Microstructured Lipid Carriers (MLC) Based on N-Acetylcysteine and Chitosan Preventing Pseudomonas aeruginosa Biofilm

The aim of this work was the development of microstructured lipid carriers (MLC) based on chitosan (CH) and containing N-acetylcysteine (NAC), a mucolytic and antioxidant agent, to inhibit the formation of Pseudomonas aeruginosa biofilm. MLC were prepared using the high shear homogenization technique. The MLC were characterized for morphology, particle size, Z potential, encapsulation efficiency and drug release. The antioxidant properties of NAC-loaded microstructured carriers were evaluated through an in vitro spectrophotometer assay. Finally, the activity of NAC-CH-MLC on biofilm production by Pseudomonas aeruginosa was also evaluated. Results obtained from this study highlighted that the use of chitosan into the inner aqueous phase permitted to obtain microstructured particles with a narrow size range and with good encapsulation efficiency. NAC-loaded MLC showed higher antioxidant activity than the free molecule, demonstrating how encapsulation increases the antioxidant effect of the molecule. Furthermore, the reduction of biofilm growth resulted extremely high with MLC being 64.74% ± 6.2% and 83.74% ± 9.95%, respectively, at 0.5 mg/mL and 2 mg/mL. In conclusion, this work represents a favorable technological strategy against diseases in which bacterial biofilm is relevant, such as cystic fibrosis.     

Nuclear Receptor Nur77 Controls Cardiac Fibrosis through Distinct Actions on Fibroblasts and Cardiomyocytes

Fibrosis is a hallmark of adverse cardiac remodeling, which promotes heart failure, but it is also an essential repair mechanism to prevent cardiac rupture, signifying the importance of appropriate regulation of this process. In the remodeling heart, cardiac fibroblasts (CFs) differentiate into myofibroblasts (MyoFB), which are the key mediators of the fibrotic response. Additionally, cardiomyocytes are involved by providing pro-fibrotic cues. Nuclear receptor Nur77 is known to reduce cardiac hypertrophy and associated fibrosis; however, the exact function of Nur77 in the fibrotic response is yet unknown. Here, we show that Nur77-deficient mice exhibit severe myocardial wall thinning, rupture and reduced collagen fiber density after myocardial infarction and chronic isoproterenol (ISO) infusion. Upon Nur77 knockdown in cultured rat CFs, expression of MyoFB markers and extracellular matrix proteins is reduced after stimulation with ISO or transforming growth factor–β (TGF-β). Accordingly, Nur77-depleted CFs produce less collagen and exhibit diminished proliferation and wound closure capacity. Interestingly, Nur77 knockdown in neonatal rat cardiomyocytes results in increased paracrine induction of MyoFB differentiation, which was blocked by TGF-β receptor antagonism. Taken together, Nur77-mediated regulation involves CF-intrinsic promotion of CF-to-MyoFB transition and inhibition of cardiomyocyte-driven paracrine TGF-β-mediated MyoFB differentiation. As such, Nur77 provides distinct, cell-specific regulation of cardiac fibrosis.     

Modulation of Tubular pH by Acetazolamide in a Ca2+ Transport Deficient Mice Facilitates Calcium Nephrolithiasis

Proximal tubular (PT) acidosis, which alkalinizes the urinary filtrate, together with Ca²⁺ supersaturation in PT can induce luminal calcium phosphate (CaP) crystal formation. While such CaP crystals are known to act as a nidus for CaP/calcium oxalate (CaOx) mixed stone formation, the regulation of PT luminal Ca²⁺ concentration ([Ca²⁺]) under elevated pH and/or high [Ca²⁺] conditions are unknown. Since we found that transient receptor potential canonical 3 (TRPC3) knockout (KO; -/-) mice could produce mild hypercalciuria with CaP urine crystals, we alkalinized the tubular pH in TRPC3-/- mice by oral acetazolamide (0.08%) to develop mixed urinary crystals akin to clinical signs of calcium nephrolithiasis (CaNL). Our ratiometric (λ340/380) intracellular [Ca²⁺] measurements reveal that such alkalization not only upsurges Ca²⁺ influx into PT cells, but the mode of Ca²⁺ entry switches from receptor-operated to store-operated pathway. Electrophysiological experiments show enhanced bicarbonate related current activity in treated PT cells which may determine the stone-forming phenotypes (CaP or CaP/CaOx). Moreover, such alkalization promotes reactive oxygen species generation, and upregulation of calcification, inflammation, fibrosis, and apoptosis in PT cells, which were exacerbated in absence of TRPC3. Altogether, the pH-induced alteration of the Ca²⁺ signaling signature in PT cells from TRPC3 ablated mice exacerbated the pathophysiology of mixed urinary stone formation, which may aid in uncovering the downstream mechanism of CaNL.     

Screening of Inhibitors Targeting Heat Shock Protein 47 Involved in the Development of Idiopathic Pulmonary Fibrosis

Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is causally related to fibrotic diseases, including idiopathic pulmonary fibrosis. The identification of Compounds that interfere with the HSP47-collagen interaction is essential for the development of relevant therapeutics. Herein, we prepared human HSP47 as a soluble fusion protein expressed in E. coli and established an assay system for HSP47 inhibitor screening. We screened a natural and synthetic Compound library established at Nagasaki University. Among 1023 Compounds, 13 exhibited inhibitory activity against human HSP47, of which three inhibited its function in a dose-dependent manner. Epigallocatechin-3-O-gallate, one of these three Compounds, is a typical polyphenol Compound derived from tea leaves. Structurally related Compounds were synthesized and examined for their activity, revealing a hydroxyl group at A-ring position 5 as important for its activity. The present findings provide valuable insight for the development of natural product-derived therapeutics for fibrotic diseases, including idiopathic pulmonary fibrosis.     

Collagen I Modifies Connexin-43 Hemichannel Activity via Integrin α2β1 Binding in TGFβ1-Evoked Renal Tubular Epithelial Cells

Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel-mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up-regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin-43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFβ1) ± the Cx43 mimetic Peptide 5 and/or an anti-integrin α2β1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell-substrate interactions. Collagen I and TGFβ1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2β1/integrin-like kinase signaling. The co-incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFβ1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin-mediated hemichannel activity through integrin α2β1 signaling, ahead of establishing Peptide 5 as a potential intervention.     

Thrombospondin-1 CD47 Signalling: From Mechanisms to Medicine

Recent advances provide evidence that the cellular signalling pathway comprising the ligand-receptor duo of thrombospondin-1 (TSP1) and CD47 is involved in mediating a range of diseases affecting renal, vascular, and metabolic function, as well as cancer. In several instances, research has barely progressed past pre-clinical animal models of disease and early phase 1 clinical trials, while for cancers, anti-CD47 therapy has emerged from phase 2 clinical trials in humans as a crucial adjuvant therapeutic agent. This has important implications for interventions that seek to capitalize on targeting this pathway in diseases where TSP1 and/or CD47 play a role. Despite substantial progress made in our understanding of this pathway in malignant and cardiovascular disease, knowledge and translational gaps remain regarding the role of this pathway in kidney and metabolic diseases, limiting identification of putative drug targets and development of effective treatments. This review considers recent advances reported in the field of TSP1-CD47 signalling, focusing on several aspects including enzymatic production, receptor function, interacting partners, localization of signalling, matrix-cellular and cell-to-cell cross talk. The potential impact that these newly described mechanisms have on health, with a particular focus on renal and metabolic disease, is also discussed.     

Attenuating Effects of Dieckol on Hypertensive Nephropathy in Spontaneously Hypertensive Rats

Hypertension induces renal fibrosis or tubular interstitial fibrosis, which eventually results in end-stage renal disease. Epithelial-to-mesenchymal transition (EMT) is one of the underlying mechanisms of renal fibrosis. Though previous studies showed that Ecklonia cava extracts (ECE) and dieckol (DK) had inhibitory action on angiotensin (Ang) I-converting enzyme, which converts Ang I to Ang II. It is known that Ang II is involved in renal fibrosis; however, it was not evaluated whether ECE or DK attenuated hypertensive nephropathy by decreasing EMT. In this study, the effect of ECE and DK on decreasing Ang II and its down signal pathway of angiotensin type 1 receptor (AT1R)/TGFβ/SMAD, which is related with the EMT and restoring renal function in spontaneously hypertensive rats (SHRs), was investigated. Either ECE or DK significantly decreased the serum level of Ang II in the SHRs. Moreover, the renal expression of AT1R/TGFβ/SMAD was decreased by the administration of either ECE or DK. The mesenchymal cell markers in the kidney of SHRs was significantly decreased by ECE or DK. The fibrotic tissue of the kidney of SHRs was also significantly decreased by ECE or DK. The ratio of urine albumin/creatinine of SHRs was significantly decreased by ECE or DK. Overall, the results of this study indicate that ECE and DK decreased the serum levels of Ang II and expression of AT1R/TGFβ/SMAD, and then decreased the EMT and renal fibrosis in SHRs. Furthermore, the decrease in EMT and renal fibrosis could lead to the restoration of renal function. It seems that ECE or DK could be beneficial for decreasing hypertensive nephropathy by decreasing EMT and renal fibrosis.     

Nasal Epithelial Cell-Based Models for Individualized Study in Cystic Fibrosis

The emergence of highly effective CFTR modulator therapy has led to significant improvements in health care for most patients with cystic fibrosis (CF). For some, however, these therapies remain inaccessible due to the rarity of their individual CFTR variants, or due to a lack of biologic activity of the available therapies for certain variants. One proposed method of addressing this gap is the use of primary human cell-based models, which allow preclinical therapeutic testing and physiologic assessment of relevant tissue at the individual level. Nasal cells represent one such tissue source and have emerged as a powerful model for individual disease study. The ex vivo culture of nasal cells has evolved over time, and modern nasal cell models are beginning to be utilized to predict patient outcomes. This review will discuss both historical and current state-of-the art use of nasal cells for study in CF, with a particular focus on the use of such models to inform personalized patient care.     

Cellular Senescence: Pathogenic Mechanisms in Lung Fibrosis

Pulmonary fibrosis is a chronic and fatal lung disease that significantly impacts the aging population globally. To date, anti-fibrotic, immunosuppressive, and other adjunct therapy demonstrate limited efficacies. Advancing our understanding of the pathogenic mechanisms of lung fibrosis will provide a future path for the cure. Cellular senescence has gained substantial interest in recent decades due to the increased incidence of fibroproliferative lung diseases in the older age group. Furthermore, the pathologic state of cellular senescence that includes maladaptive tissue repair, decreased regeneration, and chronic inflammation resembles key features of progressive lung fibrosis. This review describes regulatory pathways of cellular senescence and discusses the current knowledge on the senescence of critical cellular players of lung fibrosis, including epithelial cells (alveolar type 2 cells, basal cells, etc.), fibroblasts, and immune cells, their phenotypic changes, and the cellular and molecular mechanisms by which these cells contribute to the pathogenesis of pulmonary fibrosis. A few challenges in the field include establishing appropriate in vivo experimental models and identifying senescence-targeted signaling molecules and specific therapies to target senescent cells, known collectively as “senolytic” or “senotherapeutic” agents.