Hypotaurine Reduces Glucose-Mediated Vascular Calcification

Abstract

Aim: Vascular calcification (VC), a characteristic feature of peripheral artery disease in patients with diabetes and chronic kidney disease, has been associated with poor prognosis. We hypothesize that hyperglycemia drives VC through alterations in metabolomic and transcriptomic profiles.

Methods: Human coronary artery smooth muscle cells (SMCs) were cultured with 0, 5.5, and 25 mM glucose under calcifying conditions. Untargeted metabolomic and transcriptomic analyses were performed at different time points. Mitochondrial respiration was examined using Seahorse analysis.

Results: Glucose-treated SMCs promoted extracellular matrix (ECM) calcification in a concentration- and time-dependent manner. The absence of glucose entirely abolished SMC calcification but reduced SMC proliferation in control and calcifying conditions compared to 25 mM glucose. Multi-omics data integration revealed key players from the hypotaurine/taurine metabolic pathway as the center hub of the reconstructed network. Glucose promoted the hypotaurine secretion, while its intracellular abundance was not altered. Blocking hypotaurine production by propargylglycine increased ECM calcification, while hypotaurine treatment prevented it. Furthermore, omics data suggest energy remodeling in calcifying SMCs under hyperglycemia. Calcifying SMCs exhibited decreased oxygen consumption that was partially restored by hypotaurine. Validation of our in vitro models using the murine warfarin model demonstrated reduced hypotaurine/taurine transporter (TAUT) expression in SMCs.

Conclusions: Our multi-omics analysis revealed a role of the hypotaurine/taurine metabolic pathway in glucose-induced SMC calcification. Moreover, our data suggest a glucose-dependent energy remodeling in calcifying SMCs and that increasing glucose concentrations fuel ECM calcification. Our work highlights potential novel therapeutic targets that warrant further investigation in hyperglycemia-dependent in vitro SMC calcification.

Keywords: hypotaurine; metabolomics; transcriptomics; vascular calcification.

FIGURE 1

Glucose promoted extracellular matrix (ECM) calcification in primary human coronary artery smooth muscle cells (pSMC) concentration‐ and time‐dependently. pSMC were cultured in control (CM; 5.5 mM glucose) or calcium/phosphate (CaP)‐enriched media with 0, 5.5, or 25 mM glucose over 7 days. Mannitol served as osmotic control. (A) Representative images of ECM mineral accessed by Alizarin Red staining after treatment with glucose and mannitol in CM or CaP media for 1, 3, 5, and 7 days. Scale bars: 1000 μm. (B) Quantification of eluted Alizarin Red staining from the ECM from Figure 1A. Two‐way ANOVA with Dunnett's post hoc test. *p < 0.05 compared to all other conditions at day 7. (C) Representative images of cell viability visualized with fluorescein diacetate (FDA) and propidium iodide (PI) dual immunofluorescence staining at day 7. Negative control: Permeabilized cells with 0.05% Triton X‐100. Phase contrast shows the mineral indicated by the arrow. Scale bars: 75 μm. (D) Cell viability assessed by AlamarBlue assay. pSMC were cultured with glucose and CaP media for 7 days. (E) Apoptosis at day 7. (F) Representative images of the mitochondria‐specific dye MitoTracker Red (red) and nuclear Hoechst staining (blue) at day 7. Scale bars: 75 μm. n = 3–4 in duplicates, each n represents an independent pSMC donor. Mean ± SD. One‐way ANOVA with Dunnett's post hoc test compared to 5.5 mM glucose control (CM), n.s.; not significant.

FIGURE 2

Distinct glucose concentration‐specific gene expression profiles in primary human coronary artery smooth muscle cells. (A) Principal component analysis plot showed the segregation of the genes according to control (CM; 5.5 mM glucose), calcium/phosphate (CaP), and glucose treatment. The percentages indicate the proportion of variance explained by each feature. (B) The Venn diagram displayed the shared and unique differentially expressed genes (fold change ±1.2, p < 0.05) between different glucose treatments in CaP. n = 3, each n represents an independent pSMC donor.

FIGURE 3

Glucose concentrations and time points shape unique metabolomic profiles in calcifying primary human coronary artery smooth muscle cells (pSMC). (A) Venn diagram displayed the shared and unique metabolites between supernatant and cells (calcifying SMCs). The principal component analysis (PCA) plot showed the segregation of the metabolites according to glucose treatment in (B) supernatant and (C) cells. PCA analysis was performed to distinguish between calcifying supernatant and cell samples across different timepoints. PCA biplots illustrate the positions of metabolites, with vectors indicating their relative contributions to the principal components. (D–F) Supernatant at day 3. (G–I) Supernatant at day 5. (J–L) Cell metabolites at day 3. (M–O) Cell metabolites at day 5. n = 3, each n represents an independent pSMC donor. Fold change ±1.2, p < 0.05.

FIGURE 4

The multi‐omics network of hyperglycemia‐induced vascular calcification. (A) Multi‐omics network based on genes and metabolites differentially regulated, comparing 0 vs. 25 mM glucose treatment of calcifying human coronary artery smooth muscle cells. (B) Focused network on hypotaurine/taurine and cysteine metabolic pathways and their interactors. Blue nodes represent input molecules. Yellow nodes represent metabolites, and red nodes are based on protein–protein interactions from genes. Input: Differentially expressed genes (fold change 1.5, p < 0.05, day 3) and metabolites from the supernatant (fold change 1.2, p < 0.05, day 5) from the 0 vs. 25 mM glucose comparison. (C) Overview of the effect of glucose on the hypotaurine/taurine metabolic pathway. Blue: Decreased metabolites by glucose. Orange: Increased metabolites by glucose. White: Not regulated. Green: Enzyme gene names. (D) Abundance of secreted/extracellular hypotaurine based on an untargeted metabolomics approach. n = 3, each n represents an independent pSMC donor. Mean ± SD. One‐way ANOVA with Tukey's post hoc test.

FIGURE 5

Hypotaurine reduced extracellular matrix (ECM) calcification in calcifying hyperglycemic immortalized vascular smooth muscle cells (imSMC), in a concentration‐ and time‐dependent manner. (A–C) imSMC were cultured in control (CM; 25 mM glucose) or calcium/phospate (CaP; 25 mM glucose) conditions with or without 1 μM PAG for 7 days. (A) Representative image of Alizarin Red S staining visualizing ECM mineral. Scale bar: 1000 μm. (B) Quantification of B by the elution of Alizarin Red stain. One‐way ANOVA with Sidak post hoc test. (C) Cell viability was assessed by alamarBlue assay. (D–F) imSMC were cultured in 25 mM glucose under control (CM) or calcium/phosphate (CaP) conditions without or with hypotaurine (10, 25, 50 mM). (D) Representative image of Alizarin Red S staining visualized ECM mineral. Day 7. Scale bar: 1000 μm. (E) Quantification of A by the elution of Alizarin Red stain. (F) Cell viability was accessed by AlamarBlue at day 7. Mean ± SD. (G, H) imSMC were cultured in 0 or 25 mM glucose under control (CM) or calcium/phosphate (CaP) conditions without or with 25 mM hypotaurine. (G) Oxidative stress was assessed by the Amplex Red hydrogen/peroxidase (H₂O₂) assay after 24 h and normalized to protein. (H) Cell proliferation was assessed by BrdU assay after 7 days. B‐F) N = 3, in duplicates. One‐way ANOVA with Dunnett's post hoc test compared to CaP, n.s.; not significant. G, H). n = 4, in duplicates. One‐way ANOVA with Sidak's post hoc test. Mean ± SD. Each n represents an independent experiment.

FIGURE 6

Hypotaurine partially restores mitochondrial respiration in calcifying hyperglycemic immortalized vascular smooth muscle cells (imSMC). imSMCs were cultured in 25 mM glucose for 7 days in either control medium (CM) or calcium phosphate (CaP) conditions with or without 25 mM hypotaurine. (A) Mitochondrial oxygen consumption rate (OCR) was measured using the Seahorse XF96 flux analyzer. Mitochondrial effectors were sequentially injected: oligomycin (ATP synthase inhibitor), FCCP (uncoupling agent), and rotenone/antimycin (complex I/III inhibitors), as indicated by downward arrows. (B) Basal respiration quantified after mitochondrial effector injections. (C) ATP production derived from changes in OCR after oligomycin injection. (D) Maximal respiration measured after FCCP injection, indicating the maximal electron transport chain capacity. (E) Non‐mitochondrial respiration calculated following rotenone/antimycin injections, representing OCR from non‐mitochondrial sources. All data were normalized to the protein content. Mean ± SD. n = 3, in duplicates. Each n represents an independent experiment. One‐way ANOVA with Sidak's post hoc test.

FIGURE 7

Sodium‐ and chloride‐dependent (hypo)taurine transporter SLC6A6 (protein TAUT) expression pattern in aortas with medial calcification from warfarin‐treated mice. (A) Representative images of control (n = 6) and warfarin‐treated (n = 6) mice. Calcification is visualized by Alizarin red staining. Immunohistochemistry was performed for TAUT. Bar: 150 μm. (B) Quantification of positively stained areas for TAUT in murine aortas using ImageJ v2.0. Mean ± SD. Each dot depicts one mouse. Student's t‐test.