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.

Authorship note: KW and YG share first authorship. RRJ and KW share senior authorship. Conflict of interest: RRJ is a consultant of Boston Scientific and received a research grant from the company. He also serves on the board of directors of Ascletis Pharma. RRJ and KW also filed a patent, Methods and kits for treating pain (16/612,909), in association with Duke University.

Pulmonary arterial hypertension (PAH) is a progressive disease that, if left untreated, eventually leads to right heart failure and death. Elevated pulmonary arterial pressure (PAP) in patients with PAH is mainly caused by an increase in pulmonary vascular resistance (PVR). Sustained vasoconstriction and excessive pulmonary vascular remodeling are two major causes for elevated PVR in patients with PAH. Excessive pulmonary vascular remodeling is mediated by increased proliferation of pulmonary arterial smooth muscle cells (PASMC) due to PASMC dedifferentiation from a contractile or quiescent phenotype to a proliferative or synthetic phenotype. Increased cytosolic Ca(2+) concentration ([Ca(2+)]cyt) in PASMC is a key stimulus for cell proliferation and this phenotypic transition. Voltage-dependent Ca(2+) entry (VDCE) and store-operated Ca(2+) entry (SOCE) are important mechanisms for controlling [Ca(2+)]cyt. Stromal interacting molecule proteins (e.g., STIM2) and Orai2 both contribute to SOCE and we have previously shown that STIM2 and Orai2, specifically, are upregulated in PASMC from patients with idiopathic PAH and from animals with experimental pulmonary hypertension in comparison to normal controls. In this study, we show that STIM2 and Orai2 are upregulated in proliferating PASMC compared with contractile phenotype of PASMC. Additionally, a switch in Ca(2+) regulation is observed in correlation with a phenotypic transition from contractile PASMC to proliferative PASMC. PASMC in a contractile phenotype or state have increased VDCE, while in the proliferative phenotype or state PASMC have increased SOCE. The data from this study indicate that upregulation of STIM2 and Orai2 is involved in the phenotypic transition of PASMC from a contractile state to a proliferative state; the enhanced SOCE due to upregulation of STIM2 and Orai2 plays an important role in PASMC proliferation.

Objective: To investigate the clinical significance of IL-10 þ tumor-associated macrophages (TAMs) in gastric cancer. Background: Due to the plasticity and diversity of TAMs, it is necessary to phenotypically and functionally classify subsets of TAMs to better understand the critical role of TAMs in cancer progression. TAMs expressing interleukin-10 (IL-10) have been found to facilitate immune evasion in many malignancies, but the role of IL-10 þ TAMs in gastric cancer remains obscure. Methods: Four hundred and sixty-eight tumor tissue microarray specimens, 52 fresh tumor tissue samples of gastric cancer patients from Zhongshan Hospital, and data of 298 gastric cancer patients from the Cancer Genome Atlas (TCGA) were analyzed. IL-10 þ TAM level and immune contexture were examined by CIBERSORT, immunohistochemistry, and flow cytometry. Clinical outcomes were analyzed by Kaplan-Meier curves and Cox model. Results: Gastric cancer patients with high IL-10 þ TAM infiltration exhibited poor prognosis and inferior therapeutic responsiveness to fluorouracil-based adjuvant chemotherapy. IL-10 þ TAM infiltration yielded an immunoevasive tumor microenvironment featured by regulatory T cell infiltration and CD8 þ T cell dysfunction. The combinational analysis of IL-10 þ TAM and CD8 þ T cell infiltration stratified patients into distinct risk groups with different clinical outcomes. Moreover, IL-10 þ TAM infiltration was correlated with tumor-intrinsic characteristics including EBV status, PD-L1 expression, and genome stability in gastric cancer. Conclusions: This study revealed that IL-10 þ TAMs might drive an immunoevasive microenvironment and determine poor prognosis and inferior therapeutic responsiveness to fluorouracil-based adjuvant chemotherapy,indicating IL-10 þ TAMs could be applied as a potential target for immunotherapeutic approach in gastric cancer.

An increase in cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and a critical stimulation for PASMC proliferation and migration. Previously, we demonstrated that expression and function of calcium sensing receptors (CaSR) in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) and animals with experimental pulmonary hypertension (PH) were greater than in PASMC from normal subjects and control animals. However, the mechanisms by which CaSR triggers Ca(2+) influx in PASMC and the implication of CaSR in the development of PH remain elusive. Here, we report that CaSR functionally interacts with TRPC6 to regulate [Ca(2+)]cyt in PASMC. Downregulation of CaSR or TRPC6 with siRNA inhibited Ca(2+)-induced [Ca(2+)]cyt increase in IPAH-PASMC (in which CaSR is upregulated), whereas overexpression of CaSR or TRPC6 enhanced Ca(2+)-induced [Ca(2+)]cyt increase in normal PASMC (in which CaSR expression level is low). The upregulated CaSR in IPAH-PASMC was also associated with enhanced Akt phosphorylation, whereas blockade of CaSR in IPAH-PASMC attenuated cell proliferation. In in vivo experiments, deletion of the CaSR gene in mice (casr(-/-)) significantly inhibited the development and progression of experimental PH and markedly attenuated acute hypoxia-induced pulmonary vasoconstriction. These data indicate that functional interaction of upregulated CaSR and upregulated TRPC6 in PASMC from IPAH patients and animals with experimental PH may play an important role in the development and progression of sustained pulmonary vasoconstriction and pulmonary vascular remodeling. Blockade or downregulation of CaSR and/or TRPC6 with siRNA or miRNA may be a novel therapeutic strategy to develop new drugs for patients with pulmonary arterial hypertension.