INTRODUCTION
Yak (scientific name: Bos mutus or Bos grunniens, English name: Wild yak) is an herbivorous ruminant belonging to the Bovidae family. It is a rare cattle species native to the Qinghai-Tibet Plateau in China and its adjacent alpine and subalpine regions. Yak can adapt to the cold climate and, except for human beings, is the mammal living at the highest altitude in the world. It is distributed in areas higher than 3000 m above sea level. As all-round livestock, yak is an extremely valuable genetic pool and has social and economic significance to human beings that cannot be ignored. There are nearly 16 million yaks globally, of which 15 million are found in China. Hypoxia is a key factor affecting the survival of organisms in such an environment and plays an important role in kidney injury, pulmonary hypertension, and other organ diseases [5]. The ability of mammalian cells to adapt to high altitude and low oxygen environments is an evolutionary change that may result in considerable physiological changes related to animal viability. This has been demonstrated by some native species living in the region, including Tibetan antelope, gazelle, and yak [19]. The Tibetan Plateau, the highest plateau in the world, is known for its hypoxic environment.
Erythropoietin (EPO) is a key regulator of erythropoiesis, and its continuous production is necessary to maintain daily erythropoiesis [15]. EPO and EPO receptors (EPORs) are widely expressed in the brain, peripheral nerves, heart, kidney, skeletal muscle, bone marrow, endothelial cells, and intestinal tissues [2]. Exogenous EPO improves intestinal motility in vivo through its powerful antioxidant and inflammatory decomposition properties [2]. Hypoxia-inducible transcription factors (HIFs) induce the expression of EPO. EPO is then released into the blood and binds to EPORs on erythrocyte progenitor cells in the bone marrow, triggering erythropoiesis and leading to tissue hypoxia compensation [3]. In addition, HIF-EPO pathway deregulation mediates disorders that affect altitude adaptation. Strict regulation of erythropoietin production leads to the clearance of red blood cells and normalization of haemoglobin elevation after descent, which is a typical observation of long-term adaptation to high-altitude residents and high-altitude areas [6]. Vascular endothelial growth factor (VEGF) is a downstream target gene induced by HIF activation and plays an important role in cell adaptation to hypoxia [3]. Hypoxia also promotes VEGF expression, which has been found in many organs [3, 10]. The kidney is susceptible to hypoxia, and angiogenesis is affected under hypoxia. VEGF enhances vascular permeability, stimulates angiogenesis, and promotes blood delivery to hypoxic sites, thereby reducing tissue damage. Vascular endothelial growth factor receptor 2 (VEGFR-2) is a VEGF receptor, and many of the VEGF biological activities in endothelial cells are mediated by the tyrosine kinase receptors VEGFR-2 [13] and VEGFR-1. The binding of VEGF to the extracellular domain of these receptors results in dimerisation and phosphorylation of the tyrosine kinase domain [7]. Despite its lower affinity for ligands,VEGFR-2 is responsible for most of the biological behaviour of VEGF due to its higher ability in signal transduction [4]. The oxygen concentration in granulosa cells affects VEGF gene expression in granulosa cells [14]. Similarly, cells cultured at low oxygen concentrations, such as mouse mesenchymal cells, human trophoblast cells, and umbilical endothelial cells, produce more VEGF [12] and show higher mRNA expression of VEGF ligands and their receptors [9].
Erythropoietin can promote angiogenesis by upregulating the expression of VEGF, thereby promoting the repair of brain tissue injury and improving cardiac function and myocardial ischaemia and hypoxia, thereby decreasing the expression of HIF, which provides a certain experimental and theoretical basis for the clinical treatment of chronic myocardial infarction. The effect of EPO therapy on wound healing is attributed to EPO-mediated stimulation of cell proliferation and angiogenesis and is associated with the increased expression of VEGF, endothelial nitric oxide synthase, and inducible nitric oxide synthase. EPO and VEGF have significant genetic and functional homology, suggesting that the two cytokines play similar roles in bone repair. In addition, it has been reported that EPO can stimulate tissue regeneration after skin injury and myocardial infarction through the VEGF-dependent pathway [17]. This suggests that EPO stimulates early angiogenesis through the VEGF-dependent pathway. On the other hand, EPO has a significant inhibitory effect on the accumulation of anti-VEGF antibodies. Studies have found that increasing VEGF in fetal bovine serum medium is not sufficient to induce increased activity of EPO, highlighting the importance of VEGF in the role of epithelial cells erythropoietin. The relationship between EPO and VEGF is particularly important during normal neovascularisation and in patients treated with recombinant human erythropoietin (rhEPO) [1]. Yang et al. [20] also confirmed that rhEPO has a significant protective effect on blood-brain barrier leakage, reduces blood-brain barrier permeability, and alleviates brain oedema, usually caused by focal ischaemia in the acute stage after injury. VEGFR-1 and VEGFR-2 were significantly decreased after rhEPO treatment on day three after injury compared to VEGF upregulation. Prevention of EPO-mediated damage after ischaemia may involve endothelial cell storage, preservation of microvascular integrity, and downregulation of VEGFR-1 and VEGFR-2. EPO induces neovascularisation through expression and upregulation of VEGF/VEGFR-2, an essential growth factor and potent angiogenic factor for vascular endothelial cells.
Yak is a native species of China, mainly distributed in the plateau and its adjacent areas where the atmospheric oxygen pressure is only 53–62% of that at sea level [19]. Cattle have been domesticated for more than 4000 years and have been well adapted to hypoxia conditions through natural selection [19], making them a perfect model for studying the mechanisms related to hypoxia. In this study, the expression and distribution characteristics of EPO, VEGF and VEGFR-2 in the small intestine of yaks of three different ages in high altitude areas were analysed using an immunohistochemical method. The research results will also provide basic information for understanding the stress response mechanism in high altitude hypoxic environment.
MATERIALS AND METHODS
Animal ethics
The study was approved by the State Forestry Administration, and all procedures were performed in compliance with guidelines for the care and use of laboratory animals adopted by the Ministry of Science and Technology of the People’s Republic of China.
Materials
Wild adult, newborn, and 50-day-old yaks were collected in Hezuo City (Gansu Province, China) at an altitude of around 3000 m. Animals were euthanized with pentobarbital sodium (200 mg/kg, IV) at local slaughterhouses, complying with local regulations. Small intestine samples were collected and preserved in 4% paraformaldehyde for tissue fixation immediately after euthanasia.
Haematoxylin and eosin staining detection
Fully fixed samples were embedded in paraffin and the tissue blocks then were sliced into 5 µm sections for subsequent processes. Haematoxylin and eosin staining was used to observe the histological features of the samples.
Immunohistochemistry detection
Immunohistochemical staining was carried out based on Histostain TM-Plus Kits (Bioss, China, SP-0023). Briefly, tissue sections were deparaffinized in xylene and rehydrated in different concentration gradient of alcohol. After being rinsed in phosphate buffered saline (PBS) buffer, sections were autoclaved (15 min in a microwave oven) in 0.01 M sodium citrate buffer (pH 6.0) for antigen retrieval. The endogenous peroxidase was inactivated using 3% hydrogen peroxide at 37ºC for 10 min. The sections were then incubated with anti-EPO polyclonal antibody, anti-VEGF polyclonal antibody, anti-VEGFR-2 polyclonal antibody (Bioss, China, 1:200 dilution, bs-20398R, bs-1447R, bs-1665R and bs-0565R) at 4ºC overnight in a humid chamber. Antibody binding was coloured with DAB Substrate kit (Solarbio, China, DA1010) and tissue sections were counterstained with haematoxylin. All washing steps in-between were done in PBS. To assess the specificity of the immunolabelling, negative control slides were created using the bovine serum albumin as the primary antibody while all other steps and conditions.
Statistical analysis
Images of the stained tissue sections were observed and captured by a light microscope (Olympus CX31, Tokyo, Japan).
Image-Pro Plus (Version 6.0, Media Cybernetics, Inc.: Bethesda, MD, USA) was used to quantify the positive results of expression of VEGF, VEGFR-2 and EPO. The measurement parameters included sum area and sum integrated optical density. SPSS software (Version 19.0, SPSS Inc., Chicago, USA) was used to analyse the statistical significance.
RESULTS
Histological observation
The intestinal wall of yak consists of mucosa, submucosa, muscularis and serosal layers. The lamina propria of the mucosa extends into the lumen and is covered by intestinal epithelial cells to form intestinal villi (Figs. 1, 2A–I). Duodenal and intestinal villi are densely distributed, short and thick, in the shape of a comb; jejunum villi are sparse and foliate; and ileum villi are dense and finger-like. There are many mall intestinal glands in the lamina propria of the duodenum, jejunum, and ileum. Duodenal glands are seen in the submucosa of the duodenum, isolated lymph nodes are occasionally seen in the submucosa of the jejunum, and numerous clustered lymph nodes are seen in the submucosa of the ileum. There were some differences in the small intestine morphology in yaks at different developmental stages.
The results showed that the intestinal wall of yaks was composed of the mucosa, submucosa, muscularis, and outer membrane. The mucosa was composed of villi, lamina propria, and mucosal muscularis. Intestinal mucosa formed a large number of intestinal villi, protruding into the intestinal lumen. There were some differences in the small intestine morphology in yaks at different developmental stages.
Compared with the jejunum and ileum, the duodenal villi of newborn yaks were leaf-like and sparsely distributed. The columnar cells of the small intestinal villi are distributed in a single layer, and the nuclei, which are closely arranged in the base of the epithelial cells of the small intestinal villi, are oval or round. There are goblet cells and scattered lymphocytes between the epithelial cells. A few of the villi appear on the surface of the folds, similar to the cerebral furrows. Scattered lymphocytes were seen in the lamina propria in the central villi of the small intestine, but no aggregative lymphoid nodule was formed. Many intestinal glands were distributed in the lamina propria of the mucosa, and a small number of duodenal glands were distributed in the submucosa (Figs. 1, 2A, D, G). The jejunum villi of newborn yaks are rod-shaped, slender, and dense. The ileum and jejunum villi of newborn yaks are similar in shape but shorter in length and sparsely arranged. The columnar cells at the top of the small intestine’s villi contained a large number of vacuolar structures, and the nucleus was located at the base of the cells. The columnar cells in the villi from the centre downward had no vacuolar structures in the cytoplasm. Many intestinal glands and lymphoid aggregates were distributed in the lamina propria of the mucosa, and the lymphoid aggregates were dark and round or oval in shape (Figs. 1, 2A, D, G).
The columnar cells in the villi of the small intestine of 50-day-old yak were arranged neatly, and the cell structure was normal. The width of duodenal villi decreased, and the morphology of jejunum and ileum villi did not change significantly. The villi length the small intestine decreased compared with that at birth. Many intestinal glands were distributed in the lamina propria of the small intestine, and the number of duodenal glands in the submucosa of the duodenum increased significantly compared with that at birth. The number and volume of lymphoid aggregates in the ileum are significantly increased and enlarged, oval or long-oval, and deeply stained, accounting for approximately 1/2 of the thickness of the wall of the ileum (Figs. 1, 2B, E, G). The villi morphology in the jejunum and ileum of adult yak had noticeable changes. The nuclear volume of villi columnar cells in the jejunum and ileum decreased obviously, and the cytoplasm was darker. The length of lymph aggregation in the ileum increased, accounting for approximately 3/4 of the wall thickness (Figs. 1, 2C, F, I).
Immunohistochemical localizations
Expression of VEGF, VEGFR-2, and EPO were detected in the small intestine of 50-day-old yaks in the positive control group (Figs. 3B, E, H; 4B, E, H; 5B, E, H, 6A–I). Immunohistochemical results showed that EPO was highly expressed in the ileum and villous epithelial cells in 50-day-old yaks, but showed lower expression in newborns and adults. In newborn yak jejunum intestines and a strong positive expression in intestinal villus epithelial cells, and intestinal villus epithelial cells in adult yak’s jejunum intestines positive expression, 50 days of age in yak and jejunum was weakly positive expression in intestinal villus epithelial cells, the newborn yak duodenal glands and a strong positive expression in intestinal villus epithelial cells. Expression was high in the duodenal glands and intestinal villus epithelial cells of adult yak, but lower in those of 50-day-old yak. VEGF was highly expressed in the ileo-intestinal glands and villous epithelial cells of newborn yaks, and lower in those cells in 50-day-old and adult yaks.
It was highly expressed in the jejunal intestinal glands and villous epithelial cells of newborn yaks, and weakly positive in jejunal intestinal glands and villous epithelial cells of 50-day-old and adult yaks. It was highly expressed in duodenal glands and intestinal villus epithelial cells of 50-day-old yaks, lower in those cells in adult yaks as well as in newborn yaks. VEGFR-2 was strongly positive in duodenal gland and intestinal villi epithelial cells, strongly positive in adult yak and jejunum ileum adenoma positive in intestinal villi epithelial cells, weakly positive in newborn yak and jejunum gland, and weakly positive in duodenal gland and intestinal villi epithelial cells of adult yak. No expression was detected in the ileal intestinal glands and intestinal villus epithelial cells, or in the duodenal glands and intestinal villus epithelial cells of newborn yaks.
Optical density analysis
Results of average integrated optical density (Table 1) showed that the immunostaining intensity of VEGF was the highest in ileum of newborn yaks, followed by ileum of 50-day-old yaks and adult yaks (p < 0.05, Figs. 3, 6). The immunostaining intensity of VEGFR-2 was the highest in ileum of yaks at 50 days of age, followed by adult ileum and newborn ileum (p < 0.05, Figs. 4, 6). The immunostaining intensity of EPO was the highest in ileum of 50-day-old yaks, followed by ileum of newborn yaks and ileum of adult yaks (p < 0.05, Figs. 5, 6).
|
Species |
EPO |
VEGF |
VEGFR-2 |
|
Newborn yak |
0.1076000 ± 0.074607774 |
0.130878 ± 0.1016521 |
0.00020000 ± 0.000327872 |
|
50-day-old yak |
0.3320444 ± 0.039606651 |
0.056822 ± 0.0199790 |
0.02978889 ± 0.009988549 |
|
Adult yak |
0.03253444 ± 0.01572786 |
0.043800 ± 0.0310143 |
0.00427300 ± 0.004416746 |
DISSCUSSION
Oxygen is an important substrate for aerobic organisms to maintain metabolism and physiological functions. As a result, vertebrates evolved complex respiratory and cardiovascular systems to ensure an optimal oxygen supply to each cell. Hypoxia is a biological limiting factor in most mammals and can lead to various organ diseases, such as kidney damage and pulmonary hypertension [18]. The ability of mammals living in anoxic environments, such as the Tibetan Plateau, to maintain homeostasis depends heavily on proper respiratory regulation and blood pressure. At the same time, hypoxic conditions can activate cellular sensing mechanisms, focusing on restoring oxygen to hypoxic areas to maintain cell viability [8]. The small intestine, especially its epithelial cells, is susceptible to hypoxia. Intestinal epithelium plays an important role in nutrient absorption and nonpathogenic antigen tolerance, and is regulated by oxygen supply. In recent years, it has been increasingly recognized that tissue oxygen metabolism is the key to maintaining intestinal intraepithelial homeostasis. The unique oxygen tolerance of normal intestinal epithelium may be an adaptive adjustment to extremely low oxygenation levels. Hypoxic responses play a vital role in various biological processes in the body and are the pathogenesis of various diseases such as gastrointestinal diseases, tumours, and cardiovascular diseases. Nutrient absorption takes place mainly through small intestinal villi, and its morphology plays a great role in nutrient absorption and utilisation. Studies have shown that when the height of small intestinal villi decreases or the shape changes, it will affect the number of absorbing cells in the villi per unit area, thus affecting the digestion and absorption area, reducing the function of digestion and absorption, and seriously affecting the growth and development of animals. In this study, EPO, VEGF and VEGFR-2 showed was highly expressed in yak. Intestinal villous epithelial cells and intestinal glands.
Erythropoietin is a glycoprotein cytokine that plays a role in the proliferation, differentiation, and maturation of erythrocyte progenitors. EPO and its receptors are widely distributed in different tissues of an infant’s intestines, suggesting that EPO may play a role in gastrointestinal development. Previous studies have shown that recombinant EPO can reduce the inflammatory response, autophagy and apoptosis, and limit intestinal mucosal necrosis, thereby improving necrotizing enterocolitis damage. EPO production is induced under conditions of anaemia and hypoxia to control red blood cell production and is necessary for the maintenance of normal blood oxygen concentrations [10]. The small intestine tissue sections indicated that EPO was highly expressed in the intestinal glands, duodenal glands, and intestinal villus epithelial cells. The expression intensity in neonatal yaks ileum was higher than that in adult ileum and 50-day-old yaks ileum. The expression intensity in the jejunum of newborn yak was higher than that in the jejunum of 50-day-old yaks and higher than that in the jejunum of adult yaks. The expression intensity in 50-day-old yaks duodenum was higher than that in adult yaks duodenum and higher than that in newborn yaks duodenum. The expression level in ileal glands and villous epithelial cells of 50-day-old yaks was higher than that in adult yaks and higher than that in newborn yaks. Hypoxia stimulates the expression of EPO, further increasing red blood cells, enhancing oxygen supply capacity, and promoting angiogenesis and reconstruction. EPO is an important protein that enables yak, and other plateau animals, adapt to the low oxygen environment at high altitude VEGF and VEGFR-2 play key roles in angiogenesis and cell proliferation. VEGF can promote blood flow by promoting the formation of blood vessels in hypoxic areas, thus alleviating tissue damage caused by hypoxia [19]. In endothelial cells, much of the bioactivity of VEGF is mediated by VEGFR-2 [10]. After soft tissue injury, strong expression of VEGF can interact with platelet-derived growth factor or basic fibroblast growth factor to promote the rapid generation of mature vascular networks, and the expression of VEGF increases from the angiogenesis stage. It was observed that VEGF and VEGFR-2 were mainly expressed in the intestinal glands, duodenal glands, and intestinal villus epithelial cells in small intestine tissue sections. The expression of VEGF in the ileum and jejunum of newborn yaks was significantly higher than in adult yaks and 50 days of age. The expression of VEGFR-2 in the small intestine of 50-day-old yaks was significantly higher than in that of newborn and adult yaks. These results suggest that VEGF and VEGFR-2 play important roles in the effects of hypoxia on the small intestine of yaks of different ages. Vascular endothelial growth factor A (VEGF-A) and its receptor (VEGFR-2) are the major signalling pathways involved in tumour angiogenesis. Previous studies have shown that increased VEGFR-2 expression is associated with differentiation, metastasis/recurrence, and poor prognosis in colon cancer samples [16]. These findings suggest that VEGFR-2 is functional on the surface of endothelial cells and that VEGFR-2 has potential as an anti-angiogenic cancer therapy molecule.
Erythropoietin mediates neurovascular remodelling and neurobehaviuoral recovery in traumatic brain injury rats by increasing intracerebral VEGF expression and VEGFR-2 phosphorylation. Yang et al. [20] demonstrated that VEGF-A activates EPOR and enhances VEGFR-2-mediated retinal angiogenesis in an oxygen-induced retinopathy rat model. Nakano et al. [11] demonstrated that EPO upregulation of the VEGF/VEGFR-2 system plays an important role in the mobilisation of epithelial cells in ischaemic tissues. They showed that vascular EPOR and EPO promote postischaemic angiogenesis by increasing VEGF secretion in ischaemic muscle, mobilisation of epithelial cells, and recruitment of bone-marrow pro-angiogenic cells to ischaemic tissue [11]. This study found that when the expression level of EPO increased in the ileum and jejunum of yaks, the expression level of VEGF also increased. This confirmed that EPO regulates the formation of blood vessels in the yaks’ intestinal tissue through the VEGF/VEGFR-2 pathway, enabling yaks to adapt to the hypoxic and cold environment at high altitude.
In our study, VEGF, VEGFR-2, and EPO were mainly expressed in the small intestinal villi of yaks of different ages, reflecting the adaptation of plateau yaks to the cold and anoxic environment (Fig. 6). Combined with previous studies, this study suggests that EPO, VEGF, and VEGFR-2 may be involved in the protection, structural maintenance, and function regulation of the small intestine in yaks of different ages in hypoxic environments.
CONCLUSIONS
The results showed that the expression and distribution characteristics of VEGF, VEGFR-2, and EPO in the small intestine of yaks of different ages might be related to physiological regulation in hypoxic environments. Therefore, they are considered important potential regulatory proteins of yak adaptation. However, the underlying molecular mechanisms remain unclear. This study is a preliminary experiment, and the regulation mechanism requires further investigation. This study also provides basic data from a comparative study of the small intestine of yaks of different ages in high altitude areas.
In our study, the expression intensity of VEGF in ileum and jejunum of newborn yaks was higher than that in ileum and jejunum, ileum and intestinal glands and villous epithelial cells of 50-day-old yaks and adults. The expression level in duodenal glands and intestinal villus epithelial cells of 50-day-old yaks was much higher than that in adult yaks and higher than that in newborn yaks. These results suggest that VEGF and VEGFR-2 play important roles in the effects of hypoxia on the small intestine of yaks of different ages. The expression intensity of VEGFR-2 in intestinal glands, villous epithelial cells and duodenal glands of 50-day-old yaks was much higher than that in adult and newborn yaks. These results suggest that VEGF and VEGFR-2 play important roles in reducing the effects of high altitude and low oxygen environment on the yak small intestine.
In our study, VEGF, VEGFR-2, and EPO were mainly expressed in the small intestinal villi of yaks of different ages which reflecting the adaptation of plateau yaks to the cold and hypoxic environment.
Research shows that the small intestine is anoxic sensitive organ; the oxygen level in the blood and the oxygen concentration that diffuses into the organ play a vital role in maintaining the metabolism of intestinal mucosa epithelial cells. Hypoxic conditions can result in serious damage of the intestinal mucosa epithelium.
Acknowledgements
Supported by the National Natural Science Foundation of China (Grant No. 31860687, Grant No. 32002241), the Natural Science Foundation of Gansu Province (No. 21JR11RA024), the Fundamental Research Funds for the Central (No. 31920200004), Changjiang Scholars and Innovative Research Team in the University (IRT_17R88), Ministry of Education Animal Medicine and Innovation, entrepreneurship training program for College Students (NO.X202210742302).
