Pirfenidone and nintedanib exert additive antifibrotic effects by the SPP1-AKT pathway in macrophages and fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease with high mortality rates. It has been shown that pirfenidone (PFD) and nintedanib (Ofev) can slow down the decline in lung function of IPF patients, but their efficacy remains suboptimal. Some studies have suggested that the combination of PFD and Ofev may yield promising results. However, there is a lack of research on the combined application of these two medications in the treatment of IPF. A mouse model of bleomycin-induced (BLM) pulmonary fibrosis was established to investigate the impact of combination therapy on pulmonary fibrosis of mice. The findings demonstrated a significant reduction in lung tissue damage in mice treated with the combination therapy. Subsequent transcriptome analysis identified the differential gene secreted phosphoprotein 1 (SPP1), which was found to be associated with macrophages and fibroblasts based on multiple immunofluorescence staining results. Analysis of a phosphorylated protein microarray indicated that SPP1 plays a regulatory role in macrophages and fibroblasts via the AKT pathway. Consequently, the regulation of macrophages and fibroblasts in pulmonary fibrosis by the combination of PFD and Ofev is mediated by SPP1 through the AKT pathway, potentially offering a novel therapeutic option for IPF patients. Further investigation into the targeting of SPP1 for the treatment of pulmonary fibrosis is warranted.

Noncanonical WNT5A controls the activation of latent TGF-ß to drive fibroblast activation and tissue fibrosis.

Transforming growth factor ß (TGF-ß) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-ß remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-ß in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-ß. The activation of latent TGF-ß requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-ß, rebalanced TGF-ß signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-ß in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.

Sox9-coordinated cellular neighborhoods generate fibrosis.

Poorly regenerative organs deposit scar tissue to mend damage. Aggarwal et al. establish that transient Sox9 activity is necessary for early proximal tubule epithelial regeneration, while Trogisch et al. and Aggarwal et al. show that persistent Sox9 activity in epithelial and endothelial cells activates fibroblasts creating fibrotic microdomains in multiple organs.

Modeling acute myocardial infarction and cardiac fibrosis using human induced pluripotent stem cell-derived multi-cellular heart organoids.

Heart disease involves irreversible myocardial injury that leads to high morbidity and mortality rates. Numerous cell-based cardiac in vitro models have been proposed as complementary approaches to non-clinical animal research. However, most of these approaches struggle to accurately replicate adult human heart conditions, such as myocardial infarction and ventricular remodeling pathology. The intricate interplay between various cell types within the adult heart, including cardiomyocytes, fibroblasts, and endothelial cells, contributes to the complexity of most heart diseases. Consequently, the mechanisms behind heart disease induction cannot be attributed to a single-cell type. Thus, the use of multi-cellular models becomes essential for creating clinically relevant in vitro cell models. This study focuses on generating self-organizing heart organoids (HOs) using human-induced pluripotent stem cells (hiPSCs). These organoids consist of cardiomyocytes, fibroblasts, and endothelial cells, mimicking the cellular composition of the human heart. The multi-cellular composition of HOs was confirmed through various techniques, including immunohistochemistry, flow cytometry, q-PCR, and single-cell RNA sequencing. Subsequently, HOs were subjected to hypoxia-induced ischemia and ischemia-reperfusion (IR) injuries within controlled culture conditions. The resulting phenotypes resembled those of acute myocardial infarction (AMI), characterized by cardiac cell death, biomarker secretion, functional deficits, alterations in calcium ion handling, and changes in beating properties. Additionally, the HOs subjected to IR efficiently exhibited cardiac fibrosis, displaying collagen deposition, disrupted calcium ion handling, and electrophysiological anomalies that emulate heart disease. These findings hold significant implications for the advancement of in vivo-like 3D heart and disease modeling. These disease models present a promising alternative to animal experimentation for studying cardiac diseases, and they also serve as a platform for drug screening to identify potential therapeutic targets.

Fibroblasts' secretome from calcified and non-calcified dermis in Pseudoxanthoma elasticum differently contributes to elastin calcification.

Pseudoxanthoma elasticum (PXE) is a rare disease characterized by ectopic calcification, however, despite the widely spread effect of pro/anti-calcifying systemic factors associated with this genetic metabolic condition, it is not known why elastic fibers in the same patient are mainly fragmented or highly mineralized in clinically unaffected (CUS) and affected (CAS) skin, respectively. Cellular morphology and secretome are investigated in vitro in CUS and CAS fibroblasts. Here we show that, compared to CUS, CAS fibroblasts exhibit: a) differently distributed and organized focal adhesions and stress fibers; b) modified cell-matrix interactions (i.e., collagen gel retraction); c) imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases; d) differentially expressed pro- and anti-calcifying proteoglycans and elastic-fibers associated glycoproteins. These data emphasize that in the development of pathologic mineral deposition fibroblasts play an active role altering the stability of elastic fibers and of the extracellular matrix milieu creating a local microenvironment guiding the level of matrix remodeling at an extent that may lead to degradation (in CUS) or to degradation and calcification (in CAS) of the elastic component. In conclusion, this study contributes to a better understanding of the mechanisms of the mineral deposition that can be also associated with several inherited or age-related diseases (e.g., diabetes, atherosclerosis, chronic kidney diseases).

A novel MT-ATP6 variant associated with complicated ataxia in two unrelated Italian patients: case report and functional studies.

BACKGROUND: MT-ATP6 is a mitochondrial gene which encodes for the intramembrane subunit 6 (or A) of the mitochondrial ATP synthase, also known asl complex V, which is involved in the last step of oxidative phosphorylation to produce cellular ATP through aerobic metabolism. Although classically associated with the NARP syndrome, recent evidence highlights an important role of MT-ATP6 pathogenic variants in complicated adult-onset ataxias. METHODS: We describe two unrelated patients with adult-onset cerebellar ataxia associated with severe optic atrophy and mild cognitive impairment. Whole mitochondrial DNA sequencing was performed in both patients. We employed patients' primary fibroblasts and cytoplasmic hybrids (cybrids), generated from patients-derived cells, to assess the activity of respiratory chain complexes, oxygen consumption rate (OCR), ATP production and mitochondrial membrane potential. RESULTS: In both patients, we identified the same novel m.8777 T > C variant in MT-ATP6 with variable heteroplasmy level in different tissues. We identifed an additional heteroplasmic novel variant in MT-ATP6, m.8879G > T, in the patients with the most severe phenotype. A significant reduction in complex V activity, OCR and ATP production was observed in cybrid clones homoplasmic for the m.8777 T > C variant, while no functional defect was detected in m.8879G > T homoplasmic clones. In addition, fibroblasts with high heteroplasmic levelsof m.8777 T > C variant showed hyperpolarization of mitochondrial membranes. CONCLUSIONS: We describe a novel pathogenic mtDNA variant in MT-ATP6 associated with adult-onset ataxia, reinforcing the value of mtDNA screening within the diagnostic workflow of selected patients with late onset ataxias.

Targeting Interactions between Fibroblasts and Macrophages to Treat Cardiac Fibrosis.

Excessive extracellular matrix (ECM) deposition is a defining feature of cardiac fibrosis. Most notably, it is characterized by a significant change in the concentration and volume fraction of collagen I, a disproportionate deposition of collagen subtypes, and a disturbed ECM network arrangement, which directly affect the systolic and diastolic functions of the heart. Immune cells that reside within or infiltrate the myocardium, including macrophages, play important roles in fibroblast activation and consequent ECM remodeling. Through both direct and indirect connections to fibroblasts, monocyte-derived macrophages and resident cardiac macrophages play complex, bidirectional, regulatory roles in cardiac fibrosis. In this review, we discuss emerging interactions between fibroblasts and macrophages in physiology and pathologic conditions, providing insights for future research aimed at targeting macrophages to combat cardiac fibrosis.

Lysosomal dysfunction and overload of nucleosides in thymidine phosphorylase deficiency of MNGIE.

Inherited deficiency of thymidine phosphorylase (TP), encoded by TYMP, leads to a rare disease with multiple mitochondrial DNA (mtDNA) abnormalities, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the impact of TP deficiency on lysosomes remains unclear, which are important for mitochondrial quality control and nucleic acid metabolism. Muscle biopsy tissue and skin fibroblasts from MNGIE patients, patients with m.3243 A > G mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and healthy controls (HC) were collected to perform mitochondrial and lysosomal functional analyses. In addition to mtDNA abnormalities, compared to controls distinctively reduced expression of LAMP1 and increased mitochondrial content were detected in the muscle tissue of MNGIE patients. Skin fibroblasts from MNGIE patients showed decreased expression of LAMP2, lowered lysosomal acidity, reduced enzyme activity and impaired protein degradation ability. TYMP knockout or TP inhibition in cells can also induce the similar lysosomal dysfunction. Using lysosome immunoprecipitation (Lyso- IP), increased mitochondrial proteins, decreased vesicular proteins and V-ATPase enzymes, and accumulation of various nucleosides were detected in lysosomes with TP deficiency. Treatment of cells with high concentrations of dThd and dUrd also triggers lysosomal dysfunction and disruption of mitochondrial homeostasis. Therefore, the results provided evidence that TP deficiency leads to nucleoside accumulation in lysosomes and lysosomal dysfunction, revealing the widespread disruption of organelles underlying MNGIE.

TSPAN8+ myofibroblastic cancer-associated fibroblasts promote chemoresistance in patients with breast cancer.

Cancer-associated fibroblasts (CAFs) are abundant stromal cells in the tumor microenvironment that promote cancer progression and relapse. However, the heterogeneity and regulatory roles of CAFs underlying chemoresistance remain largely unclear. Here, we performed a single-cell analysis using high-dimensional flow cytometry analysis and identified a distinct senescence-like tetraspanin-8 (TSPAN8)+ myofibroblastic CAF (myCAF) subset, which is correlated with therapeutic resistance and poor survival in multiple cohorts of patients with breast cancer (BC). TSPAN8+ myCAFs potentiate the stemness of the surrounding BC cells through secretion of senescence-associated secretory phenotype (SASP)-related factors IL-6 and IL-8 to counteract chemotherapy. NAD-dependent protein deacetylase sirtuin 6 (SIRT6) reduction was responsible for the senescence-like phenotype and tumor-promoting role of TSPAN8+ myCAFs. Mechanistically, TSPAN8 promoted the phosphorylation of ubiquitin E3 ligase retinoblastoma binding protein 6 (RBBP6) at Ser772 by recruiting MAPK11, thereby inducing SIRT6 protein destruction. In turn, SIRT6 down-regulation up-regulated GLS1 and PYCR1, which caused TSPAN8+ myCAFs to secrete aspartate and proline, and therefore proved a nutritional niche to support BC outgrowth. By demonstrating that TSPAN8+SIRT6low myCAFs were tightly associated with unfavorable disease outcomes, we proposed that the combined regimen of anti-TSPAN8 antibody and SIRT6 activator MDL-800 is a promising approach to overcome chemoresistance. These findings highlight that senescence contributes to CAF heterogeneity and chemoresistance and suggest that targeting TSPAN8+ myCAFs is a promising approach to circumvent chemoresistance.

Creation of an Isogenic H/N/KRAS-Less Mouse Embryonic Fibroblast Cell Line Panel Derived from a Size-Sorted Diploid Clone.

Cell line panels have proven to be an invaluable tool for investigators researching a range of topics from drug mechanism or drug sensitivity studies to disease-specific etiology. The cell lines used in these panels may range from heterogeneous tumor populations grown from primary tumor isolations to genetically engineered clonal cell lines which express specific gene isoforms. Mouse embryonic fibroblast (MEF) cells are a commonly used cell line for biological research due to their accessibility and ease of genetic manipulation. This chapter will describe the process of creating a size-sorted diploid (SSDC) clonal cell panel expressing specific RAS isoforms from a previously engineered RAS-less MEF cell line pool.

Deciphering the spatial landscape and plasticity of immunosuppressive fibroblasts in breast cancer.

Although heterogeneity of FAP+ Cancer-Associated Fibroblasts (CAF) has been described in breast cancer, their plasticity and spatial distribution remain poorly understood. Here, we analyze trajectory inference, deconvolute spatial transcriptomics at single-cell level and perform functional assays to generate a high-resolution integrated map of breast cancer (BC), with a focus on inflammatory and myofibroblastic (iCAF/myCAF) FAP+ CAF clusters. We identify 10 spatially-organized FAP+ CAF-related cellular niches, called EcoCellTypes, which are differentially localized within tumors. Consistent with their spatial organization, cancer cells drive the transition of detoxification-associated iCAF (Detox-iCAF) towards immunosuppressive extracellular matrix (ECM)-producing myCAF (ECM-myCAF) via a DPP4- and YAP-dependent mechanism. In turn, ECM-myCAF polarize TREM2+ macrophages, regulatory NK and T cells to induce immunosuppressive EcoCellTypes, while Detox-iCAF are associated with FOLR2+ macrophages in an immuno-protective EcoCellType. FAP+ CAF subpopulations accumulate differently according to the invasive BC status and predict invasive recurrence of ductal carcinoma in situ (DCIS), which could help in identifying low-risk DCIS patients eligible for therapeutic de-escalation.

Synovial fibroblast gene expression is associated with sensory nerve growth and pain in rheumatoid arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We developed a machine-learning approach (graph-based gene expression module identification or GbGMI) to identify an 815-gene expression module associated with pain in synovial biopsy samples from patients with established RA who had limited synovial inflammation at arthroplasty. We then validated this finding in an independent cohort of synovial biopsy samples from patients who had early untreated RA with little inflammation. Single-cell RNA sequencing analyses indicated that most of these 815 genes were most robustly expressed by lining layer synovial fibroblasts. Receptor-ligand interaction analysis predicted cross-talk between human lining layer fibroblasts and human dorsal root ganglion neurons expressing calcitonin gene-related peptide (CGRP+). Both RA synovial fibroblast culture supernatant and netrin-4, which is abundantly expressed by lining fibroblasts and was within the GbGMI-identified pain-associated gene module, increased the branching of pain-sensitive murine CGRP+ dorsal root ganglion neurons in vitro. Imaging of solvent-cleared synovial tissue with little inflammation from humans with RA revealed CGRP+ pain-sensing neurons encasing blood vessels growing into synovial hypertrophic papilla. Together, these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA.

Heterogeneity of primary and metastatic CAFs: From differential treatment outcomes to treatment opportunities (Review).

Compared with primary tumor sites, metastatic sites appear more resistant to treatments and respond differently to the treatment regimen. It may be due to the heterogeneity in the microenvironment between metastatic sites and primary tumors. Cancer­associated fibroblasts (CAFs) are widely present in the tumor stroma as key components of the tumor microenvironment. Primary tumor CAFs (pCAFs) and metastatic CAFs (mCAFs) are heterogeneous in terms of source, activation mode, markers and functional phenotypes. They can shape the tumor microenvironment according to organ, showing heterogeneity between primary tumors and metastases, which may affect the sensitivity of these sites to treatment. It was hypothesized that understanding the heterogeneity between pCAFs and mCAFs can provide a glimpse into the difference in treatment outcomes, providing new ideas for improving the rate of metastasis control in various cancers.

Cardiomyocytes, cardiac endothelial cells and fibroblasts contribute to anthracycline-induced cardiac injury through RAS-homologous small GTPases RAC1 and CDC42.

The clinical use of the DNA damaging anticancer drug doxorubicin (DOX) is limited by irreversible cardiotoxicity, which depends on the cumulative dose. The RAS-homologous (RHO) small GTPase RAC1 contributes to DOX-induced DNA damage formation and cardiotoxicity. However, the pathophysiological relevance of other RHO GTPases than RAC1 and different cardiac cell types (i.e., cardiomyocytes, non-cardiomyocytes) for DOX-triggered cardiac damage is unclear. Employing diverse in vitro and in vivo models, we comparatively investigated the level of DOX-induced DNA damage in cardiomyocytes versus non-cardiomyocytes (endothelial cells and fibroblasts), in the presence or absence of selected RHO GTPase inhibitors. Non-cardiomyocytes exhibited the highest number of DOX-induced DNA double-strand breaks (DSB), which were efficiently repaired in vitro. By contrast, rather low levels of DSB were formed in cardiomyocytes, which however remained largely unrepaired. Moreover, DOX-induced apoptosis was detected only in non-cardiomyocytes but not in cardiomyocytes. Pharmacological inhibitors of RAC1 and CDC42 most efficiently attenuated DOX-induced DNA damage in all cell types examined in vitro. Consistently, immunohistochemical analyses revealed that the RAC1 inhibitor NSC23766 and the pan-RHO GTPase inhibitor lovastatin reduced the level of DOX-induced residual DNA damage in both cardiomyocytes and non-cardiomyocytes in vivo. Overall, we conclude that endothelial cells, fibroblasts and cardiomyocytes contribute to the pathophysiology of DOX-induced cardiotoxicity, with RAC1- and CDC42-regulated signaling pathways being especially relevant for DOX-stimulated DSB formation and DNA damage response (DDR) activation. Hence, we suggest dual targeting of RAC1/CDC42-dependent mechanisms in multiple cardiac cell types to mitigate DNA damage-dependent cardiac injury evoked by DOX-based anticancer therapy.

Nail Telocytes: Identification, Potential Physiological Function, and Role in Pathology: A Reappraisal of the So-Called Onychofibroblasts/Onychodermis.

ABSTRACT: Some authors have suggested that the fibroblasts of the nail mesenchyme (onychofibroblasts) can be distinguished from skin fibroblasts by their high expression of CD10. My 2015 study documented the presence of a relatively sparse CD34 + /CD10 + dendritic subpopulation in the dermis and hypodermis of the matrix. For some time now, my hypothesis has been that these interstitial dendritic mesenchymal cells of the matrix correspond to telocytes. Telocytes have been described as peculiar interstitial dendritic cells present in the mesenchymal tissue of numerous organs, including the skin, but their presence and characteristics in the nail unit have not been explored. This study was undertaken to more comprehensively investigate the existence and characteristics of nail telocytes. A series of 20 normal adult nail units were examined with a combination of morphological and immunohistochemical analyses. The matrix dermis contained a sparse subpopulation of CD34 + /CD10 + elongated telocytes with a higher density in the lunular region and, at this distal level, a change in their immunohistochemical profile, resulting in a progressive loss of CD34 expression. The matrix hypodermis showed CD34 + /CD10 + telocytes in their classical elongated aspect, which acquired, especially in the distal fibromyxoid area of the thumb, an oval to round morphology with multiple intracytoplasmic vacuoles. The characteristic dynamic immunophenotypic profile of the dermal telocytes with a progressive distal loss of the defining molecule CD34 was equally observed in the distal hypodermis. The nail bed dermis was thick with a dense fibrous connective tissue. A reticular network of CD34 - /CD10 + telocytes was present in the superficial dermis of the proximal nail bed. The mesenchymal cells of the deep part of the proximal nail bed dermis and the entire distal nail bed dermis were CD34 - /CD10 - . The adult nail mesenchyme is composed of 3 microanatomically distinct regions. Only the thumb has a distal hypodermis rich in mucinous material. The population of telocytes is relatively sparse compared with the fibroblastic population of the entire nail mesenchyme. The concept of onychodermis/onychofibroblasts is not valid. Nail telocytes have a dynamic immunohistochemical profile depending on whether they are located proximally or distally. The CD34 + /CD10 + profile correlates with the onychogenic epithelial region, while the CD34 - /CD10 + profile correlates with a spatial rearrangement of the nail epidermal bed.

Transcriptomic analysis identifies dysregulated pathways and therapeutic targets in PMM2-CDG.

PMM2-CDG (MIM # 212065), the most common congenital disorder of glycosylation, is caused by the deficiency of phosphomannomutase 2 (PMM2). It is a multisystemic disease of variable severity that particularly affects the nervous system; however, its molecular pathophysiology remains poorly understood. Currently, there is no effective treatment. We performed an RNA-seq based transcriptomic study using patient-derived fibroblasts to gain insight into the mechanisms underlying the clinical symptomatology and to identify druggable targets. Systems biology methods were used to identify cellular pathways potentially affected by PMM2 deficiency, including Senescence, Bone regulation, Cell adhesion and Extracellular Matrix (ECM) and Response to cytokines. Functional validation assays using patients' fibroblasts revealed defects related to cell proliferation, cell cycle, the composition of the ECM and cell migration, and showed a potential role of the inflammatory response in the pathophysiology of the disease. Furthermore, treatment with a previously described pharmacological chaperone reverted the differential expression of some of the dysregulated genes. The results presented from transcriptomic data might serve as a platform for identifying therapeutic targets for PMM2-CDG, as well as for monitoring the effectiveness of therapeutic strategies, including pharmacological candidates and mannose-1-P, drug repurposing.

Similar metabolic pathways are affected in both Congenital Myasthenic Syndrome-22 and Prader-Willi Syndrome.

Loss of prolyl endopeptidase-like (PREPL) encoding a serine hydrolase with (thio)esterase activity leads to the recessive metabolic disorder Congenital Myasthenic Syndrome-22 (CMS22). It is characterized by severe neonatal hypotonia, feeding problems, growth retardation, and hyperphagia leading to rapid weight gain later in childhood. The phenotypic similarities with Prader-Willi syndrome (PWS) are striking, suggesting that similar pathways are affected. The aim of this study was to identify changes in the hypothalamic-pituitary axis in mouse models for both disorders and to examine mitochondrial function in skin fibroblasts of patients and knockout cell lines. We have demonstrated that Prepl is downregulated in the brains of neonatal PWS-IC-p/+m mice. In addition, the hypothalamic-pituitary axis is similarly affected in both Prepl-/- and PWS-IC-p/+m mice resulting in defective orexigenic signaling and growth retardation. Furthermore, we demonstrated that mitochondrial function is altered in PREPL knockout HEK293T cells and can be rescued with the supplementation of coenzyme Q10. Finally, PREPL-deficient and PWS patient skin fibroblasts display defective mitochondrial bioenergetics. The mitochondrial dysfunction in PWS fibroblasts can be rescued by overexpression of PREPL. In conclusion, we provide the first molecular parallels between CMS22 and PWS, raising the possibility that PREPL substrates might become therapeutic targets for treating both disorders.

Molecular and cellular consequences of mevalonate kinase deficiency.

Mevalonate kinase deficiency (MKD) is an autosomal recessive metabolic disorder associated with recurrent autoinflammatory episodes. The disorder is caused by bi-allelic loss-of-function variants in the MVK gene, which encodes mevalonate kinase (MK), an early enzyme in the isoprenoid biosynthesis pathway. To identify molecular and cellular consequences of MKD, we studied primary fibroblasts from severely affected patients with mevalonic aciduria (MKD-MA) and more mildly affected patients with hyper IgD and periodic fever syndrome (MKD-HIDS). As previous findings indicated that the deficient MK activity in MKD impacts protein prenylation in a temperature-sensitive manner, we compared the subcellular localization and activation of the small Rho GTPases RhoA, Rac1 and Cdc42 in control, MKD-HIDS and MKD-MA fibroblasts cultured at physiological and elevated temperatures. This revealed a temperature-induced altered subcellular localization and activation in the MKD cells. To study if and how the temperature-induced ectopic activation of these signalling proteins affects cellular processes, we performed comparative transcriptome analysis of control and MKD-MA fibroblasts cultured at 37 °C or 40 °C. This identified cell cycle and actin cytoskeleton organization as respectively most down- and upregulated gene clusters. Further studies confirmed that these processes were affected in fibroblasts from both patients with MKD-MA and MKD-HIDS. Finally, we found that, similar to immune cells, the MK deficiency causes metabolic reprogramming in MKD fibroblasts resulting in increased expression of genes involved in glycolysis and the PI3K/Akt/mTOR pathway. We postulate that the ectopic activation of small GTPases causes inappropriate signalling contributing to the molecular and cellular aberrations observed in MKD.

Antisense oligonucleotide-mediated terminal intron retention of endoglin: A potential strategy to inhibit renal interstitial fibrosis.

TGF-ß is considered an important cytokine in the development of interstitial fibrosis in chronic kidney disease. The TGF-ß co-receptor endoglin (ENG) tends to be upregulated in kidney fibrosis. ENG has two membrane bound isoforms generated via alternative splicing. Long-ENG was shown to enhance the extent of renal fibrosis in an unilateral ureteral obstruction mouse model, while short-ENG inhibited renal fibrosis. Here we aimed to achieve terminal intron retention of endoglin using antisense-oligo nucleotides (ASOs), thereby shifting the ratio towards short-ENG to inhibit the TGF-ß1-mediated pro-fibrotic response. We isolated mRNA from kidney biopsies of patients with chronic allograft disease (CAD) (n = 12) and measured total ENG and short-ENG mRNA levels. ENG mRNA was upregulated 2.3 fold (p < 0.05) in kidneys of CAD patients compared to controls, while the percentage short-ENG of the total ENG mRNA was significantly lower (1.8 fold; p < 0.05). Transfection of ASOs that target splicing regulatory sites of ENG into TK173 fibroblasts led to higher levels of short-ENG (2 fold; p < 0.05). In addition, we stimulated these cells with TGF-ß1 and measured a decrease in upregulation of ACTA2, COL1A1 and FN1 mRNA levels, and protein expression of αSMA, collagen type I, and fibronectin. These results show a potential for ENG ASOs as a therapy to reduce interstitial fibrosis in CKD.