Endothelial dysfunction (ED) contributes to the pathologic process underlying macrovascular complications, a common complication of type 2 diabetes mellitus (T2DM). Soluble endoglin (sEng) shed from the extracellular domain of the entire endoglin molecule blocks endothelial protection mediated by transforming growth factor-beta 1 (TGF-β1). The reactive hyperemia index (RHI), which is determined by reactive hyperemia peripheral arterial tonometry (RH-PAT), is a new index with which to evaluate ED. This study determined the changes in serum sEng levels in newly-diagnosed (untreated) T2DM patients and the correlation with the RHI. The T2DM group included 34 newly-diagnosed T2DM patients, while the control group included 53 healthy adults. The clinical data from the two groups were evaluated retrospectively. The intima-media thickness (IMT) of the common carotid artery (CCA) and the ankle-brachial index (ABI) of both legs were used to assess structural vascular changes. The serum sEng level was determined using an ELISA kit. Endothelial function was assessed using RH-PAT and the RHI was computed. The serum sEng level in the T2DM group was significantly greater than the control group, although the RHI was significantly lower in the T2DM group (p < 0.05). The serum sEng level was negatively correlated with the RHI in T2DM patents (r = 0.354, p = 0.041). The serum sEng level, CCA-IMT, and ABI were not significantly correlated with T2DM (p > 0.05). In summary, among newly-diagnosed T2DM patients, the serum sEng levels were inversely correlated with the RHI, and an elevated sEng level may be associated with ED.

TYPE 2 DIABETES MELLITUS (T2DM) has intensified as a significant public health issue in China. T2DM patients have a higher risk of macrovascular complications than non-diabetic persons [1, 2]. Indeed, macrovascular complications are the leading cause of morbidity and mortality in T2DM patients [2, 3]. Atherosclerosis has been shown to be a key factor in the onset and progression of diabetic macrovascular complications; however, the mechanism underlying atherosclerosis in T2DM has not been established. Endothelial dysfunction contributes to the atherosclerosis pathologic process and is an early pathophysiologic event in diabetic macrovascular complications. Moreover, this disorder occurs before structural changes occur in the blood vessels [4, 5].

A novel metric for evaluating endothelial dysfunction (ED) is the reactive hyperemia index (RHI), which is obtained using reactive hyperemia peripheral arterial tonometry (RH-PAT). RH-PAT is a non-invasive quantitative method that provides a digital measurement of the hyperemic response. Compared with the commonly used method of forearm flow-mediated vasodilation (FMD), RH-PAT provides more standardized findings for endothelial function [6, 7]. In a study by Hu et al. [8], the RHI was shown to be significantly lower in T2DM patients than in 24 non-diabetic controls who were overweight [8]. The RHI is associated with several cardio-metabolic risk factors [9] and excess visceral adipose tissue in T2DM patients [10].

Soluble endoglin (sEng), which is derived from the extracellular domain of the complete endoglin molecule, has the potential to inhibit transforming growth factor (TGF)-mediated endothelial protection. Cardiometabolic diseases, including atherosclerosis, T2DM, and liver dysfunction, are all thought to be affected by ED, hyperglycemia, arterial hypertension, hypercholesterolemia (HCE), and obesity-related insulin resistance (IR) [11].

Recently, sEng has been identified as a marker for ED, with higher levels of serum sEng detected in patients with atherosclerosis [12], HCE [13], and pre-eclampsia [14]. It has been shown that among T1DM patients, an elevated serum sEng level occurs prior to the development of subclinical structural vascular alterations [15]. Both the microalbuminuria and normoalbuminuria groups were reported to have higher serum sEng and nitric oxide (NO) levels than the control group [15]. The microalbuminuria group had reduced flow-mediated dilatation (FMD) of the brachial artery compared to the normoalbuminuria and control groups [15]. There was a negative relationship between the serum sEng concentration and FMD %, and the serum sEng concentration and albumin excretion rate [15].

Inflammation and injury increase endoglin expression in endothelial cells (ECs). The plasma sEng level rises markedly prior to alterations in ED function and rises to a lesser amount with the development of diabetic nephropathy, thus indicating disease progression and the emergence of vascular anomalies [16]. It is uncertain, however, if the serum sEng level increases before T2DM patients undergo subclinical structural vascular alterations. An elevated serum sEng level and prolonged exposure to a high-fat diet (6 months) exacerbates ED in the aorta because sEng, in combination with HCE, may be related to modified NO production due to altered pSmad2/3/p-eNOS signaling in the heart and aorta [17]. In addition, an association between the serum sEng level and endothelial function in T2DM patients has not been established.

Therefore, in the present study the RHI was used to assess ED in newly-diagnosed diabetic patients. In addition, the correlation between the serum sEng level and the RHI was analyzed to determine the relationship between the serum sEng level and endothelial function in patients with T2DM.

This was a cross-sectional study that included T2DM patients and healthy adult volunteers. Participants were chosen from patients who underwent RH-PAT at the PLA 903rd Hospital Endocrinology Department (formerly the PLA 117th Hospital). The T2DM group included 34 newly-diagnosed T2DM patients. The World Health Organization (WHO) 1997 diagnostic standards for T2DM were satisfied by all patients. The following exclusion criteria were applied: acute complications of diabetes; complications of renal or hepatic dysfunction; use of glucocorticoids or other hormonal medications; pregnancy; cancer; infection within the past 2 months; other endocrine or metabolic diseases; and patients with incomplete data. Fifty-three healthy, non-diabetic patients (fasting blood glucose [FBG] and 2-h postprandial blood glucose [2hPBG] within the normal ranges) comprised the control group (n = 53). Following the Helsinki Declaration on Human Experimentation guidelines, the Chinese 903rd Hospital of the PLA Ethics Committee approved this study (No. 20210601/05/01/008). Additionally, written informed permission was obtained from all study participants.

All participants in the T2DM and control groups underwent thorough physical examinations and medical history evaluations. The height, weight, hip and waist circumferences, systolic (SBP) and diastolic blood pressure (DBP) were measured in both groups using the same methodology. The body mass index (BMI) was calculated by dividing the weight in kg by the height in m². The waist-to-hip ratio (WHR) was calculated by dividing the waist circumference in cm by the hip circumference in cm. Venous blood samples for use in laboratory analyses were obtained from the participants in both groups after an overnight fast of at least 10 h.

An automated biochemical analyzer (Aeroset C16000; Abbott, USA) measured the following biomarkers: FBG; total cholesterol (TC); triglycerides (TG); low-density lipoprotein-cholesterol (LDL-C); high-density lipoprotein-cholesterol (HDL-C); uric acid (UA); homocysteine (Hcy); aspartate aminotransferase (AST); and alanine aminotransferase (ALT). The fasting insulin (F-Ins) level was measured using an automated chemiluminescence immunoassay instrument (I4000; Abbott). An Afinion AS100 glycosylated hemoglobin analyzer measured the glycosylated hemoglobin (HbA1c) level (Alere, USA). The following equations were used to calculate IR using the homeostasis model assessment (HOMA) approach, as follows: HOMA-IR = fasting insulin (μIU/mL) × fasting glucose (mmol/mL)/22.5; HOMA-B = 20 × fasting insulin (μIU/mL)/fasting glucose (mmol/mL) – 3.5.

Following the manufacturer’s instructions, an ELISA kit (Westang Biotech Co., Ltd., Shanghai, China) was used to measure the serum sEng level in venous blood samples. Samples were analyzed in duplicate with the ELISA kit and the mean value was used for analysis. The coefficients of variation for intra- and inter-assay were 4.23% and 8.76%, respectively, indicating that the measurements are reliable as both values fall within an acceptable range [18].

Using the GE Logiq E9 at a transducer frequency of 6–15 MHz or the Hitachi Ascendus at a transducer frequency of 5–18 MHz, a designated experienced sonographer performed ultrasonography Doppler to measure the intima-media thickness (IMT) of the common carotid artery (CCA). The distance between the first and second echogenic line edges was used to determine the IMT. The mean value of the IMT was computed after three sets of measurements were made. The ankle pressures of both legs were measured and the ABI was calculated.

To determine endothelial function, the RHI was automatically calculated using a RH-PAT device (EndoPAT 2000; Itamar Medical Ltd., Caesarea, Israel) using the formula proposed by the manufacturer. The RH-PAT device is based on a non-invasive, user-independent technique to measure endothelial function in the hand digit microvessels [19]. The RH-PAT device tests the ability of the microcirculation to vasodilate in response to shear stress caused by the release of blood flow after a period of interruption (i.e., ischemia), a response that is dependent on the bioavailability of NO [20]. After 30 min of rest, one finger on each hand was fitted with an RH-PAT probe, and the baseline pulse amplitude was monitored for 5 min. Then, the contralateral arm was used as the control arm and a blood pressure cuff was applied to one upper arm, which served as the occluded arm. To produce reactive hyperemia, the cuff was inflated to 200 mmHg or 60 mmHg above the systolic pressure for 5 min following a 5-min equilibration interval. During cuff release, the hyperemic pulse amplitude was recorded. Hyperemia and baseline pulse amplitudes were recorded as A and B for the control arm and C and D for the occluded arm. Then, the RHI was calculated as (C/D)/(A/B). The RHI was determined by dividing the average basal pulse amplitude by the post-deflation pulse amplitude, which occurs 90–150 seconds after the cuff is released [20]. An RHI ≥ 1.67 indicates normal vascular endothelial function, whereas an RHI < 1.67 represents abnormal vascular endothelial function [21].

The data analysis was carried out using SPSS20.0 software. The data for continuous variables of the normal distribution are presented as the mean ± SD and subjected to independent samples t-tests. The results of the data that did not follow a normal distribution are reported as M (P25, P75). The data were examined using the Mann-Whitney test. Chi-square analysis was used to assess count data, which are reported as rates. Pearson analysis was used to determine the correlation between the two factors. The binary logistic regression was utilized to make predictions about the values of the variables under study. In determining the required sample size to achieve a 1-β = 0.80 and an α = 0.05, a two-sample t-test was used and the statistical power results indicated that a minimum of 21 subjects were required in each group. Statistical significance was set at a p level of 0.05.

Table 1 demonstrated that there were significant differences in WC, WHR, BMI, SBP, FBG, 2hPBG, F-Ins, HbA1c, HOMA-IR, TG, and sEng levels between the T2DM and control groups (all p < 0.05). However, there were no significant differences in age, gender, HC, DBP, 2h-Ins, TC, HDL-C, LDL-C, UA, Hcy, ALT, and AST levels between the two groups (all p > 0.05; Table 1).

The serum sEng level was significantly greater in the T2DM group than the control group (p < 0.001; Fig. 1).

Fig. 1

Comparison of serum sEng level between T2DM and control groups. The serum sEng level was determined using an ELISA kit. Compared with control group, ** p < 0.01.

There was no significant correlation between serum sEng level and age, WC, HC, WHR, BMI, SBP, DBP, FBG, 2hPBG, F-Ins, 2h-Ins, HbA1c, HOMA-IR, TG, TC, HDL-C, LDL-C, UA, Hcy, ALT, and AST levels in T2DM patients. However, in control participants, there was a positive correlation between serum sEng level and DBP (r = 0.303, p = 0.028) as well as 2hPBG level (r = 0.395, p = 0.003). No significant correlation was found between serum sEng level and age, WC, HC, WHR, BMI, SBP, FBG, F-Ins, 2h-Ins, HbA1c, HOMA-IR, TG, TC, HDL-C, LDL-C, UA, Hcy, ALT, and AST levels in control subjects (Table 2).

The study found that the RHI was significantly lower in the T2DM group compared to the control group (Fig. 2A, p < 0.001), indicating impaired endothelial function. However, there were no significant differences between the T2DM and control groups in terms of left ABI (Fig. 2B, p = 0.074), right ABI (Fig. 2C, p = 0.185), left CCA-IMT (Fig. 2D, p = 0.203), and right CCA-IMT (Fig. 2E, p = 0.054).

Fig. 2

Comparison of the RHI and IMT levels between T2DM and control groups. When compared to the control group, the RHI was considerably lower in the T2DM group (2A, p < 0.001). Left ABI (2B, p = 0.074), right ABI (2C, p = 0.185), left CCA-IMT (2D, p = 0.203), and right CCA-IMT (2E, p = 0.054) did not differ significantly between the T2DM and control groups. ** p < 0.01.

Table 3 and Fig. 3 demonstrated that the serum sEng level was positively correlated with the RHI in control subjects (Table 3 and Fig. 3A, r = 0.358, p = 0.009), but negatively correlated with the RHI in T2DM patients (Table 3 and Fig. 3B, r = –0.354, p = 0.041). However, no significant correlations were found between the serum sEng level and the left CCA-IMT, right CCA-IMT, left ABI, or right ABI in either T2DM patients or controls (Table 3).

Fig. 3

Correlation between the serum sEng level and the RHI in control (A) and T2DM groups (B).

In this study, binary logistic regression was utilized with T2DM as the dependent variable and WC, WHR, BMI, SBP, FBG, 2hPBG, F-Ins, HbA1c, HOMA-IR, TG, and sEng as independent variables. The results, presented in Table 4, revealed that in newly diagnosed T2DM patients, the odds ratios (OR) for WC (OR 1.087, 95%CI: 1.145–1.032), BMI (OR 1.332, 95%CI: 1.571–1.130), SBP (OR 1.032, 95%CI: 1.061–1.003), 2hPBG (OR 1.046, 95%CI: 1.086–1.007), F-Ins (OR 1.168, 95%CI: 1.317–1.035), HOMA-IR (OR 5.838, 95%CI: 12.803–2.281), TG (OR 1.698, 95%CI: 2.486–1.159), and sEng (OR 1.464, 95%CI: 1.821–1.077) were significantly higher compared to the control group (all p < 0.05). Therefore, the logistic regression analysis indicates a significant association between the independent variables and T2DM.

The morbidity and mortality rates of T2DM patients are increased by macrovascular complications. The IMT and ABI, as evaluated by ultrasonography Doppler, are two key indications for atherosclerosis, which is a key pathophysiologic process in the development of diabetic macrovascular complications [22]. According to the results of the current investigation there were no appreciable variations in the IMT and ABI between newly-diagnosed T2DM patients and control subjects. These findings revealed the newly-diagnosed T2DM patients included in this study did not have severe atherosclerosis and vascular changes compared to healthy adults.

Another pathophysiologic process that contributes to the emergence and progression of diabetic macrovascular complications is ED, which occurs prior to structural vascular modifications [23]. ECs are targeted by a number of substances in the early stages of diabetic macrovascular complications, including hyperglycemia, HCE, advanced glycation end products, and reactive oxygen species [24-26]. Endothelial function can be assessed using the RHI. In the current investigation newly-diagnosed T2DM patients had considerably lower RHIs than control subjects. According to this finding, newly-diagnosed T2DM patients continued to have severe ED despite no significant structural vascular modifications.

In recent years, the serum sEng level has been linked to ED as a biomarker. The endoglin cleavage product, sEng, might prevent TGF-β1 from attaching to its receptor and prevent tube formation. An in vitro investigation showed that oxidative stress increases sEng synthesis in ECs as a result of glucose variation or a chronic high glucose level [26]. According to Blazquez-Medela et al. [27], T2DM patients with retinopathy or a high likelihood of 10-year cardiovascular risk have higher sEng levels. In mouse brains, Park et al. [28] showed that sEng/VEGF-A stimulates the development of dysplastic vasculature, activates microglia, and increases the expression of inflammasome markers. Park et al. [28] concluded that sEng-stimulated microglia cause ECs to exhibit hyper-angiogenic traits, suggesting that microglia play a role in mediating sEng-induced ED and vascular malformation. Additionally, the increased circulating sEng preferentially directs BMP9, the vascular quiescence and endothelial-protective factor bone morphogenetic protein 9, signaling via cell-surface sEng at the ECs [29].

Our results indicated that newly-diagnosed T2DM patients had significantly higher serum sEng levels than the control subjects. The relationship between the serum sEng level and endothelial function was further examined because sEng is a marker for ED [30]. In our study the RHI and serum sEng levels were shown to have a negative correlation in newly-diagnosed T2DM patients, suggesting that diabetes is not significantly associated with high serum sENG levels unless there is a higher DBP. One of the major risk factors for T2DM patients is assumed to be vascular ED. Hyperglycemia, fatty acid oxidation, a decreased NO level, oxidative stress, inflammatory activation, and altered barrier function are some of the changes that diabetes causes in the endothelium [31]. By interacting with the receptor for advanced glycation end products (RAGE), which occurs during diabetes, AGEs produce an excessive amount of harmful effects in ECs, including an increase in permeability [32], inhibition of endothelial nitric oxide synthase (eNOS) activity [33], and other negative effects that influence the coagulation system [34] and stimulate NF-κB and NADPH oxidase (NOX) [35]. Glycolytic intermediates are changed into methylglyoxal during the glycation process, which changes proteins and DNA. Diabetic cardiomyopathy has been linked to upregulation of this system, which inhibits eNOS function in ECs [36]. ED is independently related to increased cardiovascular risk in hypertension and may have a role in the onset and progression of vascular inflammation, vascular remodeling, and atherosclerosis [37].

Serum sEng and RHI exhibited a positive correlation in the control patients. Additionally, the serum sEng level was not correlated with the IMT or ABI, which are markers of structural vascular modifications, in contrast to the negative relationship between the serum sEng level and RHI in newly-diagnosed T2DM patients, indicating that a higher serum sEng level can be used as a marker reflecting a lower RHI. In addition, in newly-diagnosed T2DM, the serum sEng level was inversely proportional to the RHI in newly-diagnosed T2DM. The serum sEng is elevated and proportional to the DBP, thus cannot be excluded as an early indication suggesting diabetic atherosclerosis.

Animal models and in vitro studies have been utilized to investigate the biological function of sEng. In the current work we sought to identify the variables influencing sEng production in T2DM or the process causing sEng to be altered in T2DM. Elevated and fluctuating glucose levels greatly increase the synthesis of sEng in ED based on in vitro studies [38]. Compared to normal glucose-tolerant pregnant women, pregnant women with gestational diabetes mellitus have considerably higher serum sEng levels in the omental adipose tissue [39, 40]. Our data suggest that hyperglycemia causes a rise in the serum sEng level in newly-diagnosed T2DM patients, which is related to ED. Thus, increased plasma sEng levels in diabetic patients may be an early sign of vascular changes.

Mice deficient in apoE/LDLr fed a cholesterol-rich diet attained the highest serum sEng levels and cholesterol of all the groups examined [41], suggesting that visceral adipose tissue may be an important source of sEng and that glucose, cholesterol, or LDL may promote sEng synthesis. Our study involving newly-diagnosed T2DM patients failed to confirm these reports. We assessed visceral adipose tissue based on the WC, WHR, and BMI, blood glucose based on the FBG, 2hPBG, and HbA1c levels, and based on the lipids using TC and LDL-C. The relationship between serum sEng levels and the WC, WHR, BMI, and FBG, 2hPBG, HbA1c, TC, and LDL-C levels did not show any statistically significant differences. This finding implies that the vascular changes associated with ED, as reflected by the serum sEng level, are earlier or sensitive in newly-diagnosed T2DM patients.

Regarding the cause of this correlation analysis finding, we hypothesized that the T2DM group sample size was too small because there was a total of 34 newly-diagnosed T2DM patients included in this study. Therefore, additional T2DM patients who have recently been diagnosed should be collected in the future.

Endoglin and glycemia, SBP, pulse pressure, pressure wave velocity, and electrocardiographically-evaluated left ventricular hypertrophy all have significant relationships, according to the Blázquez-Medela et al. analysis [27] of 288 patients, including 64 with T2DM, 159 with hypertension, and 65 who were healthy. Endoglin levels were significantly higher in patients with diabetes who had non-dipper and extreme dipper circadian blood pressure patterns compared to patients who had dipper patterns, patients with diabetes and hypertension who had riser patterns compared to other patients, and patients with diabetes but no hypertension who had extreme dipper patterns compared to dipper, non-dipper, and riser groups. Furthermore, there was a substantial link between the plasma sEng and lower systolic night-day ratio values. Diabetes patients with retinopathy, a high chance of 10-year cardiovascular risk, and diabetes and hypertension with three or more damaged target organs (kidney, heart, and arteries) had higher endoglin levels than patients with no organ damage. Additionally, endoglin is a marker for cardiovascular damage and ED, two vascular diseases linked to diabetes and hypertension [27]. When compared to diabetic patients without peripheral neuropathy, patients with diabetic peripheral neuropathy had considerably higher plasma sEng levels [42].

ED has an impact on cardiovascular disorders, including atherosclerosis, coronary artery disease, and hypertension, all of which are characterized by IR. IR characterizes metabolic disorders, such as T2DM and obesity, which are related to ED. According to our binary logistic regression analysis, patients with newly diagnosed T2DM had significantly higher odds ratios for HOMA-IR (OR 5.838), TG (OR 1.698), sEng (OR 1.464), and BMI (OR 1.332) compared to the control group. Notably, the highest odds ratio was found for HOMA-IR, which is a method for assessing β-cell function and insulin resistance [43]. Additionally, previous studies have indicated that sEng is involved in the pathogenesis of T2DM and is significantly associated with insulin resistance, particularly in non-obese gestational diabetes (GDM) [44]. Obesity-related insulin resistance is a major risk factor for T2DM and cardiovascular diseases, and sEng acts as a co-receptor for members of the transforming growth factor (TGF)-superfamily and is highly expressed on vascular endothelial cells. Therefore, sEng concentrations may increase in parallel with the deterioration in endothelial function before subclinical structural vascular alterations become evident [45]. Our study showed that newly diagnosed T2DM patients had 1.571, 1.698, and 1.464 times higher BMI, TC, and sEng than normal participants.

The vascular action of insulin in the endothelium, which promotes the production of the vasodilator, NO, complements the metabolic action of insulin and improves the elimination of glucose. In fact, 25%–40% of the increase in glucose absorption in response to insulin stimulation is explained by NO-dependent increases in blood flow to skeletal muscle [46]. In coronary heart disease patients, the RHI in patients with complex coronary lesions was markedly lower than patients with simple coronary lesions (0.48 ± 0.19 vs. 0.58 ± 0.16, p < 0.001). Patients with coronary lesions at several branches had a dramatically lower RHI when compared to patients with coronary lesions at a single branch (0.51 ± 0.20 vs. 0.43 ± 0.15, p = 0.01). Normal endothelial function is a basis for the maintenance of cardiovascular stability. When the EC are repeatedly injured by blood flow, tobacco, drugs, high blood lipids, high blood glucose, high blood pressure, high blood homocysteine and low estrogen, endothelial dysfunction occurs, NO secretion decreases, and endoglin secretion increases, resulting in atherosclerosis, hypertension, myocardial ischemia, cerebrovascular diseases, and diabetes. Some diseases may present endothelial dysfunction at early stage [47].

Endothelium-derived NO deficit and reactive oxygen species generation are thought to be responsible for the negative effects of both IR and altered lipid signaling on EC function [48]. The formation of reactive oxygen species and reactive nitrogen species is increased as a result of IR and metabolic cellular disruption, which functions as a negative feedback loop. ED is caused by the increased sEng levels released in aggravated inflammatory conditions [40]. The following were the study limitations. The sample size was limited because we only enrolled newly-diagnosed T2DM, and the current study was a retrospective investigation. To conduct a prospective study and try to track the incidence of structural vascular abnormalities in newly-diagnosed T2DM people with various levels of sEng, we are working to enlist more newly-diagnosed T2DM subjects.

Among newly-diagnosed T2DM patients, the serum sEng levels were inversely correlated with the RHI, and an elevated sEng level may be associated with ED.

This work was supported by the Medical Science and Technology Innovation Subject of Nanjing Region (No. 14ZX30), the Natural Science Foundation of China (No. 81701481), the Health Science and Technology Plan of Hangzhou (No. 2016B56), and the Health Science and Technology Project of Hangzhou Health Commission (No. B20210196).

The authors have no conflicts of interest to declare.

Not applicable.

The authors declare that they have no competing interests.

• 1   Viigimaa  M,  Sachinidis  A,  Toumpourleka  M,  Koutsampasopoulos  K,  Alliksoo  S, et al. (2020) Macrovascular complications of type 2 diabetes mellitus. Curr Vasc Pharmacol 18: 110–116.
• 2   He  X,  Zhang  Y,  Zhou  Y,  Dong  C,  Wu  J (2021) Direct medical costs of incident complications in patients newly diagnosed with type 2 diabetes in China. Diabetes Ther 12: 275–288.
• 3   Li  J,  Chattopadhyay  K,  Xu  M,  Chen  Y,  Hu  F, et al. (2020) Prevalence and associated factors of vascular complications among inpatients with type 2 diabetes: a retrospective database study at a tertiary care department, Ningbo, China. PLoS One 15: e0235161.
• 4   Kaur  R,  Kaur  M,  Singh  J (2018) Endothelial dysfunction and platelet hyperactivity in type 2 diabetes mellitus: molecular insights and therapeutic strategies. Cardiovasc Diabetol 17: 121.
• 5   de la Cruz-Ares  S,  Cardelo  MP,  Gutiérrez-Mariscal  FM,  Torres-Peña  JD,  García-Rios  A, et al. (2020) Endothelial dysfunction and advanced glycation end products in patients with newly diagnosed versus established diabetes: from the CORDIOPREV study. Nutrients 12: 238.
• 6   Korkmaz  H,  Onalan  O (2008) Evaluation of endothelial dysfunction: flow-mediated dilation. Endothelium 15: 157–163.
• 7   Lind  L (2013) Relationships between three different tests to evaluate endothelium-dependent vasodilation and cardiovascular risk in a middle-aged sample. J Hypertens 31: 1570–1574.
• 8   Hu  Y,  Liu  J,  Wang  G,  Xu  Y (2018) The Effects of exenatide and metformin on endothelial function in newly diagnosed type 2 diabetes mellitus patients: a case-control study. Diabetes Ther 9: 1295–1305.
• 9   Konttinen  J,  Lindholm  H,  Sinisalo  J,  Kuosma  E,  Halonen  J, et al. (2013) Association between lowered endothelial function measured by peripheral arterial tonometry and cardio-metabolic risk factors - a cross-sectional study of Finnish municipal workers at risk of diabetes and cardiovascular disease. BMC Cardiovasc Disord 13: 83.
• 10   Kurozumi  A,  Okada  Y,  Arao  T,  Tanaka  Y (2016) Excess visceral adipose tissue worsens the vascular endothelial function in patients with type 2 diabetes mellitus. Intern Med 55: 3091–3095.
• 11   Vicen  M,  Igreja Sá  IC,  Tripská  K,  Vitverová  B,  Najmanová  I, et al. (2021) Membrane and soluble endoglin role in cardiovascular and metabolic disorders related to metabolic syndrome. Cell Mol Life Sci 78: 2405–2418.
• 12   Schiopu  E,  Au  KM,  McMahon  MA,  Kaplan  MJ,  Divekar  A, et al. (2014) Prevalence of subclinical atherosclerosis is increased in systemic sclerosis and is associated with serum proteins: a cross-sectional, controlled study of carotid ultrasound. Rheumatology (Oxford) 53: 704–713.
• 13   Vitverova  B,  Blazickova  K,  Najmanova  I,  Vicen  M,  Hyspler  R, et al. (2018) Soluble endoglin and hypercholesterolemia aggravate endothelial and vessel wall dysfunction in mouse aorta. Atherosclerosis 271: 15–25.
• 14   ElMonier  AA,  El-Boghdady  NA,  Abdelaziz  MA,  Shaheen  AA (2019) Association between endoglin/transforming growth factor beta receptors 1, 2 gene polymorphisms and the level of soluble endoglin with preeclampsia in Egyptian women. Arch Biochem Biophys 662: 7–14.
• 15   Emeksiz  HC,  Bideci  A,  Damar  C,  Derinkuyu  B,  Celik  N, et al. (2016) Soluble endoglin level increase cccurs prior to development of subclinical structural vascular alterations in diabetic adolescents. J Clin Res Pediatr Endocrinol 8: 313–320.
• 16   Doghish  AS,  Bassyouni  AA,  Mahfouz  MH,  Abd El-Aziz  HG,  Zakaria  RY (2019) Plasma endoglin in Type2 diabetic patients with nephropathy. Diabetes Metab Syndr 13: 764–768.
• 17   Vitverova  B,  Najmanova  I,  Vicen  M,  Tripska  K,  Sa  ICI, et al. (2020) Long term effects of soluble endoglin and mild hypercholesterolemia in mice hearts. PLoS One 15: e0233725.
• 18   Meesters  RJ,  Voswinkel  S (2018) Bioanalytical method development and validation: from the USFDA 2001 to the USFDA 2018 guidance for industry. J Appl Bioanal 4: 67–73.
• 19   Hamburg  NM,  Benjamin  EJ (2009) Assessment of endothelial function using digital pulse amplitude tonometry. Trends Cardiovasc Med 19: 6–11.
• 20   Nobre  V,  Ataíde  TB,  Brant  LC,  Oliveira  CR,  Rodrigues  LV, et al. (2016) Use of reactive hyperemia—peripheral arterial tonometry and circulating biological markers to predict outcomes in sepsis. Rev Bras Ter Intensiva 28: 387–396.
• 21   Jedlickova  L,  Merkovska  L,  Jackova  L,  Janicko  M,  Fedacko  J, et al. (2015) Effect of ivabradine on endothelial function in patients with stable angina pectoris: assessment with the Endo-PAT 2000 device. Adv Ther 32: 962–970.
• 22   Jung  CH,  Mok  JO (2020) Recent updates on vascular complications in patients with type 2 diabetes mellitus. Endocrinol Metab (Seoul) 35: 260–271.
• 23   Kumar  V,  Jain  N,  Raizada  N,  Aslam  M,  Mehrotra  G, et al. (2021) Postprandial endothelial dysfunction and CIMT after oral fat challenge in patients with type 2 diabetes mellitus with and without macrovascular disease—a preliminary study. Diabetes Metab Syndr 15: 102317.
• 24   Love  KM,  Barrett  EJ,  Malin  SK,  Reusch  JEB,  Regensteiner  JG, et al. (2021) Diabetes pathogenesis and management: the endothelium comes of age. J Mol Cell Biol 13: 500–512.
• 25   Kattoor  AJ,  Goel  A,  Mehta  JL (2019) LOX-1: Regulation, signaling and its role in atherosclerosis. Antioxidants (Basel) 8: 218.
• 26   Domingueti  CP,  Dusse  LM,  Carvalho  Md,  de Sousa  LP (2016) Diabetes mellitus: The linkage between oxidative stress, inflammation, hypercoagulability and vascular complications. J Diabetes Complications 30: 738–745.
• 27   Blazquez-Medela  AM,  Garcia-Ortiz  L,  Gomez-Marcos  MA,  Recio-Rodriguez  JI,  Sanchez-Rodriguez  A, et al. (2010) Increased plasma soluble endoglin levels as an indicator of cardiovascular alterations in hypertensive and diabetic patients. BMC Med 8: 86.
• 28   Park  ES,  Kim  S,  Yao  DC,  Savarraj  JPJ,  Choi  HA, et al. (2022) Soluble endoglin stimulates inflammatory and angiogenic responses in microglia that are associated with endothelial dysfunction. Int J Mol Sci 23: 1225.
• 29   Lawera  A,  Tong  Z,  Thorikay  M,  Redgrave  RE,  Cai  J, et al. (2019) Role of soluble endoglin in BMP9 signaling. Proc Natl Acad Sci USA 116: 17800–17808.
• 30   Leite  AR,  Borges-Canha  M,  Cardoso  R,  Neves  JS,  Castro-Ferreira  R, et al. (2020) Novel biomarkers for evaluation of endothelial dysfunction. Angiology 71: 397–410.
• 31   Knapp  M,  Tu  X,  Wu  R (2019) Vascular endothelial dysfunction, a major mediator in diabetic cardiomyopathy. Acta Pharmacol Sin 40: 1–8.
• 32   Wautier  JL,  Zoukourian  C,  Chappey  O,  Wautier  MP,  Guillausseau  PJ, et al. (1996) Receptor-mediated endothelial cell dysfunction in diabetic vasculopathy. Soluble receptor for advanced glycation end products blocks hyperpermeability in diabetic rats. J Clin Invest 97: 238–243.
• 33   Soro-Paavonen  A,  Zhang  WZ,  Venardos  K,  Coughlan  MT,  Harris  E, et al. (2010) Advanced glycation end-products induce vascular dysfunction via resistance to nitric oxide and suppression of endothelial nitric oxide synthase. J Hypertens 28: 780–788.
• 34   Katakami  N (2018) Mechanism of development of atherosclerosis and cardiovascular disease in diabetes mellitus. J Atheroscler Thromb 25: 27–39.
• 35   Bierhaus  A,  Chevion  S,  Chevion  M,  Hofmann  M,  Quehenberger  P, et al. (1997) Advanced glycation end product-induced activation of NF-kappaB is suppressed by alpha-lipoic acid in cultured endothelial cells. Diabetes 46: 1481–1490.
• 36   Dhar  A,  Dhar  I,  Desai  KM,  Wu  L (2010) Methylglyoxal scavengers attenuate endothelial dysfunction induced by methylglyoxal and high concentrations of glucose. Br J Pharmacol 161: 1843–1856.
• 37   Gallo  G,  Volpe  M,  Savoia  C (2022) Endothelial dysfunction in hypertension: current concepts and clinical implications. Front Med (Lausanne) 8: 798958.
• 38   La Sala  L,  Pujadas  G,  De Nigris  V,  Canivell  S,  Novials  A, et al. (2015) Oscillating glucose and constant high glucose induce endoglin expression in endothelial cells: the role of oxidative stress. Acta Diabetol 52: 505–512.
• 39   Lappas  M (2014) Markers of endothelial cell dysfunction are increased in human omental adipose tissue from women with pre-existing maternal obesity and gestational diabetes. Metabolism 63: 860–873.
• 40   McElwain  CJ,  Tuboly  E,  McCarthy  FP,  McCarthy  CM (2020) Mechanisms of endothelial dysfunction in pre-eclampsia and gestational diabetes mellitus: windows into future cardiometabolic health? Front Endocrinol (Lausanne) 11: 655.
• 41   Strasky  Z,  Vecerova  L,  Rathouska  J,  Slanarova  M,  Brcakova  E, et al. (2011) Cholesterol effects on endoglin and its downstream pathways in ApoE/LDLR double knockout mice. Circ J 75: 1747–1755.
• 42   Motawi  TK,  Rizk  SM,  Ibrahim  IA,  El-Emady  YF (2014) Alterations in circulating angiogenic and anti-angiogenic factors in type 2 diabetic patients with neuropathy. Cell Biochem Funct 32: 155–163.
• 43   Minh  HV,  Tien  HA,  Sinh  CT,  Thang  DC,  Chen  CH, et al. (2021) Assessment of preferred methods to measure insulin resistance in Asian patients with hypertension. J Clin Hypertens (Greenwich) 23: 529–537.
• 44   Nergiz Avcıoğlu  S,  Demircan Sezer  S,  Altınkaya  SÖ (2022) Circulating s-Endoglin concentrations in non-obese patients with gestational diabetes mellitus. J Obstet Gynaecol 42: 1126–1131.
• 45   Emeksiz  HC,  Bideci  A,  Damar  Ç,  Derinkuyu  B,  Çelik  N, et al. (2016) Soluble endoglin level increase occurs prior to development of subclinical structural vascular alterations in diabetic adolescents. J Clin Res Pediatr Endocrinol 8: 313–320.
• 46   Kim  JA,  Montagnani  M,  Koh  KK,  Quon  MJ (2006) Reciprocal relationships between insulin resistance and endothelial dysfunction: molecular and pathophysiological mechanisms. Circulation 113: 1888–1904.
• 47   Wang  Z,  Yang  X,  Cai  J,  Shi  H,  Zhong  G, et al. (2015) Vascular endothelial function of patients with stable coronary artery disease. Pak J Med Sci 31: 538–542.
• 48   Cersosimo  E,  DeFronzo  RA (2006) Insulin resistance and endothelial dysfunction: the road map to cardiovascular diseases. Diabetes Metab Res Rev 22: 423–436.