© 2024 European Society of Medicine 1 1 L.T. Malaya Therapy National Institute of the National Academy of Medical Sciences of Ukraine, Kharkiv, Ukraine 2 V.N. Karazin Kharkiv National University, Kharkiv, Ukraine OPEN ACCESS PUBLISHED 31 August 2024 CITATION Кolesnikova, O., Vovk, K., et al., 2024. The Impact of Endothelial Dysfunction on the Course of Metabolically Associated Liver Disease in Combination with Subclinical Hypothyroidism. Medical Research Archives, [online] 12(8). https://doi.org/10.18103/mra.v12i 8.5335 COPYRIGHT © 2024 European Society of Medicine. This is an open- access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. DOI https://doi.org/10.18103/mra.v12i 8.5335 ISSN 2375-1924 ABSTRACT Background. Intrahepatic endothelial dysfunction is involved in many diseases, including fatty liver disease, and in combination with other numerous mechanisms may play a role in atherosclerosis in hypothyroidism. It is possible that endothelial dysfunction markers may be important in assessing the risk of cardiovascular events in patients with metabolic-associated fatty liver disease (MAFLD) in combination with subclinical hypothyroidism. Aims. The aim of this study was to evaluate changes in endothelial dysfunction markers in patients with MAFLD in combination with subclinical hypothyroidism. Methods. We studied 298 patients aged 40-69 years with a history of MAFLD. Of these, 128 patients had a verified diagnosis of both MAFLD and subclinical hypothyroidism. In such cases, the morphological state of the vascular endothelium was assessed by counting circulating desquamated epithelial cells (CDEC) and vascular endothelial growth factor (VEGF-A) in the blood using phase-contrast microscopy and the value of these parameters was in comparison todepending on the level of thyroid stimulating hormone, insulin, age, and cardiovascular risk. Results. It was found that the level of CDEC and VEGF-A was increased in patients with MAFLD and subclinical hypothyroidism. On the one hand, thyroid stimulating hormone levels were found to be associated with endothelial dysfunction and hyperinsulinemia. On the other hand, endothelial dysfunction indices also have links with the degree of cardiovascular risk. Patients with moderate and high cardiovascular risk have a relationship between CDEC and levels of triglycerides, C-reactive protein, and сardiovascular diseases. The levels of VEGF-A and CDEC were significantly higher in the adult age group. VEGF-A levels > 273 pg/mL were associated with abnormalities in the studied parameters, including those reflecting of the liver. Conclusion. Patients with MAFLD in combination with hypertension have elevated levels of CDEC and VEGF-A, which affects the involvement of these factors in the mechanisms of endothelial dysfunction in this category. Markers of endothelial dysfunction can be used to assess the prediction of possible cardiometabolic risk. THE EUROPEAN SOCIETY OF MEDICINE Medical Research Archives, Volume 12 Issue 8 RESEARCH ARTICLE The Impact of Endothelial Dysfunction on the Course of Metabolically Associated Liver Disease in Combination with Subclinical Hypothyroidism Olena Кolesnikova1, Kira Vovk2, Andriy Titarenko1 https://doi.org/10.18103/mra.v12i8.5335 https://doi.org/10.18103/mra.v12i8.5335 https://doi.org/10.18103/mra.v12i8.5335 https://doi.org/10.18103/mra.v12i8.5335 The Impact of Endothelial Dysfunction on the Course of Metabolically Associated Liver Disease © 2024 European Society of Medicine 2 Introduction The endothelium plays an important role in vascular biology and the regulation of liver function. Healthy endothelial cells (EC) are involved in vasodilation by releasing nitric oxide (NO), which also inhibits platelet adhesion and aggregation, as well as leukocyte adhesion. Conversely, damaged EC can develop a vasoconstrictor, proinflammatory, and procoagulant phenotype1. In addition, EC transport glucose to the cells of the blood vessel wall and to the parenchymal tissue2. Vascular endothelial growth factor as a regulator of endothelial transit of free fatty acids (FFA), controls the expression of endothelial fatty acid transport proteins and thus lipid accumulation in tissues3. Hepatic sinusoidal EC demonstrate anti-inflammatory and antifibrogenic properties, preventing Kupffer and stellate cell activation and regulating intrahepatic vascular resistance and portal pressure, so in the early stages of MAFLD, hepatic sinusoidal EC dysfunction occurs, namely, loss of the ability to generate vasodilating agents4. Endothelial dysfunction (ED) in combination with other numerous mechanisms may explain atherosclerosis in patients with hypothyroidism, although the subclinical hypothyroidism (SCH) is associated with atherosclerosis is still controversial5. Endothelial dysfunction is associated with traditional cardiovascular risk factors, such as hypertension and diabetes, and predicts the progression of atherosclerosis and cardiovascular events (CVE) in the general population1, so the assessment of ED, being an early biomarker, is useful for predicting cardiovascular risk (CVR) and evaluating treatment outcomes6. To prevent the development of CVE and its complications in patients with nonalcoholic liver steatosis in combination with SCH, it is essential to identify early predictors, which may include ED markers: VEGF-A, CDEC, the effect of which is still a subject of debate, despite the fact that over the past few years intrahepatic ED has been demonstrated in several models of liver disease, including fatty liver7. Thus, the study of markers of ED in patients with MAFLD in combination with SCH will provide an opportunity to expand our understanding of the mechanisms of CVR formation, to individualize on this basis the strategy for the prevention of CVE in a comorbid patient. At the same time, understanding the mechanisms of ED formation in patients with MAFLD in combination with SCH expands our understanding of early CVE in the combined course of diseases. Materials and methods On the basis of the State Institution «L.T. Malaya National Therapy Institute of the National Academy of Medical Sciences of Ukraine», 298 people aged 40-69 years with a history of MAFLD were studied. Of these, 128 people had a verified diagnosis of MAFLD and SCH. The diagnosis of MAFLD was established according to the recommendations of the European Association for the Study of the Liver (EASL, 2021)8. Subclinical hypothyroidism was diagnosed according to the guidelines of the European Thyroid Association (ETA, 2015)9. Waist circumference (WC) was measured with a flexible tape at the level of the navel. Cardiovascular risk was calculated using the SCORE2 scale. In all patients, the level of total cholesterol (TC), high-density lipoprotein (HDL), and triglycerides (TG) was determined by the enzymatic method on a «Humalayzer» biochemical analyzer (№ 2106-1709) using a set of reagents from «Human» (Germany). The cholesterol content of LDL cholesterol was calculated by the formula of Friedewald W.T. Glycated hemoglobin (HbA1c%) was determined from venous blood by ion-exchange chromatography using a biochemical semi-automatic analyzer «ERBA CHEM-7-RU». The concentration of C-reactive protein (CRP) and fasting insulin in the blood serum was studied by enzyme-linked immunosorbent assay on a semi- automatic enzyme-linked immunosorbent microplate analyzer «ImmunoChem - 2100» (HighTechnology, Inc., USA). The HOMA-IR index was used to quantify the severity of insulin resistance. The morphological state of the vascular endothelium was assessed by counting CDEC in the blood using phase-contrast microscopy. To diagnose the presence of thyroid pathology, ultrasound was performed using the ultrasound diagnostic system «LOGIQ-5». To assess TCIM in duplex scanning, the ultrasound diagnostic system «Philips IU» was used. Statistical analysis of the results was performed using a package of application programs for Windows. To determine differences, Student's t-test was used for dependent and independent samples. The frequency of signs in the groups was compared using the χ2 test. To determine the presence and nature of the relationship between various manifestations and pathogenetic factors of various processes, correlation analysis was performed using Pearson's test. The study was approved by the Bioethics Committee of the State Institution «L.T. Malaya National Therapy Institute» (№2 19 March 2024). Results According to the observation data in patients with combined course of MAFLD and SCH, significant changes in the vascular endothelium at the cellular level were observed, which was expressed in a significant increase in the index of CDEC in patients with MAFLD compared with the control group (10.8±1.6 cells/100 μL vs 7.5±1.2 cells/100 μL, p<0.01) and a significant increase in the desquamated fraction in patients with MAFLD in combination with SCH vs the MAFLD group - 15.4±2.2 cells/100 μL vs 10.8±1.6 cells/100 μL (p<0.01), respectively. Also, in patients with a combined course of MAFLD and SCH, there were significant changes in another marker that reflects vascular endothelial dysfunction - vascular endothelial growth factor VEGF-A: 610±112.27 pg/ml vs 428.24±74.28 pg/ml (p<0.01). The ED indices were significantly higher in the group of patients with MAFLD in combination with SCH with thyroid stimulating hormone (TSH) levels >10 mU/L compared with patients with TSH levels in the range of 4-10 mU/L: CDEC - 13.11±0.50 cells/100μL vs 8.83±1.08 cells/100μL, respectively (p=0.012); VEGF-A - 486.99±22.17 pg/mL vs 319.94±66.48 pg/mL, respectively (p=0.029) (Table 1). The Impact of Endothelial Dysfunction on the Course of Metabolically Associated Liver Disease © 2024 European Society of Medicine 3 Table 1: Comparison of ED indices depending on TSH levels in patients with MAFLD in combination with SCH ED rates in subgroups with TSH up to 4 mU/L and TSH in the range of 4-10 mU/L Indicators TSH subgroup ˂4 mU/L TSH subgroup 4-10 mU/L Significance, P CDEC, cells/100μL 8,83±1,08 13,11±0,50 p=0,012 VEGF-A, pg/mL 319,94±66,48 486,99±22,17 p=0,029 ED rates in subgroups with TSH 4-10 mU/L and TSH > 10 mU/L Indicators TSH subgroup 4-10 mU/L TSH subgroup>10 mU/L Significance, P CDEC, cells/100μL 13,11±0,50 16,50±7,50 p>0,05 VEGF-A, pg/mL 486,99±22,17 861,49±372,49 p>0,05 At the same time, the levels of CDEC and VEGF-A depended not only on TSH but also on age. Significant differences were obtained in patients aged >50 years and <50 years: CDEC - 9.88±0.52 cells/100μL vs 6.67±0.33 cells/100μL (p=0.006); VEGF-A - 398.94±25.74 pg/ml vs 97.08±19.39 pg/ml (p=0.001), i.e. there was a significant prevalence of CDEC and VEGF-A in the older age group (p<0.01), which may indicate the role of age in the development of vascular events in patients with MAFLD in combination with SCH (Table 2). Table 2: Comparison of ED rates depending on TSH of different ages in patients with MAFLD in combination with SCH Indicators TSH subgroup ˂4 mU/L TSH subgroup 4-10 mU/L Significance, P ED rates in subgroups aged <50 years CDEC, cells/100μL 6,67±0,33 9,88±0,52 p=0,006 VEGF-A, pg/mL 197,08±19,39 398,94±25,74 p=0,001 ED rates in subgroups aged >50 years CDEC, cells/100μL 11,00±1,00 13,58±0,54 p>0,05 VEGF-A, pg/mL 442,79±81,36 499,79±24,69 p>0,05 Significant changes in the level of CDEC were obtained depending on the TSH values in patients with MAFLD in combination with SCH, which indicates the influence of TSH levels on endothelial function in patients with MAFLD in combination with SH (Table 3). Table 3: Influence of TSH level on CDEC in patients with MAFLD in combination with SCH Indicators Sum of squares degree of freedom root mean square F-test Р CDEC between groups 217,791 2 108,895 4,463 0,013 within groups 3050,139 125 24,401 together 3267,930 127 Significant differences were found depending on hyperinsulinemia in the indicators reflecting ED. There was a 1.5-fold increase in the indexes of CDEC and VEGF-A in the group of patients with MAFLD in combination with SCH with an insulin level of >30 mU/L, which amounted to 17.67±2.13 cells/100μL vs 12.15±0.42 cells/100μL (p=0.003), respectively, and 718.33±106.63 pg/mL vs 449.32±19.16 pg/mL (p=0.036) (Table 4). Table 4: ED indicators depending on the level of hyperinsulinemia in patients with MAFLD in combination with SCH Indicators Insulin <30 mIU/L Insulin >30 mIU/L Significance, P CDEC, cells/100μL 12,15±0,42 17,67±2,13 Р=0,003 VEGF-A, pg/mL 449,32±19,16 718,33±106,63 Р=0,036 We analyzed the content of ED markers depending on the CVR in patients with MAFLD in combination with SCH. Comparison of moderate and low-risk groups showed significant differences in the indexes of CDEC, which amounted to 11.93±.541 cells/100μL vs 8.83±1.10 cells/100μL (p=0.060), as well as probable differences in VEGF-A - 422.82±10.01 pg/ml vs 319.94±66.47 (p=0.461). At the same time, a comparison of ED indices in the groups of high and moderate CVR demonstrated the presence of significant differences in both indicators: CDEC was 15.68±1.08 cells/100μl vs 11.93±.541 cells/100μl (p=0.004); VEGF-A - 646.44±58.11 pg/ml vs 422.82±10.01 pg/ml (p=0.001). A significant direct correlation was found in patients with low CVR between CDEC and gamma-glutamyl transpeptidase (GGT) levels - r=0.73 (p=0.099). In patients with moderate CVR, there were significant correlations of CDEC with the indicators of WC - r=0.40 (p=0.088); TG - r=0.43, (p=0.004) and CRP - r=0.30 (p=0.052). The data obtained are probably due to the fact that in the transition from the category of low to moderate CVR, its formation in patients with MAFLD in combination with SCH is influenced by a large number of factors. The analysis of correlations between patients with MAFLD in combination with SCH with high CVR revealed the presence of relationships not only with CDEC, but also with other metabolic parameters. A direct relationship between CDEC and VEGF-A was demonstrated - r=0.53 (p=0.011) and total cholesterol - r=0.48 (p=0.025). By comparing the groups of patients with MAFLD in combination with SCH and isolated MAFLD with VEGF-A The Impact of Endothelial Dysfunction on the Course of Metabolically Associated Liver Disease © 2024 European Society of Medicine 4 < 273 pg/ml did not reveal statistically significant differences in lipid and carbohydrate metabolism, liver function, TSH, CRP and TNF-α levels (p>0.01). According to the analysis of significant differences (p=0.001) obtained by comparing the groups of patients with MAFLD in combination with SCH and isolated MAFLD who had VEGF-A > 273 pg/ml, significant abnormalities in the studied parameters, including those reflecting the state of the liver, were obtained (Table 5). Table 5: Comparison of parameters in patients with MAFLD in combination with SCH with VEGF-A > 273 pg/ml and the group of isolated MAFLD Indicators MAFLD ± SCH, n=68 MAFLD, n=24 Mann– Whitney U-test Р Median Percentiles Median Percentiles 25 75 25 75 Age 60,00 52,00 67,00 54,00 47,00 58,50 33,50 0,001 TC 6,72 4,97 7,48 5,64 5,51 5,80 199,00 0,001 ТG 1,73 1,18 2,30 1,09 0,88 1,99 139,00 0,001 VLDL 0,74 0,527 1,02 0,42 0,34 0,50 164,50 0,001 HDL 1,07 0,87 1,37 1,54 1,43 1,60 185,00 0,001 LDL 3,73 3,18 4,33 2,72 2,53 2,85 195,00 0,001 AC 4,19 3,37 5,42 2,14 1,89 2,27 92,00 0,001 HbA1с 6,89 6,12 7,58 5,240 3,85 5,56 0,00 0,001 Glucose 6,50 5,31 8,11 5,30 5,09 5,54 20,00 0,001 Insulin 15,92 10,20 25,31 8,23 6,90 10,17 156,00 0,001 CDEC 11,00 8,00 14,00 8,50 6,50 9,00 204,50 0,001 VEGF-A 488,22 227,7 530,3 298,20 199,1 335,2 253,00 0,001 СRP 9,70 6,92 13,07 6,88 6,50 7,25 167,00 0,001 IMT 0,96 0,90 1,12 0,78 0,74 0,81 200,00 0,001 Discussion The results of our study indicate that patients with MAFLD in combination with SCH showed signs of ED. This is in line with the results of the study by Lekakis J. et al. in which it was found that ED is detected even within normal TSH values and worsens with increasing TSH levels, although, unlike our study, asymmetric dimethylarginine (ADMA) was used as a marker of ED and oxidized low-density lipoprotein (oxLDL)10. These data are consistent with the study by Ciccone M. et al. according to which autoimmune thyroiditis, which occurs more often in the setting of SCH, may be associated with arterial stiffness11. Moreover, Cho K. and Lee J. proved that the presence of antibodies to thyroid tissue correlated with arterial stiffness12. We have found an increase in VEGF-A in patients with MAFLD and SCH. This can be explained by the fact that VEGF-A is a profibrogenic factor13. This is consistent with the findings of Yang L. et al. according to which in a mouse model VEGF-A promoted liver fibrogenesis, as well as Shen H. et al. according to which the production of VEGF-A by hepatocytes promoted fibrosis14. At the same time, the role of VEGF-A in liver tissue repair and fibrosis disappearance remains unclear. The results of the study by Musso G. et al. indicate that in patients with MAFLD, along with a higher prevalence of traditional risk factors for CVE (obesity, DM, MS, etc.), new risk factors such as ED and thickening of the intima-media complex are found15. We observed an increase in the level of ED indicators in patients with MAFLD in combination with SCH, depending on the level of hyperinsulinemia. This is consistent with the publicly available evidence that MAFLD is closely associated with features of the metabolic syndrome, especially insulin resistance. According to Nasiri-Ansari N. et al., hyperinsulinemia, hyperglycemia and altered adipocytokine secretion can activate such harmful processes as inflammation, oxidative stress, endoplasmic reticulum stress and apoptosis, which leads to the development of MAFLD, demonstrating its multifactorial etiology16. Currently, there are two points of view regarding the relationship between ED and insulin resistance: some believe that ED is secondary to insulin resistance, i.e. it is a consequence of the factors that characterize insulin resistance, carbohydrate and lipid metabolism disorders; others believe that ED is not a consequence but a cause of insulin resistance17. According to Nasiri-Ansari N. et al., insulin resistance observed in MAFLD can lead to vascular endothelial dysfunction through various mechanisms, including an imbalance in NO production, which can lead to decreased blood flow. This in turn worsens insulin resistance, creating a vicious circle16. Significant changes in ED markers in patients with MAFLD in combination with SCH can be considered as one of the risk factors for the development of atherosclerosis and its complications, which form the background for the development of cardiovascular complications. This is confirmed by the presence of correlations between the ED markers - CDEC, VEGF-A, inflammatory markers - CRP, TNF-α and proatherogenic lipids, which indicates their involvement in the development and progression of early atherosclerotic vascular changes. This is consistent with the results of several studies that have shown that subclinical hypothyroidism is associated with atherosclerotic changes and, therefore, may increase the risk of cardiovascular disease5. Thus, the results of the study by Niknam N. et al. showed a link between SCH and ED in terms of impaired endothelium-dependent vasodilation18, and Shavdatuashvili T. reported that higher levels of serum TSH, cholesterol, and LDL are associated with greater ED. Similar findings were observed in a 10-year study in Taiwan, which included 115746 participants in the absence of thyroid disease, The Impact of Endothelial Dysfunction on the Course of Metabolically Associated Liver Disease © 2024 European Society of Medicine 5 in which SCH was defined as a serum TSH levels of 5.0- 19.96 μIU/mL with normal T4 levels, and which demonstrated that individuals with SCH have an increased risk of CVE mortality19. However, there are studies that have not found a clear association between hypothyroidism, increased risk of cardiovascular disease, and ED20. According to Lopez-Yus M. et al., pathogenetically, the above processes are associated with the activation of systemic inflammation21. Proinflammatory cytokines mediate intercellular interactions and maintain local inflammation in atherosclerotic plaque, activate endothelial cells and induce the expression of adhesion molecules, endothelial prothrombotic activity, have cardioprotective effects, increase myocardial ischemia and thus significantly change the clinical course of the disease, and are markers of poor prognosis and high CVR. However, the study by Raposo L. et al. showed that ED in patients with SCH causes a decrease in the availability of nitric oxide, which can be eliminated by levothyroxine therapy22, and according to Falkevall A. et al. the basis of ED in SCH is lipid infiltration3. Conclusion Patients with MAFLD in combination with SCH have elevated levels of CDEC and VEGF-A, which indicates the involvement of these factors in the mechanisms of ED in this category of patients. On the one hand, significant changes in the level of CDEC and VEGF-A depending on TSH and hyperinsulinemia in patients with MAFLD in combination with SCH were demonstrated, which indicates a link between TSH levels and indicators of ED and hyperinsulinemia. On the other hand, ED indices also have links with the degree of CVR. Patients with moderate and high CVR had an link between CDEC and metabolic parameters (TG, CRP, and TC). The association of CDEC and VEGF-A levels with age may indicate the influence of age in the development of CVE in patients with MAFLD in combination with SCH. The value of VEGF- A > 273 pg/mL as a marker of ED may predict the likelihood of developing CVE and can be used to assess the prediction of possible cardiometabolic risk. The Impact of Endothelial Dysfunction on the Course of Metabolically Associated Liver Disease © 2024 European Society of Medicine 6 References: 1. Cardinal H, Dieudé M, Hébert MJ. Endothelial Dysfunction in Kidney Transplantation. Front Immunol. 2018;9:1130. doi:10.3389/fimmu.2018.01130 2. Clyne AM. Endothelial response to glucose: dysfunction, metabolism, and transport. Biochem Soc Trans. 2021;49(1):313-325. doi:10.1042/BST20200611 3. Falkevall A, Mehlem A, Folestad E, et al. Inhibition of VEGF-A-B signaling prevents non-alcoholic fatty liver disease development by targeting lipolysis in the white adipose tissue. J Hepatol. 2023;78(5):901- 913. doi: 10.1016/j.jhep.2023.01.014. 4. Hammoutene A, Rautou P.E. Role of liver sinusoidal endothelial cells in non-alcoholic fatty liver disease. J Hepatol. 2019;70(6):1278-1291. doi:10.1016/j.jhep.2019.02.012 5. Saif A, Mousa S, Assem M, Tharwat N, Abdelhamid A. Endothelial dysfunction and the risk of atherosclerosis in overt and subclinical hypothyroidism. Endocr Connect. 2018;7(10):1075- 1080. doi:10.1530/EC-18-0194 6. Borges JP, Lopes GO, Verri V, et al. A novel effective method for the assessment of microvascular function in male patients with coronary artery disease: a pilot study using laser speckle contrast imaging. Braz J Med Biol Res. 2016;49(10):e5541. doi:10.1590/1414-431X20165541 7. Pasarín M, Abraldes JG, Liguori E, Kok B, La Mura V. Intrahepatic vascular changes in non-alcoholic fatty liver disease: Potential role of insulin-resistance and endothelial dysfunction. World J Gastroenterol. 2017;23(37):6777-6787. doi:10.3748/wjg.v23.i37.6777 8. Francque SM, Marchesini G, Kautz A, et al. Non- alcoholic fatty liver disease: A patient guideline. JHEP Rep. 2021;3(5):100322. doi: 10.1016/j.jhepr.2021.100322 9. Wiersinga WM. Guidance in Subclinical Hyperthyroidism and Subclinical Hypothyroidism: Are We Making Progress? Eur Thyroid J. 2015;4(3):143-148. doi:10.1159/000438909 10. Lekakis V, Papatheodoridis GV. Natural history of metabolic dysfunction-associated steatotic liver disease. Eur J Intern Med. 2024;122:3-10. doi:10.1016/j.ejim.2023.11.005 11. Ciccone MM, De Pergola G, Porcelli MT, et al. Increased carotid IMT in overweight and obese women affected by Hashimoto's thyroiditis: an adiposity and autoimmune linkage?. BMC Cardiovasc Disord. 2010;10:22. Published 2010 May 28. doi:10.1186/1471-2261-10-22 12. Cho KI, Lee JH. The impact of thyroid autoimmunity on arterial stiffness in postmenopausal patients with fibromyalgia. Int J Rheum Dis. 2017;20(12):1978- 1986. doi:10.1111/1756-185X.12257 13. Foglia B, Sutti S, Pedicini D, et al. Oncostatin M, a profibrogenic mediator overexpressed in non- alcoholic fatty liver disease, stimulates migration of hepatic myofibroblasts. Cells. 2019;9(1):28. doi:10.3390/cells9010028 14. Shen H, Yu H, Li QY, et al. Hepatocyte-derived VEGF-AA accelerates the progression of non- alcoholic fatty liver disease to hepatocellular carcinoma via activating hepatic stellate cells. Acta Pharmacol Sin. 2022;43(11):2917–2928. doi:10.1038/s41401-022-00907-5 15. Musso G, Gambino R, Cassader M. Non-alcoholic fatty liver disease from pathogenesis to management: an update. Obes Rev. 2010;11(6):430-445. doi:10.1111/j.1467- 789X.2009.00657.x 16. Nasiri-Ansari N, Androutsakos T, Flessa CM, et al. Endothelial Cell Dysfunction and Nonalcoholic Fatty Liver Disease (MAFLD): A Concise Review. Cells. 2022;11(16):2511. doi:10.3390/cells11162511 17. Meza CA, La Favor JD, Kim DH, Hickner RC. Endothelial Dysfunction: Is There a Hyperglycemia- Induced Imbalance of NOX and NOS? Int J Mol Sci. 2019;20(15):3775. doi:10.3390/ijms20153775 18. Niknam N, Khalili N, Khosravi E, Nourbakhsh M. Endothelial dysfunction in patients with subclinical hypothyroidism and the effects of treatment with levothyroxine. Adv Biomed Res. 2016;5:38. doi:10.4103/2277-9175.178783 19. Tseng FY, Lin WY, Lin CC, et al. Subclinical hypothyroidism is associated with increased risk for all-cause and cardiovascular mortality in adults. J Am Coll Cardiol. 2012;60(8):730-737. doi:10.1016/j.jacc.2012.03.047 20. Liu FH, Hwang JS, Kuo CF, Ko YS, Chen ST, Lin JD. Subclinical hypothyroidism and metabolic risk factors association: A health examination-based study in northern Taiwan. Biomed J. 2018;41(1):52-58. doi: 10.1016/j.bj.2018.02.002 21. Lopez-Yus M, Hörndler C, Borlan S, Bernal-Monterde V, Arbones-Mainar JM. Unraveling Adipose Tissue Dysfunction: Molecular Mechanisms, Novel Biomarkers, and Therapeutic Targets for Liver Fat Deposition. Cells. 2024;13(5):380. doi:10.3390/cells13050380 22. Raposo L, Martins S, Ferreira D, Guimarães JT, Santos AC. Metabolic Syndrome, Thyroid Function and Autoimmunity - The PORMETS Study. Endocr Metab Immune Disord Drug Targets. 2019;19(1):75- 83. doi: 10.2174/1871530318666180801125258