Macrovascular complications are important complications of diabetes mellitus. Endothelial dysfunction is an early pathophysiological stage of atherosclerosis and plays a key role in the occurrence and development of macrovascular complications, and oxidative stress is one of the mechanisms of atherogenesis.
Endothelial progenitor cells (EPCs) are a group of stem/progenitor cells that reside in the bone marrow and can specifically nest in the damaged endothelial area and differentiate and proliferate into mature endothelial cells. They further proliferate and differentiate into mature endothelial cells, which participate in vascular neogenesis and re-endothelialization.
Fadini GP et al. studied EPCs in people with impaired glucose regulation and T2DM and showed that in people with impaired glucose regulation, that is, in the prediabetic phase, there is already a decrease in the number of EPCs, indicating that the decrease in EPCs accompanies the natural course of diabetes, and he suggested that the increase in cardiovascular risk factors in the prediabetic phase is not only related to endothelial damage caused by hyperglycemia and other concomitant metabolic abnormalities He concluded that the increase in cardiovascular risk factors in prediabetes is not only related to endothelial damage caused by hyperglycemia and other concomitant metabolic abnormalities, but also to impaired endothelial regeneration caused by depletion of circulating progenitor cells, and that the decline in circulating progenitor cells leads to an accumulation of cardiovascular risk factors. Oxidative stress is one of the influential factors causing the decline of EPCs.
Type 2 diabetes is familially aggregated and its children are at high risk of developing diabetes, and our previous study showed that vascular endothelial dysfunction has developed in children of type 2 diabetic patients with normal glucose tolerance. Is there an altered number of EPCs in this population, is oxidative stress present, and what is the relationship between them? There are few studies in this area.
In this study, we examined the changes in endothelium-dependent vasodilatory function (FMD), the number of circulating EPCs, serum oxidants, and antioxidants in patients with type 2 diabetes mellitus (T2DM) and their first-degree relatives (FDRs), and investigated the relationship between them in order to provide a basis for the prevention and treatment of the development of T2DM and its macrovascular complications.
Data and Methods
I. Study subjects
A total of 40 patients with type 2 diabetes mellitus, 22 males and 18 females, aged (44.78±1.82) years, diagnosed by outpatient and inpatient glucose tolerance test (OGTT) from 2008-2009, were selected as the diabetic group.
Children of type 2 diabetic patients were selected as the FDRs group, with 38 cases, 20 males and 18 females, aged (46.87±1.91) years, all excluding impaired fasting glucose, impaired glucose tolerance and diabetes mellitus; normal control (NC) group 30 cases, 17 males and 13 females, aged (44.07±1.89) years, with no family history of diabetes mellitus. All subjects had normal electrocardiogram, ultrasound, blood, urine and stool examinations, excluded hypertension, coronary heart disease, cerebrovascular disease and peripheral vascular disease, no liver or kidney dysfunction, excluded taking drugs that affect the body’s metabolism, and excluded applying insulin therapy.
II. Methods
1, blood biochemical indexes determination: glucose determination using glucose oxidase method, HBAlc determination using chemical method; lipid (cholesterol, triglycerides) determination using enzymatic method, using the United States Beckman L20 automatic biochemical instrument; superoxide dismutase (SOD), malondialdehyde (MDA), glutathione – peroxidase (GSH-PX), total antioxidant capacity ( TAO-C) were determined by colorimetric method, and the kits were purchased from Nanjing Jiancheng Institute of Biological Engineering. The determination of insulin was performed by chemiluminescence method.
2, Insulin sensitivity and islet B-cell function related indexes were calculated by the formula.
Steady-state model insulin resistance index (HOMA-IR) = (FPG × FIns)/22.5.
3. Peripheral blood EPC count: Take 2mL of peripheral blood, add 150ul of whole blood to each flow-through assay tube, add 10ul of primary antibody; add 10ul of buffer to the isotype control tube. And mix gently. After incubation for 30 minutes at room temperature, add 2ml of erythrocyte lysate to each tube, mix well, incubate for 10 minutes at room temperature at 3000r/min, centrifuge for 5 minutes, and discard the supernatant. The cells were washed once with 2ml of buffer, resuspended with 500ul of buffer and then detected by flow cytometry.
CD34+KDR+ cells were identified as EPCs (kits were purchased from Invitrogen and R&D, USA). 1×105 cells were counted by analysis with FACS Calibur analyzer (BD, USA); the data were then processed with software (Macintosh CELLQuest; BD Biosciences).
4, Determination of endothelium-dependent vascular endothelial function (FMD): measured by two-dimensional ultrasound imaging scanning method (PHILIPS HDI 5000, General Motors, USA): the patient was lying down, the sphygmomanometer cuff was tied to the right forearm, the cuff was inflated to 50 mmHg above the systolic pressure and deflated after 5 min to cause reactive congestion, brachial artery images were acquired within 30~90 s and combined with The end-diastolic internal diameter (Dd1) was measured in combination with ECG within 30-90s. The change in brachial artery internal diameter after reactive congestion (FMD%)=(Dd1-Dd)/Dd×100%.
III. Statistical processing
The SPSS 13.0 statistical analysis package was used for analysis. All measurement data were expressed as mean±standard deviation, and the measured results were tested for normality and chi-square, and one-way ANOVA was used for comparison between multiple groups for measurement data, and SNK-q test was used for two-way comparison between groups, with P<0.05 as significant difference. Linear correlation analysis and multiple linear regression analysis were used for variable correlations.
Results
1. Clinical and biochemical characteristics of the three groups
As can be seen from Table 1, the three groups were matched for age and sex. there was no significant difference in BMI between the three groups. FPG and HbA1c were significantly higher in the T2DM group compared with the control group and the FDRs group (P<0.05), but there was no significant difference between the control group and the FDRs group (P>0.05). TC levels were higher in T2DM than in the FDRs and NC groups (P<0.05), and TG was not significantly different in the three groups; HOMA-IR gradually increased in the control, FDRs and T2DM groups, with significant differences (P<0.05).
2, Comparison of oxidative stress indexes and adipokines in the three groups
From Table 2, serum SOD, TAO-C and GSH-PX levels were significantly lower in the T2DM group than in the control group and FDRs (P<0.01), but there was no statistical difference between the FDRs and the control group (P>0.05); serum MDA levels gradually increased in the control group, FDRs and T2DM group (P<0.01).
3, Comparison of various parameters related to endothelial function in the three groups
As seen in Table 3, EPCs and FMD gradually decreased in the control, FDRs and T2DM groups, and the differences were statistically significant (P<0.05).
Discussion
In 1997, Asahara et al. found that there exists a precursor cell in circulating peripheral blood capable of differentiating into vascular endothelial cells (ECs), named EPCs, which are derived from bone marrow, cord blood or fetal liver and express certain specific markers such as hematopoietic progenitor cell markers: CD34 , CD133 and vascular endothelial growth factor receptor-2 (VEGFR-2) [also known as kinase functional region receptor (KDR) or fetal liver kinase-1 (FLK-1)] and endothelial markers such as CD31.
It is possible to isolate and culture EPCs in vitro and determine its properties and functions. However, they cannot be used in clinical studies on a large scale because of their high cost. Therefore, analysis of surface antigens by flow cytometry to identify progenitor cells is considered to be the gold standard. Many studies now suggest that CD34+KDR+ cells can be used as EPCs, CD34 is an important marker of hematopoietic stem cells, and KDR, as the earliest cellular marker of the vascular system, is a key receptor for embryonic blood angiogenesis and has also been used as an important molecular marker of vascular EPCs.
Some scholars believe that CD34+KDR+ EPCs count can be applied to independently predict the occurrence of cardiovascular events. Under normal conditions, the number of EPCs is relatively small, but in the case of vascular injury or tissue ischemia, EPCs present in the bone marrow can respond to locally released growth factors and cytokines and mobilize to the peripheral circulation, specifically homing to the site of injury or ischemia. They further proliferate and differentiate into mature endothelial cells, which participate in vascular neogenesis and re-endothelialization.
Once endothelial progenitor cells are damaged, the balance between endothelial damage and repair is disrupted, and the integrity of the endothelial layer is disrupted, resulting in atheromatous lesions. Therefore, EPCs play an important role in regulating the apoptosis/regeneration balance and maintaining the integrity of the endothelial layer, and they also secrete growth factors to activate mature endothelial cells in the vascular area to maintain the normal function of the endothelium.
Thus, we can conclude that changes in the number and function of EPCs are closely related to vascular endothelial function and are important influencing factors in vascular diseases, that is, the number and function of EPCs in bone marrow and peripheral blood determine the degree of endothelial repair of injured vessels, so EPCs are considered to be important biochemical factors for the treatment of various cardiovascular diseases and injured vessels.
Diabetes mellitus is an equivocal risk for cardiovascular disease, and type 2 diabetes mellitus is associated with a high incidence of cardiovascular disease and mortality. The present study showed that the number of EPCs was significantly reduced in FDRs and T2DM compared to controls, and FMD was significantly decreased, suggesting that a reduced number of EPCs and impaired vascular endothelial function already existed in first-degree relatives of diabetic patients with normal glucose tolerance.
Oxidative stress (OS) refers to the pathological process in which the excessive production of reactive oxygen groups (ROS) as well as reactive nitrogen groups (RNS), etc., and/or the reduction of the body’s antioxidant capacity leading to reduced clearance of reactive molecules under stressful conditions such as ischemia, hypoxia, and hyperglycemia, resulting in an imbalance between the oxidative and antioxidant systems, leading to elevated levels of oxygen radicals in tissues and causing tissue damage or potential damage .
Antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT), GSH-PX, etc. TAO-C represents the overall level of cellular enzymatic and non-enzymatic antioxidants. Oxidative stress is one of the mechanisms causing diabetic vascular complications, and decreased GPx-1 and MnSOD activities are now considered to be independent risk factors for cardiovascular events in patients with coronary artery disease.
The results of the present study showed that serum SOD , TAO-C and GSH-PX levels were significantly lower in the T2DM group than in the NC and FDRs groups, and serum MDA levels were significantly higher in FDRs and T2DM, suggesting that imbalance in oxidative and antioxidant systems already existed in first-degree relatives of T2DM with normal glucose tolerance, which is consistent with past reports [8].
Pearson correlation analysis showed that in FDRs, IR was positively correlated with MDA and negatively correlated with SOD, TAO-C, and GSH, suggesting that oxidative stress is closely associated with IR in first-degree relatives with diabetes. It has been shown that the presence of insulin resistance and increased intracellular lipids in myocytes in normoglycemic non-obese first-degree relatives of T2DM patients present oxidative stress due to mitochondrial dysfunction in skeletal muscle [9], and IR is a physiological defense mechanism against excess ROS at the cellular level of the body.
Compared to other tissue cells, β-cells have low levels of free radical scavenging enzymes (antioxidant enzymes) as well as ROS scavenging proteins such as thioredoxin, and thus are extremely sensitive to live ROS and most vulnerable to oxidative stress attack; therefore, when the endogenous antioxidant system is inadequately compensated, the body experiences redox imbalance, activating stress-sensitive signaling pathways, causing β-cell dysfunction and further aggravating IR, which eventually leads to the development of T2DM and its chronic complications.
From this, we can speculate that oxidative stress occurs due to genetic and environmental factors, and the two promote each other, further leading to insulin resistance and β-cell function failure, thus inducing the onset and development of diabetes and its complications.
Multiple linear regression analysis using EPCs as dependent variables showed that TOA, MDA, GSH-PX, FBG, and HbA1c entered the equation (R=0.979, P<0.05), and the difference was statistically significant, indicating that blood glucose level and oxidative stress are influencing factors of EPCs.
Hyperglycemia and its metabolites can affect EPCs and vascular endothelial function through multiple pathways, leading to the development of vascular complications through.
(1) High glucose and TNF-α downregulate the number of EPCs by activating p38-MAPK within EPCs;
(2) High glucose mediates the imbalance of FoxO transcription factor phosphorylation/acetylation, which may increase FoxO protein expression, upregulate pro-apoptotic gene expression, and mediate apoptosis in EPCs, Sir2 (silent information regulator-2 ) has an inverse regulatory effect on FoxO, and high glucose affects EPC function through Sir2 FoxO, further leading to impaired EPC function.
(3) High glucose leads to a decrease in the bioavailability of NO released from EPCs or a decrease in the cofactor of eNOS, tetrahydrobiopterin (BH4), which results in the uncoupling of eNOS, generating superoxide negative ions (O-), leading to the accumulation of reactive oxygen clusters (ROS), further reducing the number and migration capacity of EPCs.
(4) Hyperglycemia affects vascular endothelial function by impairing the function of EPCs and releasing particulate matter (MPs), which can promote thrombus and fibrin formation by initiating exogenous coagulation pathways and forming polymers with platelets; activating neutrophils, promoting monocyte-endothelial cell binding and chemotaxis to neutrophils, and participating in the development of inflammatory responses.
Oxidative stress affects the function and number of EPCs by promoting their regulation, and its molecular mechanism is as follows: the expression of Bim, a pro-apoptotic protein regulated by forkhead transcription factor, is a key signaling pathway for the apoptosis of EPCs, and oxidative stress can induce apoptosis of EPCs by inducing the upregulation of B im expression level. The HMG2CoA reductase inhibitor statins can phosphorylate forkhead transcription factors through PI3/Akt signaling pathway to inactivate them, thereby downregulating the expression level of Bim and protecting EPCs from oxidative stress-induced apoptosis;
Oxidative stress can also induce apoptosis in EPCs through the ROS-p53-Bax pathway [14], which induces the inactivation of telomerase in EPCs and promotes their senescence and regulation. It has been reported that Eric’s study also showed that the application of manganese superoxide dismutase (MnSOD) transfected EPCs promoted wounded limb healing in diabetic mice.
In conclusion, the present study suggests that insulin resistance, oxidative stress, reduction in the number of endothelial progenitor cells and impaired vascular endothelial function have been observed in first-degree relatives of type 2 diabetes with normal glucose tolerance, and that oxidative stress may be the initiating factor of diabetes and its vascular complications.