Extracellular Hsp90α is a Potential Serum Predictor of Atherosclerosis in type 2 Diabetes

Background: Atherosclerosis is the main pathological change in diabetic angiopathy, and vascular inammation plays an important role in early atherosclerosis. Heat shock protein 90, a cellular molecular chaperone, was recently determined to be secreted extracellularly, but the specic mechanism remains unclear. This study explored the relationship between Hsp90 and diabetic peripheral artery disease through serological analyses of different groups of diabetic patients and investigated the relationship between extracellular Hsp90α and vascular inammation at the cellular level. Methods: Seventy-seven selected patients were divided into three groups. The relationships among serum Hsp90, oxidative stress indexes and patient outcomes and the correlations among the indexes were analysed. An oxidative stress endothelial injury model was established under high glucose in vitro to explore the role of eHsp90 release in atherosclerosis progression. Results: Serum Hsp90 and MDA levels tended to increase in different groups with peripheral vascular disease aggravation. Hsp90α was correlated with MDA to some extent and was predictive. In vitro, high glucose and low H 2 O 2 treatment increased extracellular Hsp90 secretion, and endothelial cell conditioned medium and recombinant human Hsp90α increased monocyte migration (P<0.05). Conclusions: Extracellular Hsp90α participates in endothelial cell injury in diabetic vascular disease and initiates the inammatory response by promoting monocyte migration.

current states of the vascular endothelium and the in ammatory response that can be applied for early clinical detection, which makes early diagnosis di cult.
Heat shock protein 90 (Hsp90) is an important molecular chaperone that is widely involved in immune regulation. Lei et al. [7] proposed that Hsp90α is involved in the pathogenesis of diabetic angiopathy. In recent years, Hsp90 inhibitors have been found to have protective effects against diabetic angiopathy. For example, Lazaro et al. [8] reported that application of an Hsp90 inhibitor (17-DMAG) effectively reduced renal injury in a diabetic mouse model. Kim et al. [9] found that inhibiting Hsp90 can reduce the formation of AS by inhibiting the migration and proliferation of vascular smooth muscle cells (VSMCs). Madrigal-Matute et al. [10] found that Hsp90 is highly expressed in the thin brous cap area of human carotid atherosclerotic plaques. All the above ndings suggest that Hsp90 may be a new target for AS therapy. Extracellular Hsp90 (eHsp90) has been reported to be involved in a variety of physiological and pathological processes. Our team found that eHsp90 can be released outside the cell through exosomes under stress conditions, which in turn affects cytoskeletal proteins to promote the migration of epithelial cells, participating in damage repair. [11][12][13] Although Hsp90 has been indicated to be correlated with diabetic vascular disease, previous studies have focused more on its role as a molecular chaperone. Whether eHsp90 is involved in vascular in ammation or brosis is still unclear.
In this study, in vivo serological studies and in vitro cell models were used to explore the direct relationship between serum Hsp90 concentrations and subclinical AS in patients with T2DM and the role of eHsp90α in diabetic vascular disease in an oxidative stress injury model. The ndings will provide a new perspective for investigation into early therapeutic targets for diabetic vascular disease.

Methods
In vivo study Study subjects From August 30, 2019, to September 28, 2020, a total of 77 patients in the Endocrinology and Metabolism Department of the Nanfang Hospital of Southern Medical University were randomly enrolled in this study. The patients were divided into four groups: the DM group (12 patients), the DM+LEAD group (45 patients) and the Critical limb ischemia (CLI) group (20 patients). According to the central limit theorem, the sample size greater than 30, which can be considered as obeying normal distribution. The subjects were grouped according to their clinical presentations and the results of laboratory and imaging studies.
Inclusion criteria a. DM group: T2DM was diagnosed according to the World Health Organization (WHO) 1999 criteria. b. DM+LEAD group: Patients were diagnosed with T2DM according to the above DM diagnostic criteria and determined to have LEAD on the basis of the following: (1) Symptoms and signs of AS (intermittent claudication, resting pain, decreased or absent pulse in the dorsal foot artery, etc.); (2) An ankle-brachial arterial pressure index (ABI)<0.9, a toe-brachial arterial pressure index (TBI)<0.7, no three-phase foot pulse graph waveform, or a percutaneous oxygen partial pressure (TcPO2)<30 mmHg; or (3) Evidence of uneven thickening, AS, atherosclerotic plaques, arterial stenosis or obstruction in the carotid and/or lower extremity arteries on vascular colour Doppler ultrasonography.
c. CLI group: CLI was diagnosed for patients who met the above diagnostic criteria for DM+LEAD and who had lower extremity ischemic infection, ulceration, and/or deep tissue destruction.
The patients and/or their families were informed and agreed to participate in the study. Patients were excluded if they met any of the following exclusion criteria before admission: (1) History of diabetic ketoacidosis or hyperosmolar status within 30 days,

Cell viability assay
Cell viability was evaluated with a Cell Counting Kit-8 (CCK-8) assay, which was carried out following a standard procedure in 96-well plates. Brie y, cells were seeded into a 96-well plate at a density of 5×10 3 cells/well in 100 μl of medium and grown to 80% con uence. After treatments, the medium was replaced with fresh medium containing 10% CCK-8 reagent. The absorbance at 450 nm was measured after a 2-h incubation at 37°C. Triplicate wells were included for each group.
Detection of MDA content and SOD activity According to the manufacturer's instructions, cells were incubated with 10 μM DCFH-DA in a cell incubator with 5% CO 2 at 37°C for 20 min. Then, the uorescence intensity of the cells was detected by using a ow cytometer.

Protein extraction and protein expression analysis
HUVECs were plated in 10-cm dishes. When the cells grew to 80% con uence, the medium was replaced with fresh medium without FBS. After 24 h, conditioned medium (CM) from the serum-free cultures was collected, centrifuged and ltered through a Millipore Amicon Ultra-4 (50K) column. The total cell lysates were centrifuged at 13,000 × g for 15 min at 4°C, and the total protein concentrations were determined using a BCA Protein Assay Kit (Keygen, China). CM and cell extract samples were electrophoresed through 10% SDS polyacrylamide gels under denaturing conditions and transferred to PVDF membranes (EMD Millipore, USA). The membranes were blocked in 5% non-fat milk that was dissolved in 1× TBST and then incubated with the corresponding primary antibodies at 4°C overnight. The membranes were subsequently washed in 1× TBST and incubated with secondary antibodies for 2 h at room temperature. Speci c antigen-antibody interactions were detected with enhanced chemiluminescence.

Immuno uorescence
Cells were xed with 4% paraformaldehyde for 10 min at room temperature. Permeation was performed by incubating the pre-treated cells with ice-cold methanol at -20°C for 15 min. The cells were incubated with primary antibodies (1:100) at 4°C overnight. Then, the cells were washed with cold PBS and incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Abcam, USA) (1:200) at room temperature for 2 h. After the nuclei were stained with DAPI, images were captured using an Olympus FV1000 confocal laser scanning microscope (Tokyo, Japan).

mRNA expression analysis
Total cell RNA was isolated with RNAiso (Takara, China), and 1000 ng of total RNA was used for reverse transcription with a PrimeScript RT Reagent Kit. Real-time quantitative PCR was performed using SYBR Premix Ex Taq and Premix Taq.

Cell migration assay
A Transwell migration assay was applied to study the transmigration behaviour of THP-1 monocytes.
Twenty-four transwell inserts with pore sizes of 3 μm (Corning, USA) were employed. A total of 10 6 THP-1 monocytes in 200 μl of serum-free RPMI 1640 medium were loaded into the upper chamber of each Transwell insert. Then, 600 μl of endothelial cell CM was treated with H 2 O 2 and 17AAG, or RPMI medium was added to the lower chamber. The cells were then allowed to migrate for 8 h. The cells that migrated across the membrane to the lower chamber were counted under an inverted microscope (Nikon Eclipse TS100, Japan).

Statistical analysis
All experiments were repeated three times. SPSS 22.0 was used for statistical processing of the data. Normally distributed continuous variables are described as the mean ± standard deviation (x±s).
Continuous variables with a skewed distribution are expressed as the median (interquartile range) (Q1, Q3). Categorical variables are expressed as the number of cases (%). The differences between groups were analysed by one-way ANOVA, Dunnett's t-test, Student's t-test, K-W test and chi-square test according to the characteristics of the data. Spearman correlation analysis was used to analyse the correlations of related indexes. A receiver operating characteristic (ROC) curve was drawn to evaluate the predictive value for LEAD in diabetic patients. The signi cance level was p < 0.05.

Results
In vivo study  Comparison and correlation analyses of serum levels of Hsp90α, Hsp90β and MDA in the study subjects The serum levels of Hsp90α, Hsp90β and MDA in the different groups of patients and controls are shown in Fig. 1A-C. In pairwise comparisons, serum HSP90α levels were signi cantly higher in DM + LEAD or CLI patients than in DM patients (p < 0.01, respectively). The results showed that the serum Hsp90α and Hsp90β levels gradually increased with the progression of the disease, and T2DM patients were characterized by a state of continuous and chronic oxidative stress. The relationships among Hsp90α, Hsp90β and MDA levels in the study subjects are shown in Fig. 1D

T-test based on clinical characteristics of HSP90α levels
A T-test based on the clinical characteristics of the serum Hsp90α levels is depicted in Table 2. Except for BMI and past medical history, serum Hsp90α was not associated with other clinical characteristics of the study population. The results indicated that weight control and previous history examination are more important for patients with diabetes. However the elevated levels of Hsp90α indicated an abnormal baseline level in patients.

Histopathological sections
Both in ammatory cell in ltration and elevated Hsp90α expression were present. Tissue sections prepared from human atherosclerotic lesions were subjected to HE staining ( Fig. 2A, C and E) and immunohistochemistry (Fig. 2B, D and F). We found thickening of the vascular endothelium in CLI patients. In ammatory cells in ltrated the intima heavily, and Hsp90α was abundantly expressed in the endothelium and smooth muscle. In addition, the expression area of Hsp90α was consistent with the area of in ammatory in ltration.

In vitro study Construction of a HUVEC model of oxidative injury induced by H 2 O 2
The results of H 2 O 2 concentration screening (Fig. 3A)  μM and 300 μM exhibited signi cantly different MDA contents (Fig. 3B) and SOD activities (Fig. 3C) than control cells. ROS detection (Fig. 3D)  treatment, but the total amount of intracellular Hsp90 did not change signi cantly (Fig. 4A). After H 2 O 2 treatment, the total amount of eHsp90α and eHsp90β from HUVECs was the highest at 24 h, but the change in intracellular Hsp90 was not obvious (Fig. 4B). Treatment with H 2 O 2 and 17AAG alone or in combination signi cantly increased the secretion of Hsp90 by endothelial cells. In addition, the expression of Hsp90 mRNA was increased in the group treated with 17AAG ( Fig. 4C and D). H 2 O 2 had little effect on the expression of Hsp90AA1 and Hsp90AB1. The immuno uorescence results (Fig. 4E) showed that Hsp90α localization shifted with treatment; speci cally, the uorescence at the cell edges increased, suggesting that the expression of Hsp90α in the membrane and periphery may have increased.

Endothelial cell CM and hrHsp90α induce THP-1 migration
To study the effect of chronic oxidative stress on the development and progression of AS, we next examined the effect of endothelial cell CM on THP-1 cells. Compared with the control medium, H 2 O 2treated CM induced THP-1 migration. CM from endothelial cells treated with 17AAG alone or H 2 O 2 in combination exhibited a signi cantly decreased ability to induce THP-1 migration (Fig. 5A). We observed that hrHsp90α exposure increased the number of THP-1 cells that migrated to the lower chamber. The chemotactic effect of hrHsp90β on THP-1 monocytes was not obvious. We used 1G6-D7, a newly generated mAb that selectively targets the dual lysine region in secreted Hsp90α, to identify the speci city of Hsp90α for chemotactic THP-1 monocytes. RPMI 1640 medium pre-treated with 17AAG and 1G6-D7 for 30 min and containing hrHsp90α or hrHsp90β was added to the lower chamber. The results showed that hrHsp90α promoted the migration of monocytes to a certain extent, while the effect of recombinant Hsp90β was not obvious. The chemotactic ability of monocytes was weakened by the addition of 17AAG or 1G6-D7 (Fig. 5B).
eHsp90α activates THP-1, LRP1 and Akt kinase Next, we treated THP-1 cells with hrHsp90α, and Western blotting showed that the LRP1 protein content was signi cantly elevated in the treated cells. To con rm the involvement of the eHsp90α-Akt pathway, we determined that stimulation with eHSP90α induced time-dependent activation of p-Akt (Fig. 5C).

Discussion
In our study, we clari ed the relationships of oxidative stress with serum Hsp90α and Hsp90β levels in diabetic patients. We propose that serum Hsp90α has reference value for the early diagnosis of DM + LEAD, and our ndings indicate that low-dose and long-term ROS exposure can increase eHsp90 secretion from endothelial cells. While blocking the effect of Hsp90, we used a previously developed mAb speci c for eHSp90 to further verify the role of eHsp90. eHsp90α acts as a chemokine to induce THP-1 cell migration, which may result from a compensatory response of early in ammation and oxidative stress in AS, initiating the atherosclerotic process (Fig. 6).
The incidence and mortality rates of atherosclerotic cardiovascular disease (ASCVD) are signi cantly higher in diabetic patients than in nondiabetic patients. [14] Fifty percent of patients with T2DM had complications at the time of diagnosis. [15] However, fewer than 10% of patients have typical clinical symptoms. In most patients, the degree of vascular stenosis exceeds 50% at initial diagnosis, and the lack of effective treatments affects the patients' prognoses.
[16] Recent studies have reported several potential serum markers for the screening of diabetic vascular complications, such as CD36, YKL-40, [17] hepatocyte growth factor, [18] serum broblast growth factor 23, [19] serum sclerostin, [20] serum growth differentiation factor 15, [21] and circulating Hsp27. [22] In addition, heat shock proteins (HSPs) can be transported to the plasma membrane and released extracellularly, resulting in detectable levels of HSPs in the blood. This nding has prompted clinical studies exploring the potential use of HSPs and anti-HSP antibodies as serum biomarkers for DM complications. [23] Available data suggest that circulating Hsp27, [24] Hsp60, [25] Hsp70[26] and anti-HSP levels may be used as biomarkers for diabetic vascular disease. In T1DM, elevated serum Hsp90 levels have been found to be related to cellular autoimmunity in children. However, differences in Hsp90 levels do not predict whether individuals with positive autoantibodies will develop T1DM. [27] New ndings con rmed the role of Hsp90 as a putative autoantigen triggering in ammation within human carotid atherosclerotic plaques.
[28] However, there have been no studies on the relationships between Hsp90 and T2DM macrovascular complications.
Based on the current standardized management of clinical DM, we explored whether there were any controllable factors among the groups of patients. Clinical data of 77 patients were collected in this study. The results showed that disease severity gradually increased with age and that DF patients had ulcer infections. Low levels of HDL are also associated with an increased incidence of coronary heart disease in diabetic patients. Abnormal Hb levels may be related to renal anaemia and renal dysfunction. An elevated WBC count indicates the persistence of a chronic in ammatory response. Therefore, we sought to investigate whether there is an indicator that can be included in primary prevention strategies that has reference value for early prediction. Numerous studies have reported that patients with DM have chronic in ammation, but now many in ammation indicators are closely related to acute infection, such as CRP and PCT; [29] thus, these indicators may not truly re ect chronic in ammation. Serological studies have found that the degree of oxidative stress in diabetic patients is cumulative. Therefore, we detected differences in the levels of Hsp90α and Hsp90β in serum samples of each group (Fig. 1). Correlation analysis revealed that there was a certain correlation between HSP90α and MDA, but there was no statistical signi cance, suggesting that circulating Hsp90α may re ect a patient's oxidative stress state to a certain extent. Predictive analysis indicated that serum Hsp90α shows better performance for the initial diagnosis of LEAD in early DM patients than in diabetic patients without atherosclerotic changes. A T-test further revealed that serum Hsp90α levels in patients with abnormal BMI were signi cantly higher than those in the normal group. The serum Hsp90α levels in patients with a past medical history were also higher than those in the control group, suggesting that weight control and previous history examination are required for the prevention of DM + LEAD. Due to the small number of samples at present, confounding factors could not be well controlled, and multivariate analysis could not be carried out. In further studies, we will include more samples to make the results more representative.
AS is also related to apoptosis of macrophages, smooth muscle cells and endothelial cells. During cell activation and apoptosis, endothelial cells can release many types of extracellular vesicles (EVs), and new evidence in the eld shows that endothelial cell-derived EVs participate in the development of AS.
During this process, NO and oxidative stress can induce endothelial cell apoptosis and ROS generation before atherosclerotic plaques form, and ROS-induced apoptosis of endothelial cells has the potential to initiate AS. [30] It is generally believed that abnormal glucose metabolism damages the arterial endothelial barrier while increasing platelet and in ammatory cell aggregation, which increases local oxidative stress levels. Destruction of the barrier function of vascular endothelial cells is a crucial initial step in this process. [31] Oxidative stress is a potential mechanism for the development of endothelial cell dysfunction that is common to all risk factors. [32] Where does Hsp90 in the serum of diabetic patients come from? Previous studies have focused mostly on Hsp90 in cells and on cell membranes, and our initial ndings suggested that Hsp90α is a predictive molecule. However, the cells that release Hsp90 into the serum under conditions of high glucose and low H 2 O 2 remain unclear. Endothelial cells are the direct targets of stimulation by blood glucose and lipids in vessels and can be damaged by these components.
Thus far, no reports have suggested that endothelial cells can secrete Hsp90. Next, we veri ed cytologically that oxidative stress upregulates the expression of eHSp90 in HUVECs and that this process is not accompanied by an increase in Hsp90 gene expression or a decrease in intracellular protein expression. These ndings are in contrast to those of a study by Profumo et al. [33] in which oxidative stress upregulates Hsp90 expression on the surfaces of endothelial cells and reduces Hsp90 secretion.
There may be a link between the two ndings. The expression of eHsp90 may be related to the duration of oxidative stress in cells. After a short (2 h or 4 h) treatment, no change in the amount of eHSp90 was observed in the cell culture medium. Previous studies have rarely used indicators to simultaneously measure oxidative stress at both the population and cell levels or used such indicators as references. In our study, we used MDA to measure the oxidative stress state of the population and found that MDA levels were lower in cells after H2O2 treatment than in patient serum. These ndings suggest that endothelial cells under low-concentration H 2 O 2 treatment exhibit increased expression of eHsp90, which can simulate oxidative stress in humans.
AS is a lipid-driven in ammatory disease of the arterial intima. The balance of proin ammatory and antiin ammatory mechanisms determines the nal clinical outcome, which is characterized by gradual accumulation of lipids and in ammatory cells. [34] Two chemotactic processes are involved in AS: monocyte chemotaxis and smooth muscle broblast chemotaxis. Circulating leukocytes adhere to and migrate through the endothelial wall to the vascular smooth muscle layer of the intimal membrane. [35] The migration of monocytes is an early disease indicator. Ambade et al. [36] found that the EVs of mice with alcoholic liver disease can induce macrophage activation through Hsp90. Inhibitor treatment can reduce the formation of plaques to a certain extent by inhibiting the migration of VSMCs. [9] In addition, colocalization of in ammatory cells and Hsp90α was found in patient tissue samples, so we hypothesized that there may be a connection between Hsp90α and in ammatory cells. The results showed that eHsp90a could promote monocyte migration and initiate the in ammatory process of atherosclerosis. However, there have been no reports on the effects of eHsp90 on the LRP1 receptor and Akt kinase in monocytes. When we used recombinant Hsp90α to treat monocytes, we observed increased expression of LRP1 in a short period of time, which activated p-Akt, thereby promoting monocyte migration. Our study provides in vitro evidence of the key pathological role of eHsp90α in the control of diabetic vascular complications. This study did not go deep into the cell signalling mechanisms. In the future, we will study the pathway mechanism by knocking down or overexpressing genes, using pathway inhibitors and other means.

Conclusion
The current study shows that eHsp90α participates in the in ammatory process of AS to a certain extent and further aggravates adverse reactions at the beginning of the disease course. eHsp90α is expected to become a promising new target for disease prediction and for treatment to slow the progression of AS.

Consent for publication
If the manuscript is accepted, we approve it for publication in Cardiovascular Diabetology.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.  the expression of related proteins in HUVECs pre-treated with 17AAG and then exposed to H2O2.(n=3) (D) qPCR was employed to observe the respective Hsp90α and Hsp90β mRNA levels after HUVECs were stimulated with H2O2 and/or 17AAG for 24 h.(n=3) (E) Representative confocal images of Hsp90α subunit localization in endothelial cells after 24 h of H2O2 stimulation are shown.(n=3) All data represent the mean ±SD of three biological replicates. The differences between groups were analyzed by One-Way ANOVA, Dunnett, according to data feature. *p < 0.05, ****p < 0.0001 vs. control. Figure 5 eHsp90 promotes monocyte migration, and recombinant Hsp90α activates LRP1 receptor expression. Representative images with quanti cation of the results for a THP-1 cells transwell migration assay in the presence of H2O2-and/or 17AAG-treated endothelial cell CM (A) and different reagents (B). (n=5) * represents signi cant difference compared with untreated CM or control; (C) LRP1 and p-Akt expression was measured after hrHsp90α stimulation for different durations. (n=3) All data represent the mean ±SD of three biological replicates. The differences between groups were analyzed by One-Way ANOVA, Dunnett, according to data feature. *p < 0.05, **p < 0.01, ***p <0.001.

Figure 6
Model of extracellular Hsp90α participation in atherosclerosis.