DDAH‐1 Regulates NO‐Mediated Apoptosis and Cell Proliferation in Human Pulmonary Microvascular Endothelial Cells
Authors: Trittmann Jennifer K. , Almazroue Hanadi , Nelin Leif D. 
Published: June 2021

BACKGROUND

Nitric oxide (NO) is an endogenous pulmonary vasodilator produced by endothelial NO synthase (eNOS), and decreased eNOS‐derived NO underlies many types of pulmonary hypertension (PH). Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of eNOS, is elevated in the plasma of patients with bronchopulmonary dysplasia (BPD) prior to the onset of PH. ADMA is hydrolyzed to citrulline by dimethylarginine dimethylaminohydrolase (DDAH), and our group has previously found that a DDAH1 SNP is differentially expressed in BPD‐PH patients compared to BPD control patients.

OBJECTIVE

To test the hypothesis that siRNA‐mediated knockdown of DDAH‐1 will attenuate NO‐mediated apoptosis in human pulmonary microvascular endothelial cells (hPMVEC).

DESIGN/METHODS

hPMVEC were transfected with siRNA against DDAH‐1 or scramble control for 24h, washed and fresh media placed on them and incubated in 21% O2/5% CO2 for 24h. Protein was analyzed by western blot for DDAH‐1, eNOS, arginase I, arginase II, cleaved caspase‐3, cleaved caspase‐8, and cleaved caspase‐9, and loading controls (β‐actin, total cleaved caspase‐3, total cleaved caspase‐8, and total cleaved caspase‐9). Cell media was assayed for nitrite concentration using chemiluminescence. In a separate set of experiments after transfection, hPMVECs were washed, trypsinized, and viable cells were counted after trypan blue exclusion. In a third set of studies after transfection, hPMVECs were treated with 0.1μM of the NO donor DETA NONOate for 24h or 0.1μM DETA NONOate and 100μM of the caspase‐3 inhibitor Z‐DEVD‐FMK for 24h, and viable cell numbers were counted after trypan blue exclusion.

RESULTS

There was no difference in protein levels of eNOS, arginase II, or cleaved caspase‐9 between hPMVEC treated with DDAH‐1 siRNA and scramble controls. However, hPMVEC treated with DDAH‐1 siRNA had less DDAH‐1, arginase I, cleaved caspase‐3, and cleaved caspase‐8 protein levels than did scramble controls (p<0.05). hPMVEC treated with DDAH‐1 siRNA had lower media nitrite concentration than did scramble controls (p<0.05). hPMVEC treated with DDAH‐1 siRNA had greater viable cell numbers than did scramble controls (p<0.05). hPMVEC treated with DDAH‐1 siRNA and DETA NONOate had viable cell numbers that were less than DDAH‐1 siRNA alone (p<0.05) and similar to that seen in scramble control hPMVECs. When hPMVEC were treated with DDAH‐1 siRNA, DETA NONOate, and Z‐DEVD‐FMK, viable cell numbers were increased three fold from scramble control (p<0.05).

CONCLUSIONS

We found that knockdown of DDAH‐1 with siRNA in hPMVEC resulted in less NO production, less caspase‐3 activation and more viable cells. These data suggest that greater levels of ADMA are associated with lower levels of NO‐mediated apoptosis in hPMVEC. We speculate that enhancement of DDAH‐1 activity may be a viable therapeutic target to decrease pulmonary vascular wall proliferation and thereby ameliorate the vascular remodeling that occurs in PH.

Support or Funding Information

This research was funded by the National Heart, Lung, and Blood Institute (Grant K08HL129080), and The Research Institute, Nationwide Children's Hospital.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.