Pulmonary vascular remodeling characterized by concentric wall thickening and intraluminal obliteration is a major contributor to the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Here we report that increased hypoxia-inducible factor 2α (HIF-2α) in lung vascular endothelial cells (LVECs) under normoxic conditions is involved in the development of pulmonary hypertension (PH) by inducing endothelial-to-mesenchymal transition (EndMT), which subsequently results in vascular remodeling and occlusive lesions. We observed significant EndMT and markedly increased expression of SNAI, an inducer of EndMT, in LVECs from patients with IPAH and animals with experimental PH compared with normal controls. LVECs isolated from IPAH patients had a higher level of HIF-2α than that from normal subjects, whereas HIF-1α was upregulated in pulmonary arterial smooth muscle cells (PASMCs) from IPAH patients. The increased HIF-2α level, due to downregulated prolyl hydroxylase domain protein 2 (PHD2), a prolyl hydroxylase that promotes HIF-2α degradation, was involved in enhanced EndMT and upregulated SNAI1/2 in LVECs from patients with IPAH. Moreover, knockdown of HIF-2α (but not HIF-1α) with siRNA decreases both SNAI1 and SNAI2 expression in IPAH-LVECs. Mice with endothelial cell (EC)-specific knockout (KO) of the PHD2 gene, egln1 (egln1), developed severe PH under normoxic conditions, whereas Snai1/2 and EndMT were increased in LVECs of egln1 mice. EC-specific KO of the HIF-2α gene, hif2a, prevented mice from developing hypoxia-induced PH, whereas EC-specific deletion of the HIF-1α gene, hif1a, or smooth muscle cell (SMC)-specific deletion of hif2a, negligibly affected the development of PH. Also, exposure to hypoxia for 48-72 h increased protein level of HIF-1α in normal human PASMCs and HIF-2α in normal human LVECs. These data indicate that increased HIF-2α in LVECs plays a pathogenic role in the development of severe PH by upregulating SNAI1/2, inducing EndMT, and causing obliterative pulmonary vascular lesions and vascular remodeling.

Endothelial dysfunction is a major player in the development and progression of vascular pathology in pulmonary arterial hypertension (PAH), a disease associated with small vessel loss and obstructive vasculopathy that leads to increased pulmonary vascular resistance, subsequent right heart failure, and premature death. Over the past ten years, there has been tremendous progress in our understanding of pulmonary endothelial biology as it pertains to the genetic and molecular mechanisms that orchestrate the endothelial response to direct or indirect injury, and how their dysregulation can contribute to the pathogenesis of PAH. As one of the major topics included in the 2017 Grover Conference Series, discussion centered on recent developments in four areas of pulmonary endothelial biology: (1) angiogenesis; (2) endothelial-mesenchymal transition (EndMT); (3) epigenetics; and (4) biology of voltage-gated ion channels. The present review will summarize the content of these discussions and provide a perspective on the most promising aspects of endothelial dysfunction that may be amenable for therapeutic development.

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An increase in cytosolic free Ca concentration ([Ca]) in pulmonary artery smooth muscle cells (PASMCs) triggers pulmonary vasoconstriction and stimulates PASMC proliferation leading to vascular wall thickening. Here, we report that STIM2 (stromal interaction molecule 2), a Ca sensor in the sarcoplasmic reticulum membrane, is required for raising the resting [Ca] in PASMCs from patients with pulmonary arterial hypertension (PAH) and activating signaling cascades that stimulate PASMC proliferation and inhibit PASMC apoptosis. Downregulation of STIM2 in PAH-PASMCs reduces the resting [Ca], whereas overexpression of STIM2 in normal PASMCs increases the resting [Ca] The increased resting [Ca] in PAH-PASMCs is associated with enhanced phosphorylation (p) of CREB (cAMP response element-binding protein), STAT3 (signal transducer and activator of transcription 3), and AKT, increased NFAT (nuclear factor of activated T-cell) nuclear translocation, and elevated level of Ki67 (a marker of cell proliferation). Furthermore, the STIM2-associated increase in the resting [Ca] also upregulates the antiapoptotic protein Bcl-2 in PAH-PASMCs. Downregulation of STIM2 in PAH-PASMCs with siRNA (1) decreases the level of pCREB, pSTAT3, and pAKT and inhibits NFAT nuclear translocation, thereby attenuating proliferation, and (2) decreases Bcl-2, which leads to an increase of apoptosis. In summary, these data indicate that upregulated STIM2 in PAH-PASMCs, by raising the resting [Ca], contributes to enhancing PASMC proliferation by activating the CREB, STAT3, AKT, and NFAT signaling pathways and stimulating PASMC proliferation. The STIM2-associated increase in the resting [Ca] is also involved in upregulating Bcl-2 that makes PAH-PASMCs resistant to apoptosis, and thus plays an important role in sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in patients with PAH.

Capsaicin is an active component of chili pepper and a pain relief drug. Capsaicin can activate transient receptor potential vanilloid 1 (TRPV1) channels to increase cytosolic Ca concentration ([Ca]). A rise in [Ca] in pulmonary artery smooth muscle cells (PASMCs) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. In this study, we observed that a capsaicin-induced increase in [Ca] was significantly enhanced in PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH) compared with normal PASMCs from healthy donors. In addition, the protein expression level of TRPV1 in IPAH PASMCs was greater than in normal PASMCs. Increasing the temperature from 23 to 43°C, or decreasing the extracellular pH value from 7.4 to 5.9 enhanced capsaicin-induced increases in [Ca]; the acidity (pH 5.9)- and heat (43°C)-mediated enhancement of capsaicin-induced [Ca] increases were greater in IPAH PASMCs than in normal PASMCs. Decreasing the extracellular osmotic pressure from 310 to 200 mOsmol/l also increased [Ca], and the hypo-osmolarity-induced rise in [Ca] was greater in IPAH PASMCs than in healthy PASMCs. Inhibition of TRPV1 (with 5'-IRTX or capsazepine) or knockdown of TRPV1 (with short hairpin RNA) attenuated capsaicin-, acidity-, and osmotic stretch-mediated [Ca] increases in IPAH PASMCs. Capsaicin induced phosphorylation of CREB by raising [Ca], and capsaicin-induced CREB phosphorylation were significantly enhanced in IPAH PASMCs compared with normal PASMCs. Pharmacological inhibition and knockdown of TRPV1 attenuated IPAH PASMC proliferation. Taken together, the capsaicin-mediated [Ca] increase due to upregulated TRPV1 may be a critical pathogenic mechanism that contributes to augmented Ca influx and excessive PASMC proliferation in patients with IPAH.

The tumor-suppressive role of p53, a transcription factor that regulates the expression of many genes, has been linked to cell cycle arrest, apoptosis, and senescence. The noncanonical function or the pathogenic role of p53 has more recently been implicated in pulmonary vascular disease. We previously reported that rapid nuclear accumulation of hypoxia-inducible factor (HIF)-1α in pulmonary arterial smooth muscle cells (PASMCs) upregulates transient receptor potential channels and enhances Ca2+ entry to increase cytosolic Ca2+ concentration ([Ca2+]cyt). Also, we observed differences in HIF-1α/2α expression in PASMCs and pulmonary arterial endothelial cells (PAECs). Here we report that p53 is increased in PAECs, but decreased in PASMCs, isolated from mice with hypoxia-induced pulmonary hypertension (PH) and rats with monocrotaline (MCT)-induced PH (MCT-PH). The increased p53 in PAECs from rats with MCT-PH is associated with an increased ratio of Bax/Bcl-2, while the decreased p53 in PASMCs is associated with an increased HIF-1α. Furthermore, p53 is downregulated in PASMCs isolated from patients with idiopathic pulmonary arterial hypertension compared with PASMCs from normal subjects. Overexpression of p53 in normal PASMCs inhibits store-operated Ca2+ entry (SOCE) induced by passive depletion of intracellularly stored Ca2+ in the sarcoplasmic reticulum, while downregulation of p53 enhances SOCE. These data indicate that differentially regulated expression of p53 and HIF-1α/2α in PASMCs and PAECs and the cross talk between p53 and HIF-1α/2α in PASMCs and PAECs may play an important role in the development of PH via, at least in part, induction of PAEC apoptosis and PASMC proliferation.

Piezo is a mechanosensitive cation channel responsible for stretch-mediated Ca2+ and Na+ influx in multiple types of cells. Little is known about the functional role of Piezo1 in the lung vasculature and its potential pathogenic role in pulmonary arterial hypertension (PAH). Pulmonary arterial endothelial cells (PAECs) are constantly under mechanic stretch and shear stress that are sufficient to activate Piezo channels. Here we report that Piezo1 is significantly upregulated in PAECs from patients with idiopathic PAH and animals with experimental pulmonary hypertension (PH) compared to normal controls. Membrane stretch by decreasing extracellular osmotic pressure or by cyclic stretch (18% CS) increases Ca2+-dependent phosphorylation (p) of AKT and ERK, and subsequently upregulates expression of Notch ligands, Jagged1/2 (Jag1 and Jag-2), and Delta like-4 (DLL4) in PAECs. siRNA-mediated downregulation of Piezo1 significantly inhibited the stretch-mediated pAKT increase and Jag-1 upregulation, while downregulation of AKT by siRNA markedly attenuated the stretch-mediated Jag1 upregulation in human PAECs. Furthermore, the mRNA and protein expression level of Piezo1 in the isolated pulmonary artery, which mainly contains pulmonary arterial smooth muscle cells (PASMCs), from animals with severe PH was also significantly higher than that from control animals. Taken together, our study suggests that membrane stretch-mediated Ca2+ influx through Piezo1 is an important trigger for pAKT-mediated upregulation of Jag-1 in PAECs. Upregulation of the mechanosensitive channel Piezo1 and the resultant increase in the Notch ligands (Jag-1/2 and DLL4) in PAECs may play a critical pathogenic role in the development of pulmonary vascular remodeling in PAH and PH.

Idiopathic pulmonary arterial hypertension (PAH) is a fatal and progressive disease. Pulmonary vasoconstriction due to pulmonary arterial smooth muscle cell (PASMC) contraction and pulmonary arterial remodeling due to PASMC proliferation are causes for increased pulmonary vascular resistance in patients with PAH. We and others observed upregulation of TRPC6 channels in PASMC from patients with PAH. An increase in cytosolic Ca2+ concentration ([Ca2+]cyt) in PASMC triggers PASMC contraction and vasoconstriction, while Ca2+-dependent activation of PI3K/AKT/mTOR pathway is pivotal for cell proliferation and gene expression. Despite evidence supporting a pathological role of TRPC6, no selective and orally bioavailable TRPC6 blocker has yet been developed and tested for treatment of PAH. We sought to investigate whether block of receptor-operated Ca2+ channels or TRPC6 can reverse established PH in mice via inhibiting Ca2+-dependent activation of AKT/mTOR signaling. Here we report that intrapulmonary application of 2-aminoethyl diphenyl borniate (2-APB), a non-selective blocker of cation channels or BI-749237, a selective blocker of TRPC6, significantly and reversibly inhibited acute hypoxic pulmonary vasoconstriction. Intraperitoneal injection of 2-APB significantly attenuated the development of PH and partially reversed established PH. Oral gavage of the selective TRPC6 blocker BI-749237 reversed established PH by 50% via regression of pulmonary vascular remodeling. Furthermore, 2-APB and BI-749237 both inhibited PDGF- and serum-mediated phosphorylation of AKT and mTOR in PASMC. These results indicates that the receptor-operated and mechanosensitive TRPC6 channel is a good target for developing novel treatment for PAH. BI-749237, a selective TRPC6 blocker, is potentially a novel and effective drug for treating PAH.