Regulation of myofibroblast differentiation by cardiac glycosides.

Myofibroblast differentiation is a key process in pathogenesis of fibrotic diseases. Cardiac glycosides (ouabain, digoxin) inhibit Na(+)-K(+)-ATPase, resulting in increased intracellular [Na(+)]-to-[K(+)] ratio in cells. Microarray analysis suggested that increased intracellular [Na(+)]/[K(+)] ratio may promote the expression of cyclooxygenase-2 (COX-2), a critical enzyme in the synthesis of prostaglandins. Given antifibrotic effects of prostaglandins through activation of protein kinase A (PKA), we examined if cardiac glycosides stimulate COX-2 expression in human lung fibroblasts and how they affect myofibroblast differentiation. Ouabain stimulated a profound COX-2 expression and a sustained PKA activation, which was blocked by COX-2 inhibitor or by COX-2 knockdown. Ouabain-induced COX-2 expression and PKA activation were abolished by the inhibitor of the Na(+)/Ca(2+) exchanger, KB-R4943. Ouabain inhibited transforming growth factor-ß (TGF-ß)-induced Rho activation, stress fiber formation, serum response factor activation, and the expression of smooth muscle α-actin, collagen-1, and fibronectin. These effects were recapitulated by an increase in intracellular [Na(+)]/[K(+)] ratio through the treatment of cells with K(+)-free medium or with digoxin. Although inhibition of COX-2 or of the Na(+)/Ca(2+) exchanger blocked ouabain-induced PKA activation, this failed to reverse the inhibition of TGF-ß-induced Rho activation or myofibroblast differentiation by ouabain. Together, these data demonstrate that ouabain, through the increase in intracellular [Na(+)]/[K(+)] ratio, drives the induction of COX-2 expression and PKA activation, which is accompanied by a decreased Rho activation and myofibroblast differentiation in response to TGF-ß. However, COX-2 expression and PKA activation are not sufficient for inhibition of the fibrotic effects of TGF-ß by ouabain, suggesting that additional mechanisms must exist.

Antifibrotic effects of noscapine through activation of prostaglandin E2 receptors and protein kinase A.

Myofibroblast differentiation is a key process in the pathogenesis of fibrotic disease. We have shown previously that differentiation of myofibroblasts is regulated by microtubule polymerization state. In this work, we examined the potential antifibrotic effects of the antitussive drug, noscapine, recently found to bind microtubules and affect microtubule dynamics. Noscapine inhibited TGF-ß-induced differentiation of cultured human lung fibroblasts (HLFs). Therapeutic noscapine treatment resulted in a significant attenuation of pulmonary fibrosis in the bleomycin model of the disease. Noscapine did not affect gross microtubule content in HLFs, but inhibited TGF-ß-induced stress fiber formation and activation of serum response factor without affecting Smad signaling. Furthermore, noscapine stimulated a rapid and profound activation of protein kinase A (PKA), which mediated the antifibrotic effect of noscapine in HLFs, as assessed with the PKA inhibitor, PKI. In contrast, noscapine did not activate PKA in human bronchial or alveolar epithelial cells. Finally, activation of PKA and the antifibrotic effect of noscapine in HLFs were blocked by the EP2 prostaglandin E2 receptor antagonist, PF-04418948, but not by the antagonists of EP4, prostaglandin D2, or prostacyclin receptors. Together, we demonstrate for the first time the antifibrotic effect of noscapine in vitro and in vivo, and we describe a novel mechanism of noscapine action through EP2 prostaglandin E2 receptor-mediated activation of PKA in pulmonary fibroblasts.

Swelling rather than shrinkage precedes apoptosis in serum-deprived vascular smooth muscle cells.

Contrasting cell volume behaviours (swelling vs. shrinkage) are considered as criteria to distinguish necrosis from apoptosis. In this study, we employed a time-lapse, dual-image surface reconstruction technique to assess the volume of single vascular smooth muscle cells transfected with E1A-adenoviral protein (E1A-VSMC) and undergoing rapid apoptosis in the absence of growth factors or in the presence of staurosporine. After 30- to 60-min lag-phase, serum-deprived E1A-VSMC volume was increased by ~40%, which preceded maximal increments of caspase-3 activity and chromatin cleavage. Swollen cells underwent rapid apoptotic collapse, documented by plasma membrane budding, and terminated in 10-15 min by the formation of numerous apoptotic bodies. Suppression of apoptosis by inhibition of Na(+),K(+)-ATPase and activation of cAMP signalling with ouabain and forskolin, respectively, completely abolished the swelling of serum-deprived E1A-VSMC. In contrast to serum deprivation, apoptotic collapse of staurosporine-treated E1A-VSMC preceded attenuation of their volume by ~30%. Neither transient hyposmotic swelling nor isosmtotic shrinkage triggered apoptosis. Our results show that cell shrinkage can not be considered as ubiquitous hallmark of apoptosis. The involvement of stimulus-specific cell volume perturbations in initiation and progression of apoptosis in vascular smooth muscle cells should be examined further.

Hyperosmotic and isosmotic shrinkage differentially affect protein phosphorylation and ion transport.

In the present work, we compared the outcome of hyperosmotic and isosmotic shrinkage on ion transport and protein phosphorylation in C11-MDCK cells resembling intercalated cells from collecting ducts and in vascular smooth muscle cells (VSMC) from the rat aorta. Hyperosmotic shrinkage was triggered by cell exposure to hypertonic medium, whereas isosmotic shrinkage was evoked by cell transfer from an hypoosmotic to an isosmotic environment. Despite a similar cell volume decrease of 40%-50%, the consequences of hyperosmotic and isosmotic shrinkage on cellular functions were sharply different. In C11-MDCK and VSMC, hyperosmotic shrinkage completely inhibited Na(+),K(+)-ATPase and Na(+),P(i) cotransport. In contrast, in both types of cells isosmotic shrinkage slightly increased rather than suppressed Na(+),K(+)-ATPase and did not change Na(+),P(i) cotransport. In C11-MDCK cells, phosphorylation of JNK1/2 and Erk1/2 mitogen-activated protein kinases was augmented in hyperosmotically shrunken cells by ∼7- and 2-fold, respectively, but was not affected in cells subjected to isosmotic shrinkage. These results demonstrate that the data obtained in cells subjected to hyperosmotic shrinkage cannot be considered as sufficient proof implicating cell volume perturbations in the regulation of cellular functions under isosmotic conditions.

Prediction of standard cell types and functional markers from textual descriptions of flow cytometry gating definitions using machine learning.

BACKGROUND: A key step in clinical flow cytometry data analysis is gating, which involves the identification of cell populations. The process of gating produces a set of reportable results, which are typically described by gating definitions. The non-standardized, non-interpreted nature of gating definitions represents a hurdle for data interpretation and data sharing across and within organizations. Interpreting and standardizing gating definitions for subsequent analysis of gating results requires a curation effort from experts. Machine learning approaches have the potential to help in this process by predicting expert annotations associated with gating definitions. METHODS: We created a gold-standard dataset by manually annotating thousands of gating definitions with cell type and functional marker annotations. We used this dataset to train and test a machine learning pipeline able to predict standard cell types and functional marker genes associated with gating definitions. RESULTS: The machine learning pipeline predicted annotations with high accuracy for both cell types and functional marker genes. Accuracy was lower for gating definitions from assays belonging to laboratories from which limited or no prior data was available in the training. Manual error review ensured that resulting predicted annotations could be reused subsequently as additional gold-standard training data. CONCLUSIONS: Machine learning methods are able to consistently predict annotations associated with gating definitions from flow cytometry assays. However, a hybrid automatic and manual annotation workflow would be recommended to achieve optimal results.

Activation of P2Y receptors causes strong and persistent shrinkage of C11-MDCK renal epithelial cells.

Purinergic receptors activate diverse signaling cascades and regulate the activity of cell volume-sensitive ion transporters. However, the effects of ATP and other agonists of P2 receptors on cell volume dynamics are only scarcely studied. In the present work, we used the recently developed dual-image surface reconstruction technique to explore the influence of purinergic agonists on cell volume in the C11-Madin-Darby canine kidney cell line resembling intercalated cells from kidney collecting ducts. Unexpectedly, we found that ATP and UTP triggered very robust (55-60%) cell shrinkage that lasted up to 2 h after agonist washout. Purinergic regulation of cell volume required increases in intracellular Ca(2+) and could be partially mimicked by the Ca(2+)-ionophore ionomycin or activation of protein kinase C by 4ß-phorbol 12-myristate 13-acetate. Cell shrinkage was accompanied by strong reductions in intracellular K(+) and Cl(-) content measured using steady-state (86)Rb(+) and (36)Cl(-) distribution. Both shrinkage and ion efflux in ATP-treated cells were prevented by the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and by the BK(Ca) channel inhibitors charybdotoxin, iberiotoxin, and paxilline. To evaluate the significance of cell-volume changes in purinergic signaling, we measured the impact of ATP on the expression of the immediate-early gene c-Fos. Thirty-minute treatment with ATP increased c-Fos immunoreactivity by approximately fivefold, an effect that was strongly inhibited by charybdotoxin and completely prevented by NPPB. Overall, our findings suggest that ATP-induced cell-volume changes are partially responsible for the physiological actions of purinergic agonists.

The death of ouabain-treated renal epithelial C11-MDCK cells is not mediated by swelling-induced plasma membrane rupture.

This study examined the role of cell volume modulation in plasma membrane rupture and death documented in ouabain-treated renal epithelial cells. Long-term exposure to ouabain caused massive death of C11-MDCK (Madin-Darby canine kidney) epithelial cells, documented by their detachment, chromatin cleavage and complete loss of lactate dehydrogenase (LDH), but did not affect the survival of vascular smooth muscle cells (VSMCs) from the rat aorta. Unlike the distinct impact on cell survival, 2-h exposure to ouabain led to sharp elevation of the [Na⁺](i)/[K⁺](i) ratio in both cell types. A similar increment of Na⁺(i) content was evoked by sustained inhibition of Na⁺,K⁺-ATPase in K⁺-free medium. However, in contrast to ouabain, C11-MDCK cells survived perfectly during 24-h exposure to K⁺-free medium. At 3 h, the volume of ouabain-treated C11-MDCK cells and VSMCs, measured by the recently developed dual-image surface reconstruction technique, was increased by 16 and 12%, respectively, whereas 5-10 min before the detachment of ouabain-treated C11-MDCK cells, their volume was augmented by ~30-40%. To examine the role of modest swelling in the plasma membrane rupture of ouabain-treated cells, we compared actions of hypotonic medium on volume and LDH release. We observed that LDH release from hypoosmotically swollen C11-MDCK cells was triggered when their volume was increased by approximately fivefold. Thus, our results showed that the rupture of plasma membranes in ouabain-treated C11-MDCK cells was not directly caused by cell volume modulation evoked by Na⁺,K⁺-ATPase inhibition and inversion of the [Na⁺](i)/[K⁺](i) ratio.

HCO3-dependent impact of Na+,K+,2Cl- cotransport in vascular smooth muscle excitation-contraction coupling.

In smooth muscles, inhibition of Na(+),K(+),2Cl(-) cotransport (NKCC) by bumetanide decreased intracellular Cl(-) content ([Cl(-)](i)) and suppressed the contractions triggered by diverse stimuli. This study examines whether or not bicarbonate, a regulator of several Cl(-) transporters, affects the impact of NKCC in excitation-contraction coupling. Addition of 25 mM NaHCO(3) attenuated the inhibitory action of bumetanide on mesenteric artery contractions evoked by 30 mM KCl and phenylephrine (PE) by 5 and 3-fold, respectively. In cultured vascular smooth muscle cells, NaHCO(3) almost completely abolished inhibitory actions of bumetanide on transient depolarization and [Ca(2+)](i) elevation triggered by PE. In bicarbonate-free medium, bumetanide decreased [Cl(-)](i) by approximately 15%; this effect was almost totally abrogated by NaHCO(3). The addition of NaHCO(3) resulted in 2-fold inhibition of NKCC activity and 3-fold attenuation of [Cl(-)](i). These data strongly suggest that extracellular HCO(3)(-) diminishes the NKCC-sensitive component of excitation-contraction coupling via suppression of this carrier.

Excitation-contraction coupling in resistance mesenteric arteries: evidence for NKCC1-mediated pathway.

Bumetanide and other high-ceiling diuretics (HCD) attenuate myogenic tone and contractions of vascular smooth muscle cells (VSMC) triggered by diverse stimuli. HCD outcome may be mediated by their interaction with NKCC1, the only isoform of Na(+), K(+), 2Cl(-) cotransporter expressed in VSMC as well as with targets distinct from this carrier. To examine these hypotheses, we compared the effect of bumetanide on contractions of mesenteric arteries from wild-type and NKCC1 knockout mice. In mesenteric arteries from wild-type controls, 100 microM bumetanide evoked a decrease of up to 4-fold in myogenic tone and contractions triggered by modest [K(+)](o)-induced depolarization, phenylephrine and UTP. These actions of bumetanide were preserved after inhibition of nitric oxide synthase with NG-nitro-l-arginine methyl ester, but were absent in mesenteric arteries from NKCC1(-/-) mice. The data show that bumetanide inhibits VSMC contractile responses via its interaction with NKCC1 and independently of nitric oxide production by endothelial cells.

Augmented gene expression triggered by Na+,K+-ATPase inhibition: Role of Ca2+i-mediated and -independent excitation-transcription coupling.

In rat vascular smooth muscle cells (RVSMC), 3-h Na+,K+-ATPase inhibition by ouabain or in K+-free medium resulted in the inversion of the [Na+]i/[K+]i ratio and elevation up to 7-fold the content of Egr1, Atf3, Nr4a1 and Ptgs2 mRNAs. Ouabain increased the rate of 45Ca2+ influx by 2-fold that was abolished by L-type voltage-gated Ca2+ channel blocker nicardipine, but it was resistant to Na+/Ca2+ exchanger inhibitor KB-R7943. To study the role of Ca2+-mediated signaling in the expression of Na+i/K+i-sensitive genes we used intracellular Ca2+ chelator BAPTA and incubated RVSMC in Ca2+-free medium. The elevation of Nr4a1 and Ptgs2 expression triggered by ouabain was diminished in Ca2+-depeleted cells as well as in the presence of nicardipine and calmodulin antagonists A-7 and W-7. Ptgs2 expression was also suppressed by inhibitor of Ca2+/calmodulin-dependent protein kinase (CaMKII) KN-93 whereas increment of Nr4a1 content triggered by ouabain was attenuated by inhibitor of Ca2+/calmodulin-dependent protein phosphatase (calcineurin, CaN) cyclosporin A. Neither Ca2+ depletion nor above listed compounds had any impact on the augmented expression of Egr1 and Atf3 in ouabain-treated RVSMC. Our results strongly suggest that dissipation of transmembrane gradient of monovalent cations increases Ptgs2 and Nr4a1 transcription via augment Ca2+ influx through L-type Ca2+ channels that, in turn, leads to CaMKII-mediated phosphorylation of CREB and calcineurin-mediated dephosphorylation of NFAT, respectively. Additional experiments should be performed to identify intermediates of Na+i,K+i-mediated Ca2+-independent excitation-transcription coupling involved the regulation of Egr1 and Atf3 expression.