Endothelial cell apoptosis is an important means of regulating angiogenesis and may play a role in the pathogenesis of diseases involving the alveolar capillary septum. Small GTPases possess a C-terminal CAAX motif and undergo post-translational processing, culminating in carboxyl methylation of C-terminal cysteine by isoprenylcysteine carboxyl methyltransferase(ICMT). Based on Preliminary Results, we hypothesize that inhibition of ICMT decreases carboxyl methylation of Ras and RhoA GTPases, and causes disruption of Focal Adhesion Complexes (FAC), caspase activation, proteolysis of FAC protein components, and apoptosis. Preliminary Results indicate that ICMT inhibition changes expression and charge of GRP94, a chaperone protein that is important in the Unfolded Protein Response (UPR). Since malfunction of the UPR and endoplasmic reticulum (ER) stress response causes apoptosis, we hypothesize that decreased ICMT activity and resulting decreased Ras or RhoA activity alter GRP94 function, resulting in apoptosis due to malfunction of the UPR. 1. We will determine the effects of ICMT inhibition on FAC formation and anoikis. a. We will determine the effects of inhibitors of ICMT on methylation, localization, and activation of Ras and RhoA GTPase and on apoptosis using cultured pulmonary vascular endothelial cells, b. We will determine the role of Ras and RhoA GTPases in endothelial anoikis caused by ICMT inhibition by comparing the effects of over-expression of Ras or RhoA GTPase and downstream signaling molecules on FAC disruption and apoptosis caused by ICMT inhibition, c. We will determine the effects of ICMT inhibition on pulmonary vascular endothelial apoptosis in vivo. 2. We will determine the effect of ICMT inhibition on GRP94 and the role of the ER Stress Response in endothelial cell apoptosis caused by ICMT inhibition, a. We will determine the effects of ICMT inhibition on GRP94 expression, post-translational processing, and sub-cellular localization. b.We will determine the role of decreased small GTPase activity by assessing effects of Ras and RhoA over-expression on ICMT-induced changes in GRP94. c.We will determine the effects of ICMT inhibition on markers of the UPR/ER Stress Response. d.We will determine the effects of Ras and/or RhoA over-expression on the UPR/ER Stress Response. e.We will determine the effects of GRP94 over-expression on caspase activation, FAC disruption, proteolysis of FAC components, and anoikis induced by inhibition of ICMT. The work proposed in this application will ascertain the mechanism of GTPase methylation and the role of GRP94 in regulation of endothelial cell apoptosis. This work will improve understanding of lung diseases characterized by endothelial apoptosis, such as emphysema. In addition, understanding of apoptosis may provide clues to treatment of lung diseases dependent upon abnormal endothelial proliferation, such as Pulmonary Arterial Hypertension and cancer.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL064936-09
Application #
7589700
Study Section
Respiratory Integrative Biology and Translational Research Study Section (RIBT)
Program Officer
Moore, Timothy M
Project Start
2000-04-01
Project End
2012-03-31
Budget Start
2009-04-01
Budget End
2012-03-31
Support Year
9
Fiscal Year
2009
Total Cost
$308,778
Indirect Cost
Name
Brown University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
001785542
City
Providence
State
RI
Country
United States
Zip Code
02912
Sakhatskyy, Pavlo; Gabino Miranda, Gustavo Andres; Newton, Julie et al. (2014) Cigarette smoke-induced lung endothelial apoptosis and emphysema are associated with impairment of FAK and eIF2?. Microvasc Res 94:80-9
Lu, Qing; Sakhatskyy, Pavlo; Newton, Julie et al. (2013) Sustained adenosine exposure causes lung endothelial apoptosis: a possible contributor to cigarette smoke-induced endothelial apoptosis and lung injury. Am J Physiol Lung Cell Mol Physiol 304:L361-70
Grinnell, Katie; Duong, Huetran; Newton, Julie et al. (2012) Heterogeneity in apoptotic responses of microvascular endothelial cells to oxidative stress. J Cell Physiol 227:1899-910
Lu, Qing; Rounds, Sharon (2012) Focal adhesion kinase and endothelial cell apoptosis. Microvasc Res 83:56-63
Lu, Qing; Newton, Julie; Hsiao, Vivian et al. (2012) Sustained adenosine exposure causes lung endothelial barrier dysfunction via nucleoside transporter-mediated signaling. Am J Respir Cell Mol Biol 47:604-13
Jankowich, Matthew D; Rounds, Sharon I S (2012) Combined pulmonary fibrosis and emphysema syndrome: a review. Chest 141:222-231
Casserly, Brian; Mazer, Jeffrey M; Vang, Alexander et al. (2011) C-type natriuretic peptide does not attenuate the development of pulmonary hypertension caused by hypoxia and VEGF receptor blockade. Life Sci 89:460-6
Lu, Qing; Sakhatskyy, Pavlo; Grinnell, Katie et al. (2011) Cigarette smoke causes lung vascular barrier dysfunction via oxidative stress-mediated inhibition of RhoA and focal adhesion kinase. Am J Physiol Lung Cell Mol Physiol 301:L847-57
Jankowich, Matthew D; Rounds, Sharon (2010) Combined pulmonary fibrosis and emphysema alters physiology but has similar mortality to pulmonary fibrosis without emphysema. Lung 188:365-73
Lu, Qing; Harrington, Elizabeth O; Newton, Julie et al. (2010) Adenosine protected against pulmonary edema through transporter- and receptor A2-mediated endothelial barrier enhancement. Am J Physiol Lung Cell Mol Physiol 298:L755-67

Showing the most recent 10 out of 26 publications