Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells

Dennis J. Grab, Jose C. Garcia-Garcia, Olga V. Nikolskaia, Yuri V. Kim, Amanda Brown, Carlos A Pardo-Villamizar, Yongqing Zhang, Kevin G. Becker, Brenda A. Wilson, Ana Paula C De A Lima, Julio Scharfstein, J. Stephen Dumler

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Background: Using human brain microvascular endothelial cells (HBMECs) as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB) we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain). In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs) known as protease activated receptors (PARs) that might be implicated in calcium signaling by African trypanosomes. Methodology/Principal Findings: Using RNA interference (RNAi) we found that in vitro PAR-2 gene (F2RL1) expression in HBMEC monolayers could be reduced by over 95%. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39%-49%) and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of Gαq with Pasteurella multocida toxin (PMT). PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain) and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified. Conclusions/Significance: Together, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Gαq-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.

Original languageEnglish (US)
Title of host publicationPLoS Neglected Tropical Diseases
Volume3
Edition7
DOIs
StatePublished - Jul 2009

Fingerprint

Proteinase-Activated Receptors
Trypanosomiasis
Endothelial Cells
PAR-2 Receptor
Brain
Parasites
Blood-Brain Barrier
Peptide Hydrolases
Calcium Signaling
G-Protein-Coupled Receptors
Trypanosoma brucei rhodesiense
Cathepsin L
Cysteine Proteases
Central Nervous System Diseases
RNA Interference
Cell Communication
Permeability

ASJC Scopus subject areas

  • Infectious Diseases
  • Public Health, Environmental and Occupational Health
  • Pharmacology, Toxicology and Pharmaceutics(all)

Cite this

Grab, D. J., Garcia-Garcia, J. C., Nikolskaia, O. V., Kim, Y. V., Brown, A., Pardo-Villamizar, C. A., ... Dumler, J. S. (2009). Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells. In PLoS Neglected Tropical Diseases (7 ed., Vol. 3). [e479] https://doi.org/10.1371/journal.pntd.0000479

Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells. / Grab, Dennis J.; Garcia-Garcia, Jose C.; Nikolskaia, Olga V.; Kim, Yuri V.; Brown, Amanda; Pardo-Villamizar, Carlos A; Zhang, Yongqing; Becker, Kevin G.; Wilson, Brenda A.; De A Lima, Ana Paula C; Scharfstein, Julio; Dumler, J. Stephen.

PLoS Neglected Tropical Diseases. Vol. 3 7. ed. 2009. e479.

Research output: Chapter in Book/Report/Conference proceedingChapter

Grab, DJ, Garcia-Garcia, JC, Nikolskaia, OV, Kim, YV, Brown, A, Pardo-Villamizar, CA, Zhang, Y, Becker, KG, Wilson, BA, De A Lima, APC, Scharfstein, J & Dumler, JS 2009, Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells. in PLoS Neglected Tropical Diseases. 7 edn, vol. 3, e479. https://doi.org/10.1371/journal.pntd.0000479
Grab, Dennis J. ; Garcia-Garcia, Jose C. ; Nikolskaia, Olga V. ; Kim, Yuri V. ; Brown, Amanda ; Pardo-Villamizar, Carlos A ; Zhang, Yongqing ; Becker, Kevin G. ; Wilson, Brenda A. ; De A Lima, Ana Paula C ; Scharfstein, Julio ; Dumler, J. Stephen. / Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells. PLoS Neglected Tropical Diseases. Vol. 3 7. ed. 2009.
@inbook{5a40f822de65499aa84f9651a2982518,
title = "Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells",
abstract = "Background: Using human brain microvascular endothelial cells (HBMECs) as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB) we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain). In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs) known as protease activated receptors (PARs) that might be implicated in calcium signaling by African trypanosomes. Methodology/Principal Findings: Using RNA interference (RNAi) we found that in vitro PAR-2 gene (F2RL1) expression in HBMEC monolayers could be reduced by over 95{\%}. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39{\%}-49{\%}) and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of Gαq with Pasteurella multocida toxin (PMT). PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain) and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified. Conclusions/Significance: Together, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Gαq-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.",
author = "Grab, {Dennis J.} and Garcia-Garcia, {Jose C.} and Nikolskaia, {Olga V.} and Kim, {Yuri V.} and Amanda Brown and Pardo-Villamizar, {Carlos A} and Yongqing Zhang and Becker, {Kevin G.} and Wilson, {Brenda A.} and {De A Lima}, {Ana Paula C} and Julio Scharfstein and Dumler, {J. Stephen}",
year = "2009",
month = "7",
doi = "10.1371/journal.pntd.0000479",
language = "English (US)",
volume = "3",
booktitle = "PLoS Neglected Tropical Diseases",
edition = "7",

}

TY - CHAP

T1 - Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells

AU - Grab, Dennis J.

AU - Garcia-Garcia, Jose C.

AU - Nikolskaia, Olga V.

AU - Kim, Yuri V.

AU - Brown, Amanda

AU - Pardo-Villamizar, Carlos A

AU - Zhang, Yongqing

AU - Becker, Kevin G.

AU - Wilson, Brenda A.

AU - De A Lima, Ana Paula C

AU - Scharfstein, Julio

AU - Dumler, J. Stephen

PY - 2009/7

Y1 - 2009/7

N2 - Background: Using human brain microvascular endothelial cells (HBMECs) as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB) we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain). In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs) known as protease activated receptors (PARs) that might be implicated in calcium signaling by African trypanosomes. Methodology/Principal Findings: Using RNA interference (RNAi) we found that in vitro PAR-2 gene (F2RL1) expression in HBMEC monolayers could be reduced by over 95%. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39%-49%) and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of Gαq with Pasteurella multocida toxin (PMT). PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain) and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified. Conclusions/Significance: Together, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Gαq-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.

AB - Background: Using human brain microvascular endothelial cells (HBMECs) as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB) we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain). In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs) known as protease activated receptors (PARs) that might be implicated in calcium signaling by African trypanosomes. Methodology/Principal Findings: Using RNA interference (RNAi) we found that in vitro PAR-2 gene (F2RL1) expression in HBMEC monolayers could be reduced by over 95%. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39%-49%) and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of Gαq with Pasteurella multocida toxin (PMT). PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain) and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified. Conclusions/Significance: Together, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Gαq-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.

UR - http://www.scopus.com/inward/record.url?scp=70449601758&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=70449601758&partnerID=8YFLogxK

U2 - 10.1371/journal.pntd.0000479

DO - 10.1371/journal.pntd.0000479

M3 - Chapter

C2 - 19621073

AN - SCOPUS:70449601758

VL - 3

BT - PLoS Neglected Tropical Diseases

ER -