In this webinar, Dr. Michael Sturek reviews features of macrovascular atherosclerosis and microvascular dysfunction that underlie ischemic events and the need for appropriate animal models for optimal translation. The unabated increase in cardiometabolic disease is a main reason why coronary heart disease remains the leading cause of death worldwide. Despite the effectiveness of lipid lowering therapy in treatment of coronary atherosclerosis, calcification remains a challenging clinical problem. Lipid lowering therapy is highly effective in treating atherosclerosis, but statins and exercise have been shown to increase coronary artery calcification. Dr. Sturek will review data showing a predominance of intracellular calcium (Ca2+) release in coronary smooth muscle cells that decreases remarkably in cells from metabolic syndrome swine and humans. The early event in coronary artery calcification, i.e. the extracellular deposit of Ca2+ crystals as hydroxyapatite, may be triggered by impaired lysosomal Ca2+ signaling. Selective, novel modulation of lysosomal Ca2+ stores may alter autophagy and matrix vesicle release to treat coronary atherosclerosis and calcification.

1. Copyright 2022. All Rights Reserved. Contact Presenter for Permission
Cardiometabolic Disease
Pathophysiology & Novel Therapies
for Atherosclerosis & Calcification
Michael Sturek, MS, PhD
Professor
Anatomy, Cell Biology, & Physiology
Indiana University

2. Partners and Sponsors

3. CARDIOMETABOLIC DISEASE
PATHOPHYSIOLOGY & NOVEL THERAPIES FOR
ATHEROSCLEROSIS & CALCIFICATION
Indiana University
School of Medicine
Purdue University
Dr. Michael Sturek, Professor
Anatomy, Cell Biology, & Physiology, Indiana University School of Medicine
Biomedical Engineering, Purdue University
Co-Owner and Chief Scientific Officer, CorVus Biomedical, LLC
msturek@iu.edu

4. OVERALL QUESTIONS
1. How does dysfunctional intracellular Ca2+
signaling in coronary smooth muscle lead to
coronary artery calcification?
2. How can we translate the science to cures?

5. 1.Obesity à metabolic syndrome (MetS) àcoronary
artery disease, macrovascular atherosclerosis
2.Image early athero, calcification unstable à stable
3.Specificity: macrovascular ≠ microvascular
stiffness, voltage-gated Ca channels, SR Ca
4.↑SR Ca à progress ↓SR Ca à calcif. Pig~human
5. Endolysosome Ca à
6. Conclusions, future directions

6. National Health and Nutrition Examination Survey (NHANES)
~ 66% US adults overweight or obese
METABOLIC
SYNDROME (MetS)
TYPE 2
DIABETES (T2D)
OBESITY

7. MetS (INSULIN RESISTANCE SYNDROME,
PRE-DIABETES) à TYPE 2 DIABETES
1. Central obesity
2. Insulin resistance
3. Glucose intolerance
4. Dyslipidemia (­LDL/HDL cholesterol)
5. Dyslipidemia (­triglycerides)
6. Hypertension
Eckel et al. The Lancet 2005 PMID 15836891
WHO, 1999; European Group, 1999; ATP III, 2001
Kahn et al. Diabetologia 2005 PMID 16079964
Kahn et al. Circulation 2006 PMID 16801475

8. RELATIVE
LEVEL
BLOOD
GLUCOSE
HUMAN
-5 0 5 10 15 20 25
-10 -5 0 5 10 15 20 25 30
NORMAL GLUCOSE TOLERANCE ® INSULIN RESISTANCE, MetS ® T2D
Postprandial
Fasting
Insulin resistance —
hepatic, peripheral
Plasma insulin
Beta cell function
-10
YEARS FROM DIABETES DIAGNOSIS
COMORBIDITIES, COMPLICATIONS
MetS
T2D
Sturek, Alloosh, Sellke. Annu. Rev. Biomed. Eng. 2020 PMID 32119784
30
PIG
-0.5 0 0.5 1 1.5 2 2.5
-1 3

9. “THRIFTY GENOTYPE” (OBESITY)
OSSABAW
ACTIVE
LEAN
INACTIVE
OBESE
~18 years development model at IUSM, Purdue
à CorVus Biomedical, LLC
Kreutz et al. Diabetes Metab Syndr Obes 2011 PMID 21660293

10. MORBID OBESITY à BARIATRIC SURGERY
Flum, NIH DK084324, U. of Washington
Ethicon Endo-Surgery
Sham et al. J. Diab. Res. 2014 PMID 25215301

11. EXERCISING OBESE OSSABAW PIG
Edwards et al. Cardiovasc. Res. 2010 PMID 19744946
4 d / wk, 30 min / d, 75% max HR, 7 wk

12. EXERCISING LEAN YUCATAN PIG
4 d / wk, 30 min / d, 75% max HR, 16 wk
Poole et al. Am. J. Physiol. Heart Circ. 2020 PMID 32196357

13. LARGE ANIMAL MODEL MetS
Yucatan Ossabaw Göttingen
1. Obesity No O,G>Yuc Yes
2. Insulin resistance No Yes Yes
3. Glucose intolerance No Yes Yes
4. Dyslipid. (­LDL/HDL) Yes Yes Yes
5. Dyslipidemia (­TG) No Yes Yes
6. Hypertension No Yes No
7. Fasting hyperglycemia No Yes, variable Yes, variable
Coronary artery disease Yes O>>G,Yuc Yes
Sturek et al. Annu. Rev. Biomed. Eng. 2020 PMID 32119784
àT2D
Neeb et al. Comp. Med. 2010 PMID 20819380

14. Cluzel et al. Am J Physiol Endocrinol Metab 2022 PMID 35224983
Systematic review of 6 livestock and 7 miniature
swine breeds
“The Ossabaw miniature swine in particular
represents a highly translatable model that
develops each of the core parameters of the
syndrome with many of the associated
secondary comorbidities.”
comorbidities.”
SWINE MODEL OF HUMAN MetS

15. SWINE MODEL OF HUMAN ISCHEMIC PRECONDITIONING
Short duration myocardial ischemia and
reperfusion attenuates subsequent myocardial
infarct size in response to longer duration
ischemia.
Preconditioning ~universally shown in swine, but
randomized trials in humans are largely failures.
Bell et al. Basic Res Cardiol 2022 https://doi.org/10.1007/s00395-022-00947-2

16. SWINE MODEL OF HUMAN ISCHEMIC PRECONDITIONING
No cardioprotection in Ossabaw, but robust cardioprotection (ischemic
preconditioning) in Göttingen minipigs
à Ossabaw ~ human?!
Kleinbongard et al. Cardiovasc Res PMID 35426434; Basic Res Cardiol 2022 (In press)
ischemia / reperfusion (I/R)
ischemic preconditioning (IPC) + I/R
infarct
size
[%
of
area
at
risk]
I/R IPC+I/R
*
0
20
40
60
80
Göttingen
I/R IPC+I/R
0
20
40
60
80
infarct
size
[%
of
area
at
risk]
Ossabaw
Inflammation JAK-STAT signaling
Increase pSTAT3 during reperfusion in
Göttingen, not Ossabaw
Genomics
clusters of differences in protein
coding genes between Ossabaw
and Göttingen minipigs
Mineralization

17. PA
A
RA
LA
PVAT
LV
RV
LAD
CORONARY ATHEROSCLEROSIS
Sturek. Curr Top Membr 2022
https://doi.org/10.1016/bs.ctm.2022.09.006

18. LV
CORONARY ATHEROSCLEROSIS – HISTOLOGY
Badin et al. Curr Top Membr 2022 https://doi.org/10.1016/bs.ctm.2022.09.007

19. 1.Obesity à metabolic syndrome (MetS) àcoronary
artery disease, macrovascular atherosclerosis
2.Image early athero, calcification unstable à stable
3.Specificity: macrovascular ≠ microvascular
stiffness, voltage-gated Ca channels, SR Ca
4.↑SR Ca à progress ↓SR Ca à calcif. Pig~human
5. Endolysosome Ca à
6. Conclusions, future directions

20. VASOSPASM DILATION
Ramchandani et al. 2012
Ca channel blocker

21. INTRAVASCULAR PHOTOACOUSTIC (IVPA, IVUS)
Cao et al. Transl Biophot 2020. https://doi.org/10.1002/tbio.202000004

22. CORONARY CALCIFICATION
Ca
Ca
Branch

23. Fluorodeoxy glucose uptake
RISK OF RUPTURE
INFLAMMATION
ACTIVE CALCIFICATION
STABLE CALCIUM
TURNOVER
Coronary Artery Disease Progression
Extracellular lipid,
Proliferation,
migration
Healthy Fatty streak Complicated
lesion
Foam cells
Cell proliferation
Preatheroma Atheroma Fibroatheroma
CARDIAC MORTALITY
Outward / positive remodeling Lipid core,
start lumen occlusion
Fibrous cap,
lipid core,
positive CAC,
IVUS
Hard collagen,
calcified plaque,
high CAC,
IVUS
NaF uptake
Calcium
on CT, IVUS
IVUS
McKenney-Drake et al. Eur. J. Nucl. Med. Mol. Imaging 2018. PMID 29978245

24. CFX
RC
IVUS
L
A
D
Late
Macrocalcif.
Stenosis
Early
Molecular calcif.
Intimal thickening
Microcalcif.
Molecular calcif.
Plaque burden
Intimal thickening
Focal
Diffuse
McKenney-Drake et al. Atherosclerosis 2016. PMID 27062403
ATHERO, CALCIFICATION
A
B
C
E,F

25. Adapted from Posner. Clin Orthop Relat Res. 1985.
McKenney-Drake et al. J Am Coll Cardiol
CV Imaging 2016 PMID 26189122
EARLY CALCIFICATION – 18F-NaF PET-CT

26. CFX
RC
IVUS
L
A
D
Late
Macrocalcif.
Stenosis
Early
Molecular calcif.
Intimal thickening
Microcalcif.
Molecular calcif.
Plaque burden
Intimal thickening
Focal
Diffuse
EARLY CALCIFICATION – 18F-NaF PET-CT
McKenney-Drake et al. J Am Coll Cardiol CV Imaging 2016 PMID 26189122
McKenney-Drake et al. Eur. J. Nucl. Med. Mol. Imaging 2018. PMID 29978245
No cardiac,
microvasc. Ca

27. CFX
RC
IVUS
L
A
D
Late
Macrocalcif.
Stenosis
Early
Molecular calcif.
Intimal thickening
Microcalcif.
Molecular calcif.
Plaque burden
Intimal thickening
Focal
Diffuse
EARLY CALCIFICATION IS UNSTABLE
Ehara et al. Circulation 2004 PMID 15557374
Spotty
Nodular
Average # Ca spots by
IVUS in acute myocardial
infarction 2.8-fold > than
in stable angina patients
(p<0.005); associated
with fibrofatty plaque.
Sugane et al. Atherosclerosis 2020 PMID 33243488
Major adverse cardiac events (~Target lesion revascularization)

28. CFX
RC
IVUS
L
A
D
Late
Macrocalcif.
Stenosis
Early
Molecular calcif.
Intimal thickening
Microcalcif.
Molecular calcif.
Plaque burden
Intimal thickening
Focal
Diffuse
LATE CALCIFICATION MIGHT BE STABLE, BUT …
Van Rosendael et al. JAMA Cardiol 2020
PMID 31968065
High density Ca plaque (“1K plaque”) associated with
lower risk for future acute coronary syndrome.
BUT …
High density
High density Ca plaque à increased stiffness à
increased pulse wave velocity
Microvascular
dysfunction

29. 1.Obesity à metabolic syndrome (MetS) àcoronary
artery disease, macrovascular atherosclerosis
2.Image early athero, calcification unstable à stable
3.Specificity: macrovascular ≠ microvascular
stiffness, voltage-gated Ca channels, SR Ca
4.↑SR Ca à progress ↓SR Ca à calcif. Pig~human
5. Endolysosome Ca à
6. Conclusions, future directions

30. MACROVASCULAR ≠ MICROVASCULAR
Macro
Conduit
Growth
Sturek. Curr Top Membr 2022 https://doi.org/10.1016/bs.ctm.2022.09.006
Micro
Flow
Contraction
Relaxation

31. MACROVASCULAR ≠ MICROVASCULAR
– STIFFNESS
Trask et al. J Appl Physiol 2012 PMID 22837170

32. MACROVASCULAR ≠ MICROVASCULAR
– VOLTAGE-GATED Ca CHANNEL DENSITY
Bowles et al. AJP: Heart Circ. Physiol. 1998. PMID 9843816

33. MACROVASCULAR ≠ MICROVASCULAR
– Ca SPARKS (SR Ca RELEASE), STOCs
Mokelke et al. AJP: Heart Circ. Physiol. 2005. PMID 15528227
Control Diabetic
Dyslipid.
Diabetic
Dyslipid. Ex.

34. 1.Obesity à metabolic syndrome (MetS) àcoronary
artery disease, macrovascular atherosclerosis
2.Image early athero, calcification unstable à stable
3.Specificity: macrovascular ≠ microvascular
stiffness, voltage-gated Ca channels, SR Ca
4.↑SR Ca à progress ↓SR Ca à calcif. Pig~human
5. Ca translocation
6. Endolysosome Ca à
7. Conclusions, future directions

35. Sarcoplasmic Reticulum
Ca2+ Ca2+
Voltage-gated Ca2+ channel (VGCC)
Transient receptor potential channel (TRP)
Plasma membrane Ca2+ ATPase (PMCA)
Na+-Ca2+ exchanger (NCX)
Ryanodine receptor (RyR)
Inositol 1,4,5-trisphosphate receptor (IP3R)
Sarcoplasmic reticulum Ca2+ ATPase (SERCA)
Ca2+ SIGNALING IN HEALTHY CORONARY SM

36. DESIGN TO TEST CSM Ca2+ SIGNALING IN MetS
Badin et al. Curr Top Membr 2022 https://doi.org/10.1016/bs.ctm.2022.09.007

37. Camera
Computer
Microscope
Xenon
Lamp
Superfusion
Chamber
Vacuum
Solution
340/380 510 nm
Hill et al. J Pharmacol Exp Ther 2000 PMID 11046079
IMAGING INTRACELLULAR Ca2+ WITH FURA-2

38. TYPICAL Ca2+ SIGNALING PROTOCOL

39. BIPHASIC Ca2+ DYSREGULATION IN MetS
McKenney-Drake et al. Atherosclerosis 2016. PMID 27062403
Badin et al. Curr Top Membr 2022 https://doi.org/10.1016/bs.ctm.2022.09.007

40. Ca2+ DYSREGULATION IN HUMAN vs. PIG CSM
Badin et al. J Cardiovasc Transl Res 2021 PMID 34286469

41. Ca2+ DYSREGULATION IN HUMAN vs. PIG CSM
Badin et al. J Cardiovasc Transl Res 2021 PMID 34286469

42. 1.Obesity à metabolic syndrome (MetS) àcoronary
artery disease, macrovascular atherosclerosis
2.Image early athero, calcification unstable à stable
3.Specificity: macrovascular ≠ microvascular
stiffness, voltage-gated Ca channels, SR Ca
4.↑SR Ca à progress ↓SR Ca à calcif. Pig~human
5. Endolysosome Ca à
6. Conclusions, future directions

43. Ca2+ DYSREGULATION MetS, ENDOLYSOSOME
Badin et al. Curr Top Membr 2022 https://doi.org/10.1016/bs.ctm.2022.09.007

44. ENDOLYSOSOMES IN HUMAN CORONARY SM
Badin et al. Curr Top Membr 2022 https://doi.org/10.1016/bs.ctm.2022.09.007

45. Badin et al. Curr Top Membr 2022 https://doi.org/10.1016/bs.ctm.2022.09.007
ENDOLYSOSOME Ca2+ SIGNALING IN PIG CSM

46. ENDOLYSOSOME Ca2+ SIGNALING IN PIG CSM
Minimal response to
TRPML1 agonist
MK6-83

47. ENDOLYSOSOMES IN CALCIFICATION
Bhat et al. Sci Rep 2020 PMID 32015399
CICR
T
r
a
n
s
f
e
r
?
Neointimal calcification Medial calcification
Minimal
Matrix vesicle exocytosis is
essential for vascular calcification

48. 1.Obesity à metabolic syndrome (MetS) àcoronary
artery disease, macrovascular atherosclerosis
2.Image early athero, calcification unstable à stable
3.Specificity: macrovascular ≠ microvascular
stiffness, voltage-gated Ca channels, SR Ca
4.↑SR Ca à progress ↓SR Ca à calcif. Pig~human
5. Endolysosome Ca à
6. Conclusions, future directions

49. OVERALL QUESTIONS
1. How does dysfunctional intracellular Ca2+
signaling in coronary smooth muscle lead to
coronary artery calcification?
ANSWER: Cross-talk endolysosome with SR
suggests possible translocation Ca2+ from SR
2. How can we translate the science to cures?
ANSWER: Use models that mimic human
coronary artery disease.

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