Extracellular Hsp90α, which participates in vascular inflammation, is a novel serum predictor of atherosclerosis in type 2 diabetes

Abstract

Introduction: Atherosclerosis is the main pathological change in diabetic angiopathy, and vascular inflammation plays an important role in early atherosclerosis. Extracellular heat shock protein 90 (eHsp90) is secreted into the serum and is involved in various physiological and pathophysiological processes. However, the specific mechanism of eHsp90 in early atherosclerosis remains unclear. This study explored the relationship between Hsp90 and diabetic lower extremity arterial disease and investigated the expression of eHsp90 in vascular endothelial cells under environmental stimulation and the function and mechanism of eHsp90α involved in diabetic atherosclerosis.

Research design and methods: One hundred and three selected patients were divided into three groups: the diabetes mellitus group (n=27), the diabetic lower extremity arterial disease group (n=46), and the diabetic critical limb ischemia group (n=30). The relationships among serum Hsp90, oxidative stress indexes, and patient outcomes and the correlations among the indexes were analyzed. H&E staining and immunohistochemistry were used to observe the vasculature of amputated feet from patients with diabetic foot. An oxidative stress endothelial injury model was established under high glucose in vitro to explore the role of eHsp90 release in atherosclerosis progression.

Results: The level of serum Hsp90 was upregulated with aggravation of diabetic vascular disease. Hsp90α was correlated with malondialdehyde to some extent and was an independent risk factor in the progression of diabetic vascular disease, with predictive ability. The expression area of Hsp90α was consistent with the area of inflammatory infiltration in the vessel lumen. Vascular endothelial cells were found to increase eHsp90α secretion under stress. Then inhibition of eHsp90α can reduce the degree of cellular inflammation and damage. Endothelial cell-conditioned medium and recombinant human Hsp90α increased monocyte migration via the low-denisity lipoprotein receptor-related protein 1 (LRP1) receptor to promote disease progression.

Conclusions: eHsp90α plays a critical role in the early inflammatory injury stage of atherosclerosis.

Trial registration number: NCT04787770.

Keywords: atherosclerosis; diabetes mellitus; early diagnosis; type 2; vascular.

Conflict of interest statement

Competing interests: None declared.

© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

Comparison and correlation analyses of serum levels of MDA, Hsp90α, Hsp90β, WCC, and CRP in the study subjects. Serum MDA (A), Hsp90α (B), and Hsp90β (C) levels were measured in the different groups. *P

Figure 2

Multivariate logistic regression model and diagnostic efficacy of Hsp90α and Hsp90β for DLEAD. Histopathological changes in blood vessels. Multivariate logistic regression analysis (A): a forest plot obtained from a multiple logistic regression model as OR with 95% CI. Comparing patients with DLEAD and DM, age, Hsp90α, course, and family history were independent risk factors for the development of DLEAD. Comparing patients with DCLI and DM, age, Hsp90α, Hsp90β, and course were independent risk factors for DCLI. Receiver operating characteristic analysis of the diagnostic efficacy of Hsp90α and Hsp90β for DM and DLEAD (B). Diagnostic ability to distinguish patients with DLEAD from patients with DM. HE staining (C and E) and immunohistochemistry (D and F) (40×, 100×, 200×) of Hsp90α in the blood vessels of patients with DCLI undergoing partial amputation (n=3). CHOL, cholesterol; DCLI, diabetic critical limb ischemia; DLEAD, diabetic lower extremity arterial disease; DM, diabetes mellitus; Hb, hemoglobin; Hsp90, heat shock protein 90; LDL, low-density lipoprotein; MDA, malondialdehyd; WCC, white cell count.

Figure 3

Increased eHsp90 secretion in a vascular endothelial cell model of oxidative injury in vitro. A CCK-8 assay (A) was used to detect cell viability in HUVECs treated with different concentrations of H2O2 for 2, 4, 6, 8, and 24 hours (n=3). The levels of MDA (B) and SOD (C) were measured with assay kits (n=3). HUVECs were treated with H2O2 (100 µM, 200 µM, and 300 µM) for 24 hours, and then the intracellular levels of ROS (D), mitochondrial ROS (E), and 8-oxoG (F) and the extracellular levels of ET-1 (G) and MCP-1 (H) were detected (n=3). (I) Western blot analysis concentrated medium and total cell extracts from HUVECs cultured with the indicated concentration of H2O2. ImageJ software was used to determine the relative intensities of extracellular and intracellular Hsp90α and Hsp90β bands (n=3). (J) Extracellular and intracellular Hsp90α and Hsp90β expressions were measured after H2O2 stimulation for different durations (n=3). (K) Western blot analysis was performed to assess the expression of related proteins in HUVECs pretreated with 17AAG and then exposed to H2O2 (n=3). (L) qPCR was employed to observe the respective Hsp90α and Hsp90β mRNA levels after HUVECs were stimulated with H2O2 and/or 17AAG for 24 hours (n=3). (M) Representative confocal images of Hsp90α subunit localization in endothelial cells after 24 hours of H2O2 stimulation are shown (n=3). All data represent mean±SD of three biological replicates. Differences between groups were analyzed by one-way ANOVA and the Dunnett test, according to the data features. *P<0.05,**P<0.01,***P<0.001 ****P<0.0001 versus control. 17AAG,17-allyamino-17-demethoxygeldanamycin; ANOVA, analysis of variance; CCK-8, Cell Counting Kit-8; Ctrl, control; DAPI, 4',6-diamidino-2-phenylindole; eHsp90, extracellular heat shock protein 90; ET-1, endothelin 1; H2O2, hydrogen peroxide; Hsp90, heat shock protein 90; HUVECs, human umbilical vein endothelial cells; MCP-1, monocyte chemotactic protein 1; MDA, malondialdehyd; mRNA, messenger RNA; 8-oxoG, 8-oxoguanine; qPCR, quantitative real-time PCR; ROS, reactive oxygen species; SOD, superoxide dismutase.

Figure 4

eHsp90α enhanced the inflammatory injury state of endothelial cells and recruited monocytes through LRP1 receptors. Extracellular ET-1 (A) and MCP-1 (B) expressions were measured after hrHsp90α stimulation at different concentrations and durations (n=3). The expression of ET-1 (C) in endothelial cell-CM treated with H2O2 after 17AAG or 1G6-D7 pretreatment (n=3). Representative images with quantification of the results for a THP-1 cell Transwell migration assay in the presence of H2O2-treated and/or 17AAG-treated endothelial cell CM (D) and different reagents (E) (n=5). LRP1 and p-Akt expressions (F) were measured after hrHsp90α stimulation for different durations (n=3). All data represent mean±SD of three biological replicates. Differences between groups were analyzed by one-way ANOVA and the Dunnett test, according to the data features. *P<0.05, **P<0.01, ***P<0.001,****P<0.0001 versus control. 17AAG, 17-allyamino-17-demethoxygeldanamycin; ANOVA, analysis of variance; CM, conditioned medium; Ctrl, control; eHsp90, extracellular Hsp90; ET-1, endothelin 1; H2O2, hydrogen peroxide; hrHsp90, human recombinant Hsp90; Hsp90, heat shock protein 90; LRP1, low-denisity lipoprotein receptor-related protein 1; MCP-1, monocyte chemotactic protein 1.