EXPRESSION OF ANTI-ANGIOGENIC GENES IN SUBCUTANEOUS ADIPOSE TISSUE OF THE OBESE INDIVIDUALS WITH..

Accumulating evidence raises the hypothesis that dysregulation of different intrinsic mechanisms which control most metabolic processes are involved in the development of obesity, metabolic syndrome and type 2 diabetes mellitus, the most profound public health problems. Angiogenesis is an important component of different proliferative processes, in particular, fat tissue growth. Moreover, dysregulation of molecular components of the angiogenesis system can contributes to the development of diabetic complications. We have studied the expression levels of genes related to regulation of angiogenesis (TIMP1, TIMP2, TIMP3, TIMP4, THBS1, THBS2, THBS3, ADAMTS5, LUM, DCN, IL6 and ZEB1) in subcutaneous adipose tissue of obese individuals as well as obese patients with impaired glucose tolerance (prediabetic) and type 2 diabetes using real-time quantitative PCR. We have shown that the expression level of most of these genes with anti-angiogenic properties significantly increases in subcutaneous adipose tissue of obese individuals versus lean patients, being more intense for TIMP1, TIMP2, THBS2 and LUM genes. Increased expression level of TIMP1, TIMP2, TIMP3, ADAMTS5 as well as THBS1 and THBS2 in adipose tissue was found in obese patients with impaired glucose tolerance. At the same time, expression of genes which encode for TIMP1 and TIMP2 strongly decreased in adipose tissue of obese individuals with type 2 diabetes versus subjects with glucose intolerance. Results of this study provide strong evidence that expression of genes mostly related to suppression of angiogenesis is dysregulated in adipose tissue of obese individuals as well as in obese patients with glucose intolerance and type 2 diabetes. It is possible that these changes in the expression of TIMP and THBS genes in adipose tissue in obesity as well as in obese individuals with impaired glucose tolerance and type 2 diabetes can contribute to fat tissue storage, insulin resistance and the development of diabetic complications.


INTRODUCTION
Accumulating evidence raises the hypothesis that dysregulation of different intrinsic mechanisms which control most metabolic processes are involved in the development of obesity, metabolic syndrome and type 2 diabetes mellitus, the most profound public health problems. Angiogenesis is an important component of different proliferative processes, in particular, fat tissue growth. Moreover, dysregulation of molecular components of the angiogenesis system can contributes to the development of diabetic complications. Recently, a number of studies have provided support for a link between the altered sleep/wake patterns, namely the circadian clock, and obesity [1][2][3]. Biological rhythms are an integral component of essentially all aspects of life and regulate many physiological functions, including metabolism, insulin sensitivity and cellular proliferation and its dysregulation involved in the development of obesity, metabolic syndrome as well as type 2 diabetes mellitus [4][5][6]. Moreover, obesity is a well-known risk factor for the development of insulin resistance and secondary complications such as type 2 diabetes.
Adipose tissue growth is tightly associated with cell proliferation processes and angiogenesis regulated by different factors and are tightly interconnected; however, the vascular endothelial growth factor (VEGF) is a key factor which activity depends upon many factors. Special interest represents matrix proteins with pleiotropic roles which are linked to human obesity and diabetes complications because to reveal anti-angiogenic properties and appear to regulate cell growth and apoptosis [7][8][9][10][11].
Thrombospondin 1 (THBS1 or TSP1) is an adhesive glycoprotein that mediates cell-to-cell interactions, has anti-angiogenic properties and inhibits of cell growth [7]. It was shown that decorin (DCN) which antagonizes the angiogenic network induces the expression of thrombospondin-1 and tissue inhibitor of matrix metalloproteinase 3 (TIMP metallopeptidase inhibitor 3; TIMP3), but inhibits vascular endothelial growth factor A (VEGFA) [8]. It has recently been shown that endothelial nitric oxide synthase controls the expression of the angiogenesis inhibitor thrombospondin 2 and ADAM metallopeptidase with thrombospondin type 1 motif, 5 (ADAMTS5) [9,10].
There are four genes encoding tissue inhibitor of matrix metalloproteinase (TIMP1, TIMP2, TIMP3 and TIMP4) with different regulatory properties. TIMP1 is able to promote cell proliferation in a wide range of cell types, and may also have an anti-apopto tic function. At the same time, TIMP1 is a potent inhibitor of tumor growth and angiogenesis and is involved in cell adhesion and migration. It also controls adipogenesis in obesity both in mice and in humans as a negative regulator [11]. TIMP2 thought to be a metastasis suppressor and has a unique role among TIMP family members in its ability to directly suppress the proliferation of endothelial cells.
Moreover, Dews et al. [12] have shown that enhanced neovascularization correlated with downregulation of anti-angiogenic thrombospondin-1 and related proteins, such as connective tissue growth factor (CTGF), besides that both THBS1 and CTGF are predicted targets for repression by the miR-17-92 microRNA. Moreover, VEGFA binds connective tissue growth factor and that its angiogenic activity is inhibited in the VEGFA-CTGF complex form; however, stability of this complex as well as the angiogenic activity of VEGF depends from matrix metalloproteinases and its inhibitors [13].
Recently was shown that the endothelial dysfunction in diabetes mellitus resulting in vascular endothelial growth factor resistance despite the presence of functionally active VEGF receptor 1 [20].
The endoplasmic reticulum stress is recognized as an important determinant of type 2 diabetes and contributes to the expression profile of many regulatory genes resulting in peripheral insulin resistance and diabetic complications [21][22][23][24]; however, a detailed molecular mechanism of the development of obesity and type 2 diabetes mellitus in some representatives of obese individuals is not yet clear and remains to be determined.
The main goal of this work is to study the role of the expression of genes related to the regulation of angiogenesis (TIMP1, TIMP2, TIMP3, TIMP4, THBS2, THBS3, AD-AMTS5, LUM, DCN, IL6 and ZEB1) in subcutaneous adipose tissue of obese individuals as well as obese patients with impaired glucose tolerance and type 2 diabetes for evaluation of its significance to the development of human obesity, initial stages of meta bolic disease (prediabetes) and type 2 diabetes mellitus.

MATERIALS AND METHODS
Patients' characteristics. The 24 male subjects participate in the study. They were divided into four groups (6 men in each group): lean individuals and patients with obesity, prediabetes and type 2 diabetes mellitus.
Subjects studied were recruited from the patients' cohort within the LipidomicNet Project at Institute of Experimental Endocrinology Slovak Academy of Sciences. All participants gave written informed consent and the studies were approved by the local research bioethics committees of Institute of Experimental Endocrinology.
Clinical characteristics of the study participants are shown in Table 1. The lean (control) participants were individuals with mean age 45±8 years and mean body mass index (BMI) 23±1.4 kg/m 2 . The obese participants as well as the patients with prediabetes and type 2 diabetes were individuals with mean age (45±8; 44±7 and 48±8 years, correspondingly) and mean BMI (32±1.4; 34±1.4 and 33±2.4 kg/m 2 , correspondingly). Thus, BMI in these groups of patients was significantly higher as compared to lean individuals (Table 1).
Reverse transcription and quantitative real-time polymerase chain reaction analysis. The expression levels of genes related to regulation of an angiogenesis (TIMP1,  TIMP2, TIMP3, TIMP4, THBS1, THBS2, THBS3, ADAMTS5, LUM, DCN, IL6 and ZEB1) were measured in subcutaneous fat tissue by real-time quantitative polymerase chain reaction of complementary DNA (cDNA). QuaniTect Reverse Transcription Kit (QIAGEN, Germany) was used for cDNA synthesis. The 7900 HT Fast Real-Time PCR System (Applied Biosystems), Absolute QPCR SYBRGreen Mix (Thermo Scientific, UK) and pair of primers specific for each studied gene (Sigma, USA) were used for quantitative polymerase chain reaction. Sequences of these primers are shown in Table 2.
The expression of beta-actin mRNA was used as control of analyzed RNA quantity. An analysis of quantitative PCR was performed using special computer program "Differential expression calculator" and statistical analysis -in Excel program. Results are represented as % of control (lean individuals) and are the means ± SEM for six different samples.
The amplified DNA fragments were analyzed on a 2% agarose gel and that visualized by 5× Sight DNA Stain (EUROMEDEA).

RESULTS
In this study we have analyzed the expression levels of four genes encoded tissue inhibitor of matrix metalloproteinase (TIMP metallopeptidase inhibitor; TIMP1, TIMP2, TIMP3 and TIMP4), three genes encoded thrombospondin (THBS1, THBS2 and THBS3), ADAM metallopeptidase with thrombospondin type 1 motif, 5 (ADAMTS5), lumican (LUM), decorin (DCN), interleukin 6 (IL6) and transcription factor ZEB1 in adipose tissue from four groups of participants: lean (control), obese with normal glucose tolerance test (NGT), obese with impaired glucose tolerance (IGT; prediabetes) and obese individuals with type 2 diabetes.   As shown in Table 1, obese individuals have significantly higher body mass index (BMI), which is a main criteria of obesity, and lower insulin sensitivity index. In obese patients with impaired glucose tolerance, versus obese subjects with normal glucose tolerance, the 2 h oral glucose tolerance test (OGTT), fasting triglycerides and fasting insulin levels significantly increased, but decreased insulin sensitivity index (Table 1). This data clearly demonstrated that this group of patients has insulin resistance and impaired glucose tolerance. The group of obese individuals with type 2 diabetes has a significantly higher level of fasting glucose (9.2±1.34 mmol/l) and of 2 h oral glucose tolerance test (11.3±1.57 mmol/l) as compared to all other groups of patients as well as fasting insulin and triglycerides level, but decreased insulin sensitivity index as compared to lean or obese patients with normal glucose tolerance (Table 1) obese individuals with type 2 diabetes corresponds to the WHO (1999) criteria as fasting plasma glucose >7.0 mmol/l or 2h glucose >11.1 mmol/l during an OGTT.

DISCUSSION
In this study, we have demonstrated that obesity is associated with dysregulation of genes encoded the important regulatory factors which control angiogenesis and growth processes involved in the development of the obesity, metabolic syndrome and type 2 diabetes mellitus, the most profound public health problems. Angiogenesis is an important component of different proliferative processes, in particular, fat tissue growth; however, angiogenesis can also contribute to the development of diabetic complications. We have studied the expression of genes mostly related to suppression of an angiogenesis in subcutaneous adipose tissue of obese individuals as well as obese patients with impaired glucose tolerance and type 2 diabetes mellitus. The results of our investigation have shown that obesity leads to a significant increase of the expression of TIMP1, TIMP2, TIMP3, THBS1, THBS2, ADAMTS5, LUM, DCN and IL6 genes in subcutaneous adipose tissue, besides that, the most significant increase was shown for TIMP1, TIMP2, THBS2 and LUM genes. These findings are largely consistent with data from previous studies about the involvement of these genes in the regulation of different proliferative processes and angiogenesis [7,15,16,18]. Hence, it is possible that these genes are also involved the development of obesity, metabolic syndrome and type 2 diabetes mellitus as well as in the development of diabetic complications and VEGF resistance [8,13,14,20]. Putative model linking the dysregulation of circadian clock system with obesity and its complications; glucose intolerance and type 2 diabetes mellitus based on our results is shown in Fig. 7.
It is interesting to note that angiogenesis, like many other biological processes, is regulated by complex network of different factors which are tightly interconnected. Putative scheme of angiogenesis regulation by different factors including TIMP1, TIMP2, TIMP3 and THBS1 as well as pleiotropic functions of TIMP1 and the role of endoplasmic reticulum stress in angiogenesis is shown in Fig. 8. Thus, thrombospondin-1, a matrixbound adhesive glycoprotein, has been shown to modulate tumor progression and upregulates tissue inhibitor of metalloproteinase-1 production in human tumor cells and also up-regulates matrix metalloproteinases MMP-2 and MMP-9 [25]. This data suggested that the balance between matrix metalloproteinases and tissue inhibitors of metalloproteinases is a key determinant in different biological effects of THBS1, including tumor cell invasion, and may provide an explanation for the divergent activities reported for thrombospondin-1 in tumor progression. Thus, the THBS1 is involved in influencing the critical balance between MMPs and their inhibitors, maintaining the controlled degradation of the extracellular matrix needed to support metastasis and possibly in obesity as well as in obesity with impaired glucose tolerance where we find the overexpression of THBS1.
It is possible that ADAMTS5, LUM, DCN, IL6 and ZEB1 are also included in this network and the balance between different regulatory factors which participate in the control of angiogenesis, including vascular endothelial growth factor, a key pro-angiogenic factor, insulin, insulin-like growth factors, leptin and others, really determinate an angiogenesis both in obesity and in obesity with glucose intolerance or type 2 diabetes mellitus. Recently it was shown that the endothelial dysfunction in diabetes associates with vascular endothelial growth factor resistance and disturbed VEGF-related signal transduction pathway despite presence of functionally active VEGF receptor 1 on cell surface from diabetic individuals [52]. Hence, the interpretation of our results of the expression profile of genes, encoded anti-angiogenic factors mostly with pleiotropic func-  TIMP1, TIMP2, TIMP3, THBS1, THBS2 and LUM genes and development of obesity. Obesity with glucose intolerance demonstrated increased expression of TIMP1, TIMP2, TIMP3, THBS1 and ADAMTS5 and decreased expression of IL6 and LUM genes. Most of these genes are responsible for control of angiogenesis. Moreover, development of type 2 diabetes is associated with suppression of TIMP1 and TIMP2 gene expressions. Putative role of endoplasmic reticulum stress in the development of type 2 diabetes by modulating the gene expression is shown. Thus, we identified the changes in gene expressions, which closely related to angiogenesis in obesity and its complication: glucose intolerance and type 2 diabetes tion, in subcutaneous adipose tissue of obese individuals with normal glucose tolerance as well as with glucose intolerance and with type 2 diabetes mellitus is a little difficult because obese subjects with complications like glucose intolerance and type 2 diabetes have resistance to insulin, VEGF and possibly to many other important regulatory factors; however, this aspect of the regulatory mechanisms remains unknown yet and warrants further investigation. Endoplasmic reticulum stress is recognized as an important determinant of obesity with type 2 diabetes and contributes to the expression profile of many important regulatory factors resulting in peripheral insulin resistance and diabetic complications, although detailed molecular mechanisms cannot be ruled out. Collectively, these results demonstrate the important role of THBS, TIMP and other regulatory factors with pleiotropic function in developing the obesity and its complications, especially glucose intolerance and insulin resistance.

CONCLUSIONS
Results of this study provide strong evidence that expression of genes encoded the key regulatory factors with pleiotropic functions, related to the control of proliferation and angiogenesis, in subcutaneous adipose tissue of the obese individuals with normal glucose tolerance as well as in obese patients with glucose intolerance and type 2 diabetes is deregulated. It is possible that these changes in the expression of TIMP and THBS genes in adipose tissue in obesity as well as in obese individuals with impaired glucose tolerance and type 2 diabetes can contribute to fat tissue storage, insulin and VEGF resistance as well as the development of diabetic complications determined both by insulin resistance and endoplasmic reticulum stress. Collectively, results of this study underscore the crucial role of THBS, TIMP and some other regulatory factors in developing the obesity as well as glucose intolerance, insulin resistance and type 2 diabetes mellitus in obese individuals.