| dc.description.abstract | Wound healing is a crucial physiological process required for the healthy lifespan of an
organism. Dysregulation in the process of wound healing leads to the production of excess
extracellular matrix leading to fibrosis. Ageing is associated with increased cell death and
decreased regeneration in various organs. This results in fibrosis in multiple organs in aging
organisms, accompanied by changes in organ structure and loss in organ function. Aging is a
universal process and multiorgan fibrosis is a common factor in organ failure due to age,
ultimately leading to mortality. Sirtuins are a family of deacetylases that have been
implicated in the regulation of aging in various organisms. Although, role of some of the
sirtuins have been studied in fibrosis in an organ dependent manner, not much is understood
about the specific role of SIRT2 in multi-organ fibrosis. The present work is focused on
developing a simple and efficient in vitro model system to study fibrosis and using this model
system along with in vivo animal models to study the role of SIRT2 in multi-organ fibrosis.
Development of an in vitro model system to study fibrosis
One of the key components of a healthy heart is cardiac fibroblasts. In vitro studies on
cardiac fibrosis require access to cultured cardiac fibroblasts. The procedures used currently
for cultivating cardiac fibroblasts are laborious and call for specialized chemicals and
equipment. The isolation of primary cardiac fibroblasts is generally a byproduct of culturing
primary cardiomyocytes, as is evident by analyzing a few of the recently published and
widely cited papers. Very few protocols have been specially designed for the culture of
primary cardiac fibroblasts. The protocol described in the study helps in easily and effectively
isolating enriched cardiac fibroblasts with good viability of the cells. The yield and purity of
the cultured cardiac fibroblasts are influenced by several factors, including the quality of the
reagents used for the culture, the makeup of the digestion mixture employed, the conditions
maintained throughout the digestion of the heart tissue, and the age of the pups utilized for
culture. The current study outlines a comprehensive and streamlined procedure for isolating
and cultivating primary cardiac fibroblasts from neonatal murine pups. Using treatment with
transforming growth factor (TGF)β–1, we show that the cultured fibroblasts can be
transdifferentiated into myofibroblasts, simulating the alterations that occur in fibroblasts
during cardiac fibrosis in vivo. Studies on the numerous facets of cardiac fibrosis,
inflammation, fibroblast proliferation, and growth can be done using these cultured primary
cardiac fibroblast cells.
Investigating the role of SIRT2 in the development of tissue fibrosis
Fibrosis is an aging-associated disorder and fibrotic diseases are a major cause of multi-organ
failure with age. Multiple studies indicate that the TGF-β/SMAD signaling pathway is the
major regulator of fibrosis. However, endogenous regulators of the TGF-β/SMAD signaling
are not well understood. In our study, we identify SIRT2 as a critical regulator of TGF-
β/SMAD signaling, the transdifferentiation of fibroblasts to myofibroblasts, and resulting
fibrosis. Using in vivo and in vitro model systems, we demonstrate that SIRT2 deficiency
spontaneously transforms fibroblasts to myofibroblasts accompanied by increased expression
of α-SMA, FN1, and Col3a1 in multiple organs including heart, liver, kidney, and muscles.
On the other hand, overexpression of SIRT2 attenuates the induction of fibrosis by preventing
fibroblast transdifferentiation. Through RNA-seq analysis, we identify TGF-β/SMAD
signaling as a downstream target of SIRT2. We observe an increase in the activation of the
TGF-β/SMAD pathway in vivo, in the organs of SIRT2-/- mice including heart and liver, and
in vitro in SIRT2 deficient primary fibroblasts. Mechanistically, we show that SIRT2 binds to
and deacetylates SMAD3 to regulate its transcriptional activity. Using mass-spectrometric
analysis, we identify the residues Lys29 and Lys44 in the DNA binding domain of SMAD3
as the targets of SIRT2 for deacetylation. Interestingly, inhibition of TGF-β/SMAD signaling
rescues fibrosis in SIRT2-depleted fibroblasts. Remarkably, we observe that the levels and
activity of SIRT2 are downregulated in aged mice, accompanied by increased fibrosis and
hyperactive TGF-β/SMAD signaling. Similarly, SIRT2 levels and activity are downregulated
in failing human heart samples along with increased fibrosis. Overall, our study indicates that
SIRT2 plays a protective role against multi-organ fibrosis, and therefore may be a potential
therapeutic target for the treatment of fibrosis-related disorders | en_US |
