Functional importance of Dicer protein in the adaptive cellular response to hypoxia.

The processes by which cells sense and respond to ambient oxygen concentration are fundamental to cell survival and function, and they commonly target gene regulatory events. To date, however, little is known about the link between the microRNA pathway and hypoxia signaling. Here, we show in vitro and in vivo that chronic hypoxia impairs Dicer (DICER1) expression and activity, resulting in global consequences on microRNA biogenesis. We show that von Hippel-Lindau-dependent down-regulation of Dicer is key to the expression and function of hypoxia-inducible factor α (HIF-α) subunits. Specifically, we show that EPAS1/HIF-2α is regulated by the Dicer-dependent microRNA miR-185, which is down-regulated by hypoxia. Full expression of hypoxia-responsive/HIF target genes in chronic hypoxia (e.g. VEGFA, FLT1/VEGFR1, KDR/VEGFR2, BNIP3L, and SLC2A1/GLUT1), the function of which is to regulate various adaptive responses to compromised oxygen availability, is also dependent on hypoxia-mediated down-regulation of Dicer function and changes in post-transcriptional gene regulation. Therefore, functional deficiency of Dicer in chronic hypoxia is relevant to both HIF-α isoforms and hypoxia-responsive/HIF target genes, especially in the vascular endothelium. These findings have relevance to emerging therapies given that we show that the efficacy of RNA interference under chronic hypoxia, but not normal oxygen availability, is Dicer-dependent. Collectively, these findings show that the down-regulation of Dicer under chronic hypoxia is an adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, thereby providing an essential mechanistic insight into the oxygen-dependent microRNA regulatory pathway.

Enhanced translation of heme oxygenase-2 preserves human endothelial cell viability during hypoxia.

Heme oxygenases (HOs) -1 and -2 catalyze the breakdown of heme to release carbon monoxide, biliverdin, and ferrous iron, which may preserve cell function during oxidative stress. HO-1 levels decrease in endothelial cells exposed to hypoxia, whereas the effect of hypoxia on HO-2 expression is unknown. The current study was carried out to determine if hypoxia alters HO-2 protein levels in human endothelial cells and whether this enzyme plays a role in preserving their viability during hypoxic stress. Human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs), and human blood outgrowth endothelial cells were exposed to 21% or 1% O(2) for 48 or 16 h in the presence or absence of tumor necrosis factor-alpha (10 ng/ml) or H(2)O(2) (100 microm). In all three endothelial cell types HO-1 mRNA and protein levels were decreased following hypoxic incubation, whereas HO-2 protein levels were unaltered. In HUVECs HO-2 levels were maintained during hypoxia despite a 57% reduction in steady-state HO-2 mRNA level and a 43% reduction in total protein synthesis. Polysome profiling revealed increased HO-2 transcript association with polysomes during hypoxia consistent with enhanced translation of these transcripts. Importantly, inhibition of HO-2 expression by small interference RNA increased oxidative stress, exacerbated mitochondrial membrane depolarization, and enhanced caspase activation and apoptotic cell death in cells incubated under hypoxic but not normoxic conditions. These data indicate that HO-2 is important in maintaining endothelial viability and may preserve local regulation of vascular tone, thrombosis, and inflammatory responses during reductions in systemic oxygen delivery.

Swelling-activated Cl- currents and intracellular CLC-3 are involved in proliferation of human pulmonary artery smooth muscle cells.

BACKGROUND: Proliferation of pulmonary artery smooth muscle cells (PASMCs) leads to adverse vascular remodeling and contributes to pulmonary arterial hypertension, a condition associated with a 15% annual mortality despite treatment. We previously showed that swelling-activated Cl currents (ICl,swell) are upregulated in PASMC proliferation and that nonspecific Cl current blockers inhibit proliferation. However, the specific role of ICl,swell in PASMC proliferation and its molecular underpinning remain unknown. METHODS AND RESULTS: In the present study, we found that the specific ICl,swell blocker, DCPIB (4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy] butanoic acid), dose-dependently blocked (IC50 = 2.7 µmol/l) ICl,swell and inhibited (IC50 = 6.9 µmol/l) proliferation in isolated human PASMCs (hPASMCs). To identify the Cl channel genes underlying ICl,swell and regulating hPASMC proliferation, we measured the mRNA expression of candidate Cl channel genes (CLC-1 to CLC-7, CLC-Ka and CLC-Kb, and BEST-1 to BEST-4) in hPASMCs. CLC-2 to CLC-7 and BEST-1 are expressed in hPASMCs, with the most abundant gene being CLC-3, a channel gene previously linked to ICl,swell. Although stable expression of a microRNA-adapted shRNA targeting CLC-3 transcripts in hPASMCs selectively reduced CLC-3 mRNA by more than 80% and inhibited hPASMC proliferation (by >45%) compared with control-shRNA, it did not alter ICl,swell. Consistent with this observation, immunocytostaining studies revealed that CLC-3 protein is primarily located in intracellular areas of cultured proliferative hPASMCs. The intracellular CLC-3 protein levels were profoundly reduced by shRNA targeting CLC-3. The other molecular candidate for ICl,swell (i.e.,CLC-2) also showed a mainly intracellular distribution. CONCLUSION: Our findings support the conclusion that both ICl,swell and CLC-3 play a role in PASMC proliferation, but CLC-3 channels do not underlie ICl,swell in these cells.

Regulation of proliferation and membrane potential by chloride currents in rat pulmonary artery smooth muscle cells.

Pulmonary artery smooth muscle cell (PASMC) proliferation contributes to increased pulmonary vascular resistance and pulmonary hypertension. Because proliferation depends on membrane potential (V(m)) and because V(m) is, in part, determined by Cl(-) currents (I(Cl)), we examined the effects of I(Cl) inhibition with 4,4;-diisothiocyanatostilbene-2,2;-disulfonic acid (DIDS) on cultured rat PASMCs. DIDS (30 mumol/L) reduced cell numbers, decreased 5-bromodeoxyuridine incorporation and delayed cell cycle progression. I(Cl) inhibition with 5-Nitro-2-(3-phenylpropylamino) benzoic acid (100 mumol/L) also reduced cell numbers of cultured rat PASMCs. To test the possible involvement of I(Cl) in the regulation of PASMC proliferation, we measured V(m) and I(Cl) in both cultured (proliferating) and acutely dissociated (nonproliferating) rat PASMCs. V(m) (-39.3+/-1.4 mV) was close to the equilibrium potential of Cl(-) (-39 mV) in proliferating PASMCs but differed from equilibrium potential of Cl(-) in acutely dissociated cells (-45.3+/-0.9 mV). DIDS and substitution of extracellular Cl(-) with I(-) induced V(m) hyperpolarization in proliferating but not nonproliferating PASMCs. Consistent with V(m) recordings, DIDS-sensitive baseline and swelling-activated (Ca(2+)-independent) I(Cl)s, recorded with low Ca(2+) (<1 nmol/L) pipette solutions, were approximately 5-fold greater in proliferating than in nonproliferating PASMCs. By contrast, Ca(2+)-activated I(Cl) did not differ between proliferating and nonproliferating PASMCs. Ca(2+)-independent I(Cl)s were also increased in proliferating PASMCs acutely dissociated from rats exposed to hypoxia (10% O(2); 7 days). These findings are consistent with the conclusion that I(Cl)s regulate proliferation of PASMCs and suggest that selective I(Cl) inhibition may be useful in treating pulmonary hypertension.

Induction of matrix metalloproteinase-2 enhances systemic arterial contraction after hypoxia.

This study was carried out to determine the role of increased vascular matrix metalloproteinase-2 (MMP-2) expression in the changes in systemic arterial contraction after prolonged hypoxia. Rats and mice were exposed to hypoxia (10% and 8% O(2), respectively) or normoxia (21% O(2)) for 16 h, 48 h, or 7 days. Aortae and mesenteric arteries were either mounted in organ bath myographs or frozen in liquid nitrogen. MMP-2 inhibition with cyclic CTTHWGFTLC (CTT) reduced contraction to phenylephrine (PE) in aortae and mesenteric arteries from rats exposed to hypoxia for 7 days but not in vessels from normoxic rats. Similarly, CTT reduced contraction to Big endothelin-1 (Big ET-1) in aortae from rats exposed to hypoxia for 7 days. Responses to PE were reduced in hypoxic MMP-2(-/-) mice compared with MMP-2(+/+) mice. Increased contraction to Big ET-1 after hypoxia was observed in MMP-2(+/+) mice but not in MMP-2(-/-) mice. Rat aortic MMP-2 and membrane type 1 (MT1)-MMP protein levels and MMP activity were increased after 7 days of hypoxia. Rat aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular smooth muscle. We conclude that hypoxic induction of MMP-2 expression potentiates contraction in systemic conduit and resistance arteries. This may preserve the capacity to regulate the systemic circulation in the transition between the alterations in vascular tone and structural remodeling that occurs during prolonged hypoxic epochs.