Lactate Promotes Synthetic Phenotype in Vascular Smooth Muscle CellsNovelty and Significance
Rationale: The phenotypes of vascular smooth muscle cells (vSMCs) comprise a continuum bounded by predominantly contractile and synthetic cells. Some evidence suggests that contractile vSMCs can assume a more synthetic phenotype in response to ischemic injury, but the mechanisms that activate this phenotypic switch are poorly understood.
Objective: To determine whether lactate, which increases in response to regional ischemia, may promote the synthetic phenotype in vSMCs.
Methods and Results: Experiments were performed with vSMCs that had been differentiated from human induced pluripotent stem cells and then cultured in glucose-free, lactate-enriched (L+) medium or in standard (L−) medium. Compared with the L− medium, the L+ medium was associated with significant increases in synthetic vSMC marker expression, proliferation, and migration and with significant declines in contractile and apoptotic activity. Furthermore, these changes were accompanied by increases in the expression of monocarboxylic acid transporters and were generally attenuated both by the blockade of monocarboxylic acid transporter activity and by transfection with iRNA for NDRG (N-myc downstream regulated gene). Proteomics, biomarker, and pathway analyses suggested that the L+ medium tended to upregulate the expression of synthetic vSMC markers, the production of extracellular proteins that participate in tissue construction or repair, and the activity of pathways that regulate cell proliferation and migration. Observations in hypoxia-cultured vSMCs were similar to those in L+-cultured vSMCs, and assessments in a swine myocardial infarction model suggested that measurements of lactate levels, lactate-dehydrogenase levels, vSMC proliferation, and monocarboxylic acid transporter and NDRG expression were greater in the ischemic zone than in nonischemic tissues.
Conclusions: These results demonstrate for the first time that vSMCs assume a more synthetic phenotype in a microenvironment that is rich in lactate. Thus, mechanisms that link glucose metabolism to vSMC phenotypic switching could play a role in the pathogenesis and treatment of cardiovascular disease.
Vascular smooth muscle cells (vSMCs) are perhaps most frequently studied for their role in arterial contraction.1 vSMCs are found in a wide range of organs and participate in a variety of other physiological activities and repair mechanisms.1 The diversity of vSMC function is reflected in their phenotypes, which compose a spectrum that ranges from predominantly contractile cells at one end to predominantly synthetic cells at the other. The contractile and synthetic phenotypes are characterized by substantial differences in marker expression, morphology, and activity,2,3 and some evidence suggests that contractile cells can adopt a more synthetic phenotype in response to ischemic injury,4 but the mechanisms that activate this phenotypic switch are poorly understood.
Even under fully oxygenated conditions, vSMCs produce a substantial amount of lactate,5 and both local and systemic lactate concentrations increase in response to regional ischemia and cardiac arrest, as well as shock, burns, trauma, and other conditions.6,7 Lactate is the end product of cellular anaerobic glucose catabolism and has long been considered a metabolic waste product; however, there are reports indicating that lactate is taken up by myocytes, endothelial cells (ECs) and human cytotoxic T lymphocytes,8,9 inhibits phosphofructokinase,10 alters gene expression in L6 muscle cells,11 participates in T-cell migration,12 and contributes to tumor growth.13–16 It has been reported that the synthetic phenotype switch involves vSMC-dependent mechanisms of repair in response to injury.17,18 Here, we show that lactate promotes the synthetic phenotype of vSMCs. This mechanism maybe utilized to enhance myocardial repair in some forms of ischemia heart disease.
All the original data are available on request. Please email the request to the first author directly.
Maintenance and Differentiation of Human Induced Pluripotent Stem Cells
Human induced pluripotent stem cells (hiPSCs) were maintained on vitronectin-coated plates in DMEM-F12 (Life Science) supplemented with 20% knockout serum replacement (Invitrogen), 0.1 mmol/L MEM nonessential amino acid solution (Life Science), 2 mmol/L L glutamine (Life Science), 0.1 mmol/L b-mercaptoethanol (Sigma), and 4 ng/mL basic fibroblast growth factor (Thermo Fisher Scientific). Passage 10≈15 of hiPSC was used in this project.19 hiPSCs were differentiated into hiPSC-vSMCs and ECs as described previously.20 The standard culture medium consisted of Roswell Park Memorial Institute medium (RPMI)-1640 with 1×B27 minus insulin and a growth factor mixture (human basic fibroblast growth factor, 2 ng/mL; human epidermal growth factor, 0.5 ng/mL). The lactate-enriched (L+) medium consisted of glucose-free RPMI-1640 with 1×B27 minus insulin, growth factor mixture, and L-lactate added as needed. For the hypoxic condition, the medium was vacuumed; then, the cells were cultured for 4 hours at 37°C with 5% CO2 and 1% O2. For lactate assay, the cells were treated with 100 µmol/L CoCl2 for hypoxia condition.
Migration was evaluated across 8-µm pore size membranes in 24-well tissue culture plates with a cell-migration kit (Millipore). Cells were cultured in RPMI-1640 B27 for 24 hours, trypsinized, and then added with 300 µL RPMI-1640 B27 to the upper chamber (1×105 cells per chamber). The lower chamber contained 500 µL RPMI-1640 B27 and was coated with (positive control) or without (negative control) Matrigel. The cells were incubated for 12 hours at 37°C, and the cells that had migrated to the lower chamber were detected with CyQuant GR Dye.
Collagen gels were prepared by incubating bovine type I collagen (3 mg/mL) in 24-well culture dishes overnight at 37°C; then, the cells were plated at a density of 5×104 cells per well. On the following day, the collagen gels were treated with 1 µmol/L carbachol and detached from the sides of the wells, and the surface areas of the collagen lattices were measured from digital photographs taken 0 and 24 hours later. The decline in surface area was determined with Image J software for 3 replicates per experimental group.
Live/Dead Cell Assay
Living and dead cells were quantified via 2-color fluorescence with the LIVE/DEAD Viability/Cytotoxicity kit (Molecular Probes) as directed by the manufacturer’s instructions. The cells were cultured in medium containing calcein AM and ethidium homodimer-1, both of which easily pass from the culture medium into the cellular cytoplasm; then, viable cells were detected by intracellular esterase activity, which converts the nonfluorescent calcein AM into the intensely green fluorescent molecule calcein, and nonviable cells are detected by the binding of ethidium homodimer-1, which produces a bright red fluorescence in dead cells. Briefly, the cells were washed with PBS, incubated with 300 μL of the live/dead solution for 30 minutes at 37°C in the dark, and viewed with a microscope and appropriate filters (Leica, Germany). Each treatment group was evaluated in triplicate, and 3 representative images were taken for each triplicate. Live and dead cells were counted and quantified with Image J software.
Western Blot Analysis
Cells were washed twice in cold PBS and lysed in new RIPA (radioimunoprecipitation assay) lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris pH 8.0, 1 mmol/L EDTA, 1 mmol/L EGTA, 0.5% sodium deoxycholate, 0.1% SDS, and 1% Triton X-100) with protease inhibitors (0.1 mol/L phenylmethylsulfonyl fluoride, 5 μg/mL leupeptin, 2 μg/mL aprotinin, and 1 μg/mL pepstatin). Protein concentrations for the whole-cell lysates were determined via the bicinchoninic acid method, and equal amounts of each protein sample (15 μg) were separated on an 8% to 14% SDS-polyacrylamide gel at 80 V; then, a Turbo transfer System (Bio-Rad) was used for 8 minutes to transfer the separated proteins to a polyvinylidene difluoride membrane, and the proteins were blocked with 5% skim milk powder for 1 hour at room temperature. Membranes were incubated with primary antibodies for 1 hour at room temperature or overnight at 4°C, washed 3 times with 0.05% PBS-Tween, incubated for 1 hour at room temperature with a horseradish peroxidase-conjugated secondary antibody, and washed with 0.05% PBS-T; then, the protein bands were visualized with ECL Plus as directed by the manufacturer’s instructions and developed on film (Cell signaling).
Monocarboxylic Acid Transporter 4 and NDRG3 Knockdown
Cells were transfected with monocarboxylic acid transporter 4 (MCT4 siRNA or control siRNAs (Santa Cruz Biotechnology) by using HiperFect transfection reagent (Qiagen) as directed by the manufacturer’s instructions. Briefly, the siRNAs and reagent (in a 1:2 ratio) were dissolved in RPMI-1640 and incubated for 5 minutes at room temperature; then, the siRNA-reagent complexes were added to the cells, and the cells were incubated at 37°C. Six hours later, the transfection medium was replaced with fresh medium and the cells were cultured for 2 more days before use in subsequent experiments. Transfection with NDRG3 (N-myc downstream regulated gene 3) shRNA or control RNA particles (Origene) was performed in RPMI-1640 with 2 μg/mL Polybrene (Millipore); then, the cells were incubated overnight, the transfection medium was replaced with fresh medium, and the cells were cultured for 2 days before use in subsequent experiments.
Real-Time Reverse Transcription PCR
Total RNA was extracted with an RNeasy mini kit (Qiagen), and the PCR reactions were performed with 4 µL RT-PCR Reaction Mix (Qiagen), 1 µL iScript reverse transcriptase, 900 nmol/L forward primer, 900 nmol/L reverse primer, 250 nmol/L probe, and 50 ng RNA in 20 µL reaction volumes. Reactions were performed in an Eppendorf MastercyclerPro PCR system (Thermo Scientific) as described previously.20
The primer sequences are listed below:
GAPDH: AACTTTGGCATTGTGGAAGG (forward) and CACATTGGGGGTAGGAACA (reverse); MCT1: CTCTGGGCGCCGCGAGATAC (forward) and CAACTACCACCGCCCAGCCC (reverse); MCT4: CCAGGCCCACGGCAGGTTTC (forward) and GCCACCGTAGTC ACTGGCCG (reverse); CNN1: ATGTCCTCTGCTCACTTCAAC (forward) and CACGTTCACCTTGTTTCCTTTC (reverse); TAGLN: GAAGAAAGCCCAGGAGCATAA (forward) and CCAGGATGAGAGGAACAGTAGA (reverse); ACTA2: GATCTGGCACCACTCTTTCTAC (forward) and CAGGCAACTCGTAACTCTTCTC (reverse); MYH11: AGGCGAACCTAGACAAGAATAAG (forward) and CTGGATGTTGAGAGTGGAGATG (reverse); NDRG3: CGAGGCATACCCAAGAGGAAC (forward) and GGCACACAGCAAAATGATGAG (reverse); Vimentin: TCGTTTCGAGGTTTTCGCGTTAGAGAC (forward) and GACTAAAACTCGACCGACTCGCGA (reverse); YAP: CCTTCTTCAAGCCGCCGGAG (forward) and CAGTGTCCCAGGAGAAACAGC (reverse); mTOR: CAGAAGGTGGAGGTGTTTGAG (forward) and TGACATGACCGCTAAAGAACG (reverse); and AMPK: TGTTCCAGCAGATCCTTTCC (forward) and ATAATTGGGTGAGCCACAGC (reverse).
Cells were cultured in slide chambers for 24 to 72 hours, fixed in 4% paraformaldehyde at room temperature for 20 minutes, permeabilized in 0.1% Triton X-100 at 4°C for 10 minutes, and blocked with secondary antibody serum for 60 minutes. Primary antibodies were added to the 2% secondary antibody serum in PBS at a concentration of 1:100, and the cells were incubated at 4°C overnight; then, the labeled sections were washed and incubated with FITC- and TRITC-conjugated secondary antibodies in Lab Vision UltraV Block (Thermo Scientific) at room temperature for 1 hour, counterstained with DAPI, washed, and visualized under a fluorescence microscope (DP71; Olympus, Tokyo, Japan). Positively stained cells were quantified on serial sections by using Image J software.
Flow Cytometry Analysis
Cells were dissociated with 0.25% trypsin-EDTA, fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 (Sigma) at room temperature for 10 minutes, and incubated with antibodies (Online Table I) for 3 hours; then, the cells were washed with PBS containing 0.1% Tween 20, and incubated with Alexa Fluor 488 donkey antimouse IgG secondary antibodies (Invitrogen) at room temperature for 2 hours. Flow cytometry analyses were performed with a BD Aria II instrument (BD Biosciences).
Glucose and Lactate Uptake Assay
14C-glucose and 14C-lactate were used in the assay. The cells were cultured in RPMI-1640 with B27 minus for 18 hours and the cells were washed with PBS for 2×. The cultures were then incubated for 5 minutes in basic RPMI-1640 (no glucose, Gibco) containing 1 µCi/mL 14C-glucose or 14C-lactate (Sigma). The optimal 5 minutes time for such incubation period was established in preliminary experiments (data not shown). After washing with PBS, the protein content was determined by the bicinchoninic acid assay, and the radioactivity incorporated to the cells was measured in a liquid scintillation counter (Tri-Carb 2800 TR, Perkin Elmer). Results were expressed in dpm/µg protein.
Glucose Consumption and Lactate Turnover Assay
The cells (2×104) were seeded into 48-well plates, incubated in RPMI-1640 with 1×B27 minus insulin for 24 hours, and then changed in RPMI-1640 without B27 for 24 hours; then, the cells were lysed, and the lysate and medium were collected. Glucose and lactate levels at different time points were determined with Glucose assay (Sigma) and Lactate Assay (Biovision, Milpitas, CA) kits as directed by the manufacturer’s instructions; fluorescent and colorimetric densities were evaluated at 570 nm with a Biotek Dynax2.
ELISA, Proliferation, Apoptosis, and Lactate Concentration Measurements
Collagen I and lactate-dehydrogenase levels were measured with an ELISA kit (R&D systems, USA or Life Span biological, Seattle), cell proliferation and apoptosis were measured with proliferation and apoptosis kits (Roche), and lactate concentrations were measured with a lactate assay kit (Promokine) as directed by the manufacturers’ instructions. Measurements were quantified with a SpectraMax M3 microplate reader (Molecular Devices).
ATP Concentration Measurement
Cells were plated onto gelatin-coated, 96-well, white, and clear-bottomed culture plates (Fisher Scientific). Two days later, the cells were treated with the indicated medium, and ATP levels were measured with an ATP assay kit (Invitrogen) as directed by the manufacturer’s instructions. Briefly, 100 µL of the lysis and assay solution was added to the wells; then, the solutions were shaken for 1 minute and incubated for 20 minutes at 23°C. ATP levels were quantified with a luminometer (Synergy 2, BioTek).
Swine Model of Ischemia-Reperfusion Injury
Ischemia-reperfusion injury was surgically induced in female Yorkshire swine (≈13 kg, Manthei Hog Farm, MN) via a transient, 60-minute occlusion of the left anterior descending coronary artery as described previously.21
Protein concentrations were determined in desalted samples with Bradford reagent (Bio-Rad, Hercules, CA), and then samples containing equal amounts of protein (20 µg) were labeled with iTRAQ reagent (ABI, Foster City, CA) as directed by the manufacturer’s instructions and as described previously.22 Assessments were performed in triplicate with iTRAQ 8-plex kits.
Strong Cation Exchange Chromatography, LC-MALDI (Liquid Chromatography-Matrix-Assisted Laser Desorption/Ionization) and 4800 MS/MS, and Peptide and Protein Identification
Peptide/protein isolation and identification were conducted as described previously.23
Ingenuity Pathway Analysis
The selected proteins were imported to the Ingenuity Pathway Analysis software (http://www.ingenuity.com, May 2016) to identify their associated pathways, biological functions, and diseases. The number of genes associated with each biological function or disease was counted, and P values were calculated via Fisher exact test.
All statistical analyses were performed with Statistical Package for the Social Sciences for Windows version 17 software (SPSS, Chicago). Values are presented as mean±SD, and significance was evaluated via the Student t test. A P value of <0.05 was considered statistically significant.
Lactate Enhances hiPSC-vSMC Proliferation
hiPSCs were reprogrammed from human heart fibroblasts, engineered to express eGFP (enhanced green fluorescent protein),19 and differentiated into vSMCs (hiPSC-vSMCs) and ECs (hiPSC-ECs). Under standard (ie, glucose-containing, lactate-free) culture conditions (L−), measurements of glucose uptake (Figure 1A), glucose consumption, and lactate production (Figure 1B) in hiPSC-vSMCs did not differ significantly from those in human aortic SMCs (HA-SMCs), and glucose uptake measurements were significantly greater (P<0.05) in both hiPSC-vSMCs (3.41±0.44 normalized units) and HA-SMCs (2.98±0.42) than in hiPSC-ECs (1.00±0.24). When the cells were cultured in glucose-free, lactate-enriched (L+) medium, lactate uptake (Figure 1C) in hiPSC-vSMCs (6.30±0.23 normalized units) and HA-SMCs (5.20±0.45) was similar and significantly greater (P<0.05) than in hiPSC-ECs (1.00±0.18), and ATP levels (Figure 1D) remained steady for at least 96 hours in hiPSC-vSMCs but declined significantly (P<0.05) over 48 and 96 hours (to 36.21±3.20% and 14.2±2.10% of the original level, respectively) in hiPSC-ECs. Intracellular lactate concentrations at saturation were also greater in hiPSC-ECs than in hiPSC-vSMCs, except under hypoxic conditions, when lactate levels in both cell types were suppressed but greater in hiPSC-vSMCs than in hiPSC-ECs (Figure 1E). Furthermore, the number of living hiPSC-vSMCs or HA-SMCs increased by ≈55% to 65% when the cells were cultured for 4 days in L+ medium, compared with just 10% to 15% when the cells were cultured in L− medium (Figure 1F). The number of dead cells also increased over the 4-day culture period, but the rate of increase for cells in L+ and L− medium was not significantly different. Subsequent experiments indicated that hiPSC-vSMCs proliferated best in L+ medium when the lactate concentration was 4 to 8 mmol/L (Figure 1G).
Lactate Promotes the Synthetic Phenotype in Cultured hiPSC-vSMCs
When cultured in L+ medium, both hiPSC-vSMCs and HA-SMCs tended to become less spindle-shaped and to develop the more irregular morphology associated with synthetic vSMCs (Figure 2A). Thus, we investigated whether lactate may induce cultured vSMCs to assume a predominantly synthetic phenotype. When hiPSC-vSMCs were cultured in L+ medium (4 mmol/L lactate) for 3 days, mRNA levels (reported in relative units) for markers of the contractile phenotype decreased significantly (myosin heavy chain 11: from 2.30±0.22–0.65±0.23, P<0.05; calponin: from 1.76±0.12–0.43±0.14, P<0.05), whereas the expression of synthetic vSMC markers increased (collagen 1: from 1.30±0.24–2.81±0.33, P<0.05; vimentin: from 0.43±0.16–40.6±0.26, P<0.05; Figure 2Bi). Similar changes were observed in Western blot analyses of marker protein levels (Figure 2Bii), and L+ cultured cells were significantly more proliferative than L− cultured cells when evaluated via both the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (P<0.01) and ki67 staining (L+: 50.6±8.92 cells per field, L−: 14.50±6.11 cells per field; P<0.01; Figure 2C). Measurements of cell motility were also significantly greater when the cells were cultured in L+ medium than in L− medium (L+: 42.61±3.82%, L−: 7.88±1.82%; P<0.01; Figure 2D), but the size of gels seeded with L+ cultured cells declined by just 21.22±1.0% in response to carbachol treatment, compared with a 50.42±3.20% decline in the size of gels containing L− cultured cells (P<0.01; Figure 2E). Assessments of caspace 3 activity (L+: 4.78±0.21, L−:3.31±0.14; P<0.01; Figure 2Fi) and Western blots of caspace 3, Annexin V, and Bcl-2 levels (Figure 2Fii), both indicated that apoptosis activity was greater in L+ cultured than in L− cultured hiPSC-vSMCs, and L+ cultured hiPSC-vSMCs produced more collagen I protein (L+: 523.95±18.24 ng/mg protein, L−: 131.31±21.77 ng/mg protein; P<0.01; Figure 2G), whereas proteomics analysis indicated that a wide range of extracellular matrix proteins and transcription factors were more highly expressed in L+ hiPSC-vSMCs than in L− hiPSC-vSMCs (Figure 2H). Notably, the changes in protein expression associated with the L+ medium were similar to those observed when the cells were cultured under hypoxic conditions. Thus, lactate seems to promote the synthetic phenotype in hiPSC-vSMCs.
Lactate-Induced Phenotypic Modulation of hiPSC-vSMCs Is Mediated by MCTs and NDRG
Lactate transportation is mediated by MCTs, of which subtypes 1 and 4 are expressed in vSMCs,24,25 and compared with assessments in L− cultured hiPSC-vSMCs under normoxic conditions, MCT mRNA levels were 6.71±0.89-fold (MCT1) and 15.18±0.48-fold (MCT4) greater when the cells were cultured in L+ medium under normoxic conditions, and 5.72±1.48-fold (MCT1) and 13.21±1.68-fold (MCT4) greater when cultured in L− medium under hypoxic conditions (Figure 3A). Furthermore, the generalized MCT inhibitor α-cyano-4-hydroxycinnamate26 inhibited MCT expression by ≈50%, and the MCT1-specific inhibitor AZ3965 inhibited MCT1 expression by ≈70% in L+ cultured cells (Figure 3B), and Western blot assessments confirmed that the response of MCT1 and MCT4 protein levels to the presence of lactate, hypoxia, α-cyano-4-hydroxycinnamate, and AZ3965 were consistent with the mRNA results (Figure 3A and 3B).
The proliferation of L+ cultured cells also declined by 37.2% in the presence of α-cyano-4-hydroxycinnamate (P<0.01), by 24.5% in the presence of AZ3965 (P<0.01; Figure 3C), and by 64.2% (P<0.01) when the cells were transfected with MCT4 siRNA (Figure 3E), whereas collagen production declined by 59.4% (P<0.01), 38.2% (P<0.01), and 58.4% (P<0.01) in response to generalized MCT inhibition, MCT4-specific inhibition, and MCT4 siRNA transfection, respectively. Notably, the magnitudes of the declines in proliferation and collagen I production associated with the generalized MCT (α-cyano-4-hydroxycinnamate) and MCT1 specific (AZ3965) inhibitors were not significantly different, which suggests that the roles of MCT1 and MCT4 in lactate-induced vSMC phenotypic modulation may differ. MCT4 iRNA transfection also altered mRNA measurements of contractile (MYH11) and synthetic (collagen 1, vimentin) vSMC marker expression: during 3 days of culture in L+ medium, MYH11 mRNA levels declined (from 2.18±0.14–0.61±0.09 relative units; P<0.01), whereas collagen 1 levels increased (from 3.35±0.15–1.19±0.23 relative units, P<0.01) in cells transfected with control siRNA but did not change significantly in cells transfected with MCT4 siRNA, and vimentin mRNA levels on day 3 were 11.4-fold greater (P<0.01) in cells transfected with control iRNA than in MCT4-iRNA–transfected cells.
The L+ medium and hypoxia also upregulated the expression of NDRG3 (Figure 4A), which is stabilized by lactate to promote angiogenesis and cell growth under hypoxic conditions,27 as well as several downstream components such as Raf and ERK (extracellular signal–regulated kinase) of the NDRG3 signaling pathway (Figure 4B) that participate in cell proliferation, migration, and differentiation.28 Furthermore, when NDRG3 activity was blocked by transfecting the hiPSC-vSMCs with NDRG3 siRNA (Figure 4C), proliferation was inhibited by 43.8% (P<0.01; Figure 4D), collagen production declined by 62.2% (P<0.01; Figure 4E), and the expression of synthetic vSMC markers declined significantly, whereas contractile vSMC marker expression increased significantly (Figure 4F), in hiPSC-vSMCs that had been cultured in L+ medium.
Lactate Promotes the Synthetic vSMC Phenotype in the Hearts of Swine After Myocardial Infarction
To determine whether the results from our in vitro experiments were consistent with observations in vivo, ischemia-reperfusion injury was surgically induced in swine, and then lactate concentrations, lactate-dehydrogenase activity, and the amount of MCT1, MCT4, and NDRG3 protein were evaluated in the remote (ie, nonischemic) zone (RZ) and in the ischemic zone (IZ) 1 hour later. All 5 parameters (lactate concentration: RZ=77.72±6.71 µmol, IZ=130.12±7.89 µmol, P<0.01; lactate-dehydrogenase activity: RZ=61.14±5.73 milliunits/mg, IZ=114.88±11.19 milliunits/mg, P<0.01; and Western-blot assessments of MCT1, MCT4, and NDRG3 levels) were greater in the IZ than in the RZ (Figure 5A through 5C); furthermore, vimentin expression was observed in small vessels near the border of the infarct, and the number of cells that expressed both Ki67 and vimentin were significantly higher in the IZ than in the RZ (Figure 5D). Collectively, these results suggest that ischemia-reperfusion injury may promote the proliferation of synthetic vSMCs and that this effect could be induced by increases in lactate levels.
Proteomic, Biomarker, and Pathway Analyses
Global proteomic analyses with L− and L+ hiPSC-vSMCs identified and quantified 2713 proteins, 84.9% of which were present in both experimental groups. Within each experimental group, analyses of replicate samples were highly reproducible (ie, 91.0% of the proteins identified in L− hiPSC-vSMCs and 81.6% of the proteins identified in L+ hiPSC-vSMCs were observed in >50% of duplicates) with low variations in intensity (ie, the coefficient of variation was <10% for >95% of identified proteins). Significant differences between L− and L+ hiPSC-vSMCs were observed for 685 proteins, including 45 extracellular proteins that are involved in tissue construction or repair, most of which were expressed at higher levels in L+ than in L− cells. L+ culture conditions also upregulated the expression of 54 transcriptional or translational proteins, whereas biomarker analysis found significant differences for 15 proteins that are markers for vSMC identity, including tenascin-C and versican, which are associated with the synthetic phenotype. Furthermore, pathway analysis suggested that lactate altered the activity of mechanisms that regulate cell proliferation, survival, and migration; protein synthesis; gene transcription; and differentiation (Table)4, and the changes associated with lactate and hypoxia tended to not differ significantly (eg, both conditions increased ILK (integrin-linked kinase) signaling, which has been linked to the synthetic vSMC phenotype). Follow-up assessments via Western blot (Figure 6A) and quantitative RT-PCR (Figure 6B) confirmed that both lactate and hypoxia upregulate the expression of Yap (yes-associated protein), which participates in the Hippo signaling pathway, as well as mTOR (mechanistic target of rapamycin) and AMPK (adenosine monophosphate–activated protein kinase), which are components of pathways involved in fibrosis and injury repair. Collectively, these observations are consistent with the role of synthetic vSMCs in myocardial repair and with our observations that lactate promotes the expression of synthetic markers, as well as proliferation and migration, in hiPSC-vSMCs.
The phenotype of an individual vSMC is thought to fluctuate within a continuum bounded by predominantly contractile and synthetic cells. The synthetic phenotype is characterized by higher proliferation rates, as well as the production of extracellular matrix proteins, and likely contributes to the role of vSMCs in intimal hyperplasia, hypertension, atherosclerosis, and other disease conditions.29–31 However, synthetic vSMCs also participate in a variety of beneficial physiological processes, such as the remodeling of vessels in response to vascular injury, pregnancy, or exercise.1,32 Here, we show that the switch from a contractile to synthetic vSMC phenotype can be triggered by the presence of lactate, which has traditionally been considered a metabolic byproduct, but has more recently been shown to function as a signaling molecule13,14 with roles in wound healing, angiogenesis, chronic inflammation, cancer development, and gene expression.11,12,15,16 Thus, patients with many pathological conditions may respond to therapeutic interventions that target tissue lactate levels.33,34
Because synthetic vSMCs participate in a variety of myocardial repair processes, including angiogenesis, the switch from a contractile to synthetic vSMC phenotype may be useful for treatment of cardiovascular diseases such as increase of neovascularization. However, vSMCs also contribute to fibrosis which could be counterproductive or even harmful depending on location or microenvironment. The potential relationship between myocardial lactate levels and myocardial neovascularization has yet to be investigated. Although studies in the fields of cancer research and immune modulation have identified that changes in SMC phenotype are associated with the amount of lactate in the cellular microenvironment, which seems to be mediated by mTOR, AMPK, and PI3K-Akt,33,34 the mechanisms by which lactate levels induce vSMC phenotypic switching in cardiovascular tissues remain unclear. The results from the current study demonstrate that lactate-induced phenotypic switching requires the activity of known lactate transporters (MCT1 and MCT4) and seems to involve the stabilization of NDRG3, which has been linked to many cellular activities that are associated with the synthetic vSMC phenotype, including proliferation,28 and is activated by lactate to promote angiogenesis and cell growth under hypoxic conditions.27 Furthermore, the effects of the L+ medium on lactate concentration and on MCT1, MCT4, and NDRG3 expression were similar to those observed under hypoxic culture conditions, and myocardial infarction injury was associated with increases in lactate levels, in lactate-dehydrogenase activity, and in MCT1, MCT4, and NDRG3 expression, as well as in the number of cells that expressed the proliferation marker Ki67. Notably, many of the signaling molecules and pathways that contribute to vSMC proliferation such as protein kinases A-C, mitogen-activated protein kinase, the transforming growth factor β superfamily, the Ras-MAPK, and PI3K-Akt pathways, also seem to have a role in vSMC phenotypic switching.35 The proteomic and pathway analyses data in the current study also confirmed that lactate induces many of these same signaling mechanisms in vSMCs.
In conclusion, the results in the present study demonstrate for the first time that vSMCs assume a more synthetic phenotype in a microenvironment that is rich in lactate. Thus, mechanisms that link glucose metabolism to vSMC phenotypic switching could play a key role in the pathogenesis and treatment of cardiovascular disease.
Sources of Funding
This study was supported by the following funding sources: National Institutes of Health RO1 HL 99507, HL114120, HL 131017, and UO1 HL134764.
In September 2017, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.117.311819/-/DC1.
- Nonstandard Abbreviations and Acronyms
- adenosine monophosphate–activated protein kinase
- endothelial cells
- human aortic
- human induced pluripotent stem cells
- ischemic zone
- monocarboxylic acid transporter
- mechanistic target of rapamycin
- N-myc downstream regulated gene
- remote zone
- vascular smooth muscle cells
- yes-associated protein
- Received August 2, 2017.
- Revision received October 5, 2017.
- Accepted October 10, 2017.
- © 2017 American Heart Association, Inc.
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- Wergin MC,
- Verrax J,
- Rabbani ZN,
- De Saedeleer CJ,
- Kennedy KM,
- Diepart C,
- Jordan BF,
- Kelley MJ,
- Gallez B,
- Wahl ML,
- Feron O,
- Dewhirst MW
- Melotte V,
- Qu X,
- Ongenaert M,
- van Criekinge W,
- de Bruïne AP,
- Baldwin HS,
- van Engeland M
- Suzuki LA,
- Poot M,
- Gerrity RG,
- Bornfeldt KE
- San-Millán I,
- Brooks GA
Novelty and Significance
What Is Known?
The phenotypes of vascular smooth muscle cells (vSMCs) change within a continuum bounded by predominantly contractile and synthetic vSMCs.
Contractile vSMCs provide the mechanical force required for blood pressure regulation, whereas synthetic vSMCs produce extracellular matrix proteins, growth factors, and other molecules that contribute to vascular remodeling in response to injury, pregnancy, and other physiological conditions.
Although lactate has traditionally been considered a metabolic product, lactate levels increase in response to ischemia.
What New Information Does This Article Contribute?
When vSMCs were cultured in glucose-free, lactate-enriched (L+) medium, activities associated with the synthetic phenotype, such as proliferation, migration, and the production of proteins that participate in tissue construction or repair, and the expression of synthetic vSMC markers increased, whereas contractile activity and the expression of contractile proteins declined.
The changes induced by the L+ medium were accompanied by increases in the expression of proteins involved in lactate transport and neovascularization.
Observations in hypoxia-cultured vSMCs were similar to those in L+-cultured vSMCs, and measurements of lactate levels, lactate-dehydrogenase levels, vSMC proliferation, and monocarboxylic acid transporter and N-myc downstream regulated gene (NDRG) expression were greater in the infarct border zone than in the remote zone of infarcted porcine hearts.
The functional characteristics of vSMCs include the contractile activity, as well as participation of a variety of other physiological activities and repair mechanisms. The diversity of vSMC function is reflected in their phenotypes, which comprise a spectrum ranging from contractile cells at one end to structural synthetic cells at the other. Evidence suggests that contractile vSMCs can adopt a more synthetic phenotype in response to ischemic injury, but the mechanisms that activate this phenotypic switch are poorly understood. The results presented in this report indicate that vSMCs assume a more synthetic phenotype when cultured in the presence of lactate and, consequently, the lactate produced during ischemic injury could trigger vSMC phenotypic switching and promote vSMC-dependent repair mechanisms.