SIRT6 PROTECTS SMOOTH MUSCLE CELLS

FROM SENESCENCE AND REDUCES

ATHEROSCLEROSIS

Presented by: Naga Ayyachamy, Aisha Jamal,

Daniela Roman-Cabral, Emily Nguyen
https://doi.org/10.1161/circresaha.120.318353
 INTRODUCTION
VSMC’s play a huge role in artery plaques.
VASCULAR SMOOTH They transition from a contractile to a synthetic state and migrate
MUSCLE CELLS from the media to the intima, contributing to lesion formation.
(VSMC) Excess growth can cause the cells to stop working properly.
leading to problems like DNA damage and cell death.

A nuclear enzyme that plays a crucial role in

SIRT6 various cellular processes
Specific mechanisms by SIRT6 may protect
against atherosclerosis.

OVERALL Aimed to investigate SIRT6 expression,

function, and regulation in human VSMCs
GOAL and its impact on atherosclerosis in mice.
 METHODS
Human Atherosclerotic Plaques and Aorta:
hVSMCs were isolated --> aortas of patients
mVSMCs were isolated --> enzymatic digestion.
Treatments (hVSMCs) --> study effects
Cell Culture:
Plaque VSMCs were isolated and cultured --> aortic VSMCs.
mVSMCs cultured in DMEM/F12 medium (antibiotics)
Cloning, Transfections, and Lentiviral Infections:
Lentiviral vectors --> overexpress SIRT6 or mutant in hVSMCs.
Stable knockdown of SIRT6 in hVSMCs was achieved using GIPZ lentiviral
shRNA vectors
Lentivirus production --> transfecting HEK293FT cells
 METHODS PT 2
Quantitative Real-Time Polymerase Chain Reaction:
RNA extraction performed using kits.
cDNA synthesis & quantitative real-time PCR conducted using kits.
Western Blotting and Immunoprecipitation:
Whole cell protein lysates --> Western blotting, and immunoprecipitation
--> specific antibodies.
Antibodies --> detection, with β-actin or total histone 3 (loading controls)
 METHODS PT 3

Telomere Chromatin Immunoprecipitation:

Chromatin immunoprecipitation --> specific antibodies targeting
SIRT6.
DNA fragments --> PCR targeting telomere sequences.
Seahorse:
Oxygen consumption rate & extracellular acidification rate were
measured --> a Seahorse XF96e Flux Analyzer
 METHODS PT 4
Atherosclerosis Studies:
Calculations determined group sizes for atherosclerosis studies.
Transgenic mice (SIRT6) --> ApoE
Blood pressure, serum cytokines, lipid analysis, Oil Red O staining,
and SAbG staining (atherosclerosis progression & plaque burden)
Histology:
Oil Red O staining on thoracic aortas --> visualize lipid accumulation.
SAbG staining on brachiocephalic artery plaques --> detect
senescence-associated beta-galactosidase activity.
 METHODS PT 5
Statistical Analysis:
Animal randomization and exclusion criteria were applied
for atherosclerosis studies.
Statistical analyses were conducted to determine
significant differences between experimental groups
RESULTS

SIRT6 Protein Expression Is Reduced in VSMCs in

01
Human and Mouse Atherosclerosis and Upon
Replicative and Palmitate-Induced Senescence

02
The Ubiquitin Ligase CHIP Positively Regulates SIRT6
Stability in Human VSMCs, but Its Expression Is Reduced
in Plaque VSMCs
Figure 1. SIRT6 (Sirtuin 6) protein expression
is reduced in vascular smooth muscle cells
(VSMCs) in human atherosclerosis and
undergoing replicative and palmitate-
induced senescence
Figure 2. SIRT6 (Sirtuin 6) protein stability is
positively regulated by ubiquitin ligase CHIP
(C terminus of HSC70-interacting protein)
RESULTS

SIRT6 Delays VSMC Senescence

03

04
SIRT6 Preserves Telomere Integrity by Regulating
Telomere Histone Deacetylation and Preventing
Telomere DNA Damage

Figure 3. SIRT6 (Sirtuin 6) delays senescence,

binds to telomeres and regulates telomeric
H3K9 (histone H3 lysine 9) deacetylation and
DNA damage repair (DDR) signaling of human
vascular smooth muscle cells (hVSMCs)
RESULTS

SIRT6 Reduces Atherosclerotic Plaque Burden,

05
Inflammation and Senescence, and Preserves
Markers of Plaque Stability

06
Characterization of VSMC-Specific
SIRT6 or SIRT6H133Y Transgenic Mice
 Figure 6. Characterization of mice and mouse Figure 7. Vascular smooth muscle cell (VSMC)
vascular smooth muscle cells (VSMCs) expressing SIRT6 (Sirtuin 6) protects against development
human SIRT6 (Sirtuin 6) or SIRT6H133Y of atherosclerosis
 DISCUSSION
SIRT6 is an epigenetic regulator of genes linked to aging, inflammation and metabolism.

SIRT6 also plays a major role in DNA damage repair signaling, and its ability to repair
double-strand DNA breaks has been directly linked to longevity.

VSMCs, vascular smooth muscle cells, in human atherosclerotic plaques are characterized
by apoptosis, DNA damage, cell senescence, inflammation, and an altered energy
metabolism.

Given its distinct role in each of these processes, SIRT6 is an important target to study in
VSMCs and in atherosclerosis.
 CONCLUSION
SIRT6 protein expression is reduced in human and mouse plaque
VSMCs and is positively regulated by CHIP.

SIRT6, Sirtuin 6, regulates telomere maintenance

and VSMC lifespan and inhibits atherogenesis, all
dependent on its deacetylase activity.

Our data show that endogenous

SIRT6 deacetylase is an important
and unrecognized inhibitor of VSMC
senescence and atherosclerosis.
QUESTIONS?
 Reference Page
Grootaert MOJ;Finigan A;Figg NL;Uryga AK;Bennett MR; (n.d.). SIRT6 protects smooth muscle
cells from senescence and reduces atherosclerosis. Circulation research.
https://pubmed.ncbi.nlm.nih.gov/33353368/

U.S. National Library of Medicine. (n.d.). SIRT6 sirtuin 6 [homo sapiens (human)] - gene - NCBI.
National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/gene/51548