Glomerular and tubular effects of dapagliflozin, eplerenone and their combination in patients with chronic kidney disease: A post-hoc analysis of the ROTATE-3 study.

AIM: Sodium-glucose co-transporter 2 inhibitors and mineralocorticoid receptor antagonists reduce albuminuria and the risk of kidney failure. The aim of this study was to investigate the effects of both agents alone and in combination on markers of the glomerular endothelial glycocalyx and tubular function. METHODS: This post-hoc analysis utilized data of the ROTATE-3 study, a randomized cross-over study in 46 adults with chronic kidney disease and urinary albumin excretion ≥100 mg/24 h, who were treated for 4 weeks with dapagliflozin, eplerenone or its combination. The effects of dapagliflozin, eplerenone and the combination on outcome measures such as heparan sulphate, neuro-hormonal markers and tubular sodium handling were assessed with mixed repeated measures models. RESULTS: The mean percentage change from baseline in heparan sulphate after 4 weeks treatment with dapagliflozin, eplerenone or dapagliflozin-eplerenone was -34.8% (95% CI -52.2, -10.9), -5.9% (95% CI -32.5, 31.3) and -28.1% (95% CI -48.4, 0.1) respectively. The mean percentage change from baseline in plasma aldosterone was larger with eplerenone [38.9% (95% CI 2.8, 87.7)] and dapagliflozin-eplerenone [32.2% (95% CI -1.5, 77.4)], compared with dapagliflozin [-12.5% (95% CI -35.0, 17.8)], respectively. Mean percentage change from baseline in copeptin with dapagliflozin, eplerenone or dapagliflozin-eplerenone was 28.4% (95% CI 10.7, 49.0), 4.2% (95% CI -10.6, 21.4) and 23.8% (95% CI 6.6, 43.9) respectively. Dapagliflozin decreased proximal absolute sodium reabsorption rate by 455.9 mmol/min (95% CI -879.2, -32.6), while eplerenone decreased distal absolute sodium reabsorption rate by 523.1 mmol/min (95% CI -926.1, -120.0). Dapagliflozin-eplerenone decreased proximal absolute sodium reabsorption [-971.0 mmol/min (95% CI -1411.0, -531.0)], but did not affect distal absolute sodium reabsorption [-9.2 mmol/min (95% CI -402.0, 383.6)]. CONCLUSIONS: Dapagliflozin and eplerenone exert different effects on markers of glomerular and tubular function supporting the hypothesis that different mechanistic pathways may account for their kidney protective effects.

Calciprotein Particles Induce Endothelial Dysfunction by Impairing Endothelial Nitric Oxide Metabolism.

BACKGROUND: Calciprotein particles (CPPs) are associated with the development of vascular calcifications in chronic kidney disease. The role of endothelial cells (ECs) in this process is unknown. Here, we investigated the interaction of CPPs and ECs, thereby focusing on endothelial nitric oxide metabolism and oxidative stress. METHODS: CPPs were generated in calcium- and phosphate-enriched medium. Human umbilical vein endothelial cells were exposed to different concentrations of CPPs (0-100 µg/mL) for 24 or 72 hours. Ex vivo porcine coronary artery rings were used to measure endothelial cell-dependent vascular smooth muscle cell relaxation after CPP exposure. Serum samples from an early chronic kidney disease cohort (n=245) were analyzed for calcification propensity (measure for CPP formation) and nitrate and nitrite levels (NOx). RESULTS: CPP exposure for 24 hours reduced eNOS (endothelial nitric oxide synthase) mRNA expression and decreased nitrite production, indicating reduced nitric oxide bioavailability. Also, 24-hour CPP exposure caused increased mitochondria-derived superoxide generation, together with nitrotyrosine protein residue formation. Long-term (72 hours) exposure of human umbilical vein endothelial cells to CPPs induced eNOS uncoupling and decreased eNOS protein expression, indicating further impairment of the nitric oxide pathway. The ex vivo porcine coronary artery model showed a significant reduction in endothelial-dependent vascular smooth muscle cell relaxation after CPP exposure. A negative association was observed between NOx levels and calcification propensity (r=-0.136; P=0.049) in sera of (early) chronic kidney disease patients. CONCLUSIONS: CPPs cause endothelial cell dysfunction by impairing nitric oxide metabolism and generating oxidative stress. Our findings provide new evidence for direct effects of CPPs on ECs and pathways involved.

Inhibition of Ferroptosis Enables Safe Rewarming of HEK293 Cells following Cooling in University of Wisconsin Cold Storage Solution.

The prolonged cooling of cells results in cell death, in which both apoptosis and ferroptosis have been implicated. Preservation solutions such as the University of Wisconsin Cold Storage Solution (UW) encompass approaches addressing both. The use of UW improves survival and thus extends preservation limits, yet it remains unclear how exactly organ preservation solutions exert their cold protection. Thus, we explored cooling effects on lipid peroxidation and adenosine triphosphate (ATP) levels and the actions of blockers of apoptosis and ferroptosis, and of compounds enhancing mitochondrial function. Cooling and rewarming experiments were performed in a cellular transplantation model using Human Embryonic Kidney (HEK) 293 cells. Cell viability was assessed by neutral red assay. Lipid peroxidation levels were measured by Western blot against 4-Hydroxy-Nonenal (4HNE) and the determination of Malondialdehyde (MDA). ATP was measured by luciferase assay. Cooling beyond 5 h in Dulbecco's Modified Eagle Medium (DMEM) induced complete cell death in HEK293, whereas cooling in UW preserved ~60% of the cells, with a gradual decline afterwards. Cooling-induced cell death was not precluded by inhibiting apoptosis. In contrast, the blocking of ferroptosis by Ferrostatin-1 or maintaining of mitochondrial function by the 6-chromanol SUL150 completely inhibited cell death both in DMEM- and UW-cooled cells. Cooling for 24 h in UW followed by rewarming for 15 min induced a ~50% increase in MDA, while concomitantly lowering ATP by >90%. Treatment with SUL150 of cooled and rewarmed HEK293 effectively precluded the increase in MDA and preserved normal ATP in both DMEM- and UW-cooled cells. Likewise, treatment with Ferrostatin-1 blocked the MDA increase and preserved the ATP of rewarmed UW HEK293 cells. Cooling-induced HEK293 cell death from hypothermia and/or rewarming was caused by ferroptosis rather than apoptosis. UW slowed down ferroptosis during hypothermia, but lipid peroxidation and ATP depletion rapidly ensued upon rewarming, ultimately resulting in complete cell death. Treatment throughout UW cooling with small-molecule Ferrostatin-1 or the 6-chromanol SUL150 effectively prevented ferroptosis, maintained ATP, and limited lipid peroxidation in UW-cooled cells. Counteracting ferroptosis during cooling in UW-based preservation solutions may provide a simple method to improve graft survival following cold static cooling.

Autologous lipoaspirate as a new treatment of vulvar lichen sclerosus: A review on literature.

Lichen sclerosus (LS) is a chronic inflammatory dermatosis that mostly affects the genital and anal skin areas. Symptoms may vary from pruritis and pain to sexual dysfunction; however, LS can also be asymptomatic. LS occurs at all ages and in both sexes. Approximately 5% of all women affected by vulvar LS will develop vulvar squamous cell carcinoma. Topical treatment is safe but less effective resulting in chronic course in most patients, who suffer from persistent itching and pain. In severe cases of therapy-resistant LS, there is no adequate treatment. Fat grafting is a novel regenerative therapy to reduce dermal fibrosis. The therapeutic effect of adipose tissue grafts for LS is already investigated in various pioneering studies. This review provides an overview of these studies and the putative mechanisms-of-action of fat grafting to treat LS.

Calciprotein Particles: Balancing Mineral Homeostasis and Vascular Pathology.

[Figure: see text].

Autologous Lipofilling Improves Clinical Outcome in Patients With Symptomatic Dermal Scars Through Induction of a Pro-Regenerative Immune Response.

BACKGROUND: Autologous lipofilling is an emerging procedure to treat and possibly reverse dermal scars and to reduce scar-related pain, but its efficacy and mechanisms are poorly understood. OBJECTIVES: The aim of this study was to test the hypothesis that repeated lipografts reverse dermal scars by reinitiation of wound healing. METHODS: In a prospective, non-placebo-controlled clinical study, 27 adult patients with symptomatic scars were given 2 lipofilling treatments at 3-month intervals. As primary outcome, clinical effects were measured with the Patient and Observer Scar Assessment Scale (POSAS). Scar biopsies were taken before and after treatments to assess scar remodeling at a cellular level. RESULTS: Twenty patients completed the study. Patients' scars improved after lipofilling. The total POSAS scores (combined patient and observer scores) decreased from 73.2  [14.7] points (mean [standard deviation]) pretreatment to 46.1 [14.0] and 32.3 [13.2] points after the first and second lipofilling treatment, respectively. Patient POSAS scores decreased from 37.3 [8.8] points to 27.2 [11.3] and 21.1 [11.4] points, whereas observer POSAS scores decreased from 35.9 [9.5] points to 18.9 [6.0] and 11.3 [4.5] points after the first and second treatment, respectively. After each lipofilling treatment, T lymphocytes, mast cells, and M2 macrophages had invaded scar tissue and were associated with increased vascularization. In addition, the scar-associated epidermis showed an increase in epidermal cell proliferation to levels similar to that normal in skin. Moreover, lipofilling treatment caused normalization of the extracellular matrix organization towards that of normal skin. CONCLUSIONS: Autologous lipofilling improves the clinical outcome of dermal scars through the induction of a pro-regenerative immune response, increased vascularization, and epidermal proliferation and remodeling of scar tissue extracellular matrix.

SUL-151 Decreases Airway Neutrophilia as a Prophylactic and Therapeutic Treatment in Mice after Cigarette Smoke Exposure.

Chronic obstructive pulmonary disease (COPD) caused by cigarette smoke (CS) is featured by oxidative stress and chronic inflammation. Due to the poor efficacy of standard glucocorticoid therapy, new treatments are required. Here, we investigated whether the novel compound SUL-151 with mitoprotective properties can be used as a prophylactic and therapeutic treatment in a murine CS-induced inflammation model. SUL-151 (4 mg/kg), budesonide (500 µg/kg), or vehicle were administered via oropharyngeal instillation in this prophylactic and therapeutic treatment setting. The number of immune cells was determined in the bronchoalveolar lavage fluid (BALF). Oxidative stress response, mitochondrial adenosine triphosphate (ATP) production, and mitophagy-related proteins were measured in lung homogenates. SUL-151 significantly decreased more than 70% and 50% of CS-induced neutrophils in BALF after prophylactic and therapeutic administration, while budesonide showed no significant reduction in neutrophils. Moreover, SUL-151 prevented the CS-induced decrease in ATP and mitochondrial mtDNA and an increase in putative protein kinase 1 expression in the lung homogenates. The concentration of SUL-151 was significantly correlated with malondialdehyde level and radical scavenging activity in the lungs. SUL-151 inhibited the increased pulmonary inflammation and mitochondrial dysfunction in this CS-induced inflammation model, which implied that SUL-151 might be a promising candidate for COPD treatment.

Klotho Deficiency Induces Arteriolar Hyalinosis in a Trade-Off with Vascular Calcification.

Hyalinosis is a vascular lesion affecting the renal vasculature and contributing to aging-related renal function decline. We assessed whether arteriolar hyalinosis is caused by Klotho deficiency, a state known to induce both renal and vascular phenotypes associated with aging. Histochemistry was used to assess hyalinosis in Klotho-/- kidneys, compared with Klotho+/- and wild-type littermates. Immunohistochemistry was used to investigate vascular lesion composition and the different layers of the vascular wall. Finally, spironolactone was used to inhibit calcification in kl/kl mice, and vascular lesions were characterized in the kidney. Arteriolar hyalinosis was detected in Klotho-/- mice, which was present up to the afferent arterioles. Hyalinosis was accompanied by loss of α-smooth muscle actin expression, whereas the endothelial lining was mostly intact. Hyalinous lesions were positive for IgM and iC3b/c/d, indicating subendothelial leakage of plasma proteins. The presence of extracellular matrix proteins suggested increased production by smooth muscle cells (SMCs). Finally, in Klotho-/- mice with marked vascular calcification, treatment with spironolactone allowed for replacement of calcification by hyalinosis. Klotho deficiency potentiates both endothelial hyperpermeability and SMC dedifferentiation. In the absence of a calcification-inducing stimulus, SMCs assume a synthetic phenotype in response to subendothelial leakage of plasma proteins. In the kidney, this results in arteriolar hyalinosis, which contributes to the decline in renal function. Klotho may play a role in preventing aging-related arteriolar hyalinosis.

Human Milk Oligosaccharides Mediate the Crosstalk Between Intestinal Epithelial Caco-2 Cells and Lactobacillus PlantarumWCFS1in an In Vitro Model with Intestinal Peristaltic Shear Force.

BACKGROUND: The intestinal epithelial cells, food molecules, and gut microbiota are continuously exposed to intestinal peristaltic shear force. Shear force may impact the crosstalk of human milk oligosaccharides (hMOs) with commensal bacteria and intestinal epithelial cells. OBJECTIVES: We investigated how hMOs combined with intestinal peristaltic shear force impact intestinal epithelial cells and crosstalk with a commensal bacterium. METHODS: We applied the Ibidi system to mimic intestinal peristaltic shear force. Caco-2 cells were exposed to a shear force (5 dynes/cm2) for 3 d, and then stimulated with the hMOs, 2'-fucosyllactose (2'-FL), 3-FL, and lacto-N-triose II (LNT2). In separate experiments, Lactobacillus plantarumWCFS1 adhesion to Caco-2 cells was studied with the same hMOs and shear force. Effects were tested on gene expression of glycocalyx-related molecules (glypican 1 [GPC1], hyaluronan synthase 1 [HAS1], HAS2, HAS3, exostosin glycosyltransferase 1 [EXT1], EXT2), defensin ß-1 (DEFB1), and tight junction (tight junction protein 1 [TJP1], claudin 3 [CLDN3]) in Caco-2 cells. Protein expression of tight junctions was also quantified. RESULTS: Shear force dramatically decreased gene expression of the main enzymes for making glycosaminoglycan side chains (HAS3 by 43.3% and EXT1 by 68.7%) (P <0.01), but did not affect GPC1 which is the gene responsible for the synthesis of glypican 1 which is a major protein backbone of glycocalyx. Expression of DEFB1, TJP1, and CLDN3 genes was decreased 60.0-94.9% by shear force (P <0.001). The presence of L. plantarumWCFS1 increased GPC1, HAS2, HAS3, and ZO-1 expression by 1.78- to 3.34-fold (P <0.05). Under shear force, all hMOs significantly stimulated DEFB1 and ZO-1, whereas only 3-FL and LNT2 enhanced L. plantarumWCFS1 adhesion by 1.85- to 1.90-fold (P <0.01). CONCLUSIONS: 3-FL and LNT2 support the crosstalk between the commensal bacterium L. plantarumWCFS1 and Caco-2 intestinal epithelial cells, and shear force can increase the modulating effects of hMOs.

MicroRNA-374b induces endothelial-to-mesenchymal transition and early lesion formation through the inhibition of MAPK7 signaling.

Endothelial-mesenchymal transition occurs during intimal hyperplasia and neointima formation via mechanisms that are incompletely understood. Endothelial MAPK7 signaling is a key mechanosensitive factor that protects against endothelial-mesenchymal transition, but its signaling activity is lost in vessel areas that are undergoing pathological remodeling. At sites of vascular remodeling in mice and pigs, endothelial MAPK7 signaling was lost. The TGFß-induced microRNA-374b targets MAPK7 and its downstream effectors in endothelial cells, and its expression induces endothelial-mesenchymal transition. Gain-of-function experiments, where endothelial MAPK7 signaling was restored, precluded endothelial-mesenchymal transition. In human coronary artery disease, disease severity is associated with decreased MAPK7 expression levels and increased miR-374b expression levels. Endothelial-mesenchymal transition occurs in intimal hyperplasia and early lesion formation and is governed in part by microRNA-374b-induced silencing of MAPK7 signaling. Restoration of MAPK7 signaling abrogated these pathological effects in endothelial cells expressing miR-374b. Thus, our data suggest that the TGFß-miR-374b-MAPK7 axis plays a key role in the induction of endothelial-mesenchymal transition during intimal hyperplasia and early lesion formation and might pose an interesting target for antiatherosclerosis therapy. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Epigenetic Regulation of Endothelial-to-Mesenchymal Transition in Chronic Heart Disease.

Endothelial-to-mesenchymal transition (EndMT) is a process in which endothelial cells lose their properties and transform into fibroblast-like cells. This transition process contributes to cardiac fibrosis, a common feature of patients with chronic heart failure. To date, no specific therapies to halt or reverse cardiac fibrosis are available, so knowledge of the underlying mechanisms of cardiac fibrosis is urgently needed. In addition, EndMT contributes to other cardiovascular pathologies such as atherosclerosis and pulmonary hypertension, but also to cancer and organ fibrosis. Remarkably, the molecular mechanisms driving EndMT are largely unknown. Epigenetics play an important role in regulating gene transcription and translation and have been implicated in the EndMT process. Therefore, epigenetics might be the missing link in unraveling the underlying mechanisms of EndMT. Here, we review the involvement of epigenetic regulators during EndMT in the context of cardiac fibrosis. The role of DNA methylation, histone modifications (acetylation and methylation), and noncoding RNAs (microRNAs, long noncoding RNAs, and circular RNAs) in the facilitation and inhibition of EndMT are discussed, and potential therapeutic epigenetic targets will be highlighted.

Human adipose tissue-derived stromal cells act as functional pericytes in mice and suppress high-glucose-induced proinflammatory activation of bovine retinal endothelial cells.

AIMS/HYPOTHESIS: The immunomodulatory capacity of adipose tissue-derived stromal cells (ASCs) is relevant for next-generation cell therapies that aim to reverse tissue dysfunction such as that caused by diabetes. Pericyte dropout from retinal capillaries underlies diabetic retinopathy and the subsequent aberrant angiogenesis. METHODS: We investigated the pericytic function of ASCs after intravitreal injection of ASCs in mice with retinopathy of prematurity as a model for clinical diabetic retinopathy. In addition, ASCs influence their environment by paracrine signalling. For this, we assessed the immunomodulatory capacity of conditioned medium from cultured ASCs (ASC-Cme) on high glucose (HG)-stimulated bovine retinal endothelial cells (BRECs). RESULTS: ASCs augmented and stabilised retinal angiogenesis and co-localised with capillaries at a pericyte-specific position. This indicates that cultured ASCs exert juxtacrine signalling in retinal microvessels. ASC-Cme alleviated HG-induced oxidative stress and its subsequent upregulation of downstream targets in an NF-κB dependent fashion in cultured BRECs. Functionally, monocyte adhesion to the monolayers of activated BRECs was also decreased by treatment with ASC-Cme and correlated with a decline in expression of adhesion-related genes such as SELE, ICAM1 and VCAM1. CONCLUSIONS/INTERPRETATION: The ability of ASC-Cme to immunomodulate HG-challenged BRECs is related to the length of time for which ASCs were preconditioned in HG medium. Conditioned medium from ASCs that had been chronically exposed to HG medium was able to normalise the HG-challenged BRECs to normal glucose levels. In contrast, conditioned medium from ASCs that had been exposed to HG medium for a shorter time did not have this effect. Our results show that the manner of HG preconditioning of ASCs dictates their immunoregulatory properties and thus the potential outcome of treatment of diabetic retinopathy.

FGF2 inhibits endothelial-mesenchymal transition through microRNA-20a-mediated repression of canonical TGF-ß signaling.

Endothelial-to-mesenchymal transition (EndMT) is characterized by the loss of endothelial cell markers and functions, and coincides with de novo expression of mesenchymal markers. EndMT is induced by TGFß1 and changes endothelial microRNA expression. We found that miR-20a is decreased during EndMT, and that ectopic expression of miR-20a inhibits EndMT induction. TGFß1 induces cellular hypertrophy in human umbilical vein endothelial cells and abrogates VE-cadherin expression, reduces endothelial sprouting capacity and induces the expression of the mesenchymal marker SM22α (also known as TAGLN). We identified ALK5 (also known as TGFBR1), TGFBR2 and SARA (also known as ZFYVE9) as direct miR-20a targets. Expression of miR-20a mimics abrogate the endothelial responsiveness to TGFß1, by decreasing ALK5, TGFBR2 and SARA, and inhibit EndMT, as indicated by the maintenance of VE-cadherin expression, the ability of the cells to sprout and the absence of SM22α expression. FGF2 increases miR-20a expression and inhibits EndMT in TGFß1-stimulated endothelial cells. In summary, FGF2 controls endothelial TGFß1 signaling by regulating ALK5, TGFBR2 and SARA expression through miR-20a. Loss of FGF2 signaling combined with a TGFß1 challenge reduces miR-20a levels and increases endothelial responsiveness to TGFß1 through elevated receptor complex levels and activation of Smad2 and Smad3, which culminates in EndMT.

The 6-hydroxychromanol derivative SUL-109 ameliorates renal injury after deep hypothermia and rewarming in rats.

Background: Mitochondrial dysfunction plays an important role in kidney damage in various pathologies, including acute and chronic kidney injury and diabetic nephropathy. In addition to the well-studied ischaemia/reperfusion (I/R) injury, hypothermia/rewarming (H/R) also inflicts acute kidney injury. Substituted 6-hydroxychromanols are a novel class of mitochondrial medicines that ameliorate mitochondrial oxidative stress and protect the mitochondrial network. To identify a novel 6-hydroxychromanol that protects mitochondrial structure and function in the kidney during H/R, we screened multiple compounds in vitro and subsequently assessed the efficacy of the 6-hydroxychromanol derivatives SUL-109 and SUL-121 in vivo to protect against kidney injury after H/R in rats. Methods: Human proximal tubule cell viability was assessed following exposure to H/R for 48/4 h in the presence of various 6-hydroxychromanols. Selected compounds (SUL-109, SUL-121) or vehicle were administered to ketamine-anaesthetized male Wistar rats (IV 135 µg/kg/h) undergoing H/R at 15°C for 3 h followed by rewarming and normothermia for 1 h. Metabolic parameters and body temperature were measured throughout. In addition, renal function, renal injury, histopathology and mitochondrial fitness were assessed. Results: H/R injury in vitro lowered cell viability by 94 ± 1%, which was counteracted dose-dependently by multiple 6-hydroxychomanols derivatives. In vivo, H/R in rats showed kidney injury molecule 1 expression in the kidney and tubular dilation, accompanied by double-strand DNA breaks and protein nitrosylation. SUL-109 and SUL-121 ameliorated tubular kidney damage, preserved mitochondrial mass and maintained cortical adenosine 5'-triphosphate (ATP) levels, although SUL-121 did not reduce protein nitrosylation. Conclusions: The substituted 6-hydroxychromanols SUL-109 and SUL-121 ameliorate kidney injury during in vivo H/R by preserving mitochondrial mass, function and ATP levels. In addition, both 6-hydroxychromanols limit DNA damage, but only SUL-109 also prevented protein nitrosylation in tubular cells. Therefore SUL-109 offers a promising therapeutic strategy to preserve kidney mitochondrial function.

The decrease in histone methyltransferase EZH2 in response to fluid shear stress alters endothelial gene expression and promotes quiescence.

High uniform fluid shear stress (FSS) is atheroprotective and preserves the endothelial phenotype and function through activation of downstream mediators such as MAPK7 (Erk5). Endothelial cells respond to FSS thanks to mechanotransduction. However, how the resulting signaling is integrated and resolved at the epigenetic level remains elusive. We hypothesized that Polycomb methyltransferase EZH2 is involved in the effects of FSS in human endothelial cells. We showed that FSS decreases the expression of the Polycomb methyltransferase EZH2. Despite simultaneous activation of MAPK7, MAPK7 pathway does not directly influence the transcription of EZH2. Interestingly though, the knockdown of EZH2 activates the protective MAPK7 signaling in endothelial cells, even in the absence of FSS. To understand the influence of the FSS-decreased expression of EZH2 on endothelial transcriptome, we performed RNA-seq and differential gene expression analysis. We identified candidate groups of genes dependent on both EZH2 and FSS. Among those, Gene Ontology overrepresentation analysis revealed highly significant enrichment of the cell cycle-related genes, suggesting changes in proliferation. Indeed, the depletion of EZH2 strongly inhibited endothelial proliferation, indicating cell cycle arrest. The concomitant decrease in CCNA expression suggests the transition of endothelial cells into a quiescent phenotype. Further bioinformatical analysis suggested TXNIP as a possible mediator between EZH2 and cell cycle-related gene network. Our data show that EZH2 is a FSS-responsive gene. Decreased EZH2 levels enhance the activation of the atheroprotective MAPK7 signaling. Decrease in EZH2 under FSS mediates the decrease in the expression of the network of cell cycle-related genes, which allows the cells to enter quiescence. EZH2 is therefore important for the protective effects of FSS in endothelium.

H3K27Me3 abundance increases fibrogenesis during endothelial-to-mesenchymal transition via the silencing of microRNA-29c.

Objective: Endothelial-to-mesenchymal transition (EndMT) is a transdifferentiation process in which endothelial cells (ECs) adopt a mesenchymal-like phenotype. Over the past few years, it became clear that EndMT can contribute to several cardiovascular pathologies. However, the molecular pathways underlying the development of EndMT remain incompletely understood. Since the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2) and its concomitant mark H3K27Me3 have been shown to be elevated in many cardiovascular diseases that associate with EndMT, we hypothesized that H3K27Me3 is a determinant for the susceptibility of EndMT. Methods: To study the association between H3K27Me3 and EndMT, a knockdown model of EZH2 in human endothelial cells (HUVEC) was utilized to reduce H3K27Me3 abundance, followed by induction of EndMT using TGFß1. The expression of molecular markers of EndMT and fibrogenesis were analysed. Results: In cultured HUVECs, a reduction of H3K27Me3 abundance facilitates EndMT but mitigates fibrogenesis as shown by a decreased expression of collagen I and III. In HUVEC, H3K27Me3 abundance directly affects the expression of miR29c, a collagen-targeting miRNA. Additionally, knockdown of miR-29c in HUVEC with low H3K27Me3 abundance partly restored the expression of collagen I and III. Expectedly, in rats with perivascular fibrosis an increased abundance of H3K27Me3 associated with a decreased expression of miR-29c. Conclusion: our data shows that endothelial fibrogenesis underlies an epigenetic regulatory pathway and we demonstrate that a decreased abundance of H3K27Me3 in ECs blunts fibrogenesis in part in a miR-29c dependent manner. Therefore, a reduction of H3K27Me3 could serve as a novel therapeutical strategy to mitigate fibrogenesis and may prove to be beneficial in fibrogenic diseases including atherosclerosis, cardiac fibrosis, and PAH.

Safety, tolerability and toxicokinetics of the novel mitochondrial drug SUL-138 administered orally to rat and minipig.

Noncommunicable Chronic Diseases (NCD) are a socioeconomic burden and considered one of the major health challenges for coming decades. Mitochondrial dysfunction has been implicated mechanistically in their pathophysiology. Therefore, targeting mitochondria holds great promise to improve clinical outcomes in NCDs. SUL-138, an orally bioavailable small molecule efficacious from 0.5 mg/kg, improves mitochondrial function during disease in several preclinical animal models. As preparation for a First-in-Human (FIH) trial, SUL-138 was investigated in 30-day GLP repeated dose toxicity studies in rat and minipig, selected based on their comparability with human metabolism, to determine toxicokinetics, potential toxicity and its reversibility. Rats were allocated to either vehicle, 27, 136 or 682 mg/kg SUL-138 dose groups and minipigs were allocated to either vehicle, 16, 82 or 409 mg/kg. Treatment occurred orally for 30 days followed by a recovery period of 14 days. During these studies clinical observations, toxicokinetic, clinical pathology, necropsy and histopathology evaluations were performed. There was significant systemic exposure to SUL-138 and toxicokinetics was characterized by a rapid absorption and elimination. In the rat, toxicokinetics was dose-proportional and AUC0-tlast ratios in both species indicated that SUL-138 does not accumulate in vivo. No treatment-related adverse effects were observed for dose levels up to 136 and 82 mg/kg/day in rat and minipig respectively. In conclusion, these preclinical studies demonstrate that SUL-138 is well tolerated after repeated administration in rat and minipig, with NOAELs of 136 and 82 mg/kg/day, respectively.

Calciprotein particle counts associate with vascular remodelling in chronic kidney disease.

AIMS: Calciprotein particles (CPPs) are circulating calcium and phosphate nanoparticles associated with development of vascular calcification (VC) in chronic kidney disease (CKD). Although recent studies have been focusing on associations of CPPs with presence of VC in CKD, insights in the underlying processes and mechanisms by which CPPs might aggravate VC and vascular dysfunction in vivo are currently lacking. Here, we assessed overall burden of abdominal VC in healthy kidney donors and CKD patients, and subsequently performed transcriptome profiling in vascular tissue obtained from these subjects, linking outcome to CPP counts and calcification propensity. METHODS AND RESULTS: Calcification scores were quantified in renal arteries, iliac arteries and abdominal aorta, using computed tomography (CT) scans of kidney donors and CKD patients. Vascular tissue was collected from kidney donors (renal artery) and CKD patients (iliac artery), after which bulk RNA sequencing and gene set enrichment analysis (GSEA) was performed on a subset of patients. Calcification propensity (crystallization time, T50) was measured using nephelometry, and CPP counts with microparticle flow cytometric analysis. Increased calcification scores (based on CT) were found in CKD patients compared to kidney donors. Transcriptome profiling revealed enrichment for processes related to endothelial activation, inflammation, extracellular matrix (ECM) remodelling and ossification in CKD vascular biopsies compared to kidney donors. Calcification propensity was increased in CKD, as well as CPP counts, of which the latter significantly associated with markers of vascular remodelling. CONCLUSIONS: Our findings reveal that CKD is characterized by systemic VC with increased calcification propensity and CPP counts. Transcriptome profiling showed altered vascular gene expression with enrichment for endothelial activation, inflammation, ECM remodelling and ossification. Moreover, we demonstrate for the first time that vascular remodelling processes are associated with increased circulating CPP counts. Interventions targeting CPPs are promising avenues for alleviating vascular remodelling and VC in CKD.

Adipose stromal cells primed with hypoxia and inflammation enhance cardiomyocyte proliferation rate in vitro through STAT3 and Erk1/2.

BACKGROUND: Experimental clinical stem cell therapy has been used for more than a decade to alleviate the adverse aftermath of acute myocardial infarction (aMI). The post-infarcted myocardial microenvironment is characterized by cardiomyocyte death, caused by ischemia and inflammation. These conditions may negatively affect administered stem cells. As postnatal cardiomyocytes have a poor proliferation rate, while induction of proliferation seems even more rare. Thus stimulation of their proliferation rate is essential after aMI. In metaplastic disease, the pro-inflammatory cytokine interleukin-6 (IL-6) has been identified as potent mediators of the proliferation rate. We hypothesized that IL-6 could augment the proliferation rate of (slow-)dividing cardiomyocytes. METHODS: To mimic the behavior of therapeutic cells in the post-infarct cardiac microenvironment, human Adipose Derived Stromal Cells (ADSC) were cultured under hypoxic (2% O2) and pro-inflammatory conditions (IL-1ß) for 24h. Serum-free conditioned medium from ADSC primed with hypoxia and/or IL-1ß was added to rat neonatal cardiomyocytes and adult cardiomyocytes (HL-1) to assess paracrine-driven changes in cardiomyocyte proliferation rate and induction of myogenic signaling pathways. RESULTS: We demonstrate that ADSC enhance the proliferation rate of rat neonatal cardiomyocytes and adult HL-1 cardiomyocytes in a paracrine fashion. ADSC under hypoxia and inflammation in vitro had increased the interleukin-6 (IL-6) gene and protein expression. Similar to conditioned medium of ADSC, treatment of rat neonatal cardiomyocytes and HL-1 with recombinant IL-6 alone also stimulated their proliferation rate. This was corroborated by a strong decrease of cardiomyocyte proliferation after addition of IL-6 neutralizing antibody to conditioned medium of ADSC. The stimulatory effect of ADSC conditioned media or IL-6 was accomplished through activation of both Janus Kinase-Signal Transducer and Activator of Transcription (JAK/STAT) and Mitogen-Activated Protein (MAP) kinases (MAPK) mitogenic signaling pathways. CONCLUSION: ADSC are promising therapeutic cells for cardiac stem cell therapy. The inflammatory and hypoxic host post-MI microenvironment enhances the regenerative potential of ADSC to promote the proliferation rate of cardiomyocytes. This was achieved in paracrine manner, which warrants the development of ADSC conditioned medium as an "of-the-shelf" product for treatment of post-myocardial infarction complications.

A global downregulation of microRNAs occurs in human quiescent satellite cells during myogenesis.

During myogenesis, human satellite cells differentiate and form multinucleated myotubes, while a fraction of the human satellite cells enter quiescence. These quiescent satellite cells are able to activate, proliferate and contribute to muscle regeneration. Post-transcriptional regulation of myogenesis occurs through specific myogenic microRNAs, also known as myomiRs. Although many microRNAs are involved in myotube formation, little is known on the involvement of microRNAs in satellite cells entering quiescence. This current study aims to investigate microRNA involvement during differentiation of human satellite cells, specifically proliferating satellite cells entering quiescence. For this, clonally expanded human satellite cells were differentiated for 5 days, after which myotubes and quiescent satellite cells were separated through FACS sorting. Next, a microRNA microarray comparison of proliferating satellite cells, myotubes and quiescent satellite cells was performed and verified through qRT-PCR. We show that during human satellite cell differentiation, microRNAs are globally downregulated in quiescent satellite cells compared to proliferating satellite cells, in particular microRNA-106b, microRNA-25, microRNA-29c and microRNA-320c. Furthermore, we show that during myogenesis microRNA-1, microRNA-133, microRNA-206 and microRNA-486 are involved in myotube formation rather than satellite cells entering quiescence. Finally, we show an overall decrease in total mRNA in quiescent satellite cells, and an indication that RNaseL regulation plays a role in promoting and maintaining quiescence. Given the importance of quiescent satellite cells in skeletal muscle development and regenerative medicine, it is imperative to distinguish between myotubes and quiescent satellite cells when investigating skeletal muscle development, especially in microRNA studies, since we show that microRNAs are globally downregulated in quiescent human satellite cells.