Homocysteine metabolites inhibit autophagy by upregulating miR-21-5p, miR-155-5p, miR-216-5p, and miR-320c-3p in human vascular endothelial cells

Nutritional and genetic deficiencies in homocysteine (Hcy) metabolism lead to hyperhomocysteinemia (HHcy) and cause endothelial dysfunction, a hallmark of atherosclerosis, which is a major cause of cardiovascular disease (CVD). Impaired autophagy causes the accumulation of damaged proteins and organelles and is associated with CVD. Biochemically, HHcy is characterized by elevated levels of Hcy and its metabolites, Hcy-thiolactone and N-Hcy-protein. However, whether these metabolites can dysregulate mTOR signaling and autophagy in endothelial cells is not known. Here, we examined the influence of Hcy-thiolactone, N-Hcy-protein, and Hcy on autophagy human umbilical vein endothelial cells. We found that treatments with Hcy-thiolactone, N-Hcy-protein, or Hcy significantly downregulated beclin 1 (BECN1), autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and microtubule-associated protein 1 light chain 3 (LC3) mRNA and protein levels. We also found that these changes were mediated by upregulation by Hcy-thiolactone, N-Hcy-protein, and Hcy of autophagy-targeting microRNA (miR): miR-21, miR-155, miR-216, and miR-320c. The effects of these metabolites on levels of miR targeting autophagy as well as on the levels of BECN1, ATG5, ATG7, and LC3 mRNA and protein were abrogated by treatments with inhibitors of miR-21, miR-155, miR-216, and mir320c. Taken together, our findings show that Hcy metabolites can upregulate miR-21, miR-155, miR-216, and mir320c, which then downregulate autophagy in human endothelial cells, important for vascular homeostasis.

HUVEC can metabolize Hcy to Hcy-thiolactone and N-Hcy-protein 10 .The levels of Hcy-thiolactone and N-Hcy are regulated by extracellular Hcy, folic acid, and HDL, factors that determine the susceptibility to CVD in humans, suggesting that Hcy-thiolactone and N-Hcy-protein could be involved in endothelial dysfunction and atherosclerosis 10 .Further, Hcy-thiolactone and N-Hcy can induce pro-atherogenic patterns of gene expression, with Hcy-thiolactone upregulating LAMTOR2 mRNA, a component of the mammalian target of rapamycin (mTOR) signaling pathway in HUVEC 11 .However, mechanisms by which these metabolites can affect gene expression in vascular endothelial cells are not known.
MicroRNAs (miRs) are small non-coding RNAs playing a crucial role in cell physiology by regulating gene expression at the mRNA level 12 .miR genes are transcribed by RNA polymerase II to primary miR, which is first cleaved by DROSHA and then by DICER 13,14 , generating 21-23 nt double strand mature miRs 15 .One strand is incorporated into RISC (RNA-induced silencing complex) and regulates gene expression by binding to partially complementary mRNA sequence (mainly found in 3′ untranslated region (3′ UTR)) to induce translational repression, mRNA deadenylation or cleavage 16 .Dysregulated miR expression can lead to endothelial dysfunction 17 , CVD 18 , stroke 19 , and neuropathies 20,21 .
Autophagy is an evolutionarily conserved cellular process involving degradation and recycling damaged proteins and organelles.Autophagy occurs continually at basal levels in cells and contributes to the maintenance of cellular homeostasis.Impaired autophagy causes the accumulation of damaged proteins and organelles 22 and is associated with CVD [23][24][25] .
We hypothesize that HHcy-associated metabolites affect the expression of autophagy-related genes via a miR-mediated mechanism in human endothelial cells.To evaluate this hypothesis, we studied the influence of N-Hcy-protein, Hcy-thiolactone, and Hcy on autophagy-related protein and mRNA levels as well as on levels of miRs targeting autophagy-related mRNAs in HUVEC.We also studied how miR inhibitors affect the expression of autophagy-related genes.

Hcy-thiolactone, N-Hcy-protein, and Hcy downregulate autophagy-related proteins
In an earlier work, one of us (HJ) has shown that HUVEC can metabolize Hcy to Hcy-thiolactone and N-Hcyprotein 10 .We have also shown that these metabolites reduced autophagy flux in mouse neuroblastoma N2A cells 28 .To figure out whether each of these metabolites can affect autophagy in human endothelial cells, we treated HUVECs with N-Hcy-protein, Hcy-thiolactone, or Hcy and quantified levels of autophagy-related proteins by Western blotting.We found significantly attenuated levels of regulators of autophagosome assembly beclin 1 (BECN1) (Fig. 1A) and autophagy-related 5 (ATG5) (Fig. 1B), in HUVEC treated with N-Hcy-protein, Hcythiolactone, or Hcy, compared with untreated controls.Autophagy-related 7 (ATG7) was significantly reduced in HUVEC treated with N-Hcy-protein or Hcy-thiolactone, but not with Hcy (Fig. 1C).Levels of microtubuleassociated protein 1 light chain 3 (LC3-I), were significantly upregulated by Hcy-thiolactone and Hcy and downregulated by N-Hcy-protein (Fig. 1D).In contrast, the lipidated LC3-II was significantly downregulated (Fig. 1E).The LC3-II/LC3-I ratio, an indicator of autophagy flux, was reduced by Hcy-thiolactone and Hcy but unaffected by N-Hcy-protein (Fig. 1F).Protein p62, a receptor for the degradation of ubiquitinated substrates, known to be negatively correlated with autophagy flux 30 , was upregulated by N-Hcy-protein but unaffected by Hcy-thiolactone and Hcy (Fig. 1G).Representative images of western blots are shown in Fig. 1H.Cell viability was not significantly affected in HUVEC treated with N-Hcy-protein, Hcy-thiolactone, or Hcy, compared to untreated control (Fig. 1I), consistent with previous findings with Hcy-treated HUVEC 6 .
These findings show that Hcy-thiolactone and Hcy impair autophagy by dysregulating autophagosome assembly and autophagy flux while N-Hcy-protein dysregulates autophagosome assembly but has no effect on autophagy flux.
Treatments with miR-21 inhibitor significantly decreased p62 mRNA (P < 0.001; Fig. 5F) and p62 protein level (P < 0.01; Fig. 6J).As p62 mRNA was not affected by treatments with Hcy metabolites (Fig. 2E) and because p62 and LC3 are inversely correlated 30 , this effect is most likely due to upregulation of LC3 by the miR-21 inhibitor.

Discussion
Endothelial dysfunction, the first step in the development of atherosclerosis, plays a central role in CVD and is a common finding in HHcy in humans and in animal models 3,4 .To understand the mechanisms by which HHcy disrupts normal cellular function and causes disease, we studied in HUVECs, a widely used model of vascular cells 10,37 , how Hcy, Hcy-thiolactone and N-Hcy-protein, metabolites that accumulate in HHcy, affect the expression of miR-21, miR-155, miR-216, and miR-320c, regulators of the expression of BECN1, ATG5, ATG7, LC3, and p62 genes (Fig. 3) participating in autophagy, a process important for vascular homeostasis 37 .Our data show that Hcy, Hcy-thiolactone, and N-Hcy-protein dysregulate autophagy by upregulating the expression of miR-21, miR-155, miR-216, and miR-320c in HUVECs.The ability of Hcy, Hcy-thiolactone, and N-Hcy-protein to upregulate miR-21, miR-155, miR-216, and miR-320c expression and thus affect autophagy can explain the susceptibility of human endothelial cells to HHcy-induced endothelial dysfunction and atherosclerosis.Studies of Hcy metabolism led to the discoveries of Hcy-thiolactone and N-Hcy-protein in human cells and the proposal by one of us (HJ) that these metabolites are responsible for the pathologies associated with HHcy 38 .The metabolic conversions of Hcy to Hcy-thiolactone and N-Hcy-protein are universal and were confirmed in HUVEC 10 , mice 39,40 , and humans 39,41,42 .More recently, clinical studies by one of us (HJ) have shown that HTL predicts a risk of myocardial infarction in patients with coronary artery disease, thereby lending a support to the hypothesis that Hcy-thiolactone is mechanistically involved in CVD 43 .Our present findings that Hcythiolactone, N-Hcy-protein, and Hcy, upregulate miR-21, miR-155, miR-216, and miR-320c in HUVECs and inhibit autophagy, suggest that these miRs can also be involved in endothelial dysfunction and suggest a mechanism underlying the involvement of HHcy in CVD.
Our present findings that the expression of BECN1, ATG5, ATG7, and LC3 as well as the LC3-II/LC3-I ratio, the measure of autophagy flux, are downregulated by Hcy, Hcy-thiolactone, and N-Hcy-protein in HUVEC suggest that impaired autophagy can accelerate endothelial dysfunction and lead to vascular disease.Indeed, other investigators have shown that autophagy flux controls endothelial cells homeostasis while impaired autophagy due to factors other than HHcy can promote pro-atherogenic phenotype 37 .
Among these, miR-216a (targeting BECN1), highly expressed in arteries, is induced during endothelial aging and controls oxidized low density lipoprotein (ox-LDL)-induced autophagy in HUVEC by regulating levels of BECN1 35 ; miR-155 (targeting ATG5) that also affects LC3 and p62 expression, promotes ox-LDL-induced 34 and hydrogen peroxide-induced 33 autophagy in HUVEC; miR-320c (targeting ATG7) is associated with aging and cancer 36 .Our present findings showing that N-Hcy-protein, Hcy-thiolactone, and Hcy upregulate miR-21, miR-155, miR-216, and miR-320c suggest that miR-dependent dysregulation of autophagy can be involved in endothelial dysfunction and atherosclerosis induced by HHcy.The concentrations of Hcy and related metabolites used in the present study did not affect HUVEC viability (Fig. 1I).However, these concentrations of Hcy are known to induce the endothelial adhesion molecule VCAM-1 expression, characteristic of endothelial activation/dysfunction, without affecting cell viability 6 ; much higher Hcy concentrations can cause cell death 45 .
In conclusion, our findings define a new miR-mediated mechanism by which HHcy can induce endothelial dysfunction.In this mechanism, Hcy, Hcy-thiolactone, and N-Hcy-protein, metabolites associated with HHcy, upregulate the expression of miR-21, miR-155, miR-216, and miR-320c, which results in impaired autophagy.
For the Hcy metabolite treatment experiments, HUVEC monolayers were trypsinized, washed with PBS, seeded in 6-well plates (100,000 cells/well), and grown as above.After cells reached 70-80% confluency, the monolayers were washed with PBS (2-times) and overlaid with M199 medium without methionine (Thermo Fisher Scientific, Waltham, MA, USA) and with 5% dialyzed fetal bovine serum (FBS; Millipore Sigma).Cell cultures were treated with N-Hcy-protein (prepared as described in Ref. 11 ), L-Hcy-thiolactone, or D,L-Hcy (Millipore Sigma) (at concentrations based on earlier work 10,11 and indicated in the figures) and incubated at 37 °C in 5% CO 2 for 24 h; cells from untreated cultures were used as controls.These concentrations reflect levels of these metabolites in mice and humans (reviewed in Ref. 7 ).

HUVEC viability assay
The viability of HUVEC was assessed by using the trypan blue exclusion assay based on the principle that the dye can enter membrane-compromised dead cells but is excluded by live cells.Briefly, 10,000 cells/well were seeded into wells of a 48-well plate.After cells reached 70-80% confluency (at 24-48 h), cells were treated as described in "Hcy-thiolactone, N-Hcy-protein, and Hcy downregulate autophagy-related proteins" section Cell culture and treatments.After 24 h, cells were rinsed with PBS (2-times), trypsinized, and pelleted by centrifuged (5 min, RT, 1500 rpm).Cell pellets were suspended and incubated for 3 min in 350 µl PBS mixed with 150 µl 0.4% trypan blue solution (Millipore Sigma).For quantifying viability, cells were transferred to a hemocytometer chamber and counted using a light micrroscope at 10 × magnification.The HUVEC viability is expressed as the percentage of blue-stained dead cells in the total number of cells.

Quantification of mRNA and miR by RT-qPCR
Total RNA was isolated using Trizol reagent (Millipore Sigma).cDNA synthesis was performed using Revert Aid First cDNA Synthesis Kit (Thermo Fisher Scientific) according to manufacturer's instructions.RNA concentration was measured using NanoDrop (Thermo Fisher Scientific).RT-qPCR was performed using SYBR Green Mix and CFX96 thermocycler (Bio-Rad) and primers listed in Supplementary Table S1.The miR 1st-Strand cDNA Synthesis Kit (Agilent Technologies) was used according to manufacturer's instructions to polyadenylate and reverse-transcribe miRs from one μg of total RNA.To quantify miR levels, RT-qPCR was performed with the resulting cDNA in miRNA QPCR Master Mix (Agilent Technologies) using a universal reverse primer (Agilent Technologies) and unique miR-specific primers (same sequence as an analyzed miR) (Supplementary Table S1).Reactions were conducted on CFX96 thermocycler (Bio-Rad).18S rRNA and U6 snRNA were used as references for miR quantification.
The 2 (−ΔΔCt) method was used to calculate the relative expression levels 47 .Data analysis was performed with the CFX Manager™ Software, Microsoft Excel, and GraphPad Prism7.

Statistical analysis
Each assay was repeated three times (technical repeats) in three independent experiments (biological repeats) for each treatment and controls.Data are mean ± standard deviation (SD) of three biological repeats.Data were analyzed using one-way analysis of variance (ANOVA) with Tukey's multiple comparisons post-test or a Student t test using GraphPad Prism7 software (GraphPad Holdings LLC, San Diego CA, USA, https:// www.graph pad.com).

Figure 1 .
Figure 1.Influence of N-Hcy-protein, Hcy-thiolactone, and Hcy on the expression of autophagy-related proteins in HUVEC.Bar graphs show levels of BECN1 protein (A), ATG5 protein (B), ATG7 protein (C), LC3-I (D), LC3-II (E), LC3-II/LC3-I ratio (F), and p62 protein (G) in HUVEC treated with N-Hcy-protein (N-Hcy), Hcy-thiolactone (HTL), or Hcy in methionine-free M199/dialyzed FBS medium for 24 h.Showed proteins were quantified by Western blotting.GAPDH was used as reference for the quantification of other proteins.Panel (H) shows representative images of Western blots.The treatments with Hcy, N-Hcy, and HTL did not affect HUVEC viability (I).Each assay was repeated three times (technical repeats) in three independent experiments (biological repeats).Mean ± SD values of three biological repeats for each treatment group are shown.*P < 0.05, **P < 0.01, ***P < 0.001 from one-way ANOVA with Tukey's multiple comparisons test.

Figure 6 .
Figure 6.Influence of N-Hcy-protein, Hcy-thiolactone, and Hcy on the expression of autophagy-related proteins in HUVEC in the presence of a corresponding miR inhibitor.Bar graphs show levels of BECN1 protein (A,B), LC3-II protein (C-E), LC3-II/LC3-I ratio (F-H), LC3-I (I), p62 protein (J), ATG5 protein (K,L), and ATG7 protein (M) in HUVEC transfected with mirVana™ miRNA Mimic, Negative Control #1 (Control) or a corresponding miR inhibitor for 4 h.The cells transfected with a miR inhibitor were then untreated (Control+) or treated with N-Hcy, HTL, or Hcy in methionine-free M199/dialyzed FBS medium for 24 h.Indicated proteins were quantified by Western blotting with GAPDH protein as a reference; representative images are shown in panels (N-Q).Each assay was repeated three times (technical repeats) in three independent experiments (biological repeats).Mean ± SD values of three biological repeats for each treatment group are shown.*P < 0.05, **P < 0.01, ***P < 0.001 from a Student's two-tailed t test.