miR-21/DDAH1 pathway regulates pulmonary vascular responses to hypoxia

Abstract

The NOS (nitric oxide synthase) inhibitor ADMA (asymmetric dimethylarginine) contributes to the pathogenesis of pulmonary hypertension. Reduced levels of the enzymes metabolizing ADMA, dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2) and increased levels of miR-21 are linked to disease pathology, but the mechanisms are not understood. In the present study we assessed the potential role of miR-21 in the regulation of hypoxia-induced changes in ADMA metabolism in vitro and in vivo. Hypoxia inhibited DDAH1 and DDAH2 expression and increased ADMA levels in cultured human pulmonary endothelial cells. In contrast, in human pulmonary smooth muscle cells, only DDAH2 was reduced whereas ADMA levels remained unchanged. Endothelium-specific down-regulation of DDAH1 by miR-21 in hypoxia induced endothelial dysfunction and was prevented by overexpression of DDAH1 and miR-21 blockade. DDAH1, but not DDAH2, mRNA levels were reduced, whereas miR-21 levels were elevated in lung tissues from patients with pulmonary arterial hypertension and mice with pulmonary hypertension exposed to 2 weeks of hypoxia. Hypoxic mice treated with miR-21 inhibitors and DDAH1 transgenic mice showed elevated lung DDAH1, increased cGMP levels and attenuated pulmonary hypertension. Regulation of DDAH1 by miR-21 plays a role in the development of hypoxia-induced pulmonary hypertension and may be of broader significance in pulmonary hypertension.

Endothelial Dimethylarginine Dimethylaminohydrolase 1 Is an Important Regulator of Angiogenesis but Does Not Regulate Vascular Reactivity or Hemodynamic Homeostasis

“…These observations seem at odds with previous reports that have identified DDAH1 expression in a wide range of nonendothelial cell types, including vascular smooth muscle, renal, and neuronal cells. [18][19][20][21][22][23][24] To resolve this apparent discrepancy, we have independently generated an endothelium-specific DDAH1 −/− mouse. Using this model, we demonstrate endothelium-restricted expression of Cre activity that entirely removes DDAH1 protein in primary isolated endothelial cells.…”

Discerning functional hierarchies of microRNAs in pulmonary hypertension

“…Our first mathematical iteration identified miR-21 as controlling multiple PH target genes and pathways, including BMPR2 expression and hypoxic reprogramming, which we and others later validated in vitro and in PH rodents in vivo (Tables 1 and 2). Such experimental interrogation also revealed that actions of miR-21 in PH (28,(34)(35)(36) and elsewhere (22,83) are context specific and possibly cell specific. Thus, miR-21, while pleiotropic in its effects in PH, also carries a dynamic and shifting repertoire of actions that makes its functions as a consistent regulator of PH more challenging to define.…”

“…In turn, many of these miRNAs were confirmed to modulate PAH in vivo via use of antisense oligonucleotide inhibitor (antagomirs) (29,30). In addition, miR-21 processing is regulated by BMP signaling globally (31)(32)(33) and was shown to control PAH progression in vivo by genetic KO studies (28,34) and pharmacologic inhibition of miR-21 (35,36). Despite their modulatory activity in PH, however, actions of these miRNAs do not fully reverse disease manifestations, indicating a level of redundancy and actions as "fine tuners" consistent with their shared targeting of BMPR2.…”

Epigenetic Programming of Cardiovascular Disease by Perinatal Hypoxia and Fetal Growth Restriction

“…Thus, it has been proposed that miR-21 has a dual effect on vascular function: over a short time, it protects against hypoxia and ischemia [70,[102][103][104], and over the longer term, leads to endothelial dysfunction, apoptosis [70,102,105,106], and eNOS dysfunction. The latter would occur by targeting the expression of antioxidant enzymes [70], as well as enhancing the levels of the endogenous eNOS inhibitor asymmetric dimethyl arginine (ADMA) by downregulating the expression of the enzyme dimethyl arginine dimethylaminohydrolase 1 (DDAH1) [105,107,108]. These data suggest that the dynamic regulation of miR-21 and miR-126 could participate in the early defense of the endothelium to hypoxia and oxidative stress; nonetheless, they prime endothelial dysfunction over the long term.…”