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Epithelial-mesenchymal cell interactions and factors that control normal lung development are key players in lung injury, repair, and fibrosis. A number of studies have investigated the roles and sources of epithelial progenitors during lung regeneration; such information, however, is limited in lung fibroblasts. Thus, understanding the origin, phenotype, and roles of fibroblast progenitors in lung development, repair, and regeneration helps address these limitations. Using a combination of platelet-derived growth factor receptor α-green fluorescent protein (PDGFRα-GFP) reporter mice, microarray, real-time polymerase chain reaction, flow cytometry, and immunofluorescence, we characterized two distinct interstitial resident fibroblasts, myo- and matrix fibroblasts, and identified a role for PDGFRα kinase activity in regulating their activation during lung regeneration. Transcriptional profiling of the two populations revealed a myo- and matrix fibroblast gene signature. Differences in proliferation, smooth muscle actin induction, and lipid content in the two subpopulations of PDGFRα-expressing fibroblasts during alveolar regeneration were observed. Although CD140α(+)CD29(+) cells behaved as myofibroblasts, CD140α(+)CD34(+) appeared as matrix and/or lipofibroblasts. Gain or loss of PDGFRα kinase activity using the inhibitor nilotinib and a dominant-active PDGFRα-D842V mutation revealed that PDGFRα was important for matrix fibroblast differentiation. We demonstrated that PDGFRα signaling promotes alveolar septation by regulating fibroblast activation and matrix fibroblast differentiation, whereas myofibroblast differentiation was largely PDGFRα independent. These studies provide evidence for the phenotypic and functional diversity as well as the extent of specificity of interstitial resident fibroblasts differentiation during regeneration after partial pneumonectomy.

Many studies have investigated the source and role of epithelial progenitors during lung development; such information is limited for fibroblast populations and their complex role in the developing lung. In this study, we characterized the spatial location, mRNA expression and Immunophenotyping of PDGFRα+ fibroblasts during sacculation and alveolarization. Confocal microscopy identified spatial association of PDGFRα expressing fibroblasts with proximal epithelial cells of the branching bronchioles and the dilating acinar tubules at E16.5; with distal terminal saccules at E18.5; and with alveolar epithelial cells at PN7 and PN28. Immunohistochemistry for alpha smooth muscle actin revealed that PDGFRα+ fibroblasts contribute to proximal peribronchiolar smooth muscle at E16.5 and to transient distal alveolar myofibroblasts at PN7. Time series RNA-Seq analyses of PDGFRα+ fibroblasts identified differentially expressed genes that, based on gene expression similarity were clustered into 7 major gene expression profile patterns. The presence of myofibroblast and smooth muscle precursors at E16.5 and PN7 was reflected by a two-peak gene expression profile on these days and gene ontology enrichment in muscle contraction. Additional molecular and functional differences between peribronchiolar smooth muscle cells at E16.5 and transient intraseptal myofibroblasts at PN7 were suggested by a single peak in gene expression at PN7 with functional enrichment in cell projection and muscle cell differentiation. Immunophenotyping of subsets of PDGFRα+ fibroblasts by flow cytometry confirmed the predicted increase in proliferation at E16.5 and PN7, and identified subsets of CD29+ myofibroblasts and CD34+ lipofibroblasts. These data can be further mined to develop novel hypotheses and valuable understanding of the molecular and cellular basis of alveolarization.

BACKGROUND AND PURPOSE Ginsenosides are the main constituents for the pharmacological effects of Panax ginseng. Such effects of ginsenosides including cardioprotective and anti‐platelet activities have shown stability and bioavailability limitations. However, information on the anti‐platelet activity of ginsenoside‐Rp1 (G‐Rp1), a stable derivative of ginsenoside‐Rg3, is scarce. We examined the ability of G‐Rp1 to modulate agonist‐induced platelet activation. EXPERIMENTAL APPROACH G‐Rp1 in vitro and ex vivo effects on agonist‐induced platelet‐aggregation, granule‐secretion, [Ca2+]i mobilization, integrin‐αIIbβ3 activation were examined. Vasodilator‐stimulated phosphoprotein (VASP) and MAPK expressions and levels of tyrosine phosphorylation of the glycoprotein VI (GPVI) signalling pathway components were also studied. G‐Rp1 effects on arteriovenous shunt thrombus formation in rats or tail bleeding time and ex vivo coagulation time in mice were determined. KEY RESULT G‐Rp1 markedly inhibited platelet aggregation induced by collagen, thrombin or ADP. While G‐Rp1 elevated cAMP levels, it dose‐dependently suppressed collagen‐induced ATP‐release, thromboxane secretion, p‐selectin expression, [Ca2+]i mobilization and αIIbβ3 activation and attenuated p38MAPK and ERK2 activation. Furthermore, G‐Rp1 inhibited tyrosine phosphorylation of multiple components (Fyn, Lyn, Syk, LAT, PI3K and PLCγ2) of the GPVI signalling pathway. G‐Rp1 inhibited in vivo thrombus formation and ex vivo platelet aggregation and ATP secretion without affecting tail bleeding time and coagulation time, respectively. CONCLUSION AND IMPLICATIONS G‐Rp1 inhibits collagen‐induced platelet activation and thrombus formation through modulation of early GPVI signalling events, and this effect involves VASP stimulation, and ERK2 and p38‐MAPK inhibition. These data suggest that G‐Rp1 may have therapeutic potential for the treatment of cardiovascular diseases involving aberrant platelet activation.

GTPase of immunity-associated protein 5 (Gimap5) is linked with lymphocyte survival, autoimmunity, and colitis, but its mechanisms of action are unclear. Here, we show that Gimap5 is essential for the inactivation of glycogen synthase kinase-3β (GSK3β) following T cell activation. In the absence of Gimap5, constitutive GSK3β activity constrains c-Myc induction and NFATc1 nuclear import, thereby limiting productive CD4+ T cell proliferation. Additionally, Gimap5 facilitates Ser389 phosphorylation and nuclear translocation of GSK3β, thereby limiting DNA damage in CD4+ T cells. Importantly, pharmacological inhibition and genetic targeting of GSK3β can override Gimap5 deficiency in CD4+ T cells and ameliorates immunopathology in mice. Finally, we show that a human patient with a GIMAP5 loss-of-function mutation has lymphopenia and impaired T cell proliferation in vitro that can be rescued with GSK3 inhibitors. Given that the expression of Gimap5 is lymphocyte-restricted, we propose that its control of GSK3β is an important checkpoint in lymphocyte proliferation.

Background. Progressive diseases including cancer, metabolic, and cardiovascular disorders are marked by platelet activation and chronic inflammation. Studies suggest that dietary flavonoids such as quercetin possess antioxidant, anti-inflammatory, and antiplatelet properties, which could prevent various chronic diseases including atherosclerosis and thrombosis. However, the mechanism and the signaling pathway that links quercetin's antiplatelet activity with its anti-inflammatory property is limited and thus further exploration is required. The aim of this paper was to examine the link between antiplatelet and anti-inflammatory roles of quercetin in agonist-induced platelet activation. Methods. Quercetin effects on agonist-activated platelet-aggregation, granule-secretion, [Ca2+]i, and glycoprotein-IIb/IIIa activation were examined. Its effects on PI3K/Akt, VASP, and MAPK phosphorylations were also studied on collaged-activated platelets. Results. Quercetin dose dependently suppressed collagen, thrombin, or ADP-induced platelet aggregation. It significantly inhibited collagen-induced ATP release, P-selectin expression, [Ca2+]i mobilization, integrin-α IIb β 3 activation, and augmented cAMP and VASP levels. Moreover, quercetin attenuated PI3K, Akt, ERK2, JNK1, and p38 MAPK activations, which were supported by platelet-aggregation inhibition with the respective kinase inhibitors. Conclusion. Quercetin-mediated antiplatelet activity involves PI3K/Akt inactivation, cAMP elevation, and VASP stimulation that, in turn, suppresses MAPK phosphorylations. This result suggests quercetin may have a potential to treat cardiovascular diseases involving aberrant platelet activation and inflammation.

In mammals, the circadian clock coordinates cell physiological processes including inflammation. Recent studies suggested a crosstalk between these two pathways. However, the mechanism of how inflammation affects the clock is not well understood. Here, we investigated the role of the proinflammatory transcription factor NF-κB in regulating clock function. Using a combination of genetic and pharmacological approaches, we show that perturbation of the canonical NF-κB subunit RELA in the human U2OS cellular model altered core clock gene expression. While RELA activation shortened period length and dampened amplitude, its inhibition lengthened period length and caused amplitude phenotypes. NF-κB perturbation also altered circadian rhythms in the master suprachiasmatic nucleus (SCN) clock and locomotor activity behavior under different light/dark conditions. We show that RELA, like the clock repressor CRY1, repressed the transcriptional activity of BMAL1/CLOCK at the circadian E-box cis-element. Biochemical and biophysical analysis showed that RELA binds to the transactivation domain of BMAL1. These data support a model in which NF-kB competes with CRY1 and coactivator CBP/p300 for BMAL1 binding to affect circadian transcription. This is further supported by chromatin immunoprecipitation analysis showing that binding of RELA, BMAL1 and CLOCK converges on the E-boxes of clock genes. Taken together, these data support a significant role for NF-κB in directly regulating the circadian clock and highlight mutual regulation between the circadian and inflammatory pathways.

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