Application of linoleic acid in preparation of vascular endothelial injury reagent and zebra fish model and construction method
By constructing a vascular endothelial injury model using trans oleic acid solution in a zebrafish model, the problems of complex modeling, high cost, and insufficient visualization in existing technologies are solved, realizing a simple, low-cost, high-throughput drug screening platform suitable for the study of vascular endothelial pathological changes.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-12
- Publication Date
- 2026-04-14
AI Technical Summary
Existing vascular endothelial injury models lack stable reproduction of pathological processes in zebrafish, and existing technologies suffer from problems such as long modeling cycles, high costs, complex operations, insufficient visualization, and limited high-throughput screening applications, especially in the study of vascular injury related to lipid metabolism disorders.
Ethylened acid solution was used as a reagent for vascular endothelial injury. A vascular endothelial injury model was constructed by exposing zebrafish embryos to a specific concentration of etherified acid solution. The model was then observed and detected using fluorescence microscopy and real-time quantitative PCR to achieve visualization of the model and detection of biological markers.
It enables a simple, low-cost, easy-to-operate, and high-throughput screening model of vascular endothelial injury, and provides a real-time visualization observation and efficient drug screening platform, which is suitable for the study of vascular endothelial pathological changes and drug screening.
Smart Images
Figure CN121844994A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of animal model technology, and more specifically to the preparation of vascular endothelial injury reagents using trans oleic acid, their application in zebrafish models, and construction methods. Background Technology
[0002] Cardiovascular disease is one of the leading causes of death and disability worldwide, with persistently high morbidity and mortality rates. Numerous studies have shown that vascular endothelial injury / dysfunction is a key early event in the development and progression of cardiovascular diseases such as atherosclerosis, coronary heart disease, hypertension, and thrombosis.
[0003] As the innermost layer of the blood vessel wall in contact with blood, vascular endothelial cells are susceptible to damage. Damage to these cells can lead to an imbalance between vasodilation and vasoconstriction, activation of inflammatory responses, and increased platelet adhesion and aggregation, thereby inducing a series of vascular pathological changes and cardiovascular events. Existing models of vascular endothelial injury mainly include cellular models and mammalian models (such as high-fat diet, chemically induced, or mechanically induced injury models in rats, rabbits, or mice). While these models can simulate the pathological process of vascular endothelial injury to some extent, they still have significant limitations: cellular models lack complete vascular structure and in vivo metabolic environment, making it difficult to reflect systemic responses; mammalian models have long modeling cycles, high costs, complex methods, and difficult operations, and the visualization and high-throughput screening of model results are limited.
[0004] Zebrafish (Danio rerio) offer advantages as a vertebrate model, including high genetic conservation, rapid development, body transparency, ease of manipulation, and visualization. Their vascular system exhibits clear development, and real-time visualization of vascular structure and function can be achieved through transgenic lines with fluorescently labeled vascular endothelium. Therefore, they hold significant potential in studies of vascular development, endothelial function, and drug screening. However, a zebrafish-based model system that can stably reproduce the pathological process of vascular endothelial injury is currently lacking, particularly in research on vascular injury related to lipid metabolism disorders.
[0005] Trans fatty acids are dietary risk factors; excessive long-term intake can lead to lipid metabolism disorders and increase the risk of cardiovascular disease. Trans oleic acid is a major type of trans fatty acid used in industrial production.
[0006] Therefore, whether or not we can provide a reagent for preparing vascular endothelial injury using trans oleic acid, its application in zebrafish models, and its construction method, and overcome the above-mentioned technical deficiencies, are problems that urgently need to be solved by those skilled in the art. Summary of the Invention
[0007] In view of this, this invention provides a reagent for preparing vascular endothelial injury using trans oleic acid, its application in a zebrafish model, and a construction method. It addresses the problems of existing vascular endothelial injury models, such as long modeling cycles, high costs, complex methods, difficult operation, insufficient visualization, and lack of high-throughput application capabilities. This model provides a new experimental platform and technical means for the study of the mechanisms of vascular endothelial injury-related diseases and for high-throughput screening of preventive and therapeutic drugs.
[0008] To achieve the above objectives, the present invention adopts the following technical solution: Application of oleic acid in the preparation of reagents for zebrafish vascular endothelial injury.
[0009] Preferably, the concentration of the oleic acid solution is 20-400 μM.
[0010] This invention also provides the application of oleic acid or a zebrafish vascular endothelial injury reagent containing oleic acid in the preparation of a zebrafish vascular endothelial injury model.
[0011] Preferred concentration of oleic acid solution: 150–300 μM.
[0012] This invention also provides a method for constructing a zebrafish model of vascular endothelial injury based on trans oleic acid, comprising the following steps: S1: Prepare zebrafish embryos that are developing normally 58-60 hours after fertilization; S2: The zebrafish embryos in S1 were exposed to an oleic acid solution and cultured at a constant temperature to create a model.
[0013] Preferred concentration of the oleic acid solution is 150–300 μM; The method for preparing zebrafish embryos in step S1 is as follows: adult zebrafish that are normally cultured are separated into males and females at a ratio of 1:1 to 2:1 for 12-14 hours, and then the male and female zebrafish are mixed to start mating and spawning; the embryos are collected within 30 minutes after fertilization and spawning; then they are rinsed with embryo culture water and placed in a constant temperature culture at 27.5-28.5℃. The oleic acid solution in step S2 is an oleic acid solution prepared from zebrafish embryo culture water; The raw materials for zebrafish embryo culture water include: 0.15 M NaCl, 5 mM KCl, 0.25 mM Na2HPO4, 0.45 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4 and 4 mM NaHCO3; The conditions for isothermal culture modeling in step S2 are: culture at 27.5-28.5℃ for 72-75 h, and change the medium every 22-24 h.
[0014] Preferably, it also includes step S3: characterizing the model; including: observing and recording the endothelial state of the zebrafish tail venous plexus using a fluorescence microscope, and detecting biological markers of endothelial damage using real-time quantitative PCR and enzyme-linked immunosorbent assay.
[0015] The present invention also provides a zebrafish model of vascular endothelial injury based on trans oleic acid, wherein the model is constructed using any of the above-described construction methods.
[0016] The present invention also provides a method for drug screening using the above-mentioned zebrafish model of vascular endothelial injury based on trans oleic acid, comprising: adding candidate drugs to the zebrafish model of vascular endothelial injury, and then culturing it in a constant temperature environment of 27.5-28.5℃ for 72-75 h; changing the medium every 22-24 h; observing and statistically analyzing the abnormal expansion state of the vascular endothelium of the tail vein plexus and detecting biological markers of vascular endothelial injury, and screening out drugs with vascular endothelial protective activity and the potential to salvage vascular endothelial injury.
[0017] Preferred: Zebrafish model of vascular endothelial injury: Esteric oleic acid induces abnormal expansion of the vascular endothelium of the zebrafish tail venous plexus, preventing embryonic death and resulting in abnormal expression of biological markers of vascular endothelial injury.
[0018] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses the preparation of vascular endothelial injury reagents using trans oleic acid, its application in zebrafish models, and a construction method, achieving the following technical effects: This invention utilizes zebrafish to construct a vascular endothelial injury model, which has the advantages of short experimental cycle, low cost, simple method and easy operation, and can realize real-time visual observation of pathological changes in vascular endothelium.
[0019] This invention is the first to discover that oleic acid treatment can induce vascular endothelial damage in zebrafish, expanding the application value of oleic acid as an experimental inducing agent in the field of vascular endothelial damage research.
[0020] The model established in this invention provides a reliable experimental platform and technical support for high-throughput screening of drugs with vascular endothelial protective effects and repair potential. Attached Figure Description
[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0022] Figure 1The attached figure is a schematic diagram of the high-throughput screening of therapeutic drugs based on a zebrafish model of vascular endothelial injury provided by the present invention.
[0023] Figure 2 The attached figure shows the effects of different concentrations of oleic acid solution provided by this invention on the vascular endothelium of zebrafish embryos; where A represents the abnormal expansion of the vascular endothelium of the caudal venous plexus of zebrafish embryos induced by different concentrations of oleic acid solution (10, 20, 50, 100, 150, 300, and 400 μM); B is a statistical graph of the vascular endothelial area of the caudal venous plexus after treatment with different concentrations of oleic acid solution for 72-75 h (the sample size of each group was 20, and 10 were randomly selected for observation and recording; *** indicates that the difference compared with the control group was statistically significant (P value < 0.001); ns indicates that there was no statistically significant difference compared with the control group (P value > 0.05); statistical values are expressed as mean ± standard deviation); C represents the effect of different concentrations of oleic acid solution (10, 20, 50, 100, 150, 300, 400, 500, 600, and 700 μM) after treatment for 72-75 h. The total mortality rate of each group after h (three parallel experiments for each group, and the values are expressed as mean ± standard deviation).
[0024] Figure 3 The attached figure shows the expression changes of biological markers of vascular endothelial injury in zebrafish under different concentrations of oleic acid exposure provided by this invention; where AC represents a classic pro-inflammatory factor used to indicate the presence of an inflammatory response in the body (…). TNF- α , IL-1β and IL-6 The relative mRNA expression levels of ) and D, representing the body's anti-inflammatory factors. IL-10 The relative mRNA expression level; EG is a biological marker used to indicate the presence of damage / inflammation in the vascular endothelium of the body ( vcam1b , vwf and thbd The relative mRNA expression levels of ) ; HI represents the levels of biological markers (NO nitric oxide and ET-1 endothelin-1) used to indicate functional (dilation and contraction) impairment of vascular endothelium in the body; each group has three biological replicates; * indicates: significant difference compared to the control group, P value < 0.05; ns indicates: no significant difference compared to the control group, P value > 0.05.
[0025] Figure 4The attached figure is an experimental analysis diagram of drug screening based on a zebrafish model of endothelial injury induced by trans-oleic acid, provided by this invention. A shows the effects of several candidate drugs on the endothelial phenotype of the tail venous plexus in a 300 μM trans-oleic acid-induced model; B is a statistical graph of the endothelial area of the tail venous plexus (**** indicates: significant difference compared to the control group, P value < 0.0001; ns indicates: no significant difference compared to the model group, P value > 0.05; #### indicates: significant difference compared to the model group, P value < 0.0001); CE represents the biological markers of endothelial injury / inflammation caused by candidate drugs in the body (…). vcam1b , vwf and thbd The effect of candidate drugs on the mRNA expression level of ) and the effect of FG on the content levels of biological markers of functional (dilation and contraction) damage to vascular endothelium (NO nitric oxide and ET-1 endothelin-1). Detailed Implementation
[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0027] This invention discloses the preparation of a vascular endothelial injury reagent using trans oleic acid, its application in a zebrafish model, and its construction method.
[0028] Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the experimental materials used in the embodiments, unless otherwise specified, were all purchased from commercial channels. Further details will not be provided here.
[0029] Example 1 Construction of a zebrafish model of vascular endothelial injury based on trans oleic acid Pretreatment: Normally cultured adult zebrafish are placed in mating tanks at a male-to-female ratio of 1:1 or 2:1 and separated by a partition overnight (8:00 PM - 8:00 AM) to allow them to adapt to the new environment; the next morning (8:00 AM - 9:00 AM), the partition is removed; the male and female zebrafish begin to mate and lay eggs; within 30 minutes after fertilization and spawning, the embryos are collected, and debris, unfertilized eggs, and feces are removed with a pipette; then the embryos are rinsed three times with embryo culture water and placed in a constant temperature incubator at 27.5-28.5℃.
[0030] S1: Zebrafish embryos with normal development 58-60 h post-fertilization (i.e., 58-60 hpf) were selected under a microscope (fluorescently labeled vascular endothelial transgenic strain: Tg). Flk1 (mCherry), red fluorescently labeled vascular endothelial cells, available from the National Zebrafish Resource Center); S2: Transfer the embryos into six-well plates containing different concentrations of oleic acid solutions (150 μM and 300 μM) prepared in zebrafish embryo culture water (formula: 0.15 M NaCl, 5 mM KCl, 0.25 mM Na2HPO4, 0.45 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4 and 4 mM NaHCO3); a control group (incubated with the same volume of embryo culture water) was also set up; each group was in triplicate; and the plates were incubated in a constant temperature incubator at 27.5-28.5℃ for 72-75 h, with the medium changed every 22-24 h.
[0031] S3: The abnormal expansion of the endothelial vessels in the tail venous plexus of zebrafish after exposure to trans-oleic acid was observed and recorded using a fluorescence microscope (Leica M205 FCA, Germany). The recorded images were then analyzed using ImageJ (version 1.51k) software to statistically analyze the area of the endothelial vessels in the tail venous plexus, thereby quantifying the abnormal expansion of the endothelial vessels in the tail venous plexus.
[0032] Using conventional real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA), the pro-inflammatory factors (P-P-C) in zebrafish exposed to trans oleic acid were analyzed. TNF-α , IL-1β and IL-6 ), anti-inflammatory factors ( IL-10 ), biological markers of vascular endothelial injury ( vcam1b , vwf , thbd The primers (NO and ET-1) were used for detection. The primer sequences are shown in Table 1 below.
[0033] Statistical analysis was performed using GraphPad Prism 8.0 software to analyze the endothelial area of the tail vein plexus, the expression levels of inflammatory factors (pro-inflammatory and anti-inflammatory), and the levels of endothelial injury markers.
[0034]
[0035] Example 2 High-throughput screening of therapeutic drugs based on a zebrafish model of vascular endothelial injury. As attached Figure 1 As shown, zebrafish embryos with normal development at 58-60 hours post-fertilization (i.e., 58-60 hpf) were selected (fluorescently labeled vascular endothelial transgenic strain: Tg( Flk1(mCherry) red fluorescently labeled vascular endothelial cells); 20-25 embryos per group were aliquoted into six-well plates; zebrafish embryo culture medium containing trans-oleic acid (150-300 μM) prepared with embryo culture water was added to the six-well plates to expose the zebrafish embryos as a model group; candidate drugs were added to the model (e.g., model + drug 1, model + drug 2, model + drug 3, etc.); the well plates were then placed in a constant temperature incubator at 27.5-28.5℃ for 72-75 h; the medium was changed every 22-24 h. Finally, the abnormal dilation of the vascular endothelium of the tail vein plexus was observed and statistically analyzed, and vascular endothelial injury biomarkers were detected to screen for drugs with vascular endothelial protective activity and the potential to repair vascular endothelial damage.
[0036] Technical effectiveness verification: Comparative Experiment 1 Effects of different concentrations of oleic acid solution on the vascular endothelium of the tail venous plexus in zebrafish embryos Prior to the scheme in Example 1, firstly in the zebrafish embryonic stage (Tg( Flk1 Zebrafish embryos (mCherry, a transgenic strain of red fluorescently labeled vascular endothelial cells) were exposed to different concentrations of transoleic acid (TRA). Embryos at 58–60 hpf were selected as the treatment subjects. At this stage, the vascular network of zebrafish is basically established and functionally complete, avoiding the developmental influence of TRLA on angiogenesis and thus more accurately reflecting its damaging effect on blood vessels. Embryos were exposed to different concentrations of TRLA (10, 20, 50, 100, 150, 300, and 400 μM) prepared in embryo culture water for 72–75 h. The abnormal dilation of the vascular endothelium of the tail venous plexus was observed and statistically analyzed. Figure 2 (A, shown in the white dashed box).
[0037] The results showed that, compared with the control group, treatment with 20–400 μM oleic acid could induce significant abnormal dilation of the vascular endothelium of the tail vein plexus. Figure 2 (A and B), among which the lesion phenotype differences in the concentration range of 150–400 μM were statistically significant. P＜0.05 ).
[0038] To determine the optimal exposure range for trans-oleic acid that induces significant abnormal vascular endothelial dilation without causing mortality, the effects of different concentrations (10, 20, 50, 100, 150, 300, 400, 500, 600, and 700 μM) of trans-oleic acid solution on zebrafish embryo mortality were further investigated. The results showed that the median lethal concentration (LC50) of trans-oleic acid... 50 The concentration was 455.1 μM. When the concentration reached 400 μM, some embryos died. Figure 2 (C)
[0039] In summary, the concentration range of 150–300 μM transoleic acid was determined to be the appropriate exposure concentration range that can significantly induce abnormal dilation (lesion) of the vascular endothelium of the caudal venous plexus in zebrafish embryos without causing embryonic death.
[0040] Comparative Experiment 2 Changes in biomarkers of trans-oleic acid-induced vascular endothelial injury in zebrafish Based on the established vascular endothelial lesion phenotype, to further clarify the range of transoleic acid concentrations that can induce vascular endothelial injury in zebrafish, zebrafish embryos at 58–60 hpf were exposed to different concentrations of transoleic acid (10, 20, 50, 100, 150, and 300 μM) prepared in embryo culture water. After continuous treatment for 72–75 h, the expression of vascular endothelial injury-related biomarkers was detected by conventional real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA).
[0041] The results are as follows Figure 3 As shown: Compared with the control group, exposure to 150 μM and 300 μM transoleic acid significantly induced an inflammatory response in zebrafish embryos, manifested as pro-inflammatory factors. TNF-α , IL-1β and IL-6 Significantly upregulated ( Figure 3 (A–C), and anti-inflammatory factors IL-10 Significant downregulation ( Figure 3 (D). Simultaneously, biomarkers related to vascular endothelial injury also underwent significant changes: vascular cell adhesion molecule 1 (VCI), reflecting the inflammatory state of the vascular endothelium, was observed to be significantly altered. vcam1b ), von Willebrand factor ( vwf ) and thrombomodulin ( thbd The expression levels of ) were significantly increased ( Figure 3 The presence of EG (endothelin-1) in the blood suggests inflammation and structural damage to the vascular endothelium; a significant decrease in nitric oxide (NO) levels, reflecting vascular function, and a significant increase in endothelin-1 (ET-1) levels indicate a disorder in vasodilation and vasoconstriction. Figure 3 (China HI).
[0042] It is worth noting that the 100 μM treatment group only showed biological responses on some indicators ( Figure 3 The C, D, F, and H markers were present, while the 10, 20, and 50 μM treatment groups showed no significant differences in any of the vascular endothelial injury markers compared to the control group. Figure 3 Middle A–I).
[0043] In summary, based on a comprehensive analysis of vascular endothelial lesion phenotypes and changes in the expression of biological markers, an exposure concentration range of 150–300 μM of transoleic acid can stably induce inflammatory responses and functional impairment in zebrafish vascular endothelium, providing a reliable model for the study of vascular endothelial injury mechanisms and drug screening.
[0044] Comparative Experiment 3 Drug screening examples in zebrafish models of trans-oleic acid-induced vascular endothelial injury. Based on the method in Example 2, to further demonstrate the practical application potential of this model in drug screening, the vascular endothelial protective activity of several drug components (OPA: Ophiopogon dihydroisoflavones A, CAS No. 75239-63-3; OPH: Ophiopogon dihydroisoflavones H, CAS No. 1427086-65-4; OPF: Ophiopogon dihydroisoflavones F, CAS No. 477336-79-1; OPC: Ophiopogon dihydroisoflavones C, CAS No. 477336-77-9) was evaluated.
[0045] Based on a 300 μM transoleic acid-induced vascular endothelial injury model, all patients were administered the drug at a concentration of 10 μg / L.
[0046] The results showed that, compared with the control group, the model group had abnormal expansion of the vascular endothelium of the tail vein plexus ( Figure 4 AB, **** P ＜0.0001 Compared with the model group, OPA, OPH, and OPF did not have a rescue effect on the pathological phenotype of abnormal dilation of the tail vein endothelium. Figure 4 AB in the middle P＞0.05 Only OPC can significantly reduce the lesion phenotype (####) P＜0.0001 To clarify the effects of several drug components on the expression of vascular endothelial injury biomarkers, routine assays such as qPCR and ELISA were used for detection. Results showed that, compared with the model group, OPA, OPH, and OPF significantly affected the expression of vascular endothelial injury biomarkers (…). vcam1b , vwf , thbd The effects of (NO and ET-1) were not significantly different. Figure 4 In the context of CG, only OPC can significantly correct the abnormal expressions of these metrics. Figure 4 Medium CG).
[0047] In this drug screening case, OPC drugs were identified as having vascular endothelial protective activity and the potential to repair vascular endothelial damage.
[0048] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.
[0049] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. Application of oleic acid in the preparation of reagents for zebrafish vascular endothelial injury.
2. The application as described in claim 1, characterized in that, The concentration of the oleic acid solution is 20-400 μM.
3. Application of oleic acid or oleic acid-containing zebrafish vascular endothelial injury reagent in the preparation of zebrafish vascular endothelial injury model.
4. The application as described in claim 3, characterized in that, The concentration of the oleic acid solution is 150–300 μM.
5. A method for constructing a zebrafish model of vascular endothelial injury based on trans oleic acid, characterized in that, Includes the following steps: S1: Prepare zebrafish embryos that are developing normally 58-60 hours after fertilization; S2: The zebrafish embryos in S1 were exposed to an oleic acid solution and cultured at a constant temperature to create a model.
6. The construction method as described in claim 5, characterized in that: The concentration of the oleic acid solution is 150–300 μM; The method for preparing zebrafish embryos in step S1 is as follows: adult zebrafish that are normally cultured are separated into males and females at a ratio of 1:1 to 2:1 for 12-14 hours, and then the male and female zebrafish are mixed to start mating and spawning; the embryos are collected within 30 minutes after fertilization and spawning; then they are rinsed with embryo culture water and placed in a constant temperature culture at 27.5-28.5℃. The oleic acid solution in step S2 is an oleic acid solution prepared from zebrafish embryo culture water; The raw materials for the zebrafish embryo culture water include: 0.15 M NaCl, 5 mM KCl, 0.25 mM Na2HPO4, 0.45 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4 and 4 mM NaHCO3; The conditions for isothermal culture modeling described in step S2 are: culture at 27.5-28.5℃ for 72-75 h, with the medium changed every 22-24 h.
7. The construction method as described in claim 6, characterized in that: It also includes step S3: model characterization; including: observing and recording the endothelial status of the zebrafish tail venous plexus using a fluorescence microscope, and detecting biological markers of endothelial damage using real-time quantitative PCR and enzyme-linked immunosorbent assay.
8. A zebrafish model of vascular endothelial injury based on trans oleic acid, characterized in that, The model is constructed using any of the construction methods described in claims 5-7.
9. A method for drug screening using the zebrafish model of vascular endothelial injury based on trans-oleic acid as described in claim 8, characterized in that, include: Candidate drugs were added to a zebrafish model of vascular endothelial injury, and the fish were then cultured in a constant temperature environment of 27.5-28.5℃ for 72-75 h. The medium was changed every 22-24 h. The abnormal expansion of the vascular endothelium of the tail vein plexus was observed and statistically analyzed, and the biological markers of vascular endothelial injury were detected. Drugs with vascular endothelial protective activity and the potential to rescue vascular endothelial injury were screened out.
10. The method as described in claim 9, characterized in that, The zebrafish model of vascular endothelial injury: oleic acid induces abnormal expansion of the vascular endothelium of the zebrafish tail venous plexus, preventing embryonic death, and resulting in abnormal expression of biological markers of vascular endothelial injury.