11/9/2018

Vascular Odorant Receptors

AHW Fall Learning Community

William B. Campbell, Ph.D. David D. Gutterman, M.D. Departments of Pharmacology and Medicine (Cardiology)

November 5, 2018

Research Team Collaboration

William B. Campbell, PhD, MCW David G. Gutterman, MD, MCW Sang Kyu Park, PhD, MCW Anja Herrnreiter, MCW

David Zhang, M.D., Ph.D., MCW Mingyu Liang, M.B., Ph.D., MCW

Hiroaki Matsunami, PhD, Duke University Olivier Taboureau, PhD, Universite Paris Diderot

Changemaking

• Our long-term goal is to identify novel vascular mechanisms mediating vasodilation and inflammation that may lead to new therapies

• Our hypothesis is that specific odorant receptors are expressed in arteries and endogenous and synthetic ligands for these odorant receptors cause vasodilation and inhibit inflammation

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Odorant Receptors

1. G -coupled receptors

2. 387 Human odorant receptors (hORs)

3. 1035 Mouse odorant receptors (olfrs)

4. Detect odorants-volatile chemicals

5. Ligands are known for 30-40 ORs

6. Primarily expressed in olfactory epithelium

7. Some ORs ectopically expressed in testes, liver, heart and kidney

Odorant Receptor Signaling in Olfactory Epithelium

Mouse Aortic Vascular Odorant Receptors

A. PCR of mouse aortic ORs

A. olfr1155 B. olfr73 C. olfr376 D. olfr1020

136 nt 120 nt 141 nt 123 nt

B. Golf. C. Adenylyl Cyclase-3 A. RT-PCR A. RT-PCR Mouse Mouse Aorta Aorta

118 nt 139 nt

B. Western Blot B. Western Blot Mouse Mouse Mouse Mouse Aorta Mesentery Aorta Mesentery 77 kDa 90 kDa

46 kDa 55 kDa

OR5D16 is the human homolog of mouse olfr73. Mouse olfr73 is the eugenol OR.

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Summary of Preliminary Data in Mouse Aorta

Odorant (Ligand) Odorant Receptor (OR)

Golf

ATP AC-3

cAMP

Relaxation Other Responses

Endothelial, Vascular Smooth Muscle or other Vascular Cell (adipocytes, fibroblasts, macrophages)

RNA-Seq Analysis of Human and Mouse Vascular Cells Human Mouse 40 SMC 40 SMC EC EC 30 30

20 20

10 10 FPKM (mean) FPKM (mean)

0 0 2 3 4 8 0 1 60 W3 03 2 42 -AS1 2 103 1 1 131 1 56B1 2AE1 1 7E 2A Mouse olfrs Human ORs

12 SMC 12 SMC 10 EC 10 EC 8 8 6 6 4 4 FPKM (mean) FPKM FPKM (mean) FPKM 2 2 0 0 3 L 1 2 3 3 L 1 2 3 Y A AS A A A Y A AS A A A N N G G G N N G G G DC G G N N DC G G N N A C C CN A C C CN Human Mouse

RT-PCR for Odorant Receptors in Human and Mouse Vascular Cells

Human Mouse

OR56B1 OR2AE1 olfr1033 121212

300 bp 200 bp

1. Aortic Smooth Muscle Cells 2. Coronary Artery Endothelial Cells

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(Odora Cell Line) ☑

☑

☑ ☑

Western Blot for Odorant Receptors Transfected in Hana3A Cells

Rho Rho Lucy-Rho OR 123456 pCMV- 37 kDa OR

25 kDa

β-actin

1. Hana-3A control 2. +Rho-OR5D16 (33.4 kDa) 3. +Rho-olfr1033 (36.5 kDa) 4. +Lucy-Rho-OR5D16 (35.3 kDa) 5. +Lucy-Rho-olfr73 (38.7 kDa) 6. +Lucy-Rho-olfr1033 (38.4 kDa)

Ligand-induced Increase in Intracellular Calcium in HEK293 Cells Transfected with olfr1033

Β-Mercaptoethanol Sodium Hydrogen Sulfide Hydrate 500 500 mock +olfr1033 400 400 300 300 200 200 100 100 0 0 5 .0 .5 .0 5 .0 .5 .0 5. 5. ΔRFU veh. -6.0 - -5 -4Concentration-4 veh.[Log-6.0 M]- -5 -4 -4 Ethanethiol D-Cysteine 500 500 400 400 300 300 200 200 100 0 100 -100 0 -200 5 .0 .5 .0 h. .0 .5 .0 5. 4 veh. -6.0 - -5 -4 -4 ve -6.0 -5.5 -5 - -4 Concentration [Log M]

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Odorant-induced cAMP Production in Hana3A Cells Transfected with 1033

Hana3A + mock NECA Sodium Hydrogen Sulfide Hydrate Ethanethiol 60000 40000 D-Cysteine )) 0 30000

-T 40000

20 20000 20000 10000 0 0 h. .0 .5 .0 h. .0 .5 .0 e 7 e 6 v - -6.5 -6.0 -5 -5 v - -5.5 -5.0 -4 -4 Hana3A + olfr1033

40000 40000 30000 30000 (cAMP, Δ (T Luminescence (cAMP, 20000 20000 10000 10000 0 0 . h. .0 .5 .0 .5 .0 e 7 eh 6.0 v - -6.5 -6.0 -5 -5 v - -5 -5 -4.5 -4.0 Concentration [Log M] ODORactor Website for olfactory decoding predictions

Odorant-induced cAMP Production in Hana3A Cells Transfected with Rho- or Lucy-Rho-tagged Olfactory Receptor 1033 Hana3A + mock 40000 NECA 40000 Beta-Mercaptoethanol Eugenol 30000 30000 Sodium Hydrogen Sulfide Hydrate 20000 20000 Decanoic acid

10000 10000 )) 0 0 0 -T h. .5 .0 h. .0 .0 .5 .0 e 5 5 e 6 5.5 5 4 4 20 v - - v - - - - - Hana3A + Rho-olfr1033 40000 40000

30000 30000

20000 20000

10000 10000

0 0 h. .5 .0 h. .0 5 .0 .5 .0 e -5 -5 e -6 -5. -5 -4 -4 v Hana3A + Lucy-Rho-olfr1033v 40000 40000 (cAMP, Δ (T Luminescence (cAMP,

30000 30000

20000 20000

10000 10000

0 0 h. .5 .0 h. .0 .5 .0 .5 .0 e -5 -5 e -6 -5 -5 -4 -4 v Concentration [Logv M]

Conditioned Media (CM)-induced cAMP Production in Hana3A Cells Transfected with Olfactory Receptor 1033

Hana3A + mock NECA HAoSMC CM HUVEC CM 60000 10000 ))

0 8000

-T 40000 6000 20 4000 20000 2000 0 0 h. .0 .5 .0 % % e 7 0 3 5% v - -6.5 -6.0 -5 -5 10% 15% 20% Hana3A + olfr1033

60000 10000 8000

(cAMP, Δ (T Luminescence (cAMP, 40000 6000 4000 20000 2000 0 0 h. .0 .5 .0 % % e 7 0 3 5% v - -6.5 -6.0 -5 -5 10% 15% 20% Concentration [Log M]

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Membrane-spanning 4-Domain Family (A subfamily) of - MS4A Family

1. Located on necklace sensory neurons in olfactory epithelium 2. CD20 on B cells is a member of this family 3. Involved in chemosensory detection of fatty acids as oleic and arachidonic acids (Ms4a6d), ⍺-linolenic acid (Ms4a4b) and steroids as corticosterone (Ms4a4d) 4. Activation promotes calcium influx

Expression of Ms4a Proteins in Vascular Cells

600 SMC EC 400

200 FPKM (mean) FPKM

0 b c d a b d 4 4 4 6 6 6 a a a a a a 4 4 4 4 4 4 s s s s s s M M M M M M Mouse Ms4a

FPKM for MS4A family members was < 1 in human vascular cells

RT-PCR for Ms4a Genes in Murine Vascular Cells

Mouse

MS4a4b MS4a4d MS4a6d 1 2 1212

300 bp 200 bp

1. Aortic Smooth Muscle Cells 2. Coronary Artery Endothelial Cells

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Ligand-induced Increase in Intracellular Calcium in Mouse Coronary Artery Endothelial Cells (MCAEC) and Aortic Smooth Muscle Cells (MAoSMC) Arachidonic Acid Oleic Acid 1500 (Ms4a6d) 1500 (Ms4a6d) MCAEC MAoSMC 1000 1000

500 500

0 0 . . h .0 .5 .0 .5 .0 h .0 .5 .0 .5 .0

ΔRFU -7 -6 -6 -5 -5 -6 -5 -5 -4 -4 ve Concentration [Logve M] α-Linolenic Acid Corticosterone 1500 (Ms4a4b) 1500 (Ms4a4d)

1000 1000

500 500

0 0 . . h .0 .5 .0 .5 .0 h .0 .5 .0 .5 .0 ve -7 -6 -6 -5 -5 ve -7 -6 -6 -5 -5 Concentration [Log M]

Ligand-induced Increase in Intracellular Calcium in HEK293 Cells Transfected with Ms4a6d

+Mock 4000 Oleic Acid4000 11,12-EET +Ms4a6d 3000 3000

2000 2000

1000 1000

0 0 . . .5 .5 veh -6.0 -5.5 -5.0 -4 -4.0 veh -7.0 -6.5 -6.0 -5 -5.0

ΔRFU Concentration [Log M] Arachidonic Acid 20-HETE 4000 4000

3000 3000

2000 2000

1000 1000

0 0 . . .0 .5 veh -6.5 -6.0 -5.5 -5 -4.5 veh -7.0 -6.5 -6.0 -5 -5.0 Concentration [Log M]

Conclusions

1. Human and murine vascular endothelial cells and smooth muscle cells express specific odorant receptors.

2. These vascular cells also express odorant receptor signal proteins, Gαolf and adenylyl cyclase III but not CNG cation channels.

3. The odorant ligands for the vascular odorant receptors are unknown.

4. HeLa, Odora7, HEK293 and Hana3a cells were tested for expression of odorant receptors and Hana3a cells were superior.

5. A ligand screening methods was developed using odorant receptor overexpressing Hana3a cells.

6. No ligands for the major mouse vascular odorant receptor were found. Future studies will focus on identify ligands.

7. Vascular cells also express specific Ms4a proteins. These proteins mediate calcium entry into nasal epithelial cells in response to specific odorants. Ligands for the vascular Ms4a proteins did not increase intracellular calcium.

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Future Work

1. Screen human and mouse vascular odorant receptors for potential ligands 2. Test ligands for effects on vascular tone and inflammation

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Acknowledgements

David Gutterman, M.D.

Sang Kyu Park, Ph.D.

Anja Herrnreiter

Yuenmu Chen, Ph.D.

David Zhang, M.D., Ph.D.

Mingyu Liang, M.B., Ph.D.

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