Metabolic plasticity of vascular smooth muscle cells in vascular disease

Unlike cardiac or skeletal muscle cells, vascular smooth muscle cells (VSMCs) retain a remarkable degree of plasticity. On environmental cues they can dedifferentiate from a quiescent contractile state towards phenotypes of increased proliferation and migration as well as secretory capacity and inflammation. This ability to transition between different phenotypes is a prerequisite for physiological vascular remodeling processes, but also plays a key role in the pathogenesis of virtually all vascular diseases. These vascular diseases, above all atherosclerosis resulting in myocardial infarction or stroke, are still the leading cause of death worldwide. Based on the respective metabolic requirements of proliferating versus quiescent cells it was hypothesised that VSMCs undergo metabolic changes during dedifferentiation. Therefore, the aim of this study was to investigate the metabolic adaptions VSMCs exhibit during phenotypic transition and identify possible regulators thereof. Utilising two in vitro models and one in vivo model for VSMC dedifferentiation, this study showed that dedifferentiated VSMCs shift their energy generation from mitochondrial respiration towards elevated glycolysis and lactate production, reminiscent of the Warburg effect observed in cancer cells. Dedifferentiated VSMCs also displayed reduced expression of genes involved in mitochondrial respiration, lower mitochondrial abundance and altered mitochondrial shape, indicating a strong association between mitochondrial homeostasis and VSMC plasticity. The second objective of this study was to investigate whether intervention in VSMC metabolism would affect VSMC plasticity and vascular remodeling. Two known regulators of metabolism, Sirt6 and Sirt7, were chosen for their regulatory function in glucose and mitochondrial metabolism, respectively. The effects of VSMC specific knock-outs of Sirt6 and Sirt7 on VSMC dedifferentiation and proliferation were assessed in vitro and in vivo. Both sirtuins were expected to display atheroprotective functions. This could be confirmed for Sirt7 as the VSMC-specific knock-out of Sirt7 resulted in elevated neointima formation in a carotid artery ligation mouse model and increased plaque sizes in an ApoE-/- atherosclerosis mouse model. VSMC-specific knock-out of Sirt6 did not impact both these parameters. Contrary to expectations, the effects of the Sirt7 knock-out did not seem to be mediated by regulation of mitochondrial homeostasis. The atheroprotective role shown in this study, nevertheless renders Sirt7 an interesting target for the treatment of vascular diseases

PRAS40 suppresses atherogenesis through inhibition of mTORC1-dependent pro-inflammatory signaling in endothelial cells

Endothelial pro-inflammatory activation plays a pivotal role in atherosclerosis, and many pro-inflammatory and atherogenic signals converge upon mechanistic target of rapamycin (mTOR). Inhibitors of mTOR complex 1 (mTORC1) reduced atherosclerosis in preclinical studies, but side effects including insulin resistance and dyslipidemia limit their clinical use in this context. Therefore, we investigated PRAS40, a cell type-specific endogenous modulator of mTORC1, as alternative target. Indeed, we previously found PRAS40 gene therapy to improve metabolic profile; however, its function in endothelial cells and its role in atherosclerosis remain unknown. Here we show that PRAS40 negatively regulates endothelial mTORC1 and pro-inflammatory signaling. Knockdown of PRAS40 in endothelial cells promoted TNFα-induced mTORC1 signaling, proliferation, upregulation of inflammatory markers and monocyte recruitment. In contrast, PRAS40-overexpression blocked mTORC1 and all measures of pro-inflammatory signaling. These effects were mimicked by pharmacological mTORC1-inhibition with torin1. In an in vivo model of atherogenic remodeling, mice with induced endothelium-specific PRAS40 deficiency showed enhanced endothelial pro-inflammatory activation as well as increased neointimal hyperplasia and atherosclerotic lesion formation. These data indicate that PRAS40 suppresses atherosclerosis via inhibition of endothelial mTORC1-mediated pro-inflammatory signaling. In conjunction with its favourable effects on metabolic homeostasis, this renders PRAS40 a potential target for the treatment of atherosclerosis

Serum induces transcription of Hey1 and Hey2 genes by Alk1 but not Notch signaling in endothelial cells.

The transcriptional repressors Hey1 and Hey2 are primary target genes of Notch signaling in the cardiovascular system and induction of Hey gene expression is often interpreted as activation of Notch signaling. Here we report that treatment of primary human endothelial cells with serum or fresh growth medium led to a strong wave of Hey1 and Hey2 transcription lasting for approximately three hours. Transcription of other Notch target genes (Hes1, Hes5, ephrinB2, Dll4) was however not induced by serum in endothelial cells. Gamma secretase inhibition or expression of dominant-negative MAML1 did not prevent the induction of Hey genes indicating that canonical Notch signaling is dispensable. Pretreatment with soluble BMP receptor Alk1, but not Alk3, abolished Hey gene induction by serum. Consequently, the Alk1 ligand BMP9 stimulated Hey gene induction in endothelial cells. Several other cell types however did not show such a strong BMP signaling and consequently only a very mild induction of Hey genes. Taken together, the experiments revealed that bone morphogenic proteins within the serum of cell culture medium are potent inducers of endothelial Hey1 and Hey2 gene expression within the first few hours after medium change

Marginal activation of <i>Hey1</i>, <i>Hey2</i> and <i>ID2</i> in non-endothelial cells.

<p>HUASMC, Hela, A549 and HEK293T cells were cultured in their standard medium, before they were left untreated or the medium was changed to basal medium containing 3% FCS. The transcript levels of <i>Hey1</i>, <i>Hey2</i> and <i>ID2</i> were determined 1.5h after changing the medium and were normalized to the samples, whose medium was not changed. Mean and standard deviation, n = 3, * p<0.05, ** p<0.01.</p

Transcriptional activation of <i>Hey1</i> and <i>Hey2</i> by serum is caused by BMP-Smad-signaling.

<p>A) HUVEC were cultured in growth medium and were pretreated for 30 min with 3μM LDN193189 or a corresponding volume of DMSO. The medium was left on the cells or it was replaced with fresh growth medium (GM) or basal medium (BM) with 3% FCS, both of which were containing LDN193189 or DMSO respectively. The western blot is showing phosphorylated Smad 1/5 (Ser463/465) and VCP in cells lysed 1.5h after medium change. B) HUVEC were cultured in growth medium, which was replaced with fresh growth medium or basal medium containing 3% FCS. Relative <i>ID2</i> mRNA levels are shown 1.5 h after medium change normalized to samples not subjected to medium change (untreated). C) HUVEC were cultured in growth medium and were pretreated over night with 25μM DAPT, 30 min with 3μM LDN193189 or the corresponding volumes of DMSO. Medium was not replaced (DMSO, DAPT, LDN, DAPT + LDN) or changed to basal medium with 3% FCS complemented with the inhibitors or DMSO respectively. The transcript levels of <i>Hey1</i>, <i>Hey2</i> and <i>ID2</i> were measured 1.5h after changing the medium. D) HUVEC, HUAEC and HBMVEC were cultured in growth medium and were pretreated 30 min with 3μM LDN193189 or DMSO. The medium remained unchanged (untreated) or was replaced with basal medium with 3% FCS containing LDN193189 or DMSO. The transcript levels of <i>Hey1</i>, <i>Hey2</i> and <i>ID2</i> were measured 1.5h after changing the medium. E) HUVEC were treated with medium containing no or 3% FCS and either soluble Alk1-Fc, Alk3-Fc or control IgG (5 μg/ml) for 1.5h. Transcript levels of <i>Hey1</i>,<i>Hey2</i> or <i>Id2</i> and significances are calculated to the basal medium IgG control. F) Cells were treated with basal medium containing 3% FCS, 10 ng/ml recombinant human BMP9 or 10 ng/ml recombinant human TGFβ1 for 1.5h. Transcript levels of <i>Hey1</i>, <i>Hey2</i> and <i>Id2</i> are calculated to the sample treated with basal medium. Mean and standard deviation, n≥3, * p<0.05, ** p<0.01, *** p<0.001.</p

Smad1 binding sites in the promoter regions of <i>Hey1</i> and <i>Hey2</i>.

<p>Schematic alignment of the <i>Hey1</i> and <i>Hey2</i> promoters of <i>Homo sapiens</i> (human), <i>Pan troglodytes</i> (chimpanzee), <i>Bos Taurus</i> (cow) and <i>Rattus norvegicus</i> (rat). Potential Smad1 binding sites (GCCGnCGC) are depicted in red. For a clearer visualization, recognition sites, which are separated by less than 10 bp, are only depicted by one red bar.</p

Serum induces transcription of <i>Hey1</i> and <i>Hey2</i>.

<p>A) HUVEC were grown in full growth medium and medium was changed to fresh growth medium at t0. The chart depicts the mRNA levels of <i>Hey1</i> and <i>Hey2</i>. B) HUVEC were grown in full growth medium and medium was changed either to fresh growth medium (GM), basal medium (BM) supplemented with 3% FCS or BM supplemented with a mix of growth factors (supplements). Transcript levels of <i>Hey1</i> and <i>Hey2</i> were determined after 1.5h and are shown in relation to samples with no medium change (untreated). C) HUVEC were grown in full growth medium and medium was changed to basal medium supplemented with 3% FCS purchased from two different suppliers, charcoal filtered FCS, horse serum or basal medium complemented with 3% or 10% human serum. Transcript levels of <i>Hey1</i> and <i>Hey2</i> are shown 1.5h after medium change. n = 3 (a,b) or one experiment shown (c). Mean and standard deviation of biological (a,b) or technical (c) replicates. Significant differences were calculated between the conditions marked with asterisks and t0 (a) or untreated control respectively.* p<0.05, ** p<0.01, *** p<0.001.</p

Induction of <i>Hey</i> genes by serum does not depend on Notch signaling.

<p>A) HUVEC were grown in growth medium containing 25μM DAPT or an equivalent volume of DMSO. Medium was unchanged (DMSO, DAPT) or changed to fresh growth medium (GM) or basal medium (BM) with 3% FCS containing DAPT or DMSO respectively. Transcript levels of <i>Hey1</i> and <i>Hey2</i> were measured 1.5h after medium change and are shown in relation to the sample incubated with DMSO, whose medium was not changed. B) HUVEC were transduced with adenoviral vectors expressing GFP or dnMAML. Cells were grown in growth medium. The medium was either unchanged (GFP, dnMAML) or replaced with fresh growth medium or basal medium containing 3% FCS. Transcript levels were determined after 1.5h and are normalized to the cells transduced with GFP and unchanged medium. C) HUAEC were grown in growth medium (untreated) or the medium was substituted with fresh growth medium or basal medium containing 3% FCS. The western blot is showing the cleaved intracellular domain of Notch1 (NICD) and β-tubulin 30 minutes after medium change. The chart summarizes four independent experiments and depicts the relative levels of NICD normalized for β-tubulin. D) HUVEC were left untreated or the medium was changed to fresh growth medium or basal medium supplemented with 3% FCS. After incubating 1.5h, the cells were harvested and the mRNA levels of several established Notch target genes (<i>Hey1</i>, <i>Hey2</i>, <i>Hes1</i>, <i>Hes5</i>, <i>Dll4 EFNB2</i>) were determined. Fold changes and significances were calculated to the untreated control. Mean and standard deviation, n = 3, * p<0.05, ** p<0.01, *** p<0.001, n.s. not significant.</p

PP7L is essential for MAIL1‐mediated transposable element silencing and primary root growth

International audienceThe two paralogous Arabidopsis genes MAINTENANCE OF MERISTEMS (MAIN) and MAINTENANCE OF MERISTEMS LIKE1 (MAIL1) encode a conserved retrotransposon-related plant mobile domain and are known to be required for silencing of transposable elements (TE) and for primary root development. Loss of function of either MAIN or MAIL1 leads to release of heterochromatic TEs, reduced condensation of pericen-tromeric heterochromatin, cell death of meristem cells and growth arrest of the primary root soon after germination. Here, we show that they act in one protein complex that also contains the inactive isoform of PROTEIN PHOSPHATASE 7 (PP7), which is named PROTEIN PHOSPHATASE 7-LIKE (PP7L). PP7L was previously shown to be important for chloroplast biogenesis and efficient chloroplast protein synthesis. We show that loss of PP7L function leads to the same root growth phenotype as loss of MAIL1 or MAIN. In addition, pp7l mutants show similar silencing defects. Double mutant analyses confirmed that the three proteins act in the same molecular pathway. The primary root growth arrest, which is associated with cell death of stem cells and their daughter cells, is a consequence of genome instability. Our data demonstrate so far unrecognized functions of an inactive phosphatase isoform in a protein complex that is essential for silencing of heterochromatic elements and for maintenance of genome stability in dividing cells

Luminescent Dual Sensors Reveal Extracellular pH-Gradients and Hypoxia on Chronic Wounds That Disrupt Epidermal Repair

Wound repair is a quiescent mechanism to restore barriers in multicellular organisms upon injury. In chronic wounds, however, this program prematurely stalls. It is known that patterns of extracellular signals within the wound fluid are crucial to healing. Extracellular pH (pHe) is precisely regulated and potentially important in signaling within wounds due to its diverse cellular effects. Additionally, sufficient oxygenation is a prerequisite for cell proliferation and protein synthesis during tissue repair. It was, however, impossible to study these parameters in vivo due to the lack of imaging tools. Here, we present luminescent biocompatible sensor foils for dual imaging of pHe and oxygenation in vivo. To visualize pHe and oxygen, we used time-domain dual lifetime referencing (tdDLR) and luminescence lifetime imaging (LLI), respectively. With these dual sensors, we discovered centripetally increasing pHe-gradients on human chronic wound surfaces. In a therapeutic approach, we identify pHe-gradients as pivotal governors of cell proliferation and migration, and show that these pHe-gradients disrupt epidermal barrier repair, thus wound closure. Parallel oxygen imaging also revealed marked hypoxia, albeit with no correlating oxygen partial pressure (pO2)-gradient. This highlights the distinct role of pHe-gradients in perturbed healing. We also found that pHe-gradients on chronic wounds of humans are predominantly generated via centrifugally increasing pHe-regulatory Na+/H+-exchanger-1 (NHE1)-expression. We show that the modification of pHe on chronic wound surfaces poses a promising strategy to improve healing. The study has broad implications for cell science where spatial pHe-variations play key roles, e.g. in tumor growth. Furthermore, the novel dual sensors presented herein can be used to visualize pHe and oxygenation in various biomedical fields