Hyperglycemia-simulating environment attenuated experimentally induced calcification in cultured human aortic valve interstitial cells

Abstract

Objectives: The role of diabetes mellitus as a risk factor for the development of calcific aortic valve disease has not been fully clarified. Aortic valve interstitial cells (VICs) have been suggested to be crucial for calcification of the valve. Induced calcification in cultured VICs is a good in vitro model for aortic valve calcification. The purpose of this study was to investigate whether increased glucose levels increase experimentally induced calcification in cultured human VICs. Design: VICs were isolated from explanted calcified aortic valves after valve replacement. Osteogenic medium induced calcification of cultured VICs at different glucose levels (5, 15, and 25 mM). Calcium deposits were visualized using Alizarin Red staining and measured spectrophotometrically. Results: The higher the glucose concentration, the lower the level of calcification. High glucose (25 mM) reduced calcification by 52% compared with calcification at a physiological (5 mM) glucose concentration (correlation and regression analysis: r = −0.55, p = .025 with increased concentration of glucose). Conclusions: In vitro hyperglycemia-like conditions attenuated calcification in VICs. High glucose levels may trigger a series of events that secondarily stimulate calcification of VICs in vivo.

Keywords:

• Hyperglycemia
• glucose
• aortic valve stenosis
• valve interstitial cells
• calcification

1. Introduction

The incidence of calcific aortic valve disease (CAVD) is expected to increase with aging of the population [1]. Diabetes mellitus type 2, also a disease of the elderly, is associated with cardiovascular diseases. These include macro- and microvascular atherosclerosis, aortic stenosis, and increased cardiovascular mortality [2–7].

Diabetes is a known risk factor for the development of aortic valve calcification and stenosis [8]. Furthermore, CAVD progresses more rapidly in patients with diabetes [7,9,10]. Patients with both diabetes and CAVD have an increased prevalence of comorbidities such as hypertension, end-stage renal disease, generalized atherosclerosis, obesity, and metabolic syndrome [7]. Diabetes is also an independent risk factor for surgical or catheter-based aortic valve intervention [9,11].

The molecular and cellular mechanisms by which diabetes predisposes patients to aortic valve calcification are not yet fully understood. It is generally agreed that aortic valve interstitial cells (VICs) play a fundamental role in valve calcification irrespective of whether they acquire osteoblastic-like phenotypes [12] or undergo procalcific degeneration/death [13]. Kopytek et al. [14] suggested that high doses of glucose activated oxidative stress and induced a pro-inflammatory state in cultured VICs. Disease progression may then be accelerated and involve different pro-calcific pathways [15]. It has been previously reported that diabetes contributes to CAVD pathogenesis by promoting early mineralization of the aortic valve [16]. However, this conclusion was derived from observations in a mouse model [15]. To our knowledge, there is still no information on whether hyperglycemia directly stimulates calcification in human VICs. Induced calcification in VICs cultures is regarded as the best in vitro model of CAVD (22). We hypothesized that high levels of glucose per se have a pro-calcification effect. The purpose of the present study was to investigate whether hyperglycemia augments experimentally induced calcification in cultured human VICs.

2. Materials and methods

2.1. Ethical considerations

The aortic valves were harvested during aortic valve replacement at the Department of Cardiothoracic Surgery, Oslo University Hospital, Oslo, Norway. Written informed consent was obtained from all the patients. The project was approved by the Regional Ethics Committee of Southeast Norway (REK approval no. 2015/1003) and performed in accordance with the principles of the Declaration of Helsinki.

2.2. Isolation and cultivation of human primary aortic valve interstitial cells

This study was performed using VICs from patients with stenotic aortic valves (n = 4). These patients did not have a history of diabetes mellitus and VICs were isolated as previously described [17]. The excised aortic valve leaflets were washed with PBS, then subjected to digestion with 2 mg/mL collagenase II (Worthington Biochemical Corporation, Lakewood, NJ) overnight at 37 °C. Then, the tissue was pipetted repeatedly to break up the tissue mass and centrifuged at 300 × g for 5 min, then the pellet was washed in PBS and centrifuged at 300 × g for 5 min before the pellet containing VICs were resuspended in basic culture medium consisted of DMEM supplemented with 15% FBS (HyClone, SH30070.03, GE Healthcare, Chicago, IL) and 50 µg/mL of gentamicin (15750-037, Gibco, Waltham, MA), and plated on T75 flask. VICs were cultured in standard growing conditions at 37 °C in 5% CO₂ until confluence of 90% before passaging. Primary cells were cultured for 3–5 passages.

Cells from each donor were cultivated in either high-glucose DMEM (25 mM glucose, 41966029, Gibco, Waltham, MA) or low-glucose DMEM (5 mM glucose, 31885023, Gibco, Waltham, MA). One passage before exposure to osteogenic medium, VICs from both glucose levels were adapted to moderate-high glucose media (15 mM) by change of original media with either 5 mM or 25 mM to 15 mM glucose level media. Consequently, cultures of VICs from each patient with four different regimens of glucose were available (5 mM, 15 mM (from 5 to 15 mM), 15 mM (25–15 mM), and 25 mM) for osteogenic differentiation.

2.3. Osteogenic differentiation

To induce osteogenic differentiation of VICs, classic osteogenic medium was used for 21 days: DMEM (with different glucose concentrations) supplemented with 10% FBS, 50 µg/mL gentamicin, 50 µM ascorbic acid, 0.1 mM dexamethasone, and 10 mM β-glycerophosphate [18]. The cells were plated in 24-well tissue culture plates (33 × 10³ cells per well).

2.4. Alizarin Red staining

For the measurement of calcium deposits, after 21 days of induced osteogenic differentiation, cells were washed with PBS, fixed in 70% ethanol for 60 min, washed twice with distilled water, and stained with Alizarin Red solution (Sigma, St. Louis, MO) according to the manufacturer’s instructions. Alizarin Red staining was performed and measured spectrophotometrically. For the 24-well plates, 200 μL 10% acetic acid was added to each well and incubated for 30 min at room temperature with gentle agitation. The cells were detached using a cell scraper, and the suspension was transferred to a 1.5 mL microcentrifuge tube and vortexed vigorously for 30 s. The cells were then heated to 85 °C for 10 min before being chilled on ice for five minutes followed by centrifugation (15,000 × g) for 15 min. The supernatant was transferred to a new 1.5 mL microcentrifuge tube. To achieve a pH between 4.1 and 4.5, 75 μL of 1 M NaOH was added to each tube. Finally, 50 μL of the tube was transferred to a clear bottom 96-well plate, and the absorbance at 405 nm was measured using a plate reader (Molecular Devices, Emax, Sunnyvale, CA).

2.5. Statistics

GraphPad Prism software (La Jolla, CA) was used. The two groups with VICs cultivated with intermediate glucose concentration (15 mM) had similar calcification and were merged for analysis. Values represented as scatter plots and mean with confidence interval. Correlation and regression analysis were performed. t-test was applied with Bonferroni’s correction (p < .025 was considered significant).

3. Results

Figure 1 shows staining of calcification with Alizarin Red in wells with VICs cultured with four different glucose concentrations. The highest glucose concentration had the lowest calcification. Both cell cultures adapted to moderate-to-high glucose level (15 mM) showed similar level of calcification. In the group 5 to 15 mM and 25 to 15 mM, calcification was 70 ± 37 and 71 ± 45, respectively (mean ± SD). Consequently, these two groups were merged for presentation and analysis. Their levels of calcification were intermediate to cultures with glucose levels of 5 and 25 mM. Correlation analysis indicated a significant negative correlation between the calcification level and the glucose level of VICs cultures (Figure 2).

Figure 1. Representative microphotography pictures of Alizarin Red staining of nondifferentiated (nondif) aortic valve interstitial cells (VICs) and VICs with induced calcification. VICs were cultivated in media with different concentration of glucose (5 mM, 5–15 mM (cells adapted from 5 to 15 mM), 25–15 mM (cells adapted from 25 to 15 mM) and 25 mM).

Figure 2. High glucose levels caused lower calcification in aortic valve interstitial cells (VICs) exposed to osteogenic medium. Calcification was measured spectrophotometrically after Alizarin Red staining. One passage before exposure to osteogenic medium, VICs from both glucose levels (5 mM and 25 mM) were adapted to moderate-high glucose media (15 mM) by change of original media with either 5 mM (

) or 25 mM to 15 mM (

) glucose level media. These two groups with 15 mM were merged for presentation and analysis. Thus, cultures of VICs from each patient with three different regimens of glucose were available (5, 15 and 25 mM) for osteogenic differentiation. Values represented as scatter plots and mean with confidence interval. t-test was applied with Bonferroni’s correction (p < .025 was considered significant).

4. Discussion

There is abundant evidence that patients with diabetes mellitus have an increased occurrence of CAVD progression [9,11,19,20]. The hypothesis that hyperglycemia is a direct stimulus for calcification in VICs was not confirmed in the present study. In contrast, the higher the glucose concentration, the lower the level of experimentally induced calcification. Cell cultures adapted to moderately high glucose levels show intermediate levels of calcification. It did not matter whether they were adapted from a higher or a lower glucose level.

Most studies that employ cultured VICs are conducted using high-glucose media (25 mM glucose), which is generally common in cell culture studies [12,21–23]. Some researchers have proposed the use of glucose media with lower glucose levels to maintain a more physiological state of VICs [24,25]. However, the above-mentioned media compositions with low glucose are different not only in glucose level from the standard one, but also with the addition and concentration of other growth media components. Hence, none of the proposed methods in low-glucose media are suitable for direct comparison of how low and high glucose levels may influence calcification in cultured VICs. Therefore, VICs from both glucose levels were adapted to non-standard, moderately high-glucose media. This was performed: (1) to obtain an intermediate level of glucose during calcification; (2) to investigate whether the initial glucose level during isolation might influence induced calcification in VICs. The result was no difference between 1 and 2 groups, but a stepwise reduction in calcification when the glucose levels increased.

4.1. Possible mechanisms of aortic valve calcification in diabetes

Diabetes is known to be predictive of poor prognosis in CAVD; however, the molecular mechanisms involved in diabetes‐associated CAVD are unknown [26,27]. The present findings do not refute the established finding that diabetes mellitus is a risk factor for CAVD. However, this investigation suggests that it is not the glucose levels per se that trigger calcification of VICs. The pathological triggers may be changes in the internal milieu of the valve leaflets caused by diabetes. It has been shown that interactions between VICs and specific peptides of the extracellular matrix and endothelial cells in diseased aortic valves promote specific cellular events and subsequently trigger the calcification process [28]. However, VICs are the main functional units in the aortic valve that can differentiate into osteoblast-like cells.

The present findings are in agreement with studies suggesting that hyperglycemia per se might be less important for the severity of CAVD in diabetes [29,30]. Instead, hyperglycemia causes valvular protein glycation and leaflet accumulation of advanced glycoxidation end-products (AGEs), which may be important for the development of CAVD. A schematic overview of the possible cellular and molecular mechanisms of aortic valve calcification in diabetes mellitus, is shown in Figure 3. One important difference from calcification in patients without diabetes is the role of AGEs [29,31,32]. Accumulation of AGEs correlates with severity of CAVD [29]. AGEs activate surface receptors for AGEs (RAGE). This receptor has profound effects on cell functions [14]. The influence of diabetes on calcification is likely multifactorial, and inflammation appears to be a key factor [14,33]. Shear stress, metabolic changes in plasma, and circulating inflammatory cells may injure the endothelium [34]. The result of this process is inflammation and the creation of reactive oxygen species [35].

Figure 3. Possible pro-calcification events in aortic valve leaflets in diabetes mellitus. Accumulation of advanced glycoxidation end-products (AGEs), oxidized low-density lipoproteins (oxLDL) and invading macrophages all causing generation of reactive oxygen species (ROS) and inflammation. AGEs bind to activate the surface receptor for AGEs (RAGE). Altogether, a cascade of events develops with activation of valve interstitial cells (VICs) in a pro-osteogenic direction. This includes synthesis of cytokines (tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)). Activated valve interstitial cells (VIC) secrete bone morphogenic proteins (BMP). Signaling pathways (Notch) and transcription factors such as Runt-related transcription factor-2 (Runx2) and nuclear factor κB (NFκB) are activated. EV: extracellular vesicles; LDL: low-density lipoprotein.

Endothelial damage leads to intraleaflet accumulation of oxidized lipoproteins, causing oxidative stress and inflammation [35]. A possible mode by which high glucose levels may have a pro-calcific effect is via action on the valve endothelial cells. High glucose causes phenotype changes in human valve endothelial cells [36]. The AGEs-RAGE axis has been shown to cause endothelial-to-mesenchymal transition (EndMT) in relation to CAVD [37]. EndMT may be one factor causing CAVD [38]. Consequently, high glucose may cause CAVD by a direct action on the valve endothelial cells.

4.2. Specific features of the study

There is no optimal animal model for investigating CAVD. A good option for an experimental model for studying CAVD is VICs culture [23]. However, it is to be considered that such a model induces an osteogenesis-like process, while other models exist to induce calcification other than ossification [13]. Cultured VICs may have some differences in their phenotype compared to VICs in their native environment surrounded by the extracellular matrix [39,40]. Cultured VICs lack the influence of the natural extracellular matrix and microenvironment of the valve leaflets in vivo [41]. Furthermore, as a general rule, the results of in vitro experiments cannot be directly extrapolated to processes in the human body [23]. Calcification induced over three weeks may also have some mechanisms different from ongoing calcification in the valve in vivo, a process that develops over years.

4.3. Conclusions

Hyperglycemia itself does not directly trigger calcification of VICs in vitro. Most likely, high glucose levels initially trigger a series of events and secondary molecular and chemical modifications that drive VICs into increased calcification.

Consent form

All patients gave written informed consent.

Acknowledgements

Professor Leiv Arne Rosseland and Faiza Moghal, Oslo University Hospital, are gratefully acknowledged for administrative support.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by Helse Sør-Øst, the University of Oslo, Digital Life Norway, and Nasjonalforeningen for Folkehelsen.