Differences in vascular endothelial function and serum proteome between obese people with phlegm-dampness constitution and balanced constitution

Abstract

Objective: To evaluate the extent of vascular endothelial dysfunction and preliminary identify serum protein biomarkers associated with obese individuals at risk for cardiovascular disease (CVD).

Methods: Fifteen obese volunteers with the phlegm-dampness constitution or balanced constitution were recruited for this study respectively. The clinical baseline data was collected, and the vascular endothelial function was evaluated using the EndoPAT^TM. Blood samples were collected for the serum proteome analysis. The differences in the serum protein expression levels between the two groups were detected and the protein interaction network analysis, correlation analysis, receiver operating characteristic (ROC) curve analysis, and random forest model investigation were conducted.

Results: There were no statistical differences found in the baseline data. For vascular endothelial function, the reactive hyperemia index (RHI) of the phlegm-dampness constitution obese group was significantly lower than that of the balanced constitution obese group (1.46 ± 0.30 vs 2.82 ± 0.78, P < 0.0001), indicating vascular endothelial dysfunction. There are 66 differentially expressed serum proteins between the two groups. apolipoprotein A2 (ApoA2), angiotensin-converting enzyme 2 (ACE-2), interleukin-33 (IL-33), and forkhead box P3 (FoxP3) showed significant differences and area under curve values of their ROC curves were greater than 0.7 and correlated significantly with RHI.

Conclusion: Vascular endothelial dysfunction was present in the phlegm-dampness constitution obese group. Thus, alterations in the expression levels of key serum proteins, including ApoA2, ACE-2, IL-33, and FoxP3 could serve as potential biomarkers in the obese population at risk of CVD.

Keywords: obesity; phlegm-dampness constitution; proteome; reactive hyperemia index; vascular endothelial function.

Figure 1. Enrichment analysis based on online databases

A: KEGG enrichment analyses of the 66 DEPs; B: disease enrichment analyses of the 66 DEPs; C-E: biological process, cellular component, and molecular function enrichment analyses of the 66 DEPs. KEGG: Kyoto encyclopedia of genes and genomes; DEPs: differentially expressed proteins; c-FLIP: cellular Fas-associating death domain-like interleukin 1β converting enzyme inhibitory protein; PDGF: platelet-derived growth factor; ECM: extra cellular matrix; TRAIL: tumor necrosis factor-related apoptosis-inducing ligand; PI3K: phosphatidylinositol 3-kinase; Akt: protein kinase B; COPD: chronic obstructive pulmonary disease.

Figure 2. Network analysis of 66 differential expressed proteins via cytoscape

A: PPI network of DEPs generated via String database; B: core protein module generated via CytoHubba, demonstrating the key DEPs; C: molecular complex detection analysis results of the DEPs. PPI: protein protein interaction; DEPs: differentially expressed proteins; A2M: alpha 2 macroglobulin; ACP3: acid phosphatase 3; AHSG: alpha 2 HS glycoprotein; ALB: albumin; ANXA7: annexin 7; ApoA2: apolipoprotein A2; ApoC2: apolipoprotein C2; APP: amyloid precursor protein; AZGP1: alpha 2 glycoprotein 1; CD200: immunoregulatory protein CD200; CFHR2: complement factor H-related protein 2; CP: ceruloplasmin; CRP: C-reactive protein; FADD: Fas-associated death domain; FETUB: Fetuin B; FN1: fibronectin 1; FoxP3: forkhead box protein P3; GAL: galanin; GAST: gastrin; GRN: granulin precursor; HCRT: hypocretin neuropeptide; ICAM-2: intercellular adhesion molecule 2; IL-33: interleukin-33; ITGB3: integrin subunit beta 3; ITM2B: integral membrane protein 2B; KLK7: kallikrein-related peptidase 7; MBL: mannose-binding lectin; MDK: midkine; MME: membrane metalloendopeptidase; MMP27: matrix metalloproteinase 27; POMC: proopiomelanocortin; POU5F1: POU class 5 homeobox 1; PTX3: pentraxin 3; RET: rearranged during transfection; ROR2: receptor tyrosine kinase-like orphan receptor 2; sFRP-1: secreted frizzled-related protein 1; sFRP-4: secreted frizzled-related protein 4; SLC6A4: solute carrier family 6 member 4; SPP1: secreted phosphoprotein 1; TIMP-2: tissue inhibitor of metalloproteinases 2; TNFRSF4: tumor necrosis factor receptor superfamily member 4; TNFSF11: tumor necrosis factor superfamily member 11; TNFSF13B: tumor necrosis factor superfamily member 13B; TLR2: toll-like receptor 2; XRCC6: X-ray repair cross complementing 6; ZAP70: zeta-chain-associated protein kinase 70.

Figure 3. Receiver operating curves of screened DEPs

A-G: ROC of TLR2 (A), TIMP-2 (B), APP (C), FoxP3 (D), IL-33 (E), ApoC2 (F), and ApoA2 (G) between the PDC and BC obese groups; H-J: ROC of MMP-7 (H), ACE-2 (I), and PDGF-AA (J) between the PDC and BC obese groups. BC obese group: balanced constitution obese group consisted of 15 balanced constitution obese participants lived in Beijing for at least 1 year; PDC obese group: phlegm-dampness constitution obese group consisted of 15 balanced constitution obese participants lived in Beijing for at least 1 year. ROC: receiver operating curves; DEPs: differentially expressed proteins; PDC: phlegm-dampness constitution; BC: balanced constitution; TLR2: toll-like receptor 2; TIMP-2: tissue inhibitor of metalloproteinases 2; APP: amyloid precursor protein; FoxP3: forkhead box protein P3; IL-33: interleukin-33; ApoC2: apolipoprotein C2; ApoA2: apolipoprotein A2; MMP-7: matrix metalloproteinase-7; ACE-2: angiotensin converting enzyme 2; PDGF-AA: platelet-derived growth factor-AA; AUC: area under curve.

Figure 4. Comparison of key proteins via t-test between the PDC and BC obese groups

A-F: Box plot showing the difference in the two groups of six serum proteins significantly correlated with RHI. A: ACE-2; B: ApoA2; C: FoxP3; D: IL-33; E: TLR2; F: PDGF-AA. BC obese group: balanced constitution obese group consisted of 15 balanced constitution obese participants lived in Beijing for at least 1 year; PDC obese group: phlegm-dampness constitution obese group consisted of 15 balanced constitution obese participants lived in Beijing for at least 1 year. ACE-2: angiotensin converting enzyme 2; ApoA2: apolipoprotein A2; FoxP3: forkhead box protein P3; IL-33: interleukin-33; TLR2: toll-like receptor 2; PDGF-AA: platelet-derived growth factor-AA. PDC: phlegm-dampness constitution; BC: balanced constitution. Data were presented as mean ± 95% confident interval (n = 15). Significant differences compared with BC group were designated as ^aP < 0.05.