Association between hydrogen sulfide and OSA-associated hypertension: a clinical study

Purpose

We sought to unravel the role of hydrogen sulfide (H₂S) in the development of hypertension in patients with obstructive sleep apnea (OSA).

Methods

The study sample included 80 patients with OSA and 45 healthy controls. All subjects underwent measurement of blood pressure (BP) and serum H₂S level in the morning. Twentynine of the 39 patients with OSA and concomitant hypertension and 23 of the 41 patients with OSA but no concomitant hypertension received continuous positive alveolar pressure (CPAP) therapy for 4 weeks. Twenty-four-hour ambulatory BP and serum H₂S were determined before and after CPAP. Respiratory indices including apnea hypopnea index (AHI), lowest oxygen saturation (SaO₂), and length of time < 90% saturated (T90) were determined by polysomnography.

Results

Associations between H₂S, BP, respiratory indices, and changes with CPAP were analyzed. OSA patients had significantly higher systolic BP (p = 0.003) and diastolic BP (p = 0.009) and lower H₂S levels (p = 0.02) compared to healthy controls. H₂S negatively correlated with AHI (p = 0.005), T90 (p = 0.009), morning systolic BP (p = 0.02), and morning diastolic BP (p = 0.03). All respiratory indices were significantly improved (p < 0.05) after CPAP in OSA patients with or without hypertension. BP was significantly reduced and H₂S significantly increased after CPAP in OSA patients with hypertension (p < 0.05) but not in OSA patients without hypertension (p > 0.05).

Conclusion

Multivariate linear regression analysis demonstrated that 24h systolic BP and 24h diastolic BP correlated with H₂S as well as their changes after CPAP treatment. Reduction in H₂S may play a role in the pathogenesis of hypertension in patients with OSA.

     

Single-cell proteomic chip for profiling intracellular signaling pathways in single tumor cells

We describe a microchip designed to quantify the levels of a dozen cytoplasmic and membrane proteins from single cells. We use the platform to assess protein–protein interactions associated with the EGF-receptor-mediated PI3K signaling pathway. Single-cell sensitivity is achieved by isolating a defined number of cells (n = 0–5) in 2 nL volume chambers, each of which is patterned with two copies of a miniature antibody array. The cells are lysed on-chip, and the levels of released proteins are assayed using the antibody arrays. We investigate three isogenic cell lines representing the cancer glioblastoma multiforme, at the basal level, under EGF stimulation, and under erlotinib inhibition plus EGF stimulation. The measured protein abundances are consistent with previous work, and single-cell analysis uniquely reveals single-cell heterogeneity, and different types and strengths of protein–protein interactions. This platform helps provide a comprehensive picture of altered signal transduction networks in tumor cells and provides insight into the effect of targeted therapies on protein signaling networks.Although signal transduction inhibitors occasionally offer clinical benefit for cancer patients (1), signal flux emanating from oncogenes is often distributed through multiple pathways (2), potentially underlying the failure of most such inhibitors (3). Measuring signal flux through multiple pathways, in response to signal transduction inhibitors, may help uncover network interactions that contribute to therapeutic resistance and that are not predicted by analyzing pathways in isolation (4). The cellular and molecular complexity of a solid tumor microenvironment (5) suggests the need to study signaling in individual cancer cells.Protein–protein interactions within signaling pathways are often elucidated by assessing the levels of relevant pathway proteins in model and tumor-derived cell lines and with various genetic and molecular perturbations. Such interactions, and the implied signaling networks, may also be elucidated via quantitative measurements of multiple pathway-related proteins within single cells (6). At the single-cell level, inhibitory and activating protein–protein relationships, as well as stochastic (single-cell) fluctuations, are revealed. However, most techniques for profiling signaling pathways (7, 8) require large numbers of cells. Single-cell immunostaining (9) is promising, and some flow cytometry (6) techniques are relevant, as discussed below.We describe quantitative, multiplex assays of intracellular signaling proteins from single cancer cells using a platform called the single-cell barcode chip (SCBC). The SCBC is simple in concept: A single or defined number of cells is isolated within an approximately 2 nL volume microchamber that contains an antibody array (10) for the capture and detection of a panel of proteins. The SCBC design (11) permits lysis of each individual trapped cell.Intracellular staining flow cytometry can assay up to 11 phosphoproteins from single cells (6). Our SCBC can profile a similar size panel, but only for approximately 100 single cells per chip. Each protein is assayed twice, yielding some statistical assessment for each experiment. The SCBC is a relatively simple platform and only requires a few hundred cells per assay.We used the SCBC to study signal transduction in glioblastoma multiforme (GBM), a primary malignant brain tumor (12). GBM has been genetically characterized, yet the nature of signaling pathways downstream of key oncogenic mutations, such as epidermal growth factor receptor activating mutation (EGFRvIII) and phosphatase and tensin homolog (PTEN) tumor suppressor gene loss associated with receptor tyrosine kinase (RTK)/PI3K signaling, are incompletely understood (13–15). Single-cell experiments may also help resolve the characteristic heterogeneity of GBM.We interrogated 11 proteins directly or potentially associated with PI3K signaling (see SI Appendix, Methods I) through three isogenic GBM cell lines: U87 (expressing wild-type p53, mutant PTEN, and low levels of wild-type EGFR, no EGFRvIII) (16, 17), U87 EGFRvIII (U87 cells stably expressing EGFRvIII deletion mutant), and U87 EGFRvIII PTEN (U87 cells coexpressing EGFRvIII and PTEN) (18). Fig. 1 diagrams this biology. Each cell line was investigated under conditions of standard cell culture, in response to EGF stimulation, and after erlotinib treatment followed by EGF stimulation. The proteins assayed represented RTKs and proteins signifying activation of PI3K and MAPK signaling. They were (p- denotes phosphorylation) p-Src, p-mammalian target of rapamycin (p-mTOR), p-p70 ribosomal protein S6 kinase (p-p70S6K), p-glycogen synthase kinase-3 (p-GSK-3α/β), p-p38 mitogen activated protein kinase (p-p38α), p-extracellular regulated kinase (p-ERK), p-c-Jun N-terminal kinase (p-JNK2), p-platelet derived growth factor receptor β (p-PDGFRβ), p-vascular endothelial growth factor receptor 2 (p-VEGFR2), tumor protein 53 (P53), and total EGFR.Open in a separate windowFig. 1.The PI3K pathway activated by EGF-stimulated EGFR or by the constitutively activated EGFRvIII. All proteins in light blue with central yellow background were assayed. Orange background proteins were expressed in the cell lines U87 EGFRvIII or U87 EGFRvIII PTEN. The oval, yellow background components are the investigated molecular perturbations.