We have previously demonstrated the in vivo importance of the Akt-eNOS substrate-kinase relationship, as defective postnatal angiogenesis characteristic of global Akt1-null mice is rescued when bred to 'gain-of-function' eNOS S1176D mutant mice. While multiple studies support the vascular protective role of endothelial NO generation, the causal role of Akt1-dependent eNOS S1176 phosphorylation during atherosclerotic plaque formation is not yet clear.
We herein bred congenic 'loss-of-function' eNOS S1176A and 'gain-of-function' eNOS S1176D mutant mice to the exacerbated atherogenic Akt1-/-; ApoE-/- double knockout mice to definitively test the importance of Akt-mediated eNOS S1176 phosphorylation during atherogenesis. We find that a single amino acid substitution at the eNOS S1176 phosphorylation site yields divergent effects on atherosclerotic plaque formation, as an eNOS phospho-mimic aspartate (D) substitution at S1176 leads to favorable lipid profiles and decreased indices of atherosclerosis, even when on a proatherogenic Akt1 global deletion background. Conversely, mice harboring an unphosphorylatable mutation to alanine (S1176A) result in increased plasma lipids, increased lesion formation and cellular apoptosis, phenocopying the physiological consequence of eNOS deletion and/or impaired enzyme function. Furthermore, gene expression analyses of whole aortas indicate a combinatorial detriment from NO deficiency and Western Diet challenge, as 'loss-of-function' eNOS S1176A mice on a Western Diet present a unique expression pattern indicative of augmented T-cell activity when compared to eNOS S1176D mice.
By using genetic epistasis approaches, we conclusively demonstrate that Akt-mediated eNOS S1176 phosphorylation and subsequent eNOS activation remains to be the most physiologically relevant method of NO production to promote athero-protective effects.
© 2023 Nguyen, Rahman, Sessa and Lee.

Fibrosis is defined as an excessive accumulation of extracellular matrix (ECM) components. Many organs are subjected to fibrosis including the lung, liver, heart, skin, kidney, and muscle. Muscle fibrosis occurs in response to trauma, aging, or dystrophies and impairs muscle function. Fibrosis represents a hurdle for the treatment of human muscular dystrophies. While data on the mechanisms of fibrosis have mostly been investigated in mice, dystrophic mouse models often do not recapitulate fibrosis as observed in human patients. Consequently, the cellular and molecular mechanisms that lead to fibrosis in human muscle still need to be identified.
Combining mass cytometry, transcriptome profiling, in vitro co-culture experiments, and in vivo transplantation in immunodeficient mice, we investigated the role and nature of nonmyogenic cells (fibroadipogenic progenitors, FAPs) from human fibrotic muscles of healthy individuals (FibMCT ) and individuals with oculopharyngeal muscular dystrophy (OPMD; FibMOP ), as compared with nonmyogenic cells from human nonfibrotic muscle (MCT ).
We found that the proliferation rate of FAPs from fibrotic muscle is 3-4 times higher than those of FAPs from nonfibrotic muscle (population doubling per day: MCT 0.2 ± 0.1, FibMCT 0.7 ± 0.1, and FibMOP 0.8 ± 0.3). When cocultured with muscle cells, FAPs from fibrotic muscle impair the fusion index unlike MCT FAPs (myoblasts alone 57.3 ± 11.1%, coculture with MCT 43.1 ± 8.9%, with FibMCT 31.7 ± 8.2%, and with FibMOP 36.06 ± 10.29%). We also observed an increased proliferation of FAPs from fibrotic muscles in these co-cultures in differentiation conditions (FibMCT +17.4%, P < 0.01 and FibMOP +15.1%, P < 0.01). This effect is likely linked to the increased activation of the canonical TGFβ-SMAD pathway in FAPs from fibrotic muscles evidenced by pSMAD3 immunostaining (P < 0.05). In addition to the profibrogenic TGFβ pathway, we identified endothelin as a new actor implicated in the altered cross-talk between muscle cells and fibrotic FAPs, confirmed by an improvement of the fusion index in the presence of bosentan, an endothelin receptor antagonist (from 33.8 ± 10.9% to 52.9 ± 10.1%, P < 0.05).
Our data demonstrate the key role of FAPs and their cross-talk with muscle cells through a paracrine signalling pathway in fibrosis of human skeletal muscle and identify endothelin as a new druggable target to counteract human muscle fibrosis.
© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.

Cell surface receptors control how cells respond to their environment. Many cell surface receptors recycle from endosomes to the plasma membrane via a recently discovered pathway, which includes sorting-nexin SNX17, Retriever, WASH, and CCC complexes. Here, using mammalian cells, we discover that PIKfyve and its upstream PI3-kinase VPS34 positively regulate this pathway. VPS34 produces phosphatidylinositol 3-phosphate (PI3P), which is the substrate for PIKfyve to generate PI3,5P2. We show that PIKfyve controls recycling of cargoes including integrins, receptors that control cell migration. Furthermore, endogenous PIKfyve colocalizes with SNX17, Retriever, WASH, and CCC complexes on endosomes. Importantly, PIKfyve inhibition results in displacement of Retriever and CCC from endosomes. In addition, we show that recruitment of SNX17 is an early step and requires VPS34. These discoveries suggest that VPS34 and PIKfyve coordinate an ordered pathway to regulate recycling from endosomes and suggest how PIKfyve functions in cell migration.
© 2022, Giridharan et al.

h4>Background: /h4> The interaction of integrins and growth factor receptors in cells is tightly regulated, ensuring cell survival, proliferation, differentiation, adhesion, and migration. The IR generates reactive oxygen species, which leads to ECM remodeling and cell adhesion through the activation of proteases, soluble cytokines and growth factors. Integrins and adhesion of cells to ECM confer higher resistance to ionizing radiation and cytotoxic drug, a phenomenon known as cell adhesion mediated radiation resistance (CAM-RR) and cell adhesion mediated drug resistance (CAM-DR) . Integrins’ involvement in CML progression is well appreciated through its survival, adhesion and migration signaling. The evaluation of global genetic response (in microarray) of ionizing radiation (IR) on integrins expression has not been attempted to specify theirrole and other cell adhesion molecules (CAMs) in CML. In this study, we have compared the microarray based CAMs response in myelogenous leukemia cells onIR exposure. h4>Results: /h4>: Results revealed differential regulation of many CAMs, with strongest expression of integrin β2 (CD18), whose role has not been fully appreciated in CML due to low level of expression. However, the synergistic LiCl(GSK3β inhibitor) and IR treatment significantly upregulates CD18 expression leading to enhanced survival, cell adhesion mediated drug/radiation resistance (CAM-DR/RR) and transcription of migration related genes. These effects could be undermined in the presence of CD18 antibody. This may be one of the reasons for CML resistance to radiation therapy and its relapse upon stem cell transplantation. h4>Conclusion: /h4> This study proposes CD18 antagonist administration as an adjuvant in anti-CML therapy and other cancers in which it displays aberrant expression subsuming the contraindication of GSK3β inhibitor. Nevertheless, the CD18 mediated cell adhesion in tumor progression beckons development of improved drug regimensandidentification of diagnostic and prognostic signature for CML.

Although anti-cancer properties of the natural compound curcumin have been reported, low absorption and rapid metabolisation limit clinical use. The present study investigated whether irradiation with visible light may enhance the inhibitory effects of low-dosed curcumin on prostate cancer cell growth, proliferation, and metastasis in vitro. DU145 and PC3 cells were incubated with low-dosed curcumin (0.1-0.4 µg/mL) and subsequently irradiated with 1.65 J/cm2 visible light for 5 min. Controls remained untreated and/or non-irradiated. Cell growth, proliferation, apoptosis, adhesion, and chemotaxis were evaluated, as was cell cycle regulating protein expression (CDK, Cyclins), and integrins of the α- and β-family. Curcumin or light alone did not cause any significant effects on tumor growth, proliferation, or metastasis. However, curcumin combined with light irradiation significantly suppressed tumor growth, adhesion, and migration. Phosphorylation of CDK1 decreased and expression of the counter-receptors cyclin A and B was diminished. Integrin α and β subtypes were also reduced, compared to controls. Irradiation distinctly enhances the anti-tumor potential of curcumin in vitro and may hold promise in treating prostate cancer.