Lower plasma levels of high-density lipoproteins (HDL) in adolescents with type 2 diabetes (T2D) have been associated with a higher pulse wave velocity (PWV), a marker of arterial stiffness. Evidence suggests that HDL proteins or particle subspecies are altered in T2D and these may drive these relationships. In this work, we set out to reveal any specific proteins and subspecies that are related to arterial stiffness in youth with T2D from proteomics data. Plasma and PWV measurements were previously acquired from lean and T2D adolescents. Each plasma sample was separated into 18 fractions and evaluated by mass spectrometry. Then, we applied a validated network-based computational approach to reveal HDL subspecies associated with PWV. Among 68 detected phospholipid-associated proteins, we found that seven were negatively correlated with PWV, indicating that they may be atheroprotective. Conversely, nine proteins show positive correlation with PWV, suggesting that they may be related to arterial stiffness. Intriguingly, our results demonstrate that apoA-I and histidine-rich glycoprotein may reverse their protective roles and become antagonistic in the setting of T2D. Furthermore, we revealed two arterial stiffness-associated HDL subspecies, each of which contains multiple PWV-related proteins. Correlation and disease association analyses suggest that these HDL subspecies might link T2D to its cardiovascular-related complications.
Type 2 diabetes mellitus (T2D) is a major risk factor for development of cardiovascular disease (CVD) [
HDL-targeted therapies have significant underlying challenges because HDL is more complex than previously recognized. Proteomic studies of HDL have identified at least 95 distinct proteins associated with HDL [
Recent studies have shown that HDL is a heterogeneous group of subspecies with unique protein/lipid compositions that are not reflected in the HDL-C measurement [
Using gel filtration chromatography that separates lipoproteins by size, we previously found that adolescents with type 2 diabetes were depleted of large HDL particles compared with obese and lean adolescents [
Here we sought to determine if any proteins or groups of proteins may be related to arterial stiffness as measured by PWV. Recently, we have developed a network-based computational method to infer HDL subspecies from complex proteomics data [
Five adolescents with T2D and six lean controls aged 17–27 years were included in this analysis [
Study cohort characteristics.
Variable | Lean | T2D |
---|---|---|
| 6 | 5 |
Age (years) | 21.67 ± 2.16 | 21.6 ± 4.04 |
Body mass index (mg/kg) | 23.97 ± 2.04 | 37.89 ± 3.75 |
Total cholesterol (mg/dL) | 153.67 ± 26.38 | 215.6 ± 55.84 |
Triglycerides (mg/dL) | 73.17 ± 27.8 | 278.6 ± 344.52 |
High-density lipoprotein-cholesterol (mg/dL) | 52.67 ± 10.73 | 33.8 ± 4.44 |
Low-density lipoprotein-cholesterol (mg/dL) | 86.6 ± 32.24 | 127.8 ± 53.1 |
Systolic blood pressure (mmHg) | 119.72 ± 11.33 | 116.0 ± 8.21 |
Diastolic blood pressure (mmHg) | 71.56 ± 7.96 | 71.2 ± 5.78 |
Pulse wave velocity (m/s) | 5.74 ± 1.11 | 6.8 ± 1.12 |
Hemoglobin A1C (%) | N/A | 11.3 ± 4.65 |
Hemoglobin A1C (mmol/mol) | N/A | 100.2 ± 50.63 |
Data are mean and standard deviation (SD). N/A: not available.
For each subject, blood was fractionated by gel filtration chromatography into 18 lipid-containing fractions by size [
To investigate if any individual HDL proteins were related to arterial stiffness, we calculated Pearson correlation coefficient (PCC) between the relative abundance of HDL proteins in fractions 19–31 and PWV measurements. PCC was calculated for the lean and T2D combined dataset, the lean dataset only, and T2D dataset only. We realized that PCC of the lean and T2D combined dataset may be mainly driven by the lean group (Supplemental Figure
To infer HDL subspecies, we applied the network-based approach we previously developed for clustering comigrating proteins [
To determine whether the HDL subspecies are related to particular diseases, we performed disease association analyses using an independent analyzer, ToppCluster [
We first sought to determine if individual HDL-associated proteins in the plasma HDL fractions were associated with arterial stiffness. A total of 68 HDL-associated proteins were identified across all fractions. Figures
PCCs of peptide counts of individual proteins with PWV for the
PCCs of peptide counts of individual proteins with PWV for the
In contrast, in the T2D group, nine proteins were found to be positively correlated with PWV (Figure
Based on the correlation analysis, we wanted to know if these PWV-related proteins are associated with altered HDL particle sizes. This prompted us to investigate the distributions of these PWV-related proteins. We compared distribution patterns of all the significantly correlated proteins between the lean and T2D groups (
Next, we sought to identify potential HDL subspecies that may be related to arterial stiffness. We have previously designed a computational method to reveal putative HDL subspecies [
In Figure
Another important topological feature of a network is the number of connected components (NCC). In a graph, its connected components are the set of the largest subgraphs that are each connected. A lower NCC indicates fewer subgraphs and suggests stronger network connectivity. The NCC of lean and T2D networks are 8 and 23, respectively. In terms of number of links, the lean network has more links than the T2D network with the same PCC cutoff. This suggests that HDL proteins in the lean controls have a stronger comigration relationship than the ones in T2D subjects. Loss of the links in the T2D network is mainly within the clusters (e.g., green and yellow clusters). On the other hand, the T2D network involves additional links; for example, kininogen-1 comigrates with the pink cluster. The differences between the networks indicate that the compositional alterations of certain HDL subspecies may occur in youth with T2D.
Upon further examination, we first noted that the edge between apoA-I and apoA-II is absent in the T2D group. Although not all apoA-I containing particles include apoA-II, it is well known that apoA-I and apoA-II often reside on the same HDL complexes [
Figures
In the HDL-associated PPI network, we observed three clusters that contain at least two proteins that were highly correlated with PWV. These are the blue cluster containing four proteins (apoA-IV, antithrombin-III, kallistatin, and albumin), the green cluster containing five proteins (apoA-I, apoA-II, complement C3, complement C1s subcomponent, and Ig gamma-1 chain C region), and the pink cluster containing six proteins (hemopexin, gelsolin, plasminogen, complement C9, apoH, and alpha-2-HS-glycoprotein). The proteins in the blue cluster do not likely form a subspecies. They simply migrate together due to their similar sizes. If they formed a subspecies, their combined MW would be at least 205 kDa, without any associated lipid, and would thus elute in an earlier fraction. Therefore, this grouping will not be referred to as a subspecies. However, the remaining 2 clusters are each likely migrating together as at a subspecies, since they elute in a fraction containing proteins that are larger than any single protein in their respective clusters. The migration patterns for the 2 subspecies in both lean and T2D groups are shown in Figure
It appears that the intact subspecies containing two atheroprotective proteins are remodeled into disconnected proatherogenic elements. Lastly, in the pink cluster (Figures
Taking our analysis a step further, we sought to explore the human diseases these subspecies are associated with. We used ToppCluster [
Many pieces of evidence have suggested that alterations of certain HDL proteins or subspecies in T2D patients may be associated with arterial stiffness [
Even though previous studies have shown that HDL-associated proteins are altered in youth with T2D [
With regard to the individual HDL proteins that were correlated with arterial stiffness, several findings are noteworthy, including changes in the PCC of apoA-I, histidine-rich glycoprotein, and hemopexin with PWV. In the current analysis, apoA-I has both negative and positive correlations with arterial stiffness, which indicates that apoA-I may serve as a platform for HDL particles that can be both atheroprotective and atherogenic. Since the gel filtration technique separates HDL particles by hydrodynamic diameter, an alteration of the distribution pattern indicates a change in particle size and thus a compositional change of HDL subspecies. ApoA-I in the T2D group exhibits lower normalized abundance in the larger HDL range and an increase in the smaller particle fractions. From the HDL subspecies perspective, apoA-I is the major component and structural scaffold of HDL and is likely involved in multiple HDL subspecies. Based on the PCC in different fractions (Figures
Additionally, histidine-rich glycoprotein and hemopexin are positively correlated with PWV in the T2D group. These two proteins play roles in the regulatory functions for the blood coagulation, complement, and fibrinolysis pathways. As of now, we do not have enough information to speculate how these proteins may contribute to vascular stiffness or even if they are causal in this regard.
Our network analysis revealed clear global differences in the protein networks between lean and T2D groups. Global topological parameters of these two networks suggest that the lean network has a stronger connectivity and tends to have more clusters, while the T2D network has a more scattered topology. It is possible that, in T2D, certain proteins are missing from critical HDL subspecies, thus altering their function. Fisher et al. [
Two candidate subspecies that contain at least two PWV-correlated proteins that may be related to arterial stiffness were identified. Notably, there are some distribution pattern changes in individual proteins of the identified subspecies between the lean and T2D groups (Figure
One limitation of this work was the limited sample size. Due to the limitation of the subject recruiting as well as the relatively limited throughput of the plasma fractionation procedure, this study was carried out on a representative subject group instead of a large population. Thus, we cannot totally exclude the possibility of bias induced by individual differences. However, no other study has endeavored to subfractionate lipoproteins and track their proteome in this much detail. Another limitation of the study is that we cannot know with 100% certainty whether every protein in the analysis resided on an HDL particle. However, the study was specifically designed to increase the likelihood that we are only looking at HDL-associated proteins. The method used in this study involves a multipronged approach to fractionate HDL. First, whole plasma is size-fractionated. Second, LRA is used to isolate lipid-containing particles from each fraction. Although LRA effectively binds lipid-rich species, we cannot confidently exclude the possibility that some plasma proteins bound the LRA. Thus it is possible that, even with the LRA selection process, some plasma proteins were detected by MS analysis. However, finally, the resulting list of MS identified proteins (approximately 110 proteins) was filtered against the HDL Proteome Watch, a curated database that reports proteins that have been detected on HDL by multiple mass spectrometry studies. Thus, it is reasonable that the proteins reported in the final subspecies analysis are likely to be HDL-associated proteins. Future work will focus on validating these results in larger groups.
In summary, we identified seven HDL proteins that are negatively correlated with arterial stiffness, as well as nine proteins that positively correlated with arterial stiffness from complex proteomics data. Additionally, we constructed protein comigration networks for the lean and T2D groups, separately. Using the network-based complex identification, we discovered two PWV-related HDL subspecies that are associated with multiple complications of T2D. We caution that current findings are only based on correlation analysis. Nevertheless, the correlations are significant enough to support further experimental investigation. We found that the distribution of those proteins may provide a better understanding on how HDL proteins are altered in youth with T2D and how they are related to arterial stiffness. Our results also indicate that certain HDL proteins (e.g., apoA-I and histidine-rich glycoprotein) may reverse their protective roles and become atherogenic in T2D condition. This may explain why the HDL-C raising therapies [
High-density lipoproteins
Type 2 diabetes
Pulse wave velocity
Cardiovascular disease
Low-density lipoproteins
High-density lipoprotein-cholesterol
Trypanosome lytic factor
Apolipoprotein
Reverse cholesterol transport.
For data availability, please refer to the original study [
The study has been approved by IRB committee of Cincinnati Children’s Hospital Medical Center.
Written informed consent was obtained from participants who are 18 years old or older or from the parent or guardian with for participants below 18 years of age.
The authors declare that there are no conflicts of interest.
L. Jason Lu designed the research plan in this paper; Amy S. Shah and Scott M. Gordon performed experiments and collected clinical data; Xiaoting Zhu, Hailong Li, and Sheng Ren analyzed the data; Amy S. Shah, Debi K. Swertfeger, and John T. Melchior helped in study design; Xiaoting Zhu, Debi K. Swertfeger, Hailong Li, and L. Jason Lu wrote the manuscript; W. Sean Davidson and L. Jason Lu supervised the research.
This work was supported by National Institutes of Health (R01-HL111829 to L. Jason Lu, K23-HL118132 to Amy S. Shah, and R01-HL104136 to W. Sean Davidson), a predoctoral fellowship to Scott M. Gordon and postdoctoral fellowship to John T. Melchior from the Great Rivers Affiliate of the American Heart Association, and the National Natural Science Foundation of China (no. 31601083).
Supplemental Figure