Materials and methods
Mice
An Npr2slw/slw strain was established from a mating between a founder male spontaneously mutant mouse in the ddY mouse colony at Okayama University and a C57BL/6J female [11, 12]. The mice in the mix background of ddY and C57BL/6J used in this study was described in the previous report [14]. The mice were maintained under standard 12 h light/dark conditions. Either heterozygotes or wild-type mice were used for comparison (referred to as controls); homozygotes from the same litter for controls were used and referred to as Npr2slw/slw. Mice were anesthetized by injecting a combined anesthetic containing 7.5% medetomidine hydrochloride, 8% midazolam, and 10% butorphanol tartrate in saline (10 µl/g) into the subcutaneous of the neck or intraperitoneal using a 29G syringe. Mice were euthanized by cut the diaphragm under anesthesia and immediately removed their whole gut.
All animal experiments were carried out in accordance with the institutional guidelines regarding animal care and handling, and the experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Tokyo.
Elastica van Gieson (EVG) staining
Whole GI tissue with mesentery was fixed in 10% buffered formalin at 4 °C overnight, dehydrated, and embedded in paraffin. Six-µm-thick sections were cut, placed on glass slides, and subjected to EVG and immunofluorescence staining.
For EVG staining, tissues were deparaffinized, hydrophilized, and immersed in 1% HCl in 70% ethanol for 3 min. The tissues were then immersed in resorcin-fuchsin solution (40321, Muto Pure Chemicals, Tokyo, Japan) for 2 h. Washing was performed with 100% ethanol for 3 min three times, followed by immersion in water, and treatment with iron-hematoxylin (40341 and 40351 Muto Pure Chemicals) for 15 min. After 30 min of rinsing under running water, the tissue was immersed in 5% Sirius Red (33061, Muto Pure Chemicals) in saturated picric acid for 15 min, quickly dehydrated, permeabilized, and embedded.
Immunofluorescence staining
Immunostaining was performed as previously described [14], and the primary antibodies used were rabbit polyclonal anti-alpha-smooth muscle actin (aSMA) antibody (ab5694, Abcam, Cambridge, UK, 1:100 dilution) and goat polyclonal lymphatic vessel endothelial receptor 1 (LYVE-1) antibody (AF2125, R&D systems, Minneapolis, MN, USA, 1:100 dilution). The secondary antibodies used were donkey anti-rabbit IgG H&L Alexa Fluor 488 (A21206, Thermo Fisher, Waltham, MA, USA, 1:1,000 dilution) and donkey anti-goat IgG H&L 594 (A11058, Thermo Fisher, 1:1,000 dilution).
Microscopy and acquisition
Stained images were acquired using a BZ-X710 all in one microscope (Keyence, Osaka, Japan) or an Olympus fluorescent microscope IX73 equipped with a color camera DP73 (Olympus, Tokyo, Japan) for P8 and P15 samples or adult samples, respectively. For the observation of P8 and P15 samples, autofocus imaging was done using a ×4 lens, and z-stacked images were generated with 10 section pictures with 0.5 µm intervals when using a ×40 lens. For the observation of adult samples, single focal images were observed using ×4, ×20, and ×40 lenses. Images were edited using Fiji software [16] and Photoshop (Adobe, San Jose, CA, USA). Mesenteric artery containing two layers of lamina elastica (LE) and parallelly running veins were selected for imaging.
Blood pressure measurement for adult mice
The blood pressure of mice was measured using a BP-98A blood pressure system (Softron, Tokyo, Japan) by the tail-cuff method. Systolic, diastolic, and heart rates were measured under a condition of non-anesthesia and retention using a mouse retention device. Measurements were obtained five times for each mouse, and their average values were shown in graphs.
Statistical analysis
Data are expressed as the mean ± standard deviation (SD). Dot plots were generated using GraphPad Prism7 software (GraphPad Software, San Diego, CA, USA). The area of the lumen of the blood vessel field was calculated using the Fiji software. The statistical significance of differences in mean values was assessed using the Mann–Whitney U test. There were no criteria used for including and excluding experimental units. Randomization was not used to allocate experimental units.
Results
Vasodilation and undeveloped adipose cell in Npr2
slw/slw
In both control and Npr2slw/slw mice, a bundle of mesenteric arteries, veins, lymph vessels, and nerves were arranged and surrounded by serosa. At 1 week of age (P8), a small adipose mass covered with a capsule was found in this bundle (Fig. 1A). At 2 weeks of age (P15), the inside of the mesentery was filled with further developed adipocytes in control, whereas no developed adipocytes were found in the Npr2slw/slw mice (Fig. 1C).
The arteries in control at both P8 and P15 were covered with a developed adventitia, the inner and outer LE were contracted, and the blood vessels were thickened (Fig. 1A and C). In contrast, in Npr2slw/slw mice at both ages, the LE was relaxed, and the blood vessels were dilated (Fig. 1A and C). The LE of veins was also contracted in control but was dilated in Npr2slw/slw (Fig. 1A and C). At 1 year of age (adult), the LE of artery and vein was contracted in both control and Npr2slw/slw (Fig. 1E). The inside of the mesentery was filled with large adipocytes in control, whereas small adipocytes were filled in the Npr2slw/slw mice (Fig. 1E).
The lumen areas of arteries and veins were measured and compared between the control and Npr2slw/slw mice, and the results showed that the areas were significantly larger for Npr2slw/slw than for the control in P8 and P15 (Fig. 1B and D). In adult mice, the lumen area of arteries was larger than that of control mice, and there was no difference in the veins (Fig. 1F).
In the arteries and veins of the smooth muscle, the smooth muscle in the control mice contracted in accordance with the LE, whereas that in Npr2slw/slw was clearly elongated at P15 (Fig. 2A). However, smooth muscle in both control and Npr2slw/slw adult mice contracted in accordance with the LE (Fig. 2B). Systolic, diastolic, and heart rates were similar between control and Npr2slw/slw in adult male mice (Fig. 2C).
The villi of the entire intestine in control were developed uniformly, while Npr2slw/slw exhibited uneven development at preweaning ages but normally developed at adult age [14]. The lacteals in the partially developed villi were dilated, and the smooth muscle in the villi was undeveloped in Npr2slw/slw compared to that of the control in P15. At adult age, the lacteal and smooth muscle were recognized in villi of both control and Npr2slw/slw mice, whereas the arrangement of nuclei of mucosal epithelial cell and lamina propria mucosae was disorganized in Npr2slw/slw mice (Fig. 2D).
Discussion
The CNP/NPR-B signal is known as an endothelium-derived vasorelaxant factor and has recently been recognized as an inhibitor of adipose hypertrophy. However, despite the lack of the CNP/NPR-B signal, Npr2slw/slw unexpectedly displayed dilated blood vessels and undeveloped white adipose tissue in the mesentery. Several reasons may explain this. First, more CNP was available for binding to NPR-C, resulting in enhanced NPR-C function. NPR-C has a clearance of natriuretic peptides [17] while it also inhibits adenylyl cyclase via activation of phospholipase C by coupling with the Gi protein [18]. Indeed, NPR-C has been shown to have multiple functions: CNP/NPR-C is essential for vascular homeostasis and has a vasorelaxant effect [19, 20]. The lack of NPR-B may result in increased free CNP, which binds and activates NPR-C, leading to the dilation of mesenteric vessels and lacteals. Second, NPR-C has a binding affinity not only for CNP but also for other natriuretic peptides, such as atrial (ANP) and brain natriuretic peptide (BNP) [21]. As mentioned above, increased CNP binding to NPR-C may limit ANP and BNP binding to NPR-C, thus, enhancing the function of NPR-A, which is a receptor for ANP and BNP. In particular, ANP/NPR-A regulates blood pressure and fluid balance by relaxing major blood vessels [22,23,24,25]. This may have caused vasodilation in the Npr2slw/slw mice. Given that relaxation of blood vessels by NPR-A or NPR-C is separable action from dilation, these are speculative but maybe probable. In addition, it was recently reported that NPR-A contributes to the reduction of white adipose tissue in humans and mice [26, 27]. Therefore, the suppression of adipogenesis in Npr2slw/slw may also be due to the increased activity of NPR-A. Lastly, loss of CNP/NPR-B signal may cause the loss of its relaxing effect on the mesenteric vessels and become congested, giving the appearance of dilated vessels. Because experiments using mice have shown that CNP/NPR-B signals relax the mesenteric artery [28], it may be simply that the loss of NPR-B affected the mesenteric vessels. However, in adult Npr2slw/slw mice, vasodilation was not evidently seen, and there appeared to be no abnormalities on blood pressure. Thus, it was suggested that CNP/NPR-B signal plays an essential role in the normal functions of blood vessels during pre-weaning ages and maintenance of peripheral tissues during adult age. Small adipocytes of adult Npr2slwslw mice may have resulted from affected nutrient absorption owing to disorganized mucosal epithelial cells and lamina propria mucosae.
It remains to be answered whether the NPR-B of Npr2slw/slw, in which a premature stop codon eliminates the whole NPR-B structure under the transmembrane domain [11], contains only the extracellular ligand-binding domain that can bind to CNP but is nonfunctional or incapable of binding to CNP. That is, it remains to be determined whether the majority of CNP is cleared by clearance or CNP binds to NPR-C and enhances the NPR-C and/or NPR-A function. Further study would contribute to understanding the unique GI phenotype of Npr2slw/slw mice. Model mice, such as Npr2slw/slw mice, can be an essential source of information for understanding the function of genes and the effect of mutations. Therefore, the phenotype of the GI and vessel of Npr2slw/slw mice would provide insight for the treatment of rare GI diseases, including the GI tract itself and secondary causes, and contribute to the elucidation of the CNP/NPR-B signaling mechanism in vivo.