This document discusses a study investigating whether metformin has anti-fibrotic effects in aldosterone (Aldo) and salt-treated mouse hearts. Cardiac fibroblasts were isolated to determine the molecular mechanisms of metformin's effects on Aldo. Mice were divided into four groups: control, salt only, salt+Aldo, salt+Aldo+metformin. Metformin was found to reduce cardiac hypertrophy, fibrosis, and oxidative stress induced by Aldo in vivo. In vitro, metformin attenuated Aldo-induced fibroblast proliferation, migration, and expression of proteins involved in extracellular matrix formation through AMPK activation and reduction of TRAF3IP2 and PPM1A, which are involved in oxidative stress responses. Several

2. CIELO CAROLINA CASAS CRUZ
PAULINA CAMPO GIRÓN

3. INTRODUCTION
METFORMIN
Is widely used in the treatment of type II
diabetes. Reduces the incidence of
cardiovascular events and all-cause
mortality. It is a potent activator of
AMPK, and exerts antioxidant and anti-
inflammatory effects, independent of its
anti-hyperglycemic properties.

4. INTRODUCTION
ALDOSTERONE
Aldosterone is the principal mineralocorticoid
which is synthesized from cholesterol in the
adrenal glomerulosa, the secretion is controlled
mainly by angiotensin II and potassium.

5. INTRODUCTION
ALDOSTERONE
The most important actions of aldosterone are increase renal tubular
reabsorption of sodium and secretion of potassium. Also aldosterone can
induce the vascular muscle migration and proliferation.

6. INTRODUCTION
FIBROBLASTS
It is responsible for the synthesis and maintenance of the extracellular
matrix. Its function is the synthesis and maintenance of the extracellular
matrix, essential for maintaining the integrity of connective tissue.

7. INTRODUCTION
CARDIAC FROBROBLAST
Are key elements in the development of cardiac remodeling after acute myocardial
infarction. A controlled growth of fibroblasts and myofibroblasts is important for
proper healing and maintenance of cardiac function.

8. INTRODUCTION
IN VITRO
TRAF3 Interacting Protein 2 is an oxidative stress-responsive
cytoplasmic adapter molecule and an upstream regulator of multiple
nuclear transcription factor. Silencing TRAF3IP2 attenuates Aldo-
induced cardiomyocyte hypertrophy and fibroblast proliferation and
migration.

9. INTRODUCTION
IN VIVO
Is the development of excess fibrous connective tissue in an organ or tissue
following a reparative process. Fibrosis is caused by a chronic inflammatory
process, which triggers an increase in the production and extracellular
matrix deposition.
Four groups:
• 1: control
• 2: Salt
• 3: Salt + Aldo
• 4: Salt + Aldo + Metformin

10. INTRODUCTION
Aldosterone
Cardiac fibroblast
MigrationProliferation
In vitro
In vivo
Myocardial hypertrophy
and fibrosis
Gene
deletion
Gene
silencing

11. OBJETIVE
The overall goals of this study were to investigate whether
metformin exerts anti-fibrotic effects in aldosterone (Aldo) +
salt-treated wild type mouse hearts, and determine the
underlying molecular mechanisms in isolated adult cardiac
fibroblasts (CF).

12. MATERIALS AND METHODS
ISOLATION OF FIBROBLAST
• Células objeto de estudio:
Para determinar los mecanismos moleculares fundamentales de la
acción de la metformina sobre la aldosterona en los CF.

13. MATERIALS AND METHODS
LENTIVIRAL INFECTION
Fueron infectados con adenovirus que inhibe la AMPK y
así poder observar al tratarlos con la metformina que
es activador del AMPK los efectos que tenia, y si de esta
forma se reducían los efectos de la Aldo.

14. MATERIALS AND METHODS
Induce una cascada de eventos en las células
en respuesta a los constantes cambios de
energía de estas. Bajo isquemia o hipoxia, la
activación de la AMPK da la capacidad de
activar la actividad de PFK-2 en el tejido
cardíaco y los macrófagos en respuesta a los
condiciones isquémicas permitiendo que esas
células sigan teniendo una fuente de ATP a
través de la glucólisis anaerobia.
AMPK

15. MATERIALS AND METHODS
PPM1A
Es reguladora negativa de las vías de respuesta al estrés
celular. Induce los procesos inflamatorios.
Es un intermediario clave en la inducción de la respuesta
inflamatoria en los tejidos cardiacos.
Es medido para evidenciar la acción anti-oxidante de la metformina
H202
TRAF3IP2

16. MATERIALS AND METHODS
INMUNOBLOTTING
El análisis por inmunoblotting se
realizó usando 3 poblaciones de
fibroblastos independientes aislados
donde se evaluaron los factores
inducidos por la Aldo.
Separación electroforetica basado en
las cargas negativas de las proteínas,
después de migración, se separa la
electroforesis gracias a un soporte
solido el cual interacciones con los
antígenos específicos para identificar
esas proteínas

17. MATERIALS AND METHODS
ELISA
Análisis de citoquinas proinflamatorias como IL-6, IL 7, IL 8
se realizaron con prueba de ELISA.
Un anticuerpo o antígeno se fija a la fase solida donde interacciona con los
anticuerpos o antígenos de la muestra, formación de una conjugados enzima-
anticuerpo unido a sustratos donde hay cambios en color.

18. MATERIALS AND METHODS
IN VITRO
Activación de αSMA, Myosin-11,
vimentin, periostin, proliferación,
migración y muerte por medio de
caspasa 3 y DNA fragmentado
citoplasmatico.
El colágeno fue medido por medio
de un ensayo de hidroxiprolina.

19. MATERIALS AND METHODS
IN VIVO
Ensayo en 4 grupos algunos tratados con sal únicamente, Aldo + sal, Aldo + sal +
metformina y un grupo control.
Hipertrofia cardiaca
• Relación del peso del corazón con el peso corporal.
• El área de sección transversal de los cardiomiocitos
por medio de aglutinina de germen de trigo (WGA).
• La expresión de ANF.
El análisis de colágeno como indicador de fibrosis cardiaca
Estrés oxidativo por medio de peroxidación lipidica.

20. RESULTS

21. RESULTS

22. RESULTS

23. RESULTS

24. RESULTS

25. RESULTS

26. RESULTS

27. DISCUSSION
Y. Saisho
Activation of AMPK exerts
multiple anti-fibrotic effects in
different organs, including the
heart. For example, AMPK
activators inhibit oxidative
stress [1]
N.K. Somanna, M.
Yariswamy, J.M. Garagliano,
U. Siebenlist, S. Mummidi,
A.J. Valente
TRAF3IP2 is an oxidative
stress- responsive
cytoplasmic adapter
molecule and an activator of
multiple transcription
factors, including NF-κB,
AP-1 and C/EBPβ in various
cardiac-constituent cells,
including CF [8,31].

28. DISCUSSION
N.G. Frangogiannis In an injured heart,
activated cardiac fibroblasts
(CF) contribute to increased
synthesis, cross-linking,
and deposition of collagens,
and contractile dysfunction
[10,12,22].
N.K Somanna, M.Yariswamy It is posible that metformin
might have inhibited their
expression by reducing
oxidative stress and
blocking their transcription,
as Aldo is known to induce
their expression [8]

29. CONCLUSIONS
• Metformin has cardioprotective effects
• AMPK is a pathway of great importance that
allows anti-fibrotic effects by metformin

30. CONCLUSIONS
• Several lab tests are based on the physicochemical properties of
proteins, such as loading, molecular weight or affinity for specific
markers as antigens or antibodies which express different ways.
• Genes can be manipulated in vivo and in vitro by some methods
as silencing in vivo studies or in in vitro delation, in order to
inhibit or induce its expression.

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