Pulmonary vascular structure remodeling (PVSR) is a hallmark of pulmonary hypertension. P27^kip1, one of critical cyclin-dependent kinase inhibitors, has been shown to mediate anti-proliferation effects on various vascular cells. Beta-estradiol (β-E2) has numerous biological protective effects including attenuation of hypoxic pulmonary hypertension (HPH). In the present study, we employed β-E2 to investigate the roles of p27^kip1 and its closely-related kinase (Skp-2) in the progression of PVSR and HPH. Sprague-Dawley rats treated with or without β-E2 were challenged by intermittent chronic hypoxia exposure for 4 weeks to establish hypoxic pulmonary hypertension models, which resemble moderate severity of hypoxia-induced PH in humans. Subsequently, hemodynamic and pulmonary pathomorphology data were gathered. Additionally, pulmonary artery smooth muscle cells (PASMCs) were cultured to determine the anti-proliferation effect of β-E2 under hypoxia exposure. Western blotting or reverse transcriptional polymerase chain reaction (RT-PCR) were adopted to test p27^kip1, Skp-2 and Akt-P changes in rat lung tissue and cultured PASMCs. Chronic hypoxia significantly increased right ventricular systolic pressures (RVSP), weight of right ventricle/left ventricle plus septum (RV/LV+S) ratio, medial width of pulmonary arterioles, accompanied with decreased expression of p27^kip1 in rats. Whereas, β-E2 treatment repressed the elevation of RVSP, RV/LV+S, attenuated the PVSR of pulmonary arterioles induced by chronic hypoxia, and stabilized the expression of p27^kip1. Study also showed that β-E2 application suppressed the proliferation of PASMCs and elevated the expression of p27^kip1 under hypoxia exposure. In addition, experiments both in vivo and in vitro consistently indicated an escalation of Skp-2 and phosphorylated Akt under hypoxia condition. Besides, all these changes were alleviated in the presence of β-E2. Our results suggest that β-E2 can effectively attenuate PVSR and HPH. The underlying mechanism may partially be through the increased p27^kip1 by inhibiting Skp-2 through Akt signal pathway. Therefore, targeting up-regulation of p27^kip1 or down-regulation of Skp-2 might provide new strategies for treatment of HPH.

Immune suppressive microenvironment in tumor emerges as the main obstacle for cancer immunotherapy. In this study, we identified that HIF1α was activated in the tumor associated macrophages and acted as an important factor for the immune suppressive microenvironment. Epigenetically silencing of Hif1α via histone H3 methylation in the promoter region was achieved by CRISPR/dCas9-EZH2 system, in which histone H3 methylase EZH2 was recruited to the promoter region specifically. The Hif1α silenced macrophage, namely HERM (Hif1α Epigenetically Repressed Macrophage) manifested as inheritable tumor suppressing phenotype. In the subcutaneous B16-F10 melanoma syngeneic model, intratumoral injection of HERMs reprogrammed the immune suppressive microenvironment to the active one, reducing tumor burden and prolonging overall survival. Additionally, HERMs therapy remarkably inhibited tumor angiogenesis. Together, our study has not only identified a promising cellular and molecular target for reverting immune suppressive microenvironment, but also provided a potent strategy for reprogramming tumor microenvironment via epigenetically reprogrammed macrophages.

Hypoxic pulmonary hypertension (HPH) serves as a crucial link in the pathogenesis of chronic high-altitude diseases and chronic obstructive pulmonary disease (COPD), and ultimately may lead to right heart failure. Hypoxia triggers immune responses and inflammatory processes, which contribute to vascular remodeling and elevated pulmonary artery pressure. NLRX1, a member of the Nod-like receptor family, plays a significant regulatory role in immunity, antioxidation, and apoptosis. However, its role in the occurrence and development of HPH remains unknown. Wild type (WT) C57BL/6 mice and NLRX1 knockout (NLRX1−/−) Mice were subjected to intermittent chronic hypoxia for 6 weeks to establish a hypoxic pulmonary hypertension model that is similar to the moderate pulmonary hypertension (PH) induced by hypoxia in humans. Subsequently, data on hemodynamics and pulmonary pathomorphology were collected. Additionally, bone marrow derived macrophages (BMDMs) were cultured to determine the effects of up-regulating and down-regulating NLRX1 on cell function under hypoxia exposure. Western blotting or reverse transcription polymerase chain reaction (RT-PCR) was utilized to detect changes in inflammation factors and oxidative stress-related indicators in rat lung tissue and cultured BMDMs. Hypoxia downregulates the expression of NLRX1 in the lung tissues of mice and bone marrow-derived macrophages (BMDM). Chronic hypoxia significantly increases right ventricular systolic pressure (RVSP), the ratio of right ventricle weight to left ventricle plus septum weight (RV/LV + S), and the medial width of pulmonary arterioles in NLRX1-knockout (NLRX1−/−) mice. NLRX1 mediates the hemodynamic response and right ventricular hypertrophy in mice with hypoxic pulmonary hypertension. The deficiency of NLRX1 upregulates inflammatory mediators by activating nuclear factor-κB (NF-κB) and simultaneously promotes oxidative stress and the activation of nuclear factor E2-related factor 2 (Nrf2). The activator NX-13 of NLRX1 can reduce the production of inflammatory cytokines in BMDMs stimulated by hypoxia and mitigate oxidative stress. NLRX1 may suppress hypoxia-induced lung inflammation and alleviate hypoxia-induced pulmonary hypertension through its anti-inflammatory and antioxidant properties. Therefore, targeted up-regulation of NLRX1 may offer a new strategy for the treatment of HPH.