Novel Populations of Lung Capillary Endothelial Cells and Their Functional Significance.

The role of the lung's microcirculation and capillary endothelial cells in normal physiology and the pathobiology of pulmonary diseases is unequivocally vital. The recent discovery of molecularly distinct aerocytes and general capillary (gCaps) endothelial cells by single-cell transcriptomics (scRNAseq) advanced the field in understanding microcirculatory milieu and cellular communications. However, increasing evidence from different groups indicated the possibility of more heterogenic structures of lung capillaries. Therefore, we investigated enriched lung endothelial cells by scRNAseq and identified five novel populations of gCaps with distinct molecular signatures and roles. Our analysis suggests that two populations of gCaps that express Scn7a(Na+) and Clic4(Cl-) ion transporters form the arterial-to-vein zonation and establish the capillary barrier. We also discovered and named mitotically-active "root" cells (Flot1+) on the interface between arterial, Scn7a+, and Clic4 + endothelium, responsible for the regeneration and repair of the adjacent endothelial populations. Furthermore, the transition of gCaps to a vein requires a venous-capillary endothelium expressing Lingo2. Finally, gCaps detached from the zonation represent a high level of Fabp4, other metabolically active genes, and tip-cell markers showing angiogenesis-regulating capacity. The discovery of these populations will translate into a better understanding of the involvement of capillary phenotypes and their communications in lung disease pathogenesis.

Necrosis-Released HMGB1 (High Mobility Group Box 1) in the Progressive Pulmonary Arterial Hypertension Associated With Male Sex.

Damage-associated molecular patterns, such as HMGB1 (high mobility group box 1), play a well-recognized role in the development of pulmonary arterial hypertension (PAH), a progressive fatal disease of the pulmonary vasculature. However, the contribution of the particular type of vascular cells, type of cell death, or the form of released HMGB1 in PAH remains unclear. Moreover, although male patients with PAH show a higher level of circulating HMGB1, its involvement in the severe PAH phenotype reported in males is unknown. In this study, we aimed to investigate the sources and active forms of HMGB1 released from damaged vascular cells and their contribution to the progressive type of PAH in males. Our results showed that HMGB1 is released by either pulmonary artery human endothelial cells or human pulmonary artery smooth muscle cells that underwent necrotic cell death, although only human pulmonary artery smooth muscle cells produce HMGB1 during apoptosis. Moreover, only human pulmonary artery smooth muscle cell death induced a release of dimeric HMGB1, found to be mitochondrial reactive oxygen species dependent, and TLR4 (toll-like receptor 4) activation. The modified Sugen/Hypoxia rat model replicates the human sexual dimorphism in PAH severity (right ventricle systolic pressure in males versus females 54.7±2.3 versus 44.6±2 mm Hg). By using this model, we confirmed that necroptosis and necrosis are the primary sources of circulating HMGB1 in the male rats, although only necrosis increased circulation of HMGB1 dimers. Attenuation of necrosis but not apoptosis or necroptosis prevented TLR4 activation in males and blunted the sex differences in PAH severity. We conclude that necrosis, through the release of HMGB1 dimers, predisposes males to a progressive form of PAH.

Sex-specific stress response and HMGB1 release in pulmonary endothelial cells.

Women are known to be associated with a higher susceptibility to pulmonary arterial hypertension (PAH). In contrast, male PAH patients have a worse survival prognosis. In this study, we investigated whether the contribution of sex goes beyond the effects of sex hormones by comparing the ability of isolated male and female pulmonary endothelial cells to respire, proliferate and tolerate the stress. Mouse lung endothelial cells (MLEC) were isolated from the lungs of male and female 3-week old mice. Male MLEC showed an increased basal mitochondrial respiration rate, elevated maximal respiration, a significantly greater level of mitochondrial polarization, and a higher rate of proliferation. Exposure of cells to hypoxia (2% of O2 for 24 hours) induced a strong apoptotic response in female but not male MLEC. In contrast, treatment with mitochondrial respiratory Complex III inhibitor Antimycin A (AA, 50µM) mediated severe necrosis specifically in male MLEC, while female cells again responded primarily by apoptosis. The same effect with female cells responding to the stress by apoptosis and male cells responding by necrosis was confirmed in starved pulmonary endothelial cells isolated from human donors. Elevated necrosis seen in male cells was associated with a significant release of damage-associated alarmin, HMGB1. No stimuli induced a significant elevation of HMGB1 secretion in females. We conclude that male cells appear to be protected against mild stress conditions, such as hypoxia, possibly due to increased mitochondrial respiration. In contrast, they are more sensitive to impaired mitochondrial function, to which they respond by necrotic death. Necrosis in male vascular cells releases a significant amount of HMGB1 that could contribute to the pro-inflammatory phenotype known to be associated with the male gender.

Role of Gender in Regulation of Redox Homeostasis in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is one of the diseases with a well-established gender dimorphism. The prevalence of PAH is increased in females with a ratio of 4:1, while poor survival prognosis is associated with the male gender. Nevertheless, the specific contribution of gender in disease development and progression is unclear due to the complex nature of the PAH. Oxidative and nitrosative stresses are important contributors in PAH pathogenesis; however, the role of gender in redox homeostasis has been understudied. This review is aimed to overview the possible sex-specific mechanisms responsible for the regulation of the balance between oxidants and antioxidants in relation to PAH pathobiology.

Inositol monophosphatase 1 as a novel interacting partner of RAGE in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by progressive pulmonary vascular remodeling. The receptor for advanced glycation end products (RAGE) plays an important role in PAH by promoting proliferation of pulmonary vascular cells. RAGE is also known to mediate activation of Akt signaling, although the particular molecular mechanism remains unknown. This study aimed to identify the interacting partner of RAGE that could facilitate RAGE-mediated Akt activation and vascular remodeling in PAH. The progressive angioproliferative PAH was induced in 24 female Sprague-Dawley rats ( n = 8/group) that were randomly assigned to develop PAH for 1, 2, or 5 wk [right ventricle systolic pressure (RVSP) 56.5 ± 3.2, 63.6 ± 1.6, and 111.1 ± 4.5 mmHg, respectively, vs. 22.9 ± 1.1 mmHg in controls]. PAH triggered early and late episodes of apoptosis in rat lungs accompanied by RAGE activation. Mass spectrometry analysis has identified IMPA1 as a novel PAH-specific interacting partner of RAGE. The proximity ligation assay (PLA) confirmed the formation of RAGE/IMPA1 complex in the pulmonary artery wall. Activation of IMPA1 in response to increased glucose 6-phosphate (G6P) is known to play a critical role in inositol synthesis and recycling. Indeed, we confirmed a threefold increase in G6P ( P = 0.0005) levels in lungs of PAH rats starting from week 1 that correlated with accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), membrane translocation of PI3K, and a threefold increase in membrane Akt levels ( P = 0.02) and Akt phosphorylation. We conclude that the formation of the newly discovered RAGE-IMPA1 complex could be responsible for the stimulation of inositol pathways and activation of Akt signaling in PAH.