflammatory cell infiltration in the heart slightly improved the clinical signs, supporting its action at the cardiac level. Evidence indicates that VIP downregulates myocardial fibrosis in vitro and in experimental models of hypertension (5, 6, 67). Although this is further than the scope of the current study, these data suggest the possibility that VIP could regulate cardiac remodeling and fibrosis in late phases of the disease. In fact, a possible role for the depletion of VIP in the myocardium in the pathogenesis of myocardial fibrosis has been previously proposed, in both normotensive and hypertensive animals, in which the concentration of VIP in the heart was negatively correlated with the degree of fibrosis (6). Moreover, cardiac VIP levels decreased during the progression of diabetic myocardiopathy (68), and VIP-deficient mice have a cardiac phenotype of cardiomyopathy with pulmonary hypertension in a setting of heart failure gene upregulation (4). Although these findings support an important role of endogenous VIP in the regulation of these CVDs, we observed a significant increase in cardiac levels of VIP in animals with EAM in comparison with naive animals and a positive correlation between myocardial VIP concentration and the severity of EAM (see Fig. 11). Similarly, we found that arteries bearing atherosclerotic plaques showed increased VIP levels (Fig. 11). These new findings suggest that VIP is locally produced in response to EAM in an attempt to limit the destructive inflammatory response in the heart and artery. Because VIP is also produced by various immune cell types, mainly T cells upon activation (7, 64), it is quite possible that the increase in cardiac and arterial VIP during EAM and atherosclerosis progression is due to the increased presence of inflammatory cell infiltrates.
Finally, in this study, we confirmed the capacity of VIP to limit proliferation and migration of SMCs in response to PDGF, which is produced by endothelial cells, macrophages, and SMCs in the atherosclerotic plaque. Moreover, we described for the first time, to our knowledge, that treatment with VIP diminished the formation of neointimal lesions and prevented stenosis in arteries subjected to alterations of blood flow and vascular injury in nonatherogenic conditions. Therefore, VIP could regulate the presence of SMCs in the plaque and intimal/medial ratio in the artery and limit the outward vascular remodeling observed during the progression of atherosclerosis.
Although further pharmacological studies are needed to identify specific receptors and signaling involved in the therapeutic action of VIP in myocarditis and atherosclerosis in vivo, evidence suggests that both type 1 (VPAC1) and type 2 (VPAC2) VIP-receptors could be involved. Numerous reports indicate that binding of VIP to VPAC2, but mainly to VPAC1, in macrophages and T cells initiates a complex cAMP/PKA-mediated signaling that regulates activation of kinases and transcription factors (NF-κB, p38 MAPK, and Jak-STAT1, between others) that are critical for the production of inflammatory mediators, activation of Th1 and Th17 responses, and generation of Treg cells (reviewed by Ref. 7, 64). Moreover, various studies have involved cAMP/PKA-signaling in the induction of the PPAR-γ–ABCA1 pathway and cholesterol efflux in macrophages (38–41). Furthermore, previous studies suggest that VPAC2 is the receptor that is mainly involved in the effect of VIP in SMC proliferation, cardiomyocyte protection, and myocardial fibrosis (5, 6, 67, 68). These findings could explain controversial results describing that activation of VPAC1 with a specific agonist did not improve early signs of atherosclerosis (69) and support the notion that a coordinated signaling through both VPAC1 and VPAC2 is necessary to exert full protection by VIP in CVDs.
In summary, we provide evidence that the neuropeptide VIP could be considered an effective therapy for CVDs that course with exacerbated inflammation and autoimmunity, such as myocarditis and atherosclerosis. Its multimodal action on various components of the disease would suppose a therapeutic advantage versus current treatments. However, because the effects observed are based on mouse models of myocarditis and atherosclerosis, extrapolations to clinical practice have to be made with caution, and for example, future clinical trials with VIP-based therapies should discriminate between interventions in active and borderline myocarditis. Noteworthy is the fact that infusion of VIP has been proven safe (no side effects were observed) and effective in reducing Th1 and inflammatory responses and in inducing Treg cells in patients with an inflammatory and/or autoimmune disorder such as sarcoidosis (19).
This work was supported by the Spanish Ministry of Economy and Competitiveness and the Excellence Grant Program from the Andalusian regional government.
The online version of this article contains supplemental material.
Abbreviations used in this article:
ATP-binding cassette A1
brain natriuretic peptide
draining lymph node
experimental autoimmune myocarditis
human aortic SMC
oxidized low-density lipoprotein
platelet-derived growth factor
protein kinase A
peroxisome proliferator–activated receptor
smooth muscle cell
vasoactive intestinal peptide.
The authors have no financial conflicts of interest.