Tunable DNMT1 degradation reveals DNMT1/DNMT3B synergy in DNA methylation and genome organization.

DNA methylation (DNAme) is a key epigenetic mark that regulates critical biological processes maintaining overall genome stability. Given its pleiotropic function, studies of DNAme dynamics are crucial, but currently available tools to interfere with DNAme have limitations and major cytotoxic side effects. Here, we present cell models that allow inducible and reversible DNAme modulation through DNMT1 depletion. By dynamically assessing whole genome and locus-specific effects of induced passive demethylation through cell divisions, we reveal a cooperative activity between DNMT1 and DNMT3B, but not of DNMT3A, to maintain and control DNAme. We show that gradual loss of DNAme is accompanied by progressive and reversible changes in heterochromatin, compartmentalization, and peripheral localization. DNA methylation loss coincides with a gradual reduction of cell fitness due to G1 arrest, with minor levels of mitotic failure. Altogether, this system allows DNMTs and DNA methylation studies with fine temporal resolution, which may help to reveal the etiologic link between DNAme dysfunction and human disease.

Detection of the interactions of tumour derived extracellular vesicles with immune cells is dependent on EV-labelling methods.

Cell-cell communication within the complex tumour microenvironment is critical to cancer progression. Tumor-derived extracellular vesicles (TD-EVs) are key players in this process. They can interact with immune cells and modulate their activity, either suppressing or activating the immune system. Deciphering the interactions between TD-EVs and immune cells is essential to understand immune modulation by cancer cells. Fluorescent labelling of TD-EVs is a method of choice to study such interaction. This work aims to determine the impact of EV labelling methods on the detection by imaging flow cytometry and multicolour spectral flow cytometry of EV interaction and capture by the different immune cell types within human Peripheral Blood Mononuclear Cells (PBMCs). EVs released by the triple-negative breast carcinoma cell line MDA-MB-231 were labelled either with the lipophilic dye MemGlow-488 (MG-488), Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) or through ectopic expression of a MyrPalm-superFolderGFP reporter (mp-sfGFP), which incorporates into EVs during their biogenesis. Our results show that these labelling strategies, although analysed with the same techniques, led to diverging results. While MG-488-labelled EVs incorporate in all cell types, CFSE-labelled EVs are restricted to a minor subset of cells and mp-sfGFP-labelled EVs are mainly detected in CD14+ monocytes which are the main uptakers of EVs and other particles, regardless of the labelling method. Furthermore, our results show that the method used for EV labelling influences the detection of the different types of EV interactions with the recipient cells. Specifically, MG-488, CFSE and mp-sfGFP result in observation suggesting, respectively, transient EV-PM interaction that results in dye transfer, EV content delivery, and capture of intact EVs. Consequently, the type of EV labelling method has to be considered as they can provide complementary information on various types of EV-cell interaction and EV fate.

Autophagy protein 5 controls flow-dependent endothelial functions.

Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.

Phenotyping polarization dynamics of immune cells using a lipid droplet-cell pairing microfluidic platform.

The immune synapse is the tight contact zone between a lymphocyte and a cell presenting its cognate antigen. This structure serves as a signaling platform and entails a polarization of intracellular components necessary to the immunological function of the cell. While the surface properties of the presenting cell are known to control the formation of the synapse, their impact on polarization has not yet been studied. Using functional lipid droplets as tunable artificial presenting cells combined with a microfluidic pairing device, we simultaneously observe synchronized synapses and dynamically quantify polarization patterns of individual B cells. By assessing how ligand concentration, surface fluidity, and substrate rigidity impact lysosome polarization, we show that its onset and kinetics depend on the local antigen concentration at the synapse and on substrate rigidity. Our experimental system enables a fine phenotyping of monoclonal cell populations based on their synaptic readout.

Ret kinase-mediated mechanical induction of colon stem cells by tumor growth pressure stimulates cancer progression in vivo.

How mechanical stress actively impacts the physiology and pathophysiology of cells and tissues is little investigated in vivo. The colon is constantly submitted to multi-frequency spontaneous pulsatile mechanical waves, which highest frequency functions, of 2 s period, remain poorly understood. Here we find in vivo that high frequency pulsatile mechanical stresses maintain the physiological level of mice colon stem cells (SC) through the mechanosensitive Ret kinase. When permanently stimulated by a magnetic mimicking-tumor growth analogue pressure, we find that SC levels pathologically increase and undergo mechanically induced hyperproliferation and tumorigenic transformation. To mimic the high frequency pulsatile mechanical waves, we used a generator of pulsed magnetic force stimulation in colonic tissues pre-magnetized with ultra-magnetic liposomes. We observed the pulsatile stresses using last generation ultra-wave dynamical high-resolution imaging. Finally, we find that the specific pharmacological inhibition of Ret mechanical activation induces the regression of spontaneous formation of SC, of CSC markers, and of spontaneous sporadic tumorigenesis in Apc mutated mice colons. Consistently, in human colon cancer tissues, Ret activation in epithelial cells increases with tumor grade, and partially decreases in leaking invasive carcinoma. High frequency pulsatile physiological mechanical stresses thus constitute a new niche that Ret-dependently fuels mice colon physiological SC level. This process is pathologically over-activated in the presence of permanent pressure due to the growth of tumors initiated by pre-existing genetic alteration, leading to mechanotransductive self-enhanced tumor progression in vivo, and repressed by pharmacological inhibition of Ret.

Extracellular vesicles from adipose stromal cells combined with a thermoresponsive hydrogel prevent esophageal stricture after extensive endoscopic submucosal dissection in a porcine model.

In this study, we investigated the combination of extracellular (nano) vesicles (EVs) from pig adipose tissue-derived stromal cells (ADSCs) and a thermoresponsive gel, Pluronic® F-127 (PF-127), to prevent stricture formation after endoscopic resection in a porcine model. ADSC EVs were produced at a liter scale by a high-yielding turbulence approach from ADSCs 3D cultured in bioreactors and characterized in terms of size, morphology and membrane markers. The thermoresponsive property of the PF-127 gel was assessed by rheology. The pro-regenerative potency of ADSC EVs was investigated ex vivo in esophageal biopsies under starvation. In vivo tests were performed in a porcine model after extended esophageal endoscopic mucosal dissection (ESD). Pigs were randomized into 3 groups: control (n = 6), gel (n = 6) or a combination of 1.45 × 1012 EVs + gel (n = 6). Application of gel ± EVs was performed just after ESD with a follow-up finalized on day 21 post-ESD. There was a trend towards less feeding disorder in the EV + gel group in comparison with the gel and the control groups (16.67% vs. 66.7% vs. 83.33%, respectively) but without reaching a statistically significant difference. A significant decrease in the esophageal stricture rate was confirmed by endoscopic, radiological and histological examination for the EV + gel group. A decrease in the mean fibrosis area and larger regenerated muscularis mucosae were observed for the EV + gel group. In summary, the application of EVs + gel after extended esophageal endoscopic resection succeeded in preventing stricture formation with an anti-fibrotic effect. This nano-therapy may be of interest to tackle an unmet medical need considering that esophageal stricture is the most challenging delayed complication after extended superficial cancer resection by endoscopy.

Calpastatin prevents Angiotensin II-mediated podocyte injury through maintenance of autophagy.

The strong predictive value of proteinuria in chronic glomerulopathies is firmly established as well as the pathogenic role of angiotensin II promoting progression of glomerular disease with an altered glomerular filtration barrier, podocyte injury and scarring of glomeruli. Here we found that chronic angiotensin II-induced hypertension inhibited autophagy flux in mouse glomeruli. Deletion of Atg5 (a gene encoding a protein involved autophagy) specifically in the podocyte resulted in accelerated angiotensin II-induced podocytopathy, accentuated albuminuria and glomerulosclerosis. This indicates that autophagy is a key protective mechanism in the podocyte in this condition. Angiotensin-II induced calpain activity in podocytes inhibits autophagy flux. Podocytes from mice with transgenic expression of the endogenous calpain inhibitor calpastatin displayed higher podocyte autophagy at baseline that was resistant to angiotensin II-dependent inhibition. Also, sustained autophagy with calpastatin limited podocyte damage and albuminuria. These findings suggest that hypertension has pathogenic effects on the glomerular structure and function, in part through activation of calpains leading to blockade of podocyte autophagy. These findings uncover an original mechanism whereby angiotensin II-mediated hypertension inhibits autophagy via calcium-induced recruitment of calpain with pathogenic consequences in case of imbalance by calpastatin activity. Thus, preventing a calpain-mediated decrease in autophagy may be a promising new therapeutic strategy for nephropathies associated with high renin-angiotensin system activity.

Multidimensional Proteomic Approach of Endothelial Progenitors Demonstrate Expression of KDR Restricted to CD19 Cells.

Endothelial progenitor cells (EPCs) are involved in vasculogenesis and cardiovascular diseases. However, the phenotype of circulating EPCs remains elusive but they are more often described as CD34+KDR+. The aim of the study was to extensively characterize circulating potential vasculogenic stem cell candidates in two populations of patients with cardiovascular disease by powerful multidimensional single cell complementary cytometric approaches (mass, imaging and flow). We identified cellular candidates in one patient before and after bioprosthetic total artificial heart implantation and results were confirmed in healthy peripheral and cord blood by mass cytometry. We also quantified cellular candidates in 10 patients with different COVID-19 severity. Both C-TAH implantation and COVID-19 at critical stage induce a redistribution of circulating CD34+ and CD19+ sub-populations in peripheral blood. After C-TAH implantation, circulating CD34+ progenitor cells expressed c-Kit stem marker while specific subsets CD34+CD133-/+CD45-/dimc-Kit+KDR- were mobilized. KDR was only expressed by CD19+ B-lymphocytes and CD14+ monocytes subpopulations in circulation. We confirmed by mass cytometry this KDR expression on CD19+ in healthy peripheral and cord blood, also with a VE-cadherin expression, confirming absence of endothelial lineage marker on CD34+ subtypes. In COVID-19, a significant mobilization of CD34+c-Kit+KDR- cells was observed between moderate and critical COVID-19 patients regardless CD133 or CD45 expression. In order to better evaluate EPC phenotype, we performed imaging flow cytometry measurements of immature CD34+KDR+ cells in cord blood and showed that, after elimination of non-circular events, those cells were all CD19+. During COVID-19, a significant mobilization of CD19+KDR+ per million of CD45+ cells was observed between moderate and critical COVID-19 patients regardless of CD34 expression. CD34+c-Kit+ cells are mobilized in both cardiovascular disease described here. KDR cells in peripheral blood are CD19 positive cells and are not classic vasculogenic stem and/or progenitor cells. A better evaluation of c-Kit and KDR expressing cells will lead to the redefinition of circulating endothelial progenitors.Graphical abstract Central illustration figure. Multidimensional proteomic approach of endothelial progenitors demonstrate expression of KDR restricted to CD19 cells. Endothelial progenitor cells (EPCs) are involved in cardiovascular diseases, however their phenotype remains elusive. We elucidated here EPCs phenotype by a deep characterization by multidimensional single cell complementary cytometric approaches after Bioprosthetic total artificial heart implantation and during COVID-19. We showed a redistribution of circulating CD34+ and CD19+ sub-populations in both situations. None of the immature cell population expresses KDR. Mobilized CD34+ expressed c-Kit. Imaging flow cytometry demonstrated that CD34+KDR+ cells, after elimination of non-circular events, are all CD19+. Our results suggest a new definition of circulating EPCs and emphasize involvement of CD19 cells in cardiovascular disease.

Deletion of the myeloid endothelin-B receptor confers long-term protection from angiotensin II-mediated kidney, eye and vessel injury.

The endothelin system may be an important player in hypertensive end-organ injury as endothelin-1 increases blood pressure and is pro-inflammatory. The immune system is emerging as an important regulator of blood pressure and we have shown that the early hypertensive response to angiotensin-II infusion was amplified in mice deficient of myeloid endothelin-B (ETB) receptors (LysM-CreEdnrblox/lox). Hypothesizing that these mice would display enhanced organ injury, we gave angiotensin-II to LysM-CreEdnrblox/lox and littermate controls (Ednrblox/lox) for six weeks. Unexpectedly, LysM-CreEdnrblox/lox mice were significantly protected from organ injury, with less proteinuria, glomerulosclerosis and inflammation of the kidney compared to controls. In the eye, LysM-CreEdnrblox/lox mice had fewer retinal hemorrhages, less microglial activation and less vessel rarefaction. Cardiac remodeling and dysfunction were similar in both groups at week six but LysM-CreEdnrblox/lox mice had better endothelial function. Although blood pressure was initially higher in LysM-CreEdnrblox/lox mice, this was not sustained. A natriuretic switch at about two weeks, due to enhanced ETB signaling in the kidney, induced a hypertensive reversal. By week six, blood pressure was lower in LysM-CreEdnrblox/lox mice than in controls. At six weeks, macrophages from LysM-CreEdnrblox/lox mice were more anti-inflammatory and had greater phagocytic ability compared to the macrophages of Ednrblox/lox mice. Thus, myeloid cell ETB receptor signaling drives this injury both through amplifying hypertension and by inflammatory polarization of macrophages.

The tetraspanin CD9 controls migration and proliferation of parietal epithelial cells and glomerular disease progression.

The mechanisms driving the development of extracapillary lesions in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN) remain poorly understood. A key question is how parietal epithelial cells (PECs) invade glomerular capillaries, thereby promoting injury and kidney failure. Here we show that expression of the tetraspanin CD9 increases markedly in PECs in mouse models of CGN and FSGS, and in kidneys from individuals diagnosed with these diseases. Cd9 gene targeting in PECs prevents glomerular damage in CGN and FSGS mouse models. Mechanistically, CD9 deficiency prevents the oriented migration of PECs into the glomerular tuft and their acquisition of CD44 and ß1 integrin expression. These findings highlight a critical role for de novo expression of CD9 as a common pathogenic switch driving the PEC phenotype in CGN and FSGS, while offering a potential therapeutic avenue to treat these conditions.

MicroRNA-21 Deficiency Alters the Survival of Ly-6Clo Monocytes in ApoE-/- Mice and Reduces Early-Stage Atherosclerosis-Brief Report.

Objective- To determine the role of microRNA-21 (miR-21) on the homeostasis of monocyte subsets and on atherosclerosis development in ApoE-/- (apolipoprotein E) mice. Approach and Results- In ApoE-/- mice, miR-21 expression was increased in circulating Ly-6Clo nonclassical monocytes in comparison to Ly-6Chi monocytes. The absence of miR-21 significantly altered the survival and number of circulating Ly-6Clo nonclassical monocytes in ApoE-/- mice. In the early stages of atherosclerosis, the absence of miR-21 limited lesion development both in the aortic sinus (by almost 30%) and in the aorta (by almost 50%). This was associated with less monocyte availability in circulation and increased apoptosis of local macrophages in plaques. At later stages of atherosclerosis, lesion size in the aortic root was similar in ApoE-/- and ApoE-/- miR-21-/- mice, but plaques showed a less stable phenotype (larger necrotic cores) in the latter. The loss of protection in advanced stages was most likely because of excessive inflammatory apoptosis related to an impairment of local efficient efferocytosis. Conclusions- Gene deletion of miR-21 in ApoE-/- mice alters Ly-6Clo nonclassical monocytes homeostasis and contribute to limit early-stage atherosclerosis.

Selective EGFR (Epidermal Growth Factor Receptor) Deletion in Myeloid Cells Limits Atherosclerosis-Brief Report.

OBJECTIVE: To determine the consequences of specific inhibition of EGFR (epidermal growth factor receptor) in myeloid cells in atherosclerosis development. APPROACH AND RESULTS: Atherosclerotic lesion size was significantly reduced in irradiated Ldlr-/- mice reconstituted with LysMCre+Egfrlox/lox bone marrow, compared with chimeric Ldlr-/- mice reconstituted with LysMCre-Egfrlox/lox bone marrow, after 4 (-43%; P<0.05), 7 (-34%; P<0.05), and 12 weeks (-54%; P<0.001) of high-fat diet. Reduction of lesion size was associated with marked reduction in macrophage accumulation and necrotic core size. Specific deletion of Egfr in myeloid cells reduced TNF-α (tumor necrosis factor-α) and IL (interleukin)-6 production by stimulated macrophages but had no effect on IL-10 and IL-12p70 secretion. Finally, we found that myeloid deletion of Egfr limited cytoskeletal rearrangements and also lipid uptake by macrophages through a downregulation of the scavenger receptor CD36 (cluster of differentiation 36). CONCLUSIONS: Gene deletion of Egfr in myeloid cells limits IL-6 and TNF-α production, lipid uptake, and consecutively reduces atherosclerosis development.

Endothelial Epas1 Deficiency Is Sufficient To Promote Parietal Epithelial Cell Activation and FSGS in Experimental Hypertension.

FSGS, the most common primary glomerular disorder causing ESRD, is a complex disease that is only partially understood. Progressive sclerosis is a hallmark of FSGS, and genetic tracing studies have shown that parietal epithelial cells participate in the formation of sclerotic lesions. The loss of podocytes triggers a focal activation of parietal epithelial cells, which subsequently form cellular adhesions with the capillary tuft. However, in the absence of intrinsic podocyte alterations, the origin of the pathogenic signal that triggers parietal epithelial cell recruitment remains elusive. In this study, investigation of the role of the endothelial PAS domain-containing protein 1 (EPAS1), a regulatory α subunit of the hypoxia-inducible factor complex, during angiotensin II-induced hypertensive nephropathy provided novel insights into FSGS pathogenesis in the absence of a primary podocyte abnormality. We infused angiotensin II into endothelial-selective Epas1 knockout mice and their littermate controls. Although the groups presented with identical high BP, endothelial-specific Epas1 gene deletion accentuated albuminuria with severe podocyte lesions and recruitment of pathogenic parietal glomerular epithelial cells. These lesions and dysfunction of the glomerular filtration barrier were associated with FSGS in endothelial Epas1-deficient mice only. These results indicate that endothelial EPAS1 has a global protective role during glomerular hypertensive injuries without influencing the hypertensive effect of angiotensin II. Furthermore, these findings provide proof of principle that endothelial-derived signaling can trigger FSGS and illustrate the potential importance of the EPAS1 endothelial transcription factor in secondary FSGS.

Nuclear Factor Erythroid 2-Related Factor 2 Drives Podocyte-Specific Expression of Peroxisome Proliferator-Activated Receptor γ Essential for Resistance to Crescentic GN.

Necrotizing and crescentic rapidly progressive GN (RPGN) is a life-threatening syndrome characterized by a rapid loss of renal function. Evidence suggests that podocyte expression of the transcription factor peroxisome proliferator-activated receptor γ (PPARγ) may prevent podocyte injury, but the function of glomerular PPARγ in acute, severe inflammatory GN is unknown. Here, we observed marked loss of PPARγ abundance and transcriptional activity in glomerular podocytes in experimental RPGN. Blunted expression of PPARγ in podocyte nuclei was also found in kidneys from patients diagnosed with crescentic GN. Podocyte-specific Pparγ gene targeting accentuated glomerular damage, with increased urinary loss of albumin and severe kidney failure. Furthermore, a PPARγ gain-of-function approach achieved by systemic administration of thiazolidinedione (TZD) failed to prevent severe RPGN in mice with podocyte-specific Pparγ gene deficiency. In nuclear factor erythroid 2-related factor 2 (NRF2)-deficient mice, loss of podocyte PPARγ was observed at baseline. NRF2 deficiency markedly aggravated the course of RPGN, an effect that was partially prevented by TZD administration. Furthermore, delayed administration of TZD, initiated after the onset of RPGN, still alleviated the severity of experimental RPGN. These findings establish a requirement for the NRF2-PPARγ cascade in podocytes, and we suggest that these transcription factors have a role in augmenting the tolerance of glomeruli to severe immune-complex mediated injury. The NRF2-PPARγ pathway may be a therapeutic target for RPGN.

Endothelial cell and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis.

The glomerulus is a highly specialized capillary tuft, which under pressure filters large amounts of water and small solutes into the urinary space, while retaining albumin and large proteins. The glomerular filtration barrier (GFB) is a highly specialized filtration interface between blood and urine that is highly permeable to small and midsized solutes in plasma but relatively impermeable to macromolecules such as albumin. The integrity of the GFB is maintained by molecular interplay between its 3 layers: the glomerular endothelium, the glomerular basement membrane and podocytes, which are highly specialized postmitotic pericytes forming the outer part of the GFB. Abnormalities of glomerular ultrafiltration lead to the loss of proteins in urine and progressive renal insufficiency, underlining the importance of the GFB. Indeed, albuminuria is strongly predictive of the course of chronic nephropathies especially that of diabetic nephropathy (DN), a leading cause of renal insufficiency. We found that high glucose concentrations promote autophagy flux in podocyte cultures and that the abundance of LC3B II in podocytes is high in diabetic mice. Deletion of Atg5 specifically in podocytes resulted in accelerated diabetes-induced podocytopathy with a leaky GFB and glomerulosclerosis. Strikingly, genetic alteration of autophagy on the other side of the GFB involving the endothelial-specific deletion of Atg5 also resulted in capillary rarefaction and accelerated DN. Thus autophagy is a key protective mechanism on both cellular layers of the GFB suggesting autophagy as a promising new therapeutic strategy for DN.