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Introduction Lymphedema is characterized by a chronic state of lymphatic vascular insufficiency with interstitial edema, inflammation, and dermal pathology; it affects 100-250 million individuals globally, but lacks effective pharmacological therapies [...], Pathologic lymphatic remodeling in lymphedema evolves during periods of tissue inflammation and hypoxia through poorly defined processes. In human and mouse lymphedema, there is a significant increase of hypoxia inducible factor 1[alpha] (HIF-1[alpha]), but a reduction of HIF-2[alpha] protein expression in lymphatic endothelial cells (LECs). We questioned whether dysregulated expression of these transcription factors contributes to disease pathogenesis and found that LEC-specific deletion of Hif2[alpha] exacerbated lymphedema pathology. Even without lymphatic vascular injury, the loss of LEC-specific Hif2[alpha] caused anatomic pathology and a functional decline in fetal and adult mice. These findings suggest that HIF-2[alpha] is an important mediator of lymphatic health. HIF-2[alpha] promoted protective phosphorylated TIE2 (p-TIE2) signaling in LECs, a process also replicated by upregulating TIE2 signaling through adenovirus-mediated angiopoietin-1 (AngptT) gene therapy. Our study suggests that HIF-2[alpha] normally promotes healthy lymphatic homeostasis and raises the exciting possibility that restoring HIF-2[alpha] pathways in lymphedema could mitigate long-term pathology and disability.

Chronic hypoxia causes remodeling and alters contractile responses in both pulmonary arteries and pulmonary veins. Although pulmonary arteries have been studied extensively in these disorders, the mechanisms by which pulmonary veins respond to hypoxia and whether these responses contribute to chronic hypoxic pulmonary hypertension remain poorly understood. In pulmonary arterial smooth muscle, we have previously demonstrated that influx of [Ca.sup.2+] through store-operated calcium channels (SOCC) thought to be composed of transient receptor potential (TRP) proteins is likely to play an important role in development of chronic hypoxic pulmonary hypertension. To determine whether this mechanism could also be operative in pulmonary venous smooth muscle, we measured intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) by fura-2 fluorescence microscopy in primary cultures of pulmonary venous smooth muscle cells (PVSMC) isolated from rat distal pulmonary veins. In cells perfused with [Ca.sup.2+]-free media containing cyclopiazonic acid (10 [micro]M) and nifedipine (5 [micro]M) to deplete sarcoplasmic reticulum [Ca.sup.2+] stores and block voltage-dependent [Ca.sup.2+] channels, restoration of extracellular [Ca.sup.2+] (2.5 mM) caused marked increases in [[[Ca.sup.2+]].sub.i], whereas Mn[Cl.sub.2] (200 [micro]M) quenched fura-2 fluorescence, indicating store-operated [Ca.sup.2+] entry (SOCE). SKF-96365 and Ni[Cl.sub.2], antagonists of SOCC, blocked SOCE at concentrations that did not alter [Ca.sup.2+] responses to 60 mM KCl. Of the seven known canonical TRP (TRPC1-7) and six vanilloid-related TRP channels (TRPV1-6), real-time PCR revealed mRNA expression of TRPC1 > TRPC6 > TRPC4 > TRPC2 [approximately equal to] TRPC5 > TRPC3, TRPV2 > TRPV4 > TRPV1 in distal PVSMC, and TRPC1 > TRPC6 > TRPC3 > TRPC4 [approximately equal to] TRPC5, TRPV2 [approximately equal to] TRPV4 > TRPV1 in rat distal pulmonary vein (PV) smooth muscle. Western blotting confirmed protein expression of TRPC1, TRPC6, TRPV2, and TRPV4 in both PVSMC and PV. Our results suggest that SOCE through [Ca.sup.2+] channels composed of TRP proteins may contribute to [Ca.sup.2+] signaling in rat distal PV smooth muscle. calcium signaling; canonical transient receptor potential; intracellular calcium concentration doi: 10.1152/ajplung.00176.2009.

Am J Physiol Cell Physiol 298:C114-C123, 2010. First published November 4, 2009; doi:10.1152/ajpcell.00629.2008.--In pulmonary arterial smooth muscle cells (PASMCs), [Ca.sup.2+] influx through store-operated [Ca.sup.2+] channels thought to be composed of canonical transient receptor potential (TRPC) proteins is an important determinant of intracellular free calcium concentration ([[Ca.sup.2+]].sub.i]) and pulmonary vascular tone. Sildenafil, a type V phosphodiesterase inhibitor that increases cellular cGMP, is recently identified as a promising agent for treatment of pulmonary hypertension. We previously demonstrated that chronic hypoxia elevated basal [[Ca.sup.2+]].sub.i] in PASMCs due in large part to enhanced store-operated [Ca.sup.2+] entry (SOCE); moreover, ex vivo exposure to prolonged hypoxia (4% [O.sub.2] for 60 h) upregulated TRPC1 and TRPC6 expression in PASMCs. We examined the effect of sildenafil on basal [[Ca.sup.2+]].sub.i], SOCE, and the expression of TRPC in PASMCs under prolonged hypoxia exposure. We also examined the effect of sildenafil on TRPC1 and TRPC6 expression in pulmonary arterial smooth muscle (PA) from rats that developed chronically hypoxic pulmonary hypertension (CHPH). Compared with vehicle control, treatment with sildenafil (300 nM) inhibited prolonged hypoxia induced increases of 1) basal [[Ca.sup.2+]].sub.i], 2) SOCE, and 3) mRNA and protein expression of TRPC in PASMCs. Moreover, sildenafil (50 mg x [kg.sup.-1] x [day.sup.-1]) inhibited mRNA and protein expression of TRPC1 and TRPC6 in PA from chronically hypoxic (10% [O.sub.2] for 21 days) rats, which was associated with decreased right ventricular pressure and right ventricular hypertrophy. Furthermore, we found, in PASMCs exposed to prolonged hypoxia, that knockdown of TRPC1 or TRPC6 by their specific small interference RNA attenuated the hypoxic increases of SOCE and basal [[Ca.sup.2+]].sub.i], suggesting a cause and effect link between increases of TRPC1 and TRPC6 expression and the hypoxic increases of SOCE and basal [[Ca.sup.2+]].sub.i]. These results suggest that sildenafil may alter basal [[Ca.sup.2+]].sub.i] in PASMCs by decreasing SOCE through downregulation of TRPC1 and TRPC6 expression, thereby contributing to decreased vascular tone of pulmonary arteries during the development of CHPH. calcium signaling; store-operated [Ca.sup.2+] entry; basal intracellular calcium

Stromal interaction molecule 1 (STIM1) is a recently discovered membrane-spanning protein thought to sense lumenal [Ca.sup.2+] in sarco(endo)plasmic reticulum (SR/ER) and transduce activation of [Ca.sup.2+]-permeable store-operated channels (SOC) in plasmalemma in response to SR/ER [Ca.sup.2+] depletion. To evaluate the role of STIM1 and a closely related protein, STIM2, in [Ca.sup.2+] signaling of rat distal pulmonary arterial smooth muscle cells (PASMC) during hypoxia, we used fluorescent microscopy and the [Ca.sup.2+]-sensitive dye, fura 2, to measure basal intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+].sub.i]), store-operated [Ca.sup.2+] entry (SOCE), and increases in [[[Ca.sup.2+].sub.i] caused by acute hypoxia (4% [O.sub.2]) or depolarization (60 mmol/l KCl) in cells treated with small interfering RNA targeted to STIM1 (siSTIM1) or STIM2 (siSTIM2). As determined by real-time quantitative PCR analysis (qPCR), STIM1 mRNA was 200-fold more abundant than STIM2 in untreated control PASMC. siSTIM1 and siSTIM2 caused specific and significant knockdown of both mRNA measured by qPCR and protein measured by Western blotting, siSTIM1 markedly inhibited SOCE and abolished the sustained [[[Ca.sup.2+].sub.i] response to hypoxia but did not alter the initial transient [[[Ca.sup.2+].sub.i] response to hypoxia, the [[[Ca.sup.2+].sub.i] response to depolarization, or basal [[[Ca.sup.2+]].sub.i]. The only effect of siSTIM2 was a smaller inhibition of SOCE. We conclude that STIM1 was quantitatively more important than STIM2 in activation of SOC in rat distal PASMC and that the increase in [[[Ca.sup.2+]].sub.i] induced by acute hypoxia in these cells required SR [Ca.sup.2+] release and STIM1-dependent activation of SOC. STIM2; TRPC1; hypoxic pulmonary vasoconstriction; calcium signaling; vascular smooth muscle; KCl

Background: Chronic obstructive pulmonary disease (COPD) is characterized by airway remodeling with airway smooth muscle (ASM) hypertrophy and hyperplasia. Since tobacco use is the key risk factor for the development of COPD and intracellular Ca2+ concentration ([Ca2+]i) plays a major role in both cell proliferation and differentiation, we hypothesized that nicotinic acetylcholine receptor (nAChR) activation plays a role in the elevation of [Ca2+]i in airway smooth muscle cells (ASMCs). Methods: We examined the expression of nAChR and characterized the functions of α7-nAChR in ASMCs. Results: RT-PCR analysis showed that α2-7, β2, and β3-nAChR subunits are expressed in rat ASMCs, with α7 being one of the most abundantly expressed subtypes. Chronic nicotine exposure increased α7-nAChR mRNA and protein expression, and elevated resting [Ca2+]i in cultured rat ASMCs. Acute application of nicotine evoked a rapid increase in [Ca2+]i in a concentration-dependent manner, and the response was significantly enhanced in ASMCs cultured with 1 µM nicotine for 48 hours. Nicotine-induced Ca2+ response was reversibly blocked by the α7-nAChR nicotinic antagonists, methyllycaconitine and α-bungarotoxin. Small interfering RNA suppression of α7-nAChR also substantially blunted the Ca2+ responses induced by nicotine. Conclusion: These observations suggest that nicotine elevates [Ca2+]i in ASMCs through α7-nAChR-mediated signals pathways, and highlight the possibility that α7-nAChR can be considered as a potential target for the treatment of airway remodeling.that nicotine elevates [Ca2+]i in ASMCs through α7-nAChR-mediated signals pathways, and highlight the possibility that α7-nAChR can be considered as a potential target for the treatment of airway remodeling. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Pulmonary hypertension (PH) is a progressive disease affecting the lung vasculature that is characterized by sustained vasoconstriction and leads to vascular remodeling. The lung microbiome contributes to PH progression, but the function of the gut microbiome and the correlation between the gut microbiome and metabolome remain unclear. We have analyzed whether chronic hypoxia-induced PH alters the rat fecal microbiota., Purpose: We explored hypoxia-induced pulmonary hypertension model rats to find out the characteristic changes of intestinal microorganisms and metabolites of hypoxia-induced pulmonary hypertension, and provide a theoretical basis for clinical treatment., Methods: In the current study, a chronic hypoxia-induced PH rat model was used to investigate the role of the gut microbiome and metabolome as a potential mechanism contributing to the occurrence and development of PH. 16S ribosomal ribonucleic acid (16S rRNA), short-chain fatty acid (SCFA) measurements, mass spectrometry (MS) metabolomics analysis and metatranscriptome were performed to analyze stool samples. The datasets were analyzed individually and integrated for combined analysis using bioinformatics approaches., Results: Our results suggest that the gut microbiome and metabolome of chronic hypoxia-induced PH rats are distinct from those of normoxic rats and may thus aid in the search for new therapeutic or diagnostic paradigms for PH., Conclusion: The gut microbiome and metabolome are altered as a result of chronic hypoxia-induced PH. This imbalanced bacterial ecosystem might play a pathophysiological role in PH by altering homeostasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Cao, Wang, Mo, Peng, Hong, Zhou, Sun, Li, Liang, Zhao, Zheng, Li and Peng.)

Rationale: Exposure to acute hypoxia causes vasoconstriction in both pulmonary arteries (PA) and pulmonary veins (PV). The mechanisms on the arterial side have been studied extensively. However, bare attention has been paid to the venous side., Objectives: To investigate if acute hypoxia caused the increase of intracellular Ca(2+) concentration ([Ca(2+)]i), and Ca(2+) influx through store-operated calcium channels (SOCC) in pulmonary venous smooth muscle cells (PVSMCs)., Methods: Fluorescent microscopy and fura-2 were used to measure effects of 4% O2 on [Ca(2+)]i and store-operated Ca(2+) entry (SOCE) in isolated rat distal PVSMCs., Measurements and Main Results: In PVSMCs perfused with Ca(2+)-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca(2+) stores in the sarcoplasmic reticulum (SR) and nifedipine to prevent Ca(2+) entry through L-type voltage-depended Ca(2+) channels (VDCC), hypoxia markedly enhanced both the increase in [Ca(2+)]i caused by restoration of extracellular [Ca(2+)] and the rate at which extracellular Mn(2+) quenched fura-2 fluorescence. Moreover, the increased [Ca(2+)]i in PVSMCs perfused with normal salt solution was completely blocked by SOCC antagonists SKF-96365 and NiCl2 at concentrations that SOCE >85% was inhibited but [Ca(2+)]i responses to 60 mM KCl were not altered. On the contrary, L-type VDCC antagonist nifedipine inhibited increase in [Ca(2+)]i to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca(2+)]i caused by hypoxia was completely abolished by perfusion with Ca(2+)-free KRBS., Conclusions: These results suggest that acute hypoxia enhances SOCE via activating SOCCs, leading to increased [Ca(2+)]i in distal PVSMCs.