Generation and characterization of inducible knockout mice for adrenomedullin

Resumen

Adrenomedullin (AM) and proadrenouiedullin N-terminal 20 peptide (PAMP) are twO biologically active peptides synthesized by the same gene, adm. Human AM consists of '52 amino acids, has an interna!ring of 6 amino acids and a disulfide bond. Because of these two characteristics, this hormone shares sorne structural homology with caicitonin gene-related peptide (CGRP), as well as with amylin, so it was englobed within the CGRP su,perfamily. Using different immunoreactivity and in situ hybridization techniques, AM has been found to be synthesized in adult organisms in numerous tissues and cell types including adrenal medulla and cortex, kidney, lung, heart, pituitary, hypothalamus, skeletal muscle, the entire digestive system. and bone, among others. The presence of AM and PAMP has also been detected in almost all body secretions and fluids such as saliva, sweat, urine, breast milk, etc. IIi addition, AM has also been locaiized in fetal tissues and in the placenta. The wide tissue distribution and gene expression of this peptide is a reflection of the great variety of physiological functions that it is able to carry out. Under physiological conditions AM is a potent vasodilator and bronchodilator peptide, acts as an endogenous antimicrobial agent, regulates electrolyte balance, has diuretic and natriuretic effects, regulates the secretion of other hormones such as insulin, progesterone, or angiotensin II, modulates the inflammatory response, is a potent angiogenic agent, promotes tissue regeneration aft:er damage, acts through its receptors in the central Ílervous system to regulate nociceptive and stress responses, through distinct cellular signaling pathways it controls the organization of the cytoskeleton, regulates mitosis and promotes or inhibits apoptosis depending on the cell type. And these are just a few examples of what AM is able to do under normal conditions. Related with its implication in many physiological functions, AM is also directly involved in many pathological processes, some of them with a high social and economic impact for healtli systems. Among the diseases in which this hormone has a main role are cardiovascular diseases (myocardial infarction, heart failure or hypertension), cerebrovascular diseases, diabetes mellitus, Alzheimer's disease, chronic renal failure, glomerulonephritis, septic shock, cancer, and many others. The modulation of AM levels has a therapeutic value in all these pathologies and new therapies using this hormone or based on it are already being tested in di:fferent hospitals arolUld the world. Apart from the diseases mentioned above, there are other pathologies in which AM is not directly related to the etiology of the process, but its exogenous administration or its blockade (depending on the case) has demonstrated to have therapeutic effects. These include diseases with an immune component such as inflammatory bowel disease (IBD) or arthritis,hyperalgesia, resistance to analgesics, retinopathy, gastric ulcer, asthma, or atherosclerosis, among others. The value that this hormone has in biomedicine is evident and thus, having an adequate knowledge about its functions and the effects of increasing or decreasing its circulating levels in the body is becoming very relevant. Since this hormone is of vital importance during embryonic development, and the early abrogation of the adm gene causes the death of the embryos in the maternal uterus, until now it has only been possible to study the role of AM and / or PAMP using conditional knock-out (KO) animal models for specific tissues or cell types. Although these genetic models have allowed to obtain a broad knowledge of these two peptides, they do not seem to be entirely adequate. The major problem with these models is that AM is a ubiquitous and circulating hormone, which travels through the blood bound to complement factor H, therefore, although we can completely delete it from a certain organ, sorne amounts of AM produced in other parts of the body will reach the organ of study through the bloodstream. If we take into account the contradictory results that exist on the role of AM in several physiological processes, it seems evident that something is wrong with the models that currently exist to study this hormone. The main objective of this Thesis has been to generate the fust KO model for the adm gene in which it is deleted from the whole organism. And then, use that model to contrast the knowledge accumulated so far on the main physiological and pathological processes in which AM is involved. The second airo of this study was to evaluate the efficacy of new therapies based on the modulation of AM levels using synthetic small molecules, taking as reference the results obtained from the characterization of the KO model for the adm gene. lt is possible to generate complete adm KO mice using the Cre/loxP technology associated with a tet-On temporary recombination system, where expression of the Cre recombinase is induced after administration of doxycycline, a tetracycline­ like antibiotic. After a pilot study, it was found that it is necessary to administer a dose of 2 mg/mL doxycycline in drinking water, supplemented with 5% sucrose, for at least 14 days to achieve the complete deletion of the gene in all organs, including those with blood-barriers such as the brain or testicles. Induction of deletion in adult mice results in KO animals for AM that are viable, do not exhibit any major abnormality, and do not show significant phenotypic differences when compared with their wild-type (WT) litterrnates; with the exception of a significant weight gain in males, and occasional episodes of rapid weight gain and loss (usually more than 10 grams intwo days). The analysis of the animals at the time they reach the maximum weight peak confirmed that these episodes correspond to a generalized edema, a process known as anasarca. On the other hand, the constant weight gain experienced by AM deficient mice is partly due to an increase in bone density. According to the results obtained, this increase in bone mass seems to be explained by an indirect mechanism that implies the regulation of glycemia, ghrelin, and CGRP secretion. ' Following on these results, the efficacy of a small molecule that negatively modulates AM, 16311, was tested to treat osteoporosis. It was confirmed that the 16311 molecule is able to prevent bone loss in a mouse model of osteoporosis without causing any measurable toxicity, making this molecule a good candidate for a frrst-in-class osteoporosis preventing drug. On the other hand, the lack of AM leads to changes in the composition of intestinal microbiota. The absence of AM is associated with a decrease in the abundance of two beneficia! bacteria such as Lactobacillus gasseri and Bifidobacterium choerinum. In contrast, there was a significant increase in the abundance of two bacteria belonging to the Firmicutes filum (Clostridium scindens and Christensenella minuta) as well as the levels of B. vulgatus, all of them are known inductors of intestinal inflammation. In addition, in KO mice there is an alteration in the normal cornmunication between the colonic mucosa and the intestinal bacteria, due to a greater expression of Toll-like Receptor 4 (TLR4), the main bacterial receptor of the colon. This dysbiosis could explain that KO mice are extremely sensitive to the process of colitis caused by both the administration of 2.5% sodium dextran sulfate (DSS) in drinking water and by the rectal instillation of 3 mg of 2,4,6- trinitrobeozenesulphate (TNBS). The lack of endogenous AM correlates with an exacerbation of colitis symptoms at ali levels, suggesting that endogenous AM is able to delay the development of IBD and prevent the onset of serious clinical signs such as anorexia, severe diarrhea, dehydration, and rectal bleeding. In addition, AM collaborates to maintain basal levels of inflammatory cytokines and slightly modulates the levels of sorne adhesion molecules such as e-cadherin. AM also promotes the expression of certain molecules directly related to the•regeneration of colonic epithelium after damage. Surprisingly, tbe total Iack of AM seems to cause different effects in males and in females. Inthe case of acute colitis, the absence of AM causes more severe clinical signs and greater microscopic damage infemales, suggesting a more important protective role of this bormone inthis sex. According with its important protective action in the colon and the conflicting results published on the subject, we decided to study the involvement of AM and PAMP in the development and progression of colitis- associated coloreetal cancer by testing four small molecules to fmd out if they could be considered as an altemative treatment in this pathology. Modulators of PAMP had no effect on the development of colitis and cancer, indicating that this peptide .either has no major role incolon cancer, or that the treatment based on the modulators of PAMP is not very effective. However, AM inhibitors worsened the pbenotype whereas AM positive modulators reduced tumor burden, suggesting that AM may play a protective role during tbe progression phase of colon cancer, and that AM modulator therapy may represent a new treatment for colitis-associated colon cancer. In conclusion, our inducible KO for the adm gene constitutes a new model, more adequate and reliable than the previous genetic models, to study the functions of this hormone in different physiological and pathological processes. The information acquired with this genetic model may imply a new advance in the understanding of certain diseases in which AM is directly involved. Furthermore, if we consider that there is a mutation whose carriers have significantly Iower AM Ievels than the normal population., converting them into a natural AM "knock-down" , our KO model could shed light into the etíology of sorne important diseases, sucb as IBD, which still remains obscure. In addition, new therapies based on the modulation of AM levels through small molecules should be considered for the treatment of those pathologies in whicb this hormone is involved in sorne way, since accorcilng to our results they have demonstrated its efficacy in the treatment of osteoporosis and colitis-associated colorectal cancer.