Biomarker comprising limch1

Abstract

To provide a novel biomarker and applications thereof.SOLUTION: The present invention relates to a biomarker for preeclampsia, comprising LIMCH1 or a gene encoding LIMCH1.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention broadly relates to LIMCH1 or substances targeting LIMCH1, uses thereof, and particularly to biomarkers containing LIMCH1. [Background technology]

[0002] Preeclampsia is a serious pregnancy complication that affects 5-10% of pregnant women and is a leading cause of maternal mortality. Globally, preeclampsia accounts for nearly 50,000 mothers' and 500,000 infant deaths each year.

[0003] Preeclampsia is a major clinical problem because it causes intrauterine growth retardation and therapeutic preterm delivery is a factor in extremely low birth weight and neonatal death.

[0004] Preeclampsia is treated symptomatically with antihypertensive drugs, but the ultimate treatment is limited to terminating the pregnancy, i.e., expelling the fetus and placenta. The problem with preeclampsia is that there is no effective treatment that allows women to continue their pregnancy safely.

[0005] Soluble fms-like tyrosine kinase 1 (sFlt-1) / placental growth factor (PlGF) is known to be an indicator for predicting the onset of preeclampsia. The significance of the sFlt-1 / PlGF ratio has been established, and when the ratio is 38 or less, it is considered that the possibility of developing PE within one week is low, and it is diagnosed as having a high negative predictive value. It has been reported that the higher the sFlt-1 / PlGF ratio, the more likely it is that severe complications such as pulmonary edema will develop in a short period of time (Harald Zeisler et al., “Predictive Value of the sFlt-1:PlGF Ratio in Women with Suspected Preeclampsia”, N Engl J Med. 2016 Jan 7;374(1):13-22., Stefan Verlohren et al., “Clinical interpretation and implementation of the sFlt-1 / PlGF ratio in the prediction, diagnosis and management of preeclampsia”, Pregnancy Hypertens. 2022 Mar;27:42-50.) [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Harald Zeisler et al., N Engl J Med, 2016 [Non-Patent Document 2] Stefan Verlohren et al., Pregnancy Hypertens, 2022 Summary of the Invention [Problem to be solved by the invention]

[0007] Preeclampsia is a pregnancy complication that can cause serious complications for both mother and baby, but there is currently no way to treat it while allowing pregnant women to continue their pregnancies safely.

[0008] The present invention aims to provide a novel indicator for the diagnosis of preeclampsia and a novel target for the treatment thereof. [Means for solving the problem]

[0009] The present inventors identified LIMCH1 through functional analysis of serum exosomes derived from patients with preeclampsia, and completed the present invention.

[0010] That is, the present application includes the following inventions. [1] A biomarker for preeclampsia, comprising LIMCH1 (LIM and calponin homology domains 1) or the gene encoding LIMCH1. [2] The biomarker described in [1], wherein the preeclampsia is early-onset or late-onset. [3] 1. A method for analyzing a sample from a subject suffering from or likely to suffer from preeclampsia, the method comprising determining the presence or absence of LIMCH1 or the gene encoding it in the sample. [4] The method according to [3], wherein a sample in which LIMCH1 is present is determined to be derived from a subject suffering from or likely to suffer from preeclampsia. [5] The method according to [4], wherein the sample contains exosomes. [6] The method according to any of [3] to [5], wherein LIMCH1 is expressed on exosomes. [7] The method according to [6], wherein the sample containing exosomes is derived from a biological material selected from the group consisting of whole blood, serum, plasma, saliva, urine, sputum, lymph, amniotic fluid, placental tissue, and cells. [8] The method according to any one of [3] to [7], further comprising a step of determining the presence or absence of soluble fms-like tyrosine kinase-1 (sFlt-1). [9] The method according to [8], in which sFlt-1 is measured as a ratio to placental growth factor (PIGF).

[10] A pharmaceutical composition for treating or preventing preeclampsia, comprising as an active ingredient a substance that suppresses the expression of LIMCH1 or the gene encoding same. Effect of the Invention

[0011] The present inventors have demonstrated for the first time that LIMCH1 is involved in the pathogenesis of preeclampsia. Therefore, the present invention makes it possible to provide a novel method for diagnosing preeclampsia using LIMCH1 as an indicator and a novel method for treating preeclampsia by targeting LIMCH1.

[0012] Disruption of endothelial cell-cell adhesion induced by placenta-derived LIMCH1-loaded exosomes may be associated with pulmonary edema and reversible occipital leukoencephalopathy through increased vascular permeability, and may be a potential new target. [Brief description of the drawings]

[0013] [Figure 1] Part of the procedure for identifying LIMCH1 as a preeclampsia-associated exosomal protein by proteomic analysis (left) and the normalized LIMCH1 expression level in EV proteomic data (right) are shown. [Diagram 2] The results of verifying LIMCH1 expression by placental immunohistochemical staining are shown. [Diagram 3] The results of placental immunohistochemical staining were further analyzed using HALO software. Blue (1): negative; yellow (2): weakly positive; orange (3): moderately positive; red (4): strongly positive. [Figure 4] The results of quantification of staining intensity are shown. [Diagram 5] 1 shows the results of a time-course analysis of LIMCH1 expression in JAR cells cultured under 20% oxygen and 1% oxygen (hypoxia). [Figure 6] The results of analyzing the cell proliferation ability of HUVEC (human umbilical vein endothelial cells) to which exosomes derived from LIMCH1-overexpressing JAR were added are shown. [Figure 7] 1 shows the results of a FITC-dextran permeability assay using HUVECs to which exosomes derived from LIMCH1-overexpressing JAR were added. [Figure 8] The results of RNA-Seq analysis of HUVECs exposed to LIMCH1-loaded exosomes are shown. [Figure 9] The results of GSEA (Gene Set Enrichment Analysis) analysis of HUVECs exposed to LIMCH1-loaded exosomes are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hereinafter, the embodiments or implementations of the present invention will be described in detail, however, the present invention is not limited to these and various modifications are possible without departing from the gist of the present invention.

[0015] Biomarkers In a first embodiment, the present invention provides a biomarker for diagnosing preeclampsia, comprising LIM and calponin homology domains 1 (LIMCH1). The term "biomarker" refers to a characteristic that can be objectively measured and evaluated as an indicator of normal biological processes, pathological processes, or pharmacological responses to therapeutic intervention, and as used herein refers to a biological molecule, such as a protein or gene, having such a characteristic.

[0016] In one embodiment, the biomarker consists of LIMCH1.

[0017] LIMCH1 was identified as an actin stress fiber-associated protein that regulates nonmuscle myosin II (NM-II) activity (Yu-Hung Lin et al., “LIMCH1 regulates nonmuscle myosin-II activity and suppresses cell migration”, Mol Biol Cell. 2017 Apr 15; 28(8): 1054-1065). Unless otherwise specified, LIMCH1 refers to the protein, and the gene encoding it is referred to as the LIMCH1 gene.

[0018] An example of human LIMCH1 is a protein consisting of 1057 amino acids as shown in SEQ ID NO: 1. The base sequence encoding the protein shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2. This sequence is isoform b that we used for overexpression, and there are 21 other variants, all of which are encompassed by the present invention.

[0019] LIMCH1 need not be full-length, but may be a fragment thereof, or a nucleic acid encoding it, a nucleic acid complementary thereto, or a transcription product thereof, so long as it can be used as an indicator for diagnosing preeclampsia or as a target thereof.

[0020] LIMCH1 can be detected in any biological material derived from a subject suffering from or likely to suffer from preeclampsia. The biological material is not particularly limited, but examples thereof include whole blood, serum, plasma, saliva, urine, sputum, lymph, amniotic fluid, placental tissue, and cells. LIMCH1 can be detected by known means. Specifically, the detection method includes a method of detecting LIMCH1 or a fragment thereof, or a complex by an immunological method such as ELISA using an antibody that recognizes LIMCH1 or a fragment thereof, or a complex, a method of isolating LIMCH1 or a fragment thereof, or a complex from a sample, cleaving it directly or as necessary with an enzyme, etc., and detecting it using a sequencer, and a method of detecting LIMCH1 or a fragment thereof, or a complex by mass spectrometry. The measurement of LIMCH1 fragments can be quantitatively measured by ELISA using antibodies that specifically recognize the LIMCH1 fragments, or liquid chromatography mass spectrometry.

[0021] When detecting a nucleic acid encoding LIMCH1, transcription products such as mRNA, cDNA, etc. may be measured. Methods for such measurements are not particularly limited, but include, for example, PCR, microarray, Northern blot, spectrophotometry, fluorometry, etc.

[0022] Preeclampsia is diagnosed after the 20th week of pregnancy due to high blood pressure and protein in the urine, or organ damage. Preeclampsia is broadly classified as early onset or late onset, with onset before 34 weeks being early onset (Eo) and onset after 34 weeks being late onset. It has been suggested that the mechanisms of onset of the two types may be different. Eo is more severe, with Eo-related maternal mortality rates ten times higher than in normal pregnancies, and in recent years, Eo-PE has been on the rise.

[0023] As used herein, the term "transcription product" refers not only to an RNA strand transcribed using the DNA of a gene as a template (an RNA strand synthesized by RNA polymerase), but also to an RNA strand modified within a cell after transcription. RNA strands as transcription products include a wide range of RNA strands, including, for example, messenger RNA (mRNA), as well as non-coding RNA that is not translated into proteins, such as ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA). These RNA strands may be processed within a cell after transcription.

[0024] In the present invention, other indicators for detecting preeclampsia can also be used to diagnose the disease in a subject with high accuracy. For example, soluble fms-like tyrosine kinase-1 (sFlt-1) or fragments thereof, or nucleic acids encoding them, complementary nucleic acids, or transcription products thereof, can be used in combination with LIMCH1 to analyze samples from subjects who are suffering from or may be suffering from preeclampsia.

[0025] sFlt-1 is an inhibitor of PlGF (placental growth factor), an angiogenic factor involved in placental formation, and these factors are involved in the pathogenesis of preeclampsia. The sFlt-1 / PlGF ratio is known to be an indicator for predicting the onset of PE.

[0026] When the sFlt-1 / PlGF ratio is below 38, the possibility of developing PE within one week is considered low, and the negative predictive value is considered high. The higher the sFlt-1 / PlGF ratio, the more likely it is that serious complications such as pulmonary edema will develop in a short period of time.

[0027] Detection kit In a second embodiment, the present invention provides a kit for detecting biomarkers for diagnosing preeclampsia, including LIMCH1.

[0028] The kit of the present invention includes a reagent for measuring the presence or expression level of a biomarker. For example, when measuring the expression level of a biomarker by immunoassay, an antibody against each marker is used as a reagent. If necessary, the kit may also include a diluent for a body fluid derived from a subject, an antibody-immobilized solid phase, a reaction buffer, a washing solution, a labeled secondary antibody or an antibody fragment thereof, a reagent for detecting a labeled substance, a standard substance, and the like. Examples of the diluent include physiological saline, a surfactant, an aqueous solution containing a protein such as bovine serum albumin (BSA) or casein in a buffer solution, and the like.

[0029] In one embodiment, the reagent for measuring the presence or expression level of LIMCH1 is an anti-LIMCH1 antibody.

[0030] The antibody-immobilized solid phase is prepared by molding various polymeric materials to suit the purpose, and immobilizing antibodies or antibody fragments thereof on the material. The shape of the immobilized phase may be a tube, cup, bead, plate, stick, etc., and the material may be a polymeric material such as polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin, agarose, cellulose, polyethylene terephthalate, glass, ceramics, metal, etc. Methods for immobilizing antibodies include known methods such as physical methods, chemical methods, or a combination of these.

[0031] The reaction buffer may be any that provides a solvent environment for the binding reaction between the antibody on the antibody-immobilized solid phase and the antigen in the body fluid derived from the test animal, and examples of the reaction buffer include reaction buffers containing surfactants, buffer solutions, proteins such as BSA and casein, preservatives, stabilizers, reaction promoters, etc.

[0032] The labeled secondary antibody or antibody fragment thereof can be an antibody or antibody fragment thereof that recognizes an antibody against the code for the biomarker, which is labeled with a labeling enzyme such as horseradish peroxidase (HRP), bovine intestinal alkaline phosphatase, or β-galactosidase, or which is mixed with a buffer solution, a protein such as BSA or casein, a preservative, etc.

[0033] Reagents for detecting the labeled substance may vary depending on the labeling enzyme. For example, when horseradish peroxidase is used, examples of the reagent include absorbance measurement substrates such as tetramethylbenzidine and orthophenylenediamine, fluorescent substrates such as hydroxyphenylpropionic acid and hydroxyphenylacetic acid, and luminescent substrates such as luminol. When alkaline phosphatase is used, examples of the reagent include absorbance measurement substrates such as 4-nitrophenyl phosphate and fluorescent substrates such as 4-methylumbelliferyl phosphate.

[0034] In addition, when the expression level of a biomarker is measured by PCR, the kit may contain, for example, a primer capable of specifically amplifying a part or the whole of the biomarker. In addition, reverse transcriptase, Taq polymerase, a reaction buffer, etc. may be contained as necessary. In the case of real-time PCR, a fluorescent reagent, etc. may also be contained.

[0035] The kit of the present invention may also appropriately include a package insert showing the method of use, etc. Furthermore, the kit of the present invention may broadly include an apparatus, etc. used for detecting the biomarker.

[0036] Analysis method In a third embodiment, the present invention provides a method for analyzing a sample from a subject who is suffering from or may be suffering from preeclampsia. The method of the present invention can be used by a clinical laboratory technician to evaluate in vitro test information related to the disease. Thus, the method of the present invention can also be used by a physician to properly diagnose or treat preeclampsia, directly or indirectly.

[0037] The detection methods of the present invention comprise the step of detecting or quantifying one or more biomarkers in a sample derived from a subject.

[0038] In one embodiment, detecting or quantifying the biomarker comprises determining the presence or absence of LIMCH1 or the gene encoding it.

[0039] As used herein, "subject" refers to any vertebrate, including humans and non-human primates (e.g., monkeys such as cynomolgus monkeys, rhesus monkeys, chimpanzees, etc.), and other mammals, such as cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice). In some embodiments, the subject may be a human or a non-human animal. In some embodiments, the subject may be a patient at risk of developing preeclampsia or a patient already suffering from such disease.

[0040] As used herein, a "specimen" broadly refers to a biological material believed to contain a target such as LIMCH1. Any cell, tissue, or body fluid may be used to obtain a specimen. Such cells, tissues, and body fluids may include tissue sections such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood (such as whole blood), plasma, serum, red blood cells, platelets, sputum, stool, tears, mucus, saliva, gynecological fluids, amniotic fluid, urine, bronchoalveolar lavage (BAL) fluid, interstitial fluid, ocular lens fluid, cerebrospinal fluid, sweat, nasal secretions, synovial fluid, leucorrhea, and the like. Cells and tissues may include lymph, ascites, urine, peritoneal fluid, cerebrospinal fluid, and the like. A specimen may be a culture of biological material derived from a subject. Isolation or purification of a target protein, etc. may be performed from the sample depending on the purpose.

[0041] In one embodiment, the specimen comprises extracellular vesicles, preferably exosomes.

[0042] Extracellular vesicles and exosomes are isolated from samples by known methods such as ultracentrifugation, density gradient ultracentrifugation (DGUC), coprecipitation, ultrafiltration, immunoaffinity purification, size exclusion chromatography purification, microfluidic separation techniques, etc. The sample containing extracellular vesicles or exosomes is selected from the group consisting of, for example, whole blood, serum, plasma, saliva, urine, sputum, lymph, amniotic fluid, placental tissue, and cells.

[0043] In one embodiment, LIMCH1 is expressed on an exosome.

[0044] The sample collected from the subject can be appropriately pretreated as required for detecting the biomarker. For example, it is preferable to appropriately dilute the sample before use. The solution used for dilution is not particularly limited as long as it does not affect the detection of the biomarker.

[0045] If a biomarker is detected in a sample or its concentration is elevated compared to that of a healthy individual, it can be determined that the subject is susceptible to preeclampsia, has the disease, or is progressing with the disease.

[0046] Screening Methods In a fourth embodiment, the present invention provides a method for screening a drug for treating or preventing preeclampsia. The screening method of the present invention may comprise the step of selecting a substance that inhibits expression of LIMCH1 or a fragment thereof, or a nucleic acid encoding the same, a nucleic acid complementary thereto, or a transcription product thereof, as a drug candidate.

[0047] The pharmaceutical obtained by the screening method of the present invention is not particularly limited in terms of its administration route, dosage, usage, dosage form, etc., so long as it exerts the desired effect, particularly the effect of inhibiting LIMCH1 expression, and ultimately the effect of treating or preventing preeclampsia.

[0048] Pharmaceutical Compositions In a fifth embodiment, the present invention provides a pharmaceutical composition for treating or preventing preeclampsia, which comprises, as an active ingredient, LIMCH1 or a fragment thereof, or a nucleic acid encoding the same, a nucleic acid complementary thereto, or a substance that inhibits the expression of a transcription product thereof.

[0049] The pharmaceutical composition is not particularly limited in terms of its administration route, dosage, usage, dosage form, etc., so long as it exerts the effect of treating or preventing preeclampsia.

[0050] In one embodiment, the active ingredient is an anti-LIMCH1 antibody.

[0051] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to these. EXAMPLES

[0052] <Specimen and sample collection> Among women who provided written informed consent, we used serum from seven women with early-onset preeclampsia (PE) who delivered at our hospital between 2014 and 2022 after PE onset, as well as serum from nine women who did not later develop PE, all of which were matched for gestational age. The serum was centrifuged at 3,000 rpm (500 g) at 4°C for 10 minutes, after which the supernatant was collected and stored at -80°C until use.

[0053] Subject information is shown in Table 1. [Table 1]

[0054] <Isolation of extracellular vesicles (EVs)> One milliliter of each sample was centrifuged at 10,000 g for 40 minutes at 4° C. D-PBS was added to the supernatant, which was then filtered through a 0.45 μm filter (Millex-HV 13 mm, Millipore) and ultracentrifuged at 110,000 g for 70 minutes at 4° C. using an MLS50 rotor (Beckman Coulter Inc, USA).

[0055] The supernatant was discarded, D-PBS was added, and albumin and IgG were removed using the ProteoExtract Albumin / IgG Removal Kit (Merck & Co. Inc, USA).D-PBS was further added, and the cells were subjected to ultracentrifugation at 110,000g, 4℃, and 70 min to isolate small EVs.

[0056] <lc-ms ms> Small EVs is a registered trademark of EasyPep ) Peptides were prepared using a Mini MS Sample Prep Kit (Thermo Fisher Scientific Inc., USA) and subjected to LC-MS / MS.

[0057] Peptide analysis was performed by LC-MS via a nanoelectrospray ion source using an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) coupled with an UltiMate3000 RSLCnano LC system (Dionex Co., The Netherlands) and a nano high-performance LC capillary column (150 mm i.d. × 75 μm) (Nikkyo Technos Co., Japan).

[0058] Reversed-phase chromatography was performed with a linear gradient of solvent A (2% acetonitrile containing 0.1% formic acid) and solvent B (95% acetonitrile containing 0.1% formic acid) (0 min, 5% B; 100 min, 40% B) at an estimated flow rate of 300 nl / min. A precursor ion scan with mass-to-charge ratio (m / z) from 400 to 1600 was performed prior to MS / MS analysis. MS / MS was performed using an isolation width of 1.6 m / z using a quadrupole, high-energy C trap dissociation fragmentation with a normalized collision energy of 35%, and rapid-scan MS analysis in the ion trap. Only precursors with charge states 2–6 were sampled in MS2. The dynamic exclusion time was set to 15 s and the tolerance was set to 10 ppm (parts per million). The instrument was run in top speed mode with 3-s cycles.

[0059] <Proteomics data analysis> Raw data were processed using Proteome Discover (version 1.4, Thermo Fisher Scientific) alone or in combination with the MASCOT search engine (version 2.7.0, Matrix Science Inc., Boston, MA, USA) to identify proteins. Peptides and proteins were identified using the UniProt (2021_02) database with a precursor mass tolerance of 10 ppm and a fragment ion mass tolerance of 0.8 Da. The fixed modification was carbamidomethylation of cysteine, and the variable modification was oxidation of methionine. Two missed cleavages by trypsin were allowed.

[0060] A total of 16 women, including 7 with early-onset preeclampsia (PE) and 9 without PE, were analyzed. 356 proteins that were detected in 8 or more cases were analyzed, and the quantitative values ​​obtained were logarithmic and corrected by the Quantile method. Proteins that were not detected in 9 or more cases were excluded.

[0061] Because PE patients were divided into two groups, comparison with controls was performed for cluster 1 and cluster 2. 76 proteins that overlapped between cluster 1 and cluster 2 were selected as PE-associated EV proteins.

[0062] Among the 76 proteins that were selected as PE-associated EV proteins and whose expression was elevated in PE patient serum EVs, we selected LIMCH1, whose expression was also elevated in PE placentas in two GEO datasets (GSE114691 and GSE148241: placental RNA-seq) (Figure 1, left). The normalized LIMCH1 expression levels in the EV proteomics data are shown in a graph in comparison with the control (Figure 1, right).

[0063] <Immunohistochemical staining> LIMCH1 expression was examined by placental immunohistochemical staining. Seven cesarean section placentas from patients with PE and five cesarean section placentas from patients with induced preterm birth without PE were used for serum proteomics analysis. Paraffin sections were deparaffinized and antigen retrieval was performed with 10 mM citrate buffer in a microwave oven. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide / methanol for 30 minutes. Sections were blocked with 10% goat serum for 30 minutes, and anti-LIMCH1 antibody (ab96178; Abcam, Oxford, UK) was added at a 1 / 200 dilution and incubated overnight at 4°C. After washing with PBS, sections were treated with Histofine Simple Stain MAX-PO® kit (Nichirei Bioscience Inc., Tokyo, Japan), visualized with diaminobenzidine (DAB; Nichirei Bioscience), and counterstained with Mayer's hematoxylin solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Image analysis was performed using HALO software (Indica Labs, Albuquerque, NM, USA).

[0064] The results of immunostaining are shown in Figure 2. The results of immunohistochemical staining indicate that LIMCH1 is expressed mainly in syncytiotrophoblasts.

[0065] Analysis by HALO software is shown in Figure 3. Blue (1) is negative, yellow (2) is weakly positive, orange (3) is moderately positive, and red (4) is strongly positive. H-score was calculated as 1 x the percentage of weakly positive cells (%) + 2 x the percentage of moderately positive cells (%) + 3 x the percentage of strongly positive cells (%).

[0066] The results of quantifying the staining intensity are shown in Figure 4. The H-score was significantly higher in the PE placenta than in the normal placenta (p<0.05).

[0067] Next, we confirmed LIMCH1 expression in JAR cells, a choriocarcinoma cell line. After culturing JAR cells under 20% oxygen and 1% oxygen (hypoxia) for 8, 24, 48, and 72 hours, RNA was extracted and reverse transcribed, and quantitative PCR was performed to examine the change in LIMCH1 expression due to 20% and 1% oxygen using GAPDH as an internal standard. Total RNA was extracted using QIAzol and miRNeasy Mini Kit (Qiagen, Hilden, Germany). The results are shown in Figure 5. It was revealed that LIMCH1 expression in JAR cells was significantly increased by hypoxia (p<0.05). Since it has been reported that PE placentas are exposed to hypoxia, it was suggested that the increase in LIMCH1 expression in PE placentas may be related to hypoxia.

[0068] Next, FLAG-LIMCH1 or only the FLAG tag was introduced into JAR using a retrovirus to create a LIMCH1 constitutively overexpressing line and a control line. Exosomes were extracted from each cell line using ultracentrifugation, and it was confirmed that LIMCH1 was successfully overexpressed in exosomes. The cell proliferation ability of HUVECs (human umbilical vein endothelial cells) to which LIMCH1-overexpressing JAR-derived exosomes had been added was examined, and a FITC-dextran permeability assay was performed. The cell proliferation ability was evaluated using an Incucyte SX5 (SARTORIUS, Göttingen, Germany). For the FITC-dextran permeability assay, HUVECs were seeded on a 12-well transwell with 0.4 μm pores, and when they reached confluence, EV was added. After 24 hours, 40 kDa FITC-dextran was added, and the OD value of the FITC in the lower layer was measured over time using a plate reader.

[0069] EVs derived from LIMCH1-overexpressing JARs showed no change in the cell proliferation ability of HUVECs (human umbilical vein endothelial cells) compared to control EVs (Figure 6), but a fluorescein isothiocyanate (FITC) dextran permeability assay showed a significant increase in permeability with the addition of LIMCH1-loaded EVs (p<0.01) (Figure 7).

[0070] We performed RNA-Seq analysis and Gene Set Enrichment Analysis (GSEA) on HUVECs exposed to LIMCH1-carrying EVs. Total RNA was extracted from HUVECs exposed to LIMCH1-carrying EVs or control EVs for 24 hours using QIAzol and miRNeasy Mini Kit (Qiagen). RNA-Seq was performed using NovaSeq (Illumina, CA, USA). GSEA analysis was performed using the website (http: / / www.broadinstitute.org / gsea / index.jsp).

[0071] As a result, the gene groups related to cell-cell adhesion and fluid volume regulation were significantly changed between HUVECs exposed to LIMCH1-loaded EVs and those exposed to control EVs (Figures 8 and 9). These results suggest that the disruption of endothelial cell-cell adhesion caused by LIMCH1-loaded EVs may be a mechanism for the onset of PE complications such as pulmonary edema and reversible occipital leukoencephalopathy through increased vascular permeability.

Claims

1. A biomarker for preeclampsia, comprising LIMCH1 (LIM and calponin homology domains 1) or the gene encoding LIMCH1.

2. The biomarker of claim 1, wherein the preeclampsia is early-onset or late-onset.

3. A method for analyzing a sample from a subject suffering from or likely to suffer from preeclampsia, the method comprising determining the presence or absence of LIMCH1 or the gene encoding it in the sample.

4. 4. The method of claim 3, wherein the sample in which LIMCH1 is present is determined to be from a subject suffering from or likely to suffer from preeclampsia.

5. The method of claim 4, wherein the sample contains exosomes.

6. The method of claim 5, wherein LIMCH1 is expressed on exosomes.

7. 7. The method of claim 6, wherein the exosome-containing sample is derived from a biological material selected from the group consisting of whole blood, serum, plasma, saliva, urine, sputum, lymph, amniotic fluid, placental tissue, and cells.

8. The method according to claim 3 or 4, further comprising the step of determining the presence or absence of soluble fms-like tyrosine kinase-1 (sFlt-1).

9. The method of claim 8, wherein sFlt-1 is measured as a ratio to placental growth factor (PIGF).

10. A pharmaceutical composition for treating or preventing preeclampsia, comprising as an active ingredient a substance that suppresses the expression of LIMCH1 or a gene encoding same.

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