Vascular Aging – New Lessons From a Novel "Knock-In" Mouse Model
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This document summarizes a presentation about a novel "knock-in" mouse model that expresses a catalytically inactive form of tissue transglutaminase (TG2). The TG2-C277S mouse model was developed using CRISPR/Cas9 gene editing to mutate the active site cysteine residue of TG2. Biochemical analysis showed the mutant TG2 lacked crosslinking activity but retained GTP binding ability. Preliminary analysis found the TG2-C277S mice expressed normal levels of TG2 protein and its interaction with integrins and fibronectin were preserved. This novel mouse model will provide insights into TG2 functions in vascular aging independent of its enzymatic crosslinking activity.
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Vascular Aging – New Lessons From a Novel "Knock-In" Mouse Model
- 1. Lakshmi Santhanam, Ph.D. Associate Professor Anesthesiology and Critical Care Medicine Scintica Instrumentation Phone: +1 (519) 914 5495 sales@scintica.com Vascular Aging – New Lessons From a Novel “Knock-In” Mouse Model
- 2. Vascular Aging – New Lessons From a Novel “Knock-In” Mouse Model Lakshmi Santhanam, Ph.D. Department of Anesthesiology & CCM Johns Hopkins University School of Medicine
- 3. Objectives • Mechanical perspective of vascular structure and function • Understand the biophysical, biochemical, and cellular hallmarks of vascular stiffening • Tissue transglutaminase as a target in vascular aging • Determine TG2’s mechanisms of action in the vasculature • Understand cellular vs. matrix contributions to overall PWV in mouse models.
- 4. Vascular Stiffness – Mechanical Perspective The aorta as a non-linelarly viscoelastic material Systole - Expansion energy storage Diastole – elastic recoil unidirectional blood flow 𝑆𝑡𝑟𝑒𝑠𝑠; 𝜎 = 𝐹𝑜𝑟𝑐𝑒 𝐴𝑟𝑒𝑎 𝑆𝑡𝑟𝑎𝑖𝑛; 𝜆 = 𝐶ℎ𝑎𝑛𝑔𝑒 𝑖𝑛 𝑑𝑖𝑚𝑒𝑛𝑠𝑖𝑜𝑛 𝑂𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑑𝑖𝑚𝑒𝑛𝑠𝑖𝑜𝑛 𝑆𝑡𝑟𝑎𝑖𝑛 𝑟𝑎𝑡𝑒 = 𝑑𝜆 𝑑𝑡 Loading – increasing stress Unloading – decreasing stress Einc depends on Distending pressureWang et al (2016)
- 5. Load-bearing elements of the vascular wall Elastic part: ECM • Elastin fiber integrity • Elastin/Collagen ratio Elastin engages at low distension Collagen engages at high distention Physiologic range of pressure engages both Viscous part: Incompletely understood • Cell content • VSMC tone Shadwick (1995) J. Exp. Biol 202:3305–3313 Wolinsky and Glagov (1964) Circ Res. 14:400–413
- 6. Vascular Compliance – An Essential Element of Overall Cardiovascular Health Compliant Stiff Windkessel effect Pulsatile flow input Steady flow • Minimizes pump work needed • Avoids cyclic loading/unloading of end organs – protects from flow induced damage • Optimal transport across capillaries
- 7. Pulse Wave Velocity – an Index of in vivo Vascular Stiffness Image from indusinstruments.com 𝑃𝑊𝑉 = 𝐸𝑖𝑛𝑐ℎ 𝐷𝜌 Where Einc = incremental elastic modulus h = wall thickness D= lumen diameter 𝜌 = blood density Moens Korteweg equation: • Longitudinal studies • Minimal discomfort to mouse • No damage to vasculature can be used for ex vivo measures Doppler PWV: Non-invasive PWV measurement
- 8. 20 40 60 80 100 120 3.0 3.5 4.0 4.5 5.0 MAP (mmHg) PWV(m/s) SNP infusion PE infusion L Transducer 1 Transducer 2 Catheter Catheter Drug infusion (PE to 120 mm Hg; SNP to 50 mm Hg) ECG d2P/dt2 PWV = L/Δt Δt Proximal wave Distal wave Einc = Incremental elastic modulus h = wall thickness r = radius of vessel ρ = blood density Moens – Korteweg Equation: MAP dependence of Pulse Wave Velocity: Simultaneous PWV-MAP Measurement (Invasive) If HTN Is noted, PWV must be corrected for or normalized against MAP
- 9. Vascular Stiffness Increases with Aging
- 10. European Heart Journal, Volume 31, Issue 19, October 2010, Pages 2338–2350, EUROPEAN CONSORTIUM Pulse Wave Velocity Increases with Age and Hypertension N = 11,092 subjectsN = 1455 subjects; Normal PWV Numbers at the tops of bars represent the overall percentage distribution of all subtypes of untreated hypertension in that age group. IDH (SBP <140 mm Hg and DBP ≥90 mm Hg) – Pharmacologics available SDH (SBP ≥140 mm Hg and DBP ≥90 mm Hg) – IDH drugs + Lifestyle modifications ISH (SBP ≥140 mm Hg and DBP <90 mm Hg) – NO PHARMACOLOGICAL AGENTS! Franklin et al. Hypertension. 2001 Mar;37(3):869-74
- 11. PWV and Aging in Rodents Mice Rats Steppan et al, AJP Heart and Circ Physiol., 2019 Steppan et al, JAHA, 2014
- 12. PWV is a valuable predictor of cardiovascular risk Ben Shlomo et al (2014); JACC 63:636-636 17,635 subjects • aPWV predicts future cardiovascular events and mortality, even after accounting for other established cardiovascular risk factors. • Predictor of coronary heart disease and stroke after adjusting for traditional risk factors • Predictive value is stronger in younger versus older subjects – improved classification particularly for younger individuals at intermediate risk • Predictive value was not modified by hypertension, smoking, sex, diabetes, or kidney disease. • Improved the overall 10-year classification by 13%. Lifestyle Vs. GeneticsAge Diabetes Hypertension Obesity Co-morbidities Exercise Diet Stress
- 13. Endothelial Cells (EC) Smooth Muscle Cells (VSMC) Nitric Oxide (NO) Blood pressure (cGMP) Protein function (S-nitrosylation) Protein secretion (“secretome”) Gene expression ↑ Matrix Stiffness (↑collagen, ↑ elastin fragmentation, ↑remodeling) ↑ Cell stiffness Molecular Events Structural Changes ↑ Vascular Stiffness ↑ VSMC motility, proliferation, de-differentiation Cell behavior Vascular Function Molecular/ Cellular Hallmarks of Vascular Stiffening Aging Endothelial dysfunction: ↓ NO Bioavailability ↓ Barrier function Aberrant Cell-matrix interaction/mechano-transduction
- 14. Audience Poll
- 15. Tissue transglutaminase (TG2): A Matrix Remodeling Enzyme “Open” “Closed” GTP Ca2+ https://pdb101.rcsb.org/motm/209 b-sandwich Catalytic core Barrel 1 Barrel 2 Full length TG2(4 domains) Transamidation Reaction = Classical Transglutaminase Function • Member of transglutaminase family • 1 of 8 catalytically active members • >80% TG2 is cytosolic • <20% at cell membrane/ ECM • Secretion occurs via Golgi-independent pathway • Activated in wound healing, fibrosis Lysine Glutamine Isopeptide bond
- 16. Bakker et al, Circ Res, 2006 Eftekhari et al, J Vasc Res, 2007 Tissue Transglutaminase (TG2) Background So…Does TG2 play a role in regulating stiffness in the aorta? TG2 mediates resistance vessel remodeling Low flow induced remodeling PE induced vasoconstriction Purified TG2 + CysNO Lai TS et al, Biochemistry 40(16), 4904-4910 (2001)
- 17. TG2 secretion is NO dependent = TG2 Red = Intracellular Green = Extracellular NO? GSNO = NO donor Cyst = TG inhibitor L-NAME = eNOS inhibitor DTT = reducing agent Santhanam et al, Circ Res, 2010
- 18. Endothelial NO regulates TG2 secretion and activity Santhanam et al, Circ Res, 2009 Santhanam et al, Chem Biol, 2011 Jandu et al, Amino Acids, 2011 Co-culture approach HAEC = endothelial cells HASMC = smooth muscle cells L-NAME = NOS inhibitor HAEC = human aortic endothelial cells HASMC = human aortic SMC IMR-90 = human Fibroblast
- 19. Hypothesis Increased Central Vascular Stiffness TG2 NO SNO Inhibits cross-linking function eNOS Hypothesis ROSNO eNOS H2O2 O2-. AGINGDecreased NO bioavailability Increased ROS Disrupt cell signaling Impair vascular function NOX enzymes Mitochondrial enzymes
- 20. TG2-SNO decreases and transamidation activity increases with age Decellularize Homogenize tissue scaffold Determine Matrix TG2 by Western Blotting = TG2 = VSMC = EC L-NAME BASE E- YOUNG OLD O1 O2 Y1 Y2 Santhanam et al; Circ Res 2009 Jandu et al, Amino Acids, 2011 Cystamine - + - + Young Old BPA Total TG2 RAT HUMAN 33-49 Years 62-101 Years TG activity increases with age in human aorta
- 21. TG2 is the primary vascular TGase WT TG2-/-± L-NAME 20 mg/kg/day 4 weeks Santhanam et al, Circ Res, 2010 -L682.777 + L682.777 80% TGase activity in the aorta is TG2 derived (Green = TG2 activity; blue = nucleus) WT aorta becomes stiffer; but TG2 KO mice are protected in L-NAME induced endothelial dysfunction. L-NAME = NOS inhibitor Doppler PWV Pressure myography L-NAME = eNOS inhibitor Compliance = inverse of stiffness
- 22. 0.0 0.5 1.0 1.5 2.0 0 1000 2000 3000 Strain Stress,mN/mm2 TG2-/- WT ** A) B) TG2-/- mice: In vivo and ex vivo stiffness TG2 GAPDH WT TG2-/- Young mice (Decellularized matrix) Tensile testing Decellularize Steppan et al, JAHA; 2017 Feb 3;6(2):e004161. Armstrong et al Acta Cir Bras. 2018 Nov;33(11):991-999. C)
- 23. TG2-/- mouse aorta is more compliant in vitro but not in vivo D) E) 50 70 90 110 130 150 2.0 2.5 3.0 3.5 4.0 4.5 5.0 MAP (mmHg) PWV,m/s WT TG2-/- 0.0 0.5 1.0 1.5 2.0 0 500 1000 1500 2000 Strain Stress,mN/mm2 TG2-/- WT ** Intact vessels VSMCs compensate for more compliant matrix to maintain baseline physiological stiffness Steppan et al, JAHA; 2017 Feb 3;6(2):e004161.
- 24. b-sandwich Catalytic core Barrel 1 Barrel 2 Full length TG2 (4 domains) What is the mechanism? CYTOSOL EXTRACELLULAR MATRIX (Protein crosslinking – classic TGase reaction) CELL SURFACE (Cell adhesion – Fibronectin, Integrin, syndecan, binding) INTRACELLULAR/ CELL MEMBRANE (INNER) (GTPase/ GTP binding) = GTP = Ca2+ 1. Gundemir, Biochim. Biophys. Acta, 2012: 1823(2):406-19 2. Mehta K. Transglutaminases. 2015:215-228. 3. Kang et al, Biochem Biophys Res Commun. 2002 Apr 26;293(1):383-90 Ca2+ GTP Closed conformation Open conformation TG2: A molecular “Swiss-army knife” • GTPase PLC VSMC tone • TG2-adhesive function Strength/number of focal adhesions • Traction force • VSMC stiffness 4. Feng et al, Biochemistry. 1999 Feb 16;38(7):2224-32 5. Belkin and co-workers – TG2-integrin/Fn interaction 6. Griffin and co-workers – TG2- syndecan interaction
- 25. IP: Integrin β1 WB: TG2 WB: Integrin β1 WT eNOSKO WT eNOS KO 0 100 200 300 400 TG2/Integrin (NormalizedtoWT) * (n = 5) IgGcontrol Jung et al, AJP Heart, 2013 Steppan et al, JAHA, 2017 What is the Mechanism? Magnetic twisting cytometry 𝑆𝑡𝑖𝑓𝑓𝑛𝑒𝑠𝑠 ∝ 1 𝐷𝑖𝑠𝑝𝑙𝑎𝑐𝑒𝑚𝑒𝑛𝑡 1. Tighter TG2 mediated cell adhesion 2. Cell Stiffness WT TG2-/- 0.0 0.2 0.4 0.6 0.8 1.0 CellStiffness(Pa/nm) P=0.0019
- 26. Exogenous TG2 recovers passive stiffnessand vasoreactivity in TG2-/- mice independent of crosslinking function L682.777 = Specific TG2 inhibitor gpTG2 = purified guinea pig liver transglutaminase rTG2 = recombinant TG2
- 27. TGM2-C277S mouse: A novel knock-in mouse sgRNA Cas9 nickase HDR Oligo Fertilized oocytes Surrogate mother Screen to ID F0 (Founders) sgRNA Target C277 active site cysteine HDR Oligo sequence bearing desired mutation Santhanam Lab, unpublished results
- 28. TG2 GAPDH WT TG2-/- TG2C277SHOM TG2C277S-HET TGM2-WT TGM2-C277S TGM2-C277S mouse model development Sanger sequencing – identified 2 founders One founder yielded robust colony Mice are born with expected Mendelian-frequency with Het x Het mating strategy Santhanam Lab, unpublished results
- 29. WT TG2-/- HOM HET 0 50 100 150 TG2/GAPDH (%AvWT) TG2 GAPDH WT HOM TG2-/- HET WT TG2-/- HOM HET 0 50 100 150 TG2activity(FP) (%AvWT) ** *** * ** WT TG2-/- HOM HET 0 50 100 150 TG2activity(BPA) (%AvWT) * *** ** Ai) Aii) Aiii) Liver WT C) Fibronectin Integrin b1 IP:TG2 Integrin b1 IP: Int.b1 IB: Fibronectin Input TG2 IP:FN TG2 Integrin b1 TG2 Fibronectin TG2-/- HOM WT TG2-/- C277S 0 50 100 150 GTP-bound/TotalTG2 (%AvWT) B) GTP-bound TG2 Total TG2 WT TG2-/- HOM GAPDH Aiv) WT TGM2-C277S TG2KO WT TG2-/- HOM Biochemical characterization of TG2-C277S A) Crosslinking activity is lost B) GTP binding is preserved C) Fn/integrinb1 binding is preserved Santhanam Lab, unpublished results
- 30. Matrix remodeling and VSMCs are both shareholders in vascular stiffening • Aortic stiffness increases with age in WT mice • TG2-/- mice are protected from aging associated stiffening • TGM2-C277S mice Stiffer than TG2-/-, but marked protection vs. WT A) B) ECMTG2 Total TG2 GAPDH Y O Y O WT TGM2-C277S 3-6mo >15mo 3-6mo >15mo 3-6mo >15mo 0 2 4 6 8 PWV(m/s) WT C277S **** TG2-/- **** **** ** ** Santhanam Lab, unpublished results
- 31. Matrix and VSMC both contribute to passive stiffness **** **** **** **** Santhanam Lab, unpublished results
- 32. Targeting TG2 crosslinking function interrupts vascular stiffening but does not reverse it ± Cystamine (TG inhibitor) 40 mg/kg/day 3 months Santhanam et al, Circ Res., 2010 Cystamine = TG inhibitor (non-specific)
- 33. Audience Poll
- 34. Conclusions • TG2 is an important target in aging-associated vascular stiffening • TG2 is regulated in the vasculature by an NO-dependent mechanism • The novel TGM2-C277S knock-in mouse shows matrix and VSMC are both important shareholders in vascular stiffening • VSMC-Matrix interaction/biomechanical input from the matrix is an important determinant of VSMC tone/stiffness in vivo • Targeting individual functions of TG2 can yield distinct changes to overall stiffness by altering the VSMC stiffness and/or VSMC-ECM crosstalk • This model can be used to determine the specific role of TG2’s crosslinking function in other diseases.
- 35. Acknowledgements Dr. Dan Berkowitz Dr. Daniel Nyhan Dr. Steven An Dr. Marc Halushka Dr. Alberto Avolio Dr. Alexey Belkin Dr. Mark Butlin Dr. Jochen Steppan Dr. Sungmee Jung Dr. Dinani Armstrong Dr. Young Jun Oh Funding: NIH R01-HL105296 AHA BGIA2220181 ACCM StAAR awards Dr. Simran Jandu Dr. Alanah Webb James Chen Sean Melucci Ivy Wang Sandeep Jandu Kavitha Nandakumar Mentors/collaborators Postdoctoral Mentees/Fellows Pre-doc Mentees/ Technicians
- 36. To ask a question, click the Q&A Button, type your question and click send. Any questions that are not addressed during the live webinar will be answered following the event. Please indicate whom you want to address your question. Thank you for participating! Q&A SESSION: Lakshmi Santhanam, Ph.D. Associate Professor Anesthesiology and Critical Care Medicine