Metabolic syndrome is defined as a combination of obesity, elevated triglycerides, decreased high-density lipoproteins, hypertension and insulin resistance. It is at least partially caused by sedentary life style and unhealthy dietary habits and is a major risk factor for development and progression of cardiovascular disease and type 2 diabetes. Growing medical and socioeconomic impact of the metabolic syndrome warrants further active search for novel risk markers and therapeutic targets. Recent experimental and epidemiological studies have demonstrated the multiple roles of the endogenous methylarginines, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) as wells as the enzymes, which are involved in their catabolism, dimethyarginine dimethylaminohydrolases (DDAHs) and alanine:glyoxylate aminotransferase 2 (AGXT2) in the pathogenesis of metabolic syndrome and its complications. ADMA is thought to exhibit its pathological effects by inhibiting and uncoupling nitric oxide synthases (NOS), while SDMA can inhibit transport of L-arginine. DDAHs, namely DDAH1 and DDAH2, have been thought as the major enzymes metabolizing ADMA to citrulline, while being inactive towards SDMA. Experimental studies with upregulation of DDAH1 in animal models showed that lowering ADMA results in protection against endothelial dysfunction, atherosclerosis, ischemia/reperfusion injury and vascular remodeling, acceleration of angiogenesis in the settings of ischemia and improvement of insulin sensitivity. Unfortunately, all the attempts to upregulate DDAH1 using small drugs have not been successful. The data regarding the role of DDAH2 are contradictory, with some studies showing that it can metabolize ADMA under certain conditions and other studies questioning its enzymatic activity towards ADMA. AGXT2 is a mitochondrial aminotransferase, which can metabolize, among its other substrates, both ADMA and SDMA. It is a large protein with possible allosteric regulatory sites, suggesting that, in contrast to DDAH1, it could be upregulated by small molecules. The role of AGXT2 in different pathophysiological processes involving ADMA and SDMA is poorly understood. It has been recently discovered in the offspring cohort of the Framingham Heart Study participants that a composite compound, consisting of the products of metabolism of ADMA and SDMA by AGXT2 (asymmetric dimethylguanidino valeric acid (ADGV) and symmetric dimethylguanidino valeric acid (SDGV), correspondingly) is an independent biomarker of CT (computed tomography)-defined NAFLD (non-alcoholic fatty liver disease) and a predictor of future diabetes up to 12 years before disease, suggesting that AGXT2 may play a key role in development of metabolic disease and its progression. We and other have recently identified several other metabolically active substrates of AGXT2, such as a marker of cardiovascular and overall mortality homoarginine and a regulator of fatty acid oxidation and browning of adipose tissue beta-amino-isobutyric acid (BAIBA), which further supports the importance of AGXT2 in pathogenesis of cardiovascular and metabolic diseases. The data presented in the current thesis enable answering the two research aims: 1) Identification of the tissue and intracellular expression pattern of human AGXT2 and 2) Testing the hypothesis that ubiquitous transgenic overexpression of AGXT2 protects from ADMA-induced vascular damage in vivo. The first research aim provided a thorough characterization of AGXT2 expression in humans using multiple complimentary techniques and addressed the current discrepancy in the literature with previous demonstration of comparable levels of Agxt2 expression by RT-PCR and Western Blot in the kidneys and liver in mice, and previous reports on detection of predominant Agxt2 expression in the kidneys by Northern Blot and in-situ RNA-hybridization in rats. In our current study we analyzed AGXT2 expression in human tissues from a normal tissue bank by RT-PCR and further validated the results by Western Blot. We also performed immunohistochemical staining for AGXT2 and double fluorescent staining with an anti-AGXT2 antibody and a monoclonal anti-mitochondrial antibody. We saw the strongest expression of AGXT2 in the kidney and liver both on the mRNA and protein levels. Our immunohistochemistry stainings showed that AGXT2 is present in the convoluted tubule in the kidney and in the liver hepatocytes. The double fluorescent staining revealed the intracellular localization of AGXT2 in mitochondria. In the second research aim we investigated whether long-term upregulation of AGXT2 is safe and can protect from ADMA- mediated vascular damage in the setting of DDAH1 deficiency, which is commonly observed in cardiovascular pathologies. We generated AGXT2 transgenic (TG) mice with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of ADGV, the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling. The work, included into this thesis demonstrates that both hepatocytes and kidney tubular epithelial cells are the major sources of AGXT2 in humans, where the enzyme is localized in mitochondria. The expression of AGXT2 in the liver is consistent with the proposed role of AGXT2 in development and progression of NAFLD and is consistent with our previous discovery of hepatocyte nuclear factor 4 alpha (HNF4α) as the major regulator of Agxt2 expression in the mouse liver. Chronic upregulation of AGXT2 in mice lowered systemic ADMA levels without any obvious effects on viability, development, growth and fertility, suggesting potential safety of this ADMA-lowering approach. Overexpression of AGXT2 protected from ADMA-induced vascular damage in the highly clinically relevant settings of DDAH1 deficiency, suggesting that the observed vascular damage was indeed caused by ADMA itself, rather than by some ADMA-independent effects of DDAH1 deficiency. The observed protective effects of AGXT2 upregulation are especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful. The current study, therefore, provides the basis for the future screens to identify small molecules, which would upregulate AGXT2 activity.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:86452 |
| Date | 12 July 2023 |
| Creators | Jarzebska, Natalia |
| Contributors | Weiss, Norbert, Schwedhelm, Edzard, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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