Regulation of Partial Endothelial-to-Mesenchymal Transition by circATXN1 in Ischemic Diseases

doi:10.21203/rs.3.rs-4997868/v1

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Regulation of Partial Endothelial-to-Mesenchymal Transition by circATXN1 in Ischemic Diseases

https://doi.org/10.21203/rs.3.rs-4997868/v1

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We conducted circRNA sequencing of endothelial cells (ECs) subjected to hypoxia and serum deprivation to ascertain molecules linked to partial or transient mesenchymal activation in endothelial cells (ECs) following tissue ischemia/hypoxia. The outcome demonstrated that the circATXN1 expression was significantly raised. Herein, we estimated the circATXN1 function in regulating mesenchymal characteristics during the partial endothelial-to-mesenchymal transition (EndMT) and its impact on angiogenesis. Over-expression of circATXN1 inhibited mesenchymal traits in endothelial cells, whereas circATXN1 knockdown promoted partial EndMT by upregulating mesenchymal markers and partially reducing endothelial markers. In vivo, circATXN1 knockdown enhanced angiogenesis and functional recovery post-ischemia, while circATXN1 overexpression impeded these processes. Mechanistically, circATXN1 specifically stabilized SLUG mRNA by interacting with ALKBH5, an mRNA demethylase, without altering its transcription. CircATXN1 regulated the subcellular localization of ALKBH5, preventing its nuclear entry and thus stabilizing SLUG mRNA via decreased m6A methylation. This interaction was confirmed through RNA pulldown, LC-MS/MS, and RIP assays. Our findings position circATXN1 as a crucial regulator in ischemic diseases, particularly in angiogenesis. CircATXN1’s downregulation enhances SLUG-mediated partial EndMT, leading to increased ECs proliferation, migration, and sprouting. Targeting circATXN1 with RNA interference (RNAi) represents a potential therapeutic strategy for ischemic disorders.

Health sciences/Biomarkers/Diagnostic markers

Biological sciences/Cell biology/Cell signalling/Stress signalling

Partial Endothelial-to-Mesenchymal Transition

Ischemic Diseases

Endothelial Cells

m6A Methylation

Non-coding RNA

Ischemic conditions, encompassing ischemic heart disease, peripheral artery disease, and cerebral ischemia, are marked by the blockage or limitation of blood flow in different tissues and organs caused by hypoxia/ischemia [1–3]. These disorders have a significant impact on world health, causing high rates of the disorders and death. Endothelial-to-mesenchymal transition (EndMT) is a dynamic biological process occurring in endothelial cells (ECs) that involves the development and progression of ischemic conditions [4–6]. This process is distinguished by alterations in the genes and proteins expression, encompassing the hindrance in endothelial markers (encompassing VE-cadherin and CD31) and a rise in mesenchymal markers (encompassing SM22α and α-smooth muscle actin (αSMA)). The phenotypic switches associated with EndMT are consistent with molecular changes, leading to the loss of endothelial properties involving cytokine-induced leukocyte adhesion and tube formation and the emergence of mesenchymal traits, including matrix invasion and migratory capacity. EndMT exemplifies endothelium plasticity [7]. The initiation and magnitude of EndMT are impacted by intrinsic factors, such as embryonic lineage [7–9] and genetic makeup [10], as well as external elements, including hemodynamics, cytokines, and hypoxia, which can trigger EndMT independently or in combination [7].

Partial or reversible EndMT, unlike a complete mesenchymal transition, leads to an intermediate phenotype in endothelial cells (ECs) that are undergoing a transition toward a mesenchymal state [11]. This transition retains or partially loses endothelial traits while acquiring mesenchymal properties. Angiogenesis processes, such as tubulogenesis and sprouting, necessitate a partial or reversible EndMT [12]. During this stage, cells have a temporary loss of apical-basal polarity and acquire the ability to migrate, but they do not fully develop all mesenchymal characteristics or totally lose cell adhesion [11, 13, 14]. Angiogenesis is also crucial for postischemic revascularization and functional recovery in ischemic diseases. Recent studies using single-cell transcriptomics have shown the temporary and changing partial EndMT nature in ischemic models [15], encompassing myocardial infarction (MI) and hindlimb ischemia (HLI). These results demonstrate that there is an increase in mesenchymal gene enrichment in vessel sites that have undergone clonal expansion. This indicates that hypoxia-stimulated partial EndMT may help promote neovascularization under ischemic conditions.

Circular RNAs (circRNAs) are a distinct kind of non-coding RNAs that are often produced via the process of backsplicing [16, 17]. These entities are distinguished by their strong stability and a covalently sealed uninterrupted loop, without 5' to 3' polarity and a polyadenylated tail. This allows them to withstand RNA exonucleolytic digestion [18]. CircRNAs have been suggested as important regulators in the processes of growth [19], development [16, 20], and tissue regeneration [21] and are the key to revealing the pathological mechanism of diseases and search for therapeutic targets. Their role in the initiation and progression of ischemic diseases has been extensively studied. Emerging evidence indicates that circRNAs may impact ischemic diseases by regulating EndMT. For instance, circDLGAP4 could improve ischemic stroke outcomes by targeting miR-143 to regulate EndMT related to blood-brain barrier integrity [22]. Additionally, circUCK2 was proved to upregulate HECTD1 expression by binding with FUS, inhibiting EndMT to mitigate BBB damage in ischemic stroke [23]. However, few investigations have been conducted on the connection between circRNAs and hypoxia-induced partial EndMT in ischemic disease. Considering the similarities in biological characteristics between angiogenesis and partial EndMT, it is important to investigate the molecular mechanism behind hypoxia-induced partial EndMT and its impact on revascularization in both diseases.

Herein, we reveal that a circRNA called circATXN1, which originates from ATXN1 is extremely conserved and is strongly activated by hypoxia. Furthermore, it is significantly elevated in the ischemic tissue of HLI and MI models. Besides, circATXN1 potently hampers the partial EndMT process and inhibits revascularization and functional recovery in these ischemic tissues, while knocking down circATXN1 achieves quite the opposite effects. Moreover, circATXN1 directly binds ALKBH5 to prevent it from entering the nucleus and exerting demethylation, resulting in the destabilization of SLUG mRNA, which acts as a master regulator of partial EndMT. Overall, our research suggests that the circATXN1 gene has a pivotal contribution in progressing hypoxia and ischemia, resulting in a very potential target for treating ischemic disorders.

All human studies and animal procedures conformed to the National Institutes of Health (NIH) guidelines, and were approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, China, and conducted in accordance with the Declaration of Helsinki.

Cell culture

The HEK293T cells were purchased from Zhongqiaoxinzhou Biotechnology (Shanghai, China) and cultivated in high-glucose DMEM enriched with 10% FBS and 100U/mL penicillin-streptomycin (P/S). Human cardiac microvascular endothelial cells (HCMECs) at passage 3, sourced from Lonza Bioscience (Basel, Switzerland), were cultivated in endothelial culture media (ECM; ScienCell, Carlsbad, CA, USA). Primary mouse CMECs (MCMECs) were obtained from the left ventricles of C57BL/6 mice using a previously established method [24, 25]. After the epicardial, endocardial, and coronary arteries were removed, the left ventricular tissue was chopped and digested with liberase. The cell suspension was incubated for 30 min at 4°C with microbeads conjugated to anti-CD31 antibody (ab7388, Abcam, UK; Thermo Fisher Scientific, USA) under gentle rotation. The first step was the isolation of primary MCMECs using a magnetic separator. These cells were subsequently cultivated on fibronectin-coated plates using a complete ECM (ScienCell, USA). Following the removal of serum, the cells were cultivated in hypoxic conditions consisting of 5% CO₂, 1% O₂, and the remaining balance of N₂ for a period of 1–3 days, and the cells in the control group were kept in the normoxia mixture (5% CO₂ and 21% O₂), both the hypoxic cells and normoxic cells were derived from the same maternal generation.

Recombinant adenovirus and adeno-linked virus generation

In vitro experiments, recombinant adenovirus was used to infect endothelial cells to interfere with cirAXTN1, SLUG, and ALKBH5. Additionally, recombinant adeno-associated virus (AAV) type 9 systems, including vectors with control/circATXN1 driven by an endothelial-specific promoter (Tie promoter) or scramble shRNA/circATXN1 shRNA, were administrated into the mice tail veins to modify circATXN1 levels in the heart. The AAV2 vector genomes were administered to the mice via tail vein injection, with 100 µl of the virus containing 2 × 10¹¹ VG.

Mice lower limb ischemia model

After anesthesia with inhaled 2% isoflurane, C57/BL6J mice (8–10 weeks, male) were subjected to a femoral artery surgery in the right hind limb. There was one ligation site at the opening of the profundal femoris artery and another near the opening of the saphenous artery. Then, the femoral artery between the two points was cut off, and finally, sew up the wound. For gain and loss of function experiments, the mice were injected with the AAV virus in the gastrocnemius muscle 1 week before HLI surgery. The blood flow was calculated by laser Doppler scanning system (Perimed, Sweden) on postoperative days 1, 3, 7, and 14. The perfusion ratio was quantified from the ischemic limb (right) and the control limb (left), which reflected the recovery of blood flow.

Mice myocardial infarction model

After anesthesia with inhaled 2% isoflurane, C57/BL6J mice (6–8 weeks, male) received a thoracotomy with the assistance of a small animal respirator. Under the microscope, the left anterior descending coronary artery was ligation with 10–0 nylon wire, and the color of the surrounding myocardial tissue turned immediately pale. Mice in the sham group received all the surgical procedures induced by MI except the ligation step. For gain and loss of function experiments, the mice were administered with AAV virus via the tail vein 1 week before MI surgery. Left ventricular ejection fraction was measured on pre- and postoperative days 7,14, and 28 by a small animal ultrasound instrument.

RT-qPCR

Aligning with manufacturer recommendations, TRIzol reagent (Takara, Japan) was implemented to obtain total RNA. The PrimeScriptTM RT reagent Kit (Takara, Japan) was implemented to convert 1 µg of total RNA into cDNA with reverse transcription, and random hexamer primers were used specifically for the reverse transcription of circRNAs. If RNase R pretreatment is required, 5 µg of total RNAs and 10 units of RNase R were incubated at 37°C for 15 min. The qPCR experiment was conducted with the SYBR Green master mix (Vazyme, China) and examined using the QuantStudio 6 Operating Software (Life Technologies, USA). Primer sequences are shown in the Supplementary table 3.

RNA sequencing for circRNA

Total RNA was obtained with the TRIZOL reagent (Takara, Japan) to find differentially expressed circRNAs in HCMECs under normoxic and hypoxic conditions. rRNA Depletion and VAHTS Universal V6 RNA-seq Library Prep Kits (Vazyme, China) were utilized to produce circRNA-sequencing libraries based on the manufacturer's recommendations. The CIRC2 algorithm was employed to identify circRNAs, which were then annotated by comparing sequences in circBase. circRNAs with a log fold change greater than 1 and a p-value less than 0.05 between the two conditions were considered to be significantly differentially expressed.

The mRNA half-life determination

The cells were treated with 5 µM actinomycin D (Sigma, USA) and afterward digested at specified time intervals for the purpose of RNA extraction. At each time point, the initial level (0 h) was implemented to adjust the remaining RNA levels. The decay kinetics of mRNA were analyzed by GraphPad Prism software, and the result was rendered with a one-phase exponential decay curve.

Cytosolic/Nuclear fractionation

Typically, 1×10⁷ cells were gathered with trypsin-EDTA buffer. After washing and centrifugation 2–3 times, the cell pellets were operated according to the instructions of the NE-PER Nuclear and Cytoplasmic Extraction Reagents step by step. The obtained nuclear/cytosolic extracts were used for subsequent Western blot (WB) analysis. As references for the cytoplasmic and nuclear fractions, GAPDH and LMNB were chosen, respectively.

RNA fluorescence in situ hybridization (RNA-FISH)

A biotin-labeled oligonucleotide probe was developed in TSINGKE, either antisense (AS) or sense (S), that specifically targets the circ-ATXN1 junction. The biotin RNA Labeling Mix and T7 RNA polymerase (Roche, Germany) were employed to generate the probes for 18S and U6 through in vitro transcription of PCR fragments. The hybridization occurred inside a humidified chamber at 37°C for 16 h, either with or without treatment with RNase R at a concentration of 3 U/mg. The Fluorescent In Situ Hybridization kit (RiboBio, Guangzhou, China) was implemented for signal detection, whereas 4′,6-diamidino-2-phenylindole (DAPI, Sigma) was implemented for nuclei counterstaining. Primer sequences are shown in the Supplementary table 3.

Immunofluorescence assays

HCMECs were planted on the cell slide to a suitable density and treated with 4% paraformaldehyde for 15 min to fix them. The cell specimens were afterward rinsed three rounds with pre-chilled PBS and subjected to 0.1% Triton X-100 at ambient temperature for 15 min to allow for permeabilization. Following a 1–2 h blocking with a 1% BSA solution at ambient temperature, the slides were treated with primary antibodies and kept in a humidified container overnight at 4°C. After rinsing the cell slides with PBS three rounds for 5 min each. They were treated with fluorescent dye-labeled secondary antibodies for 1 h at ambient temperature in the darkness. Afterward, the slides were incubated with DAPI for 15 min at ambient temperature without exposure to light. For immunofluorescence staining (IFS) of tissues, the tissues need to be fixed, paraffin-embedded, and sectioned. Sections were successively deparaffinized, rehydrated, and treated in a pressure cooker with sodium citrate antigen repair solution. The subsequent steps, including blocking, primary and secondary antibody incubation, and DAPI staining, refer to the procedures described above. A fluorescence microscope (Zeiss, Germany) was used to visualize and acquire images, and ImageJ software was deployed to analyze images.

Immunohistochemistry

Immunohistochemical staining was conducted as earlier established [26]. Tissue samples were embedded in paraffin and sectioned, Vimentin immunohistochemical staining was performed. Photographs were taken using an optical microscope (Olympus, Japan).

Western Blot analysis

The cells were gathered and disrupted with RIPA buffer containing a protease inhibitor. Typically, a quantity of 20 µg of protein was isolated with a 10% SDS-PAGE method and then electro-transferred onto PVDF membranes (Millipore, USA). The membranes were obstructed with a 5% solution of skim milk and thereafter incubated overnight at 4°C with primary antibodies. Following the washing step with TBS-T solution, HRP-conjugated secondary antibodies were introduced and incubated at ambient temperature for 2 h. The blots were processed with Super ECL plus reagents (Vazyme, China) and were displayed using automated chemiluminescence image analysis equipment (Tanon 5200, China). Antibodies used are shown in the Supplementary table 2.

Migration assays

After 24h infection, HCMECs were enzymatically broken down and resuspended in serum-free ECM. A suspension containing 1×10⁴ cells was placed into the upper chambers, while a full medium with 10%FBS was introduced to a 24-well plate lower chambers. The entire Transwell® device (Corning, USA) was incubated in the cell incubator for 24h, and the upper chamber was successively transferred to 4% paraformaldehyde for fixation and 0.1% crystal violet for staining. Next, a cotton swab was utilized to delicately cleanse the non-migratory cells located inside the top cavity, while the migrated cells were left intact on the underside of the polycarbonate membrane. The migrating cells were captured with microscopy and quantified.

FUCCI reporter cell line and Proliferation assays

The plasmid pBOB-EF1‐FastFUCCI‐Puro (RRID: Addgene 86849) was employed to create the cell cycle reporter (FastFUCCI reporter). This plasmid encodes two fluorescent probes (mKO2‐hCDT1 and mAG‐hGEM) that are used to distinguish different phases of the cell cycle. The FastFUCCI reporter was stably transduced into endothelial cells by lentivirus to form the FUCCI reporter cell line. The indicator cells exhibited red-, yellow-, and green-emitting populations, representing cells in the G1, G1/S-, and S- or G2-to-M phases, respectively. Through the gain- or loss-of-function experiments on the FUCCI reporter cell line, we could explore the effect of targeted gene(s) on cell cycle and cell proliferation.

Spheroid Sprouting Assay

HCMECs were seeded in a round bottom 96-well plate and cultured with a complete medium comprising 0.25% (w/v) methylcellulose for 24 h using hanging drops to create spheroids. The spheroids were embedded in a mixed collagen solution (formula as described previously), and the volume of the solution was calculated based on the number of spheroids, resulting in an approximate concentration of 50 spheroids/ml. The mixture was then introduced to the 24-well plate (1 ml/well) and polarized for 30min. After incubation with ECM containing 50 ng/ml VEGF for 24 h, the collagen with spheroids was fixed with 4% paraformaldehyde, photographed, and analyzed.

Dual-luciferase reporter assay

The genomic DNA was implemented to amplify the SLUG promoter region (–2089/ + 235) while the One Step Cloning Kit (C112-02, Vazyme Biotech Ltd., China) was deployed to insert the pGL3-Basic vector (Promega). The pRL-TK plasmid (Renilla luciferase reporter plasmid, Promega), which operates as an internal control, was co-transfected into HEK293T cells along with the Luciferase reporter structures. The Dual-Luciferase Reporter Assay Kit (Promega) was implemented to ascertain the luciferase activity, aligning with the manufacturer's recommendations.

Pulldown assay with Biotin-labeled RNA probes and mass spectrometry

Typically, 3 µg of biotin-labeled sense and antisense RNA probes were incubated at 4°C for 1 h with streptavidin magnetic beads. Subsequent to a first mild wash with the buffer solution (20 mM Tri-HCl, pH7.5, 1 mM EDTA, and 450 mM NaCl), the beads were then exposed to cell lysates at 4°C for an interval of 4 h. This was afterward followed by five further washes. Following that, the binding proteins were subjected to immunoblot analysis or mass spectrometry (Bioprofile Technology Co., Ltd, Shanghai, China). Probe sequences are shown in the Supplementary table 3.

RNA-IP assay

The cells were cross-linked using UV radiation at a dosage of 200 J/cm² and a wavelength of 254 nm. Next, the cells were lysed with a solution comprising protease inhibitors and RNase inhibitors. The resulting mixture was then homogenized and centrifuged. The supernatant, was afterward incubated with anti-ALKBH5 and rabbit IgG for a duration of 4 h with gentle rotation. Next, Protein A/G Agarose beads (Millipore, USA) were introduced into the mixes and left to rotate for an additional 1 h. The immunoprecipitated RNA was purified using Trizol Reagent after being washed three times. It was then subjected to RT-qPCR analysis using primers. All processes were conducted on ice or at a temperature of 4°C.

MeRIP-qPCR

The MeRIP test was derived from a previously published methodology [27]. To summarize, the undamaged poly-A-purified RNA was denatured at 70°C for 10 min. Subsequently, the specimen was rapidly chilled with ice and mixed with m6A antibody in a 1ml solution comprising RNasin Plus RNase inhibitor 400 U (Promega, USA), 50 mM Tris-HCl, 750 mM NaCl, and 0.5% (vol/vol) Igepal CA-630 (Sigma Aldrich, USA). The combination was incubated for 2 h at 4°C. The mixture was incubated at 4°C with rotation for 2 h, following the Dynabeads Protein G (Invitrogen, USA) were purified and introduced to it. The m6A RNA was eluted two times using a solution containing 6.7 mM N6-methyladenosine 5′-monophosphate sodium salt. The rinses were conducted at 4°C for 1 h. Afterward, it was precipitated with 5 µg of glycogen, one-tenth of the amount of 3 M sodium acetate, and 2.5 times the volume of 100% ethanol. The precipitation procedure was conducted overnight at − 80°C. The m6A enrichment degree was evaluated using qPCR analysis. The fragmented mRNA was immediately incubated with a solution comprising m6A antibodies and subjected to comparable treatment. Primer sequences are shown in the Supplementary table 3.

Study population

Patients with acute myocardial infarction (AMI) and lower limb ischemia, comparing them with healthy volunteers. All participants were recruited from the First Affiliated Hospital of Zhengzhou University. The control group consisted of healthy volunteers. These volunteers had no significant systemic diseases, such as ischemic heart disease, cancer, pulmonary disease, or infectious diseases. Patients diagnosed with acute myocardial infarction (AMI) were included in the study. Blood samples were collected prior to any surgical intervention, primarily on the first day of admission. Patients with clinically diagnosed lower limb ischemia were also included. Blood samples were taken upon admission and prior to any surgical or interventional treatments. For all groups, blood samples were collected under standard clinical conditions. Clinical characteristics, vital signs, laboratory test results, and radiological reports were extracted from electronic medical records to complement the blood sample data. A total of 32 healthy volunteers, 32 acute myocardial infarction patients and 32 nondiabetic patients with a car accident were recruited in this study as well. Clinical characteristics, vital sign, laboratory tests, radiological reports were extracted from electronic medical records, between January 2023 and March 2024. The main clinical and laboratory data of human subjects were summarized in Supplementary Table 5.

Extraction of exosomes

Extraction of exosomes was performed as described previously with minor modifications [28]. Briefly, Cellular components in fresh blood samples were eliminated with centrifugation (two times 2,500×g, 4°C, 15 min). Supernatant (i.e., platelet-free plasma) was diluted to 2× with phosphate-buffered saline (PBS), and was filtered through a 0.8 µm filter (Merck, Darmstadt, Germany) by hydrostatic pressure to remove remaining platelets and apoptotic bodies. Then, the samples were centrifuged at 13,200 × g for 22 min at 4°C. The supernatant was filtered through 0.22 µm filters, and exosomes were pelleted by ultracentrifugation at 120,000×g for 1h, 3h, 6h, or 14h at 4°C. Additional conditions included ultracentrifugation at 37°C with protease inhibitors or using 5× diluted plasma. The pellets were washed with PBS and re-centrifuged at 120,000×g. Exosomes were used fresh to maintain integrity.

Statistical analysis

The data were presented as the mean value ± the standard error of the mean (s.e.m.). The cutoff values for gene expression were determined based on the median or mean levels. We used Shapiro-Wilk test to evaluate data normality. The differences were evaluated using Student's t-test, analysis of variance (ANOVA). For non-normally distributed data across multiple groups, we employed Kruskal-Wallis test. The expression correlation was assessed with the Pearson correlation coefficient analysis. The Log-rank test was deployed to evaluate the disparity in survival analysis. The two-sided Student’s t-test was implemented as to evaluate the expression difference, and the p-values below 0.05 were deemed significant.

CircATXN1 was upregulated in partial EndMT endothelial cells

Prior investigations have shown that transient or partial EndMT possesses an essential function in the occurrence and progression of ischemic conditions [13, 29]. Here, we selected two ischemic animal models, MI and HLI, to verify the biological process of partial EndMT. The blocking of the left anterior descending coronary artery permanently induces the MI. The sham-operated mice received a comparable treatment, but without the closure of the artery, in order to function as a control group. Furthermore, the right superficial and deep femoral arteries were securely tied and removed in order to cause HLI, whereas the left side received a comparable surgical operation without tying and served as the control group. Aligned with a prior investigation, IFS demonstrated a significant rise in double-positive cells expressing CD31 (an endothelial marker) and αSMA (a mesenchymal marker) in both the myocardial infarction and HLI ligation groups (Supplementary Figs. 1A-B). This confirms the presence of partial EndMT following the ischemic disease. Subsequently, we examined whether ECs received partial EndMT in vitro under hypoxic circumstances. HCMECs were precisely regulated in hypoxic conditions (1% O₂, 5% CO₂, and balanced N₂) and deprived of serum for a duration of 1 day in order to replicate the very challenging microenvironment of tissue ischemia. In order to induce complete EndMT, HCMECs were treated to transforming growth factor β (TGF-β) at a concentration of 20 ng/mL, serving as a positive control. IFS revealed that hypoxic HCMECs exhibited a progressive elevation in the mesenchymal indicator αSMA expression and a hindrance in the endothelial marker CD31 expression (Fig. 1A), suggesting a transition from an endothelial to a mesenchymal phenotype under hypoxic conditions. As a result, exposure to hypoxia significantly elevated the expression of some proteins that indicate the presence of mesenchymal cells, such as fibronectin, αSMA, and fibroblast-specific protein 1 (FSP1). However, the proteins CD31 and VE-cadherin, which are associated with endothelial cells, were reduced but not totally eliminated (Fig. 1B). These results indicate that mesenchymal ECs maintained some characteristics of ECs despite undergoing a partial EndMT. Conversely, the administration of TGF-β resulted in the total disappearance of endothelium markers, a condition known as complete EndMT. The occurrence of circRNA and hypoxia-induced partial EndMT in ischemic disorders is hardly documented. Bioinformatic transcriptome analysis of normoxia- and hypoxia-treated ECs was performed to identify relevant circRNAs (Supplementary Table 1). Among the upregulated circRNAs, there was one circRNA circATXN1 highly conserved and met the screening criteria (p < 0.05, logFC > 1, CPM > 0.2) (Fig. 1C). Sequence alignment of human and mouse circATXN1 showed that the homology was as high as 81.90% (Supplementary Fig. 2A). We detected circATXN1 level by RT-qPCR experiments to verify these findings. The outcomes demonstrated that circATXN1 was significantly raised in both hypoxic-treated homo HCMECs and mice MCMECs (Fig. 1D). Next, we examined the circATXN1 expression in ischemic animal models, and compared to the respective controls, RT-PCR reported that both ischemic myocardial and gastrocnemius had a significant rise in circATXN1 expression (Fig. 1E, Supplementary Fig. 3A). Furthermore, immunofluorescence reported an elevation in circATXN1 expression in the CD31-positive cells in the ischemic disease model (Fig. 1F, Supplementary Fig. 3B).

Characterization of the hypoxia-induced circATXN1

The bioinformatics analysis revealed that circATXN1 originated from exon 7 of the ATXN1 host gene and had a length of 2078 nucleotides (Fig. 2A). The validity of this discovery was confirmed by experimental verification using RT-PCR with several primers and Sanger sequencing (Fig. 2B). We also identified homologous variants of circATXN1 in mice (circATXN1) to ascertain the conservation of circATXN1 (Supplementary Fig. 4A). Additionally, the amplification of circATXN1 was achieved exclusively by utilizing divergent primers and cDNAs as templates rather than genomic DNAs. This effectively rules out the possibility that circATXN1 was generated by genomic rearrangements or PCR artifacts (Fig. 2C). When comparing the linear transcripts of ATXN1 with circATXN1, it was shown that circATXN1 was not affected by the RNA exonuclease RNase R (Fig. 2C). This provides more evidence that circATXN1 is a circular RNA. The subcellular location of circATXN1 was determined by IFS, revealing its predominant localization in the cytoplasm of HCMEC (Fig. 2D). Simultaneously, RNAs from the cytoplasm and nucleus were isolated for RT-qPCR analysis. Figure 2E and Supplementary Fig. 4C clearly demonstrates that the circATXN1 expression level in the cytoplasm was very high. Considering that an elevated transcription of the host gene would result in an upregulation of circRNA expression, we assessed the circATXN1 host transcript ATXN1 mRNA expression levels in response to hypoxic stress. However, hypoxia did not increase the expression of ATXN1, revealing that the rise of circATXN1 was not raised from the change in host transcript but by another regulatory mechanism (Fig. 2F, Supplementary Fig. 4B). According to reports, around 33% of many circRNAs are subject to dynamic regulation by the alternative splicing factor, Quaking (QKI) [30]. We wondered if QKI was involved in circATXN1 biogenesis, so we infected HCMECs with Ad QKI-sh, and the knockdown efficiency is shown in Fig. 2G. Then, the RT-qPCR results indicated that QKI knockdown significantly reduced circATXN1 abundance in both normoxic and hypoxic circumstances (Fig. 2H), indicating that QKI may contribute to regulating circATXN1 expression. circSMAD2 was used as a positive control downstream of QKI regulation, and circGNB1 as a negative control.

CircATXN1 inhibits hypoxia-stimulated Partial EndMT in vitro

Partial EndMT possesses an essential contribution to ischemic diseases and angiogenesis [12, 29]. Hence, the relevance of circATXN1 to ischemic/hypoxic signaling prompted us to ascertain the connection between circATXN1 and partial EndMT. In partial mesenchymal transition instances, ECs not only retent or partially lose their original characteristics but also gain some mesenchymal traits, leading to increased motility that could be involved in the (patho) physiological growth of blood vessels [31]. Therefore, we used adenovirus to achieve the intervention of circATXN1 in vitro so as to clarify the influence of circATXN1 on cell proliferation, migration, and sprouting. The overexpression/knockdown efficiency of circATXN1 and changes in the host gene are shown in Supplementary Figs. 5A-C. The FUCCI system was successfully integrated into main ECs with overexpression of circATXN1, as well as the control cells. The cell cycle was analyzed with FUCCI imaging. Following culture with hypoxic circumstances for a duration of 24 h, the S/G2-M cells population in the Ad circATXN1 group had a significant decline contrasted with the control group (Fig. 3A), while the population at the proliferative stage was significantly increased when circATXN1 was knocked down (Fig. 3D). These facts showed that circATXN1 could inhibit hypoxia-induced endothelial cell proliferation. Moreover, the circATXN1 implication on cell migration during partial EndMT was estimated with transwell assays. The overexpression of circATXN1 reduced cell migration (Fig. 3B), and the deficiencies of circATXN1 further increased the number of transmembrane cells under hypoxic conditions (Fig. 3E). Collective migration is the coordinated migration of endothelial tip and stalk cells, which collaborate to facilitate sprouting angiogenesis. Throughout the process of sprouting angiogenesis, angiogenic ECs have the potential to experience a partial EndMT [12] [32, 33]. This transition allows the ECs to acquire mesenchymal properties, which are essential for developing new vascular sprouts. We conducted spheroid sprouting tests to estimate the circATXN1 consequence of this phenomenon. A decrease in total sprout numbers was observed in circATXN1-overexpressed endothelial cells (Fig. 3C), whereas circATXN1 knockdown reinforced hypoxia-induced sprouting (Fig. 3F). The overexpression of circATXN1 suppressed the development of mesenchymal indicators caused by hypoxia and enhanced the expression of the endothelial marker (Figs. 3G-H). In contrast, the circATXN1 knockdown resulted in an upregulation of mesenchymal markers (fibronectin, aSMA, and FSP1) and a downregulation of endothelial indicators (CD31 and VE-cadherin). However, there was partial preservation of the CD31 and VE-cadherin expression (Figs. 3I-J). Under normal oxygen circumstances, both the overexpression and knockdown of circATXN1 did not have a significant impact on the expression of these markers. The above indicates that circATXN1 may hamper the acquisition of mesenchymal characteristics in HCMECs during partial EndMT.

CircATXN1 knockdown could enhance partial EndMT in vivo

We selected two ischemic disease models, MI and HLI, and achieved circATXN1 intervention by injecting adeno-associated virus to ascertain the in vivo circATXN1 impact on partial EndMT throughout responding to ischemia and its contribution to the restoration of blood flow after ischemic damage. By RT-qPCR and IFS, we detected the overexpression and knockdown efficiency of circATXN1 in the ischemic tissue of these two models, and staining results suggested that the intervention effect of AAV was mainly manifested in endothelial cells (Supplementary Figs. 6A-D, Supplementary Figs. 8A-D). The results from laser Doppler ultrasound showed a reduced blood flow recovery of 7 days in the circATXN1 overexpression group after the HLI surgery (Supplementary Fig. 7A), whereas the circATXN1 knockdown group showed improved perfusion recovery (Fig. 4A). Immunohistochemical and IFS revealed that circATXN1 restrained partial EndMT following ischemic injury, which was marked by downregulated mesenchymal marker (Vimentin) expression in circATXN1-overexpressing mice (Supplementary Fig. 7B). On the contrary, circATXN1 knockdown up-regulated the expression of mesenchymal marker (Fig. 4B). We used an MI model in the adult phase to assess the circATXN1 function in ischemic heart disease. Cardiac function was assessed 4 weeks after MI, and then the hearts were collected for histological section preparation and immunostaining. Knockdown of circATXN1 significantly enhanced cardiac function and remodeling after MI (Fig. 4C). In comparison to the AAV Scr-sh group, the AAV circATXN1-sh mice exhibited smaller infarct sizes in the infarct area (Fig. 4D). Remarkably, circATXN1 knockdown significantly increased the overall survival rate after MI in comparison to the control group of mice. The survival rates were 90% (18/20) and 65% (13/20), respectively, during 4 weeks of monitoring. (Fig. 4E). As predicted, immunohistochemical results showed that mesenchymal marker were upregulated (Fig. 4F). We injected AAV Control or AAV circATXN1 into a tail vein before MI to test whether circATXN1 overexpression could aggravate the effects. Contrasted with the AAV Control group, the AAV circATXN1 group reported a weaker heart function, a larger infarct size, worser survival rate (with 50% (10/20) and 70% (14/20) survival), and downregulated mesenchymal marker (Supplementary Figs. 7C-F). Collectively, our outcomes demonstrate that knockdown circATXN1 could promote partial EndMT in vivo, improving the recovery of blood flow and function after ischemic injury.

CircATXN1 hinders partial EndMT by inhibiting SLUG

There are many mechanisms that might induce EndMT, and these pathways converge on various signaling proteins, including SNAIL, TWIST, SLUG, and ZEB1 [33–36]. These transcription factors may be stimulated by intracellular signals and ultimately induce EndMT. The concepts of "EndMT" and "partial EndMT" are not independent, and there is a big interlinked nature of similarity. Subsequently, we first screened these 6 transcription factors to study the mechanism of circATXN1 regulating partial EndMT. WB analysis indicated that under hypoxic conditions, SLUG protein levels were negatively correlated with circATXN1 (Figs. 5A and Supplementary Fig. 9A), but the expression of the other 5 transcription factors was not significantly altered. Hughes et al. discovered that SLUG induces EC to undergo a partial EndMT [13]. They proposed that this process is contingent on the presence of endothelial SLUG, suggesting that SLUG levels influence the extent to which cells commit to a mesenchymal identity-determining whether cells undergo partial or complete EndMT. Later, we examined whether SLUG knockdown may restore the partial EndMT caused by circATXN1 lack in a hypoxic environment. The function assays demonstrated that after infection with Ad SLUG-sh, proliferation, migration, and the sprouting effect of circATXN1 knockdown on HCMECs significantly decreased (Figs. 5B–D). Furthermore, alterations in the levels of endothelial and mesenchymal markers were found (Fig. 5E). These results prompted us to believe that the reduced partial EndMT and impaired blood flow recovery caused by circATXN1 in ischemic disease are due to poor SLUG expression.

CircATXN1 Inhibits Partial EndMT via interaction with ALKBH5

Elevating proof reveals that circRNAs have a role in several biological processes in multiple ways, including functioning as regulating transcription, miRNA sponges, being translated into functional peptides or interacting with proteins [17, 37]. Luciferase reporters containing the SLUG promoter sequences were constructed to confirm whether circATXN1 can directly regulate SLUG transcription. The result showed that circATXN1 overexpression possessed no significant impact on the reporter plasmid luciferase activity (Supplementary Fig. 10A), which suggested that circATXN1 does not regulate SLUG expression by affecting SLUG transcriptional activity. Then we knocked down AGO2, the core protein of the miRNA-stimulated silencing complex (miRISC) [38–40], and had no effect on circATXN1 mediated SLUG reduction (Supplementary Fig. 10B), suggesting that circATXN1 might not act as a miRNA sponge to control SLUG expression. As shown in Supplementary Fig. 10C, analysis of the circATXN1 sequence revealed the presence of two ATG initiation codons, meaning that there may be two encoded peptides, one containing 186 amino acids and the other 640 amino acids. Next, mCherry was added to the front of the termination codons of these two possible encoding peptide segments, respectively, and then the constructed plasmids were transfected into 293T cells, and finally, the expression was detected with WB. Contrasted with the mCherry group, no bands were shown in the two recombinant plasmids, indicating that circATXN1 does not encode peptides.

We conducted RNA pulldown tests in order to discover the circATXN1-interacting proteins. This included introducing biotinylated DNA oligomers, either in the sense (S) or antisense (AS) direction, that crossed the back-splicing junction site of circATXN1 into cells. Subsequently, the circATXN1-bound proteins were separated using SDS-PAGE and visualized using silver staining. Figure 6A demonstrates the particular capture of a protein band with a molecular weight ranging from 40 to 55 kDa (shown by a black arrow) using the AS probe and excised for LC-MS/MS analysis (Supplementary Table 4). Among them, ALKBH5, a demethylase that often acts as an eraser for mRNA m6A modification [41–43], was of great interest to us (Supplementary Fig. 11A). The association of circATXN1 with ALKBH5 was validated in circATXN1 pulldown samples; the amounts of pulled circRNA and the interacting protein ALKBH5 were both positively correlated with the concentration of antisense probes used for incubation (Fig. 6B). In addition, the association between ALKBH5 and circATXN1 was further validated using RNA immunoprecipitation (RIP) tests employing an anti-ALKBH5 antibody (Fig. 6C). Immunofluorescence assay reported that circATXN1 was localized in the cytoplasm, and ALKBH5 was expressed in both the nucleus and cytoplasm (Fig. 6D). By knocking down ALKBH5, the SLUG-mediated partial EndMT effect induced by down-regulated circATXN1 was effectively weakened (Fig. 6E), which means that circATXN1 regulates the expression of SLUG and downstream effectors through ALKBH5. On this basis, when we supplement the SLUG protein, the impaired proliferation, migration, and sprouting ability of endothelial cells are restored (Figs. 6F-H). These results suggest that the diminished partial EndMT and impaired endothelial function caused by circATXN1 in ischemic conditions are attributed to reduced SLUG expression mediated through its interaction with ALKBH5.

CircATXN1 reduced SLUG mRNA stability by Preventing ALKBH5 Nuclear Translocation

As shown in Figs. 7A-C, not only the mRNA expression of SLUG but also the protein level was altered in ALKBH5-intervened cells. Previous results reminded us that circATXN1 does not collaborate with ALKBH5 in the transcriptional regulation of SLUG (Supplementary Fig. 9A). Actinomycin D was used to evaluate the stability of SLUG mRNA, and ALKBH5 knockdown significantly shortened the half-life of SLUG mRNA (Fig. 7D). Since ALKBH5 often acts as an m6A demethylase, we wondered if ALKBH5 was involved in circATXN1-induced downregulation of SLUG expression in this way. Indeed, as indicated by methylation quantification assay, ALKBH5 knockdown could increase m6A modification of SLUG mRNA (Fig. 7E); while the increased expression of the wild-type ALKBH5 gene decreased the m6A content, the mutant ALKBH5 H204A, which lacks catalytic activity [44], did not have the same effect (Fig. 7F). Meanwhile, the wild-type ALKBH5 overexpression, but not the mutated ALKBH5 H204A gene, was capable of bringing back SLUG expression (Figs. 7C and Fig. 7G). This suggests that ALKBH5 primarily influences SLUG expression via its demethylation activity. Besides, immunofluorescence colocalization analysis and nuclear plasma separation experiment found that when circATXN1 expression was down-regulated, ALKBH5 was translocated to the nucleus, whereas when circATXN1 expression was increased, ALKBKH was more localized in the cytoplasm (Figs. 7H, Fig. 7I and Supplementary Figs. 12A-B). Notably, circATXN1 only regulated the space and time change of ALKBH5 but had no effect on its expression (Fig. 6E). In aggregate, circATXN1 directly binds ALKBH5, preventing it from entering the nucleus and exerting demethylation to destabilize SLUG mRNA, which impedes the partial EndMT process.

CircATXN1 expression was up-regulated in plasma of patients with ischemic disease

We identified the presence of circATXN1 in exosomes derived from human plasm and serum a in the public RNA-seq NCBI GEO Dataset GSE100206, showing widespread expression across different individuals (Fig. 8A). Building on the findings from our study, which demonstrated that circATXN1 is predominantly expressed in endothelial cells, we hypothesized that circATXN1 detected in plasma may originate from endothelial cells and be secreted into peripheral blood in the form of exosomes (Supplementary Fig. 13). To investigate whether circATXN1 expression is significantly elevated in the endothelial cells of patients with ischemic disease and subsequently secreted into the peripheral blood, we collected peripheral blood samples from healthy volunteers, patients with myocardial infarction and non-diabetic patients with a car accident. We extracted exosomes from these samples and measured circATXN1 expression using RT-qPCR. Our results revealed that circATXN1 expression in peripheral blood exosomes was significantly higher in patients with myocardial infarction and lower limb ischemia compared to the control group (Figs. 8B-C). These findings suggest that circATXN1 might serve as a valuable biomarker for predicting disease progression in ischemic conditions.

We conducted a sequencing analysis on endothelial cells with hypoxia and serum deprivation in order to discover molecules that are linked to the partial or temporary activation of mesenchymal cells in ECs following tissue ischemia/hypoxia. The findings revealed a significant rise in the circATXN1 expression. A partial mesenchymal transition can potentially contribute to the growth of blood vessels in (patho) physiological conditions by promoting a pro-migratory and -invasive phenotype in endothelial cells [7, 11, 14, 45]. Endothelial tip cells, which guide the formation of new sprouts, can also be considered to have undergone a partial EndMT [12, 15, 46]. This transition involves the loss of apical-basal polarity, separation from the endothelium of established blood vessels and guiding the invading sprout. Therefore, by overexpressing circATXN1, we examined the proliferation, migration, and sprouting phenotypes of endothelial cells, and the outcomes revealed that circATXN1 hampered the acquisition of mesenchymal characteristics in HCMECs during partial EndMT. In contrast, circATXN1 knockdown raised the expression of the mesenchymal marker and mitigated but partially preserved the expression of endothelial markers, which drove ECs toward a partial EndMT. Subsequently, in vivo, outcomes suggested that circATXN1 predictably hampered angiogenesis postischemia and function recovery, while circATXN1 knockdown had an improved effect.

Then, we examined the effects of circATXN1 on several specific transcription factors recognized to be involved in EndMT regulation, and results showed that, compared with other transcription factors, cirATXN1 only affected SLUG expression and was negatively correlated. Nevertheless, the luciferase reporter experiment demonstrated that circATXN1 did not impact the SLUG promoter activity, suggesting that circATXN1 did not directly regulate SLUG transcription. CircATXN1 also did not affect ATXN1 expression, largely avoiding other potential regulatory pathways mediated by the host gene. Since circRNAs regulate various biological processes in different ways, and here other possible mechanisms of action (such as miRNA sponges, translating into functional peptides) were excluded one by one through RNA pulldown assays and LC-MS/MS analysis, we ultimately identified a protein that interacts with circATXN1 to mediate the regulation of SLUG, which was ALKBH5. The interaction was further confirmed by RIP assays.

Since ALKBH5 usually acts as an eraser for mRNA m6A modification, we hypothesized that it may also regulate SLUG mRNA expression in this way [47, 48]. With the addition of actinomycin D, which interfered with mRNA synthesis, ALKBH5 could indeed weaken the m6A modification of SLUG mRNA, thereby consolidating the mRNA stability and increasing the mRNA expression, while the reported catalytic inactive mutant ALKBH5 H204A abolished this function. We conducted immunofluorescence co-localization analysis and nuclear plasma separation experiment in order to explore further how circATXN1 regulates ALKBH-mediated SLUG mRNA demethylation, and the results proved that circATXN1 precisely regulated the space and time change of ALKBH5, but had no effect on its expression. More specially, circATXN1 worked through its direct interaction with ALKBH5, leading ALKBH5 to remain in the cytoplasm and unable to enter the nucleus for demethylation, thus destabilizing downstream SLUG mRNA. Therefore, when the expression of circATXN1 was down-regulated, more ALKBH5 entered the nucleus to work. At this time, by knocking down ALKBH5, the SLUG-mediated partial EndMT effect induced by down-regulated circATXN1 is effectively weakened, and if we supplement SLUG protein, the impaired angiogenic abilities (such as proliferation, migration, and sprouting) of endothelial cells were restored.

Our observations show that circATXN1, expressed in ECs, has control of ischemia conditions, namely in the biological process of angiogenesis. Upon interaction with ALKBH5 (a demethylase), cicATXN1 downregulates SLUG, which is a vital transcript factor in partial EndMT. CircATXN1 knockdown induces the activation of a partial EndMT program, leading to the transition of EC into an active, mesenchymal-like state. This state is marked by increased proliferation, migration, and sprouting. Hence, circATXN1 possesses the capacity to operate as a valuable target for the management of ischemia disorders. By use of complementary base pairs, small interfering RNA has the ability to suppress the ncRNA expression. One of the most appealing benefits of using siRNAs to target ncRNAs is their ability to expand the range of targets that may be effectively addressed. This is particularly advantageous since ncRNAs are difficult to target using standard small chemical compounds and antibody medicines. The recent FDA clearance of patisiran [49] and givosiran [50] underscores the viability of the RNAi method. Further investigation is required to determine the practicality and therapeutic benefits of circATXN1 in ischemia disorders. Considering circRNA has a covalently closed loop structure, which is highly stable and resistant to exonuclease, detection of circRNA expression by noninvasive sampling in body fluids could serve as a reliable biomarker, making it useful for routine clinical testing [51, 52]. We found that circATXN1 was widely expressed in human plasma, and circATXN1 levels in peripheral blood exosomes were significantly elevated in the peripheral blood exosomes of patients with ischemic disease. These findings suggest that circATXN1 could potentially serve as a valuable biomarker for predicting disease progression in ischemic conditions.

This research was supported by the National Natural Science Foundation of China (Grant Nos. 82200953, 81970201, 82070284), the Funding for Scientific Research and Innovation Team of The First Affiliated Hospital of Zhengzhou University (Grant No. QNCXTD2023013), Henan Province Science and Technology Project Plan (Grant No. 222102310128), the Medical Science and Technology Program of Henan Province (Nos: LHGJ20220286 and LHGJ20220297). Henan Province Young and Middle-aged Health Science and Technology Innovation Talent Training Project (YXKC2022017).

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There is NO Competing Interest.

SupplementaryFigures.pdf
Supplementary Figures 1-13
Supplementarytable1.FoldchangesofcircRNAsinnormoxicandhypoxicHCMECs.xlsx
Supplementary table 1.
Supplementarytable235.docx
Supplementary table 2 3 5
Supplementarytable4.IdentifiedcircATXN1bindingproteinbymassspectrometry.xlsx
Supplementary table 4.

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Regulation of Partial Endothelial-to-Mesenchymal Transition by circATXN1 in Ischemic Diseases

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Abstract

Figures

Introduction

Materials and Methods

Cell culture

Recombinant adenovirus and adeno-linked virus generation

Mice lower limb ischemia model

Mice myocardial infarction model

RT-qPCR

RNA sequencing for circRNA

The mRNA half-life determination

Cytosolic/Nuclear fractionation

RNA fluorescence in situ hybridization (RNA-FISH)

Immunofluorescence assays

Immunohistochemistry

Western Blot analysis

Migration assays

FUCCI reporter cell line and Proliferation assays

Spheroid Sprouting Assay

Dual-luciferase reporter assay

Pulldown assay with Biotin-labeled RNA probes and mass spectrometry

RNA-IP assay

MeRIP-qPCR

Study population

Extraction of exosomes

Statistical analysis

Results

CircATXN1 was upregulated in partial EndMT endothelial cells

Characterization of the hypoxia-induced circATXN1

CircATXN1 inhibits hypoxia-stimulated Partial EndMT in vitro

CircATXN1 hinders partial EndMT by inhibiting SLUG

CircATXN1 Inhibits Partial EndMT via interaction with ALKBH5

CircATXN1 reduced SLUG mRNA stability by Preventing ALKBH5 Nuclear Translocation

CircATXN1 expression was up-regulated in plasma of patients with ischemic disease

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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