In this study, we analyzed a cohort of six colorectal cancer patients harboring KRAS mutations and with wild-type BRAF from a transcriptional perspective, with the aim of elucidating the role of the stromal cells in tumor progression. Specifically, paraffin-embedded specimens were subjected to microdissection and hybridized on Agilent-026652 microarrays to compare the gene expression of tumor samples composed of neoplastic epithelial samples against the neighboring stromal tissue. A paired rank-product test led to the detection of 193 differentially expressed genes. Subsequent functional enrichment analysis pointed to extracellular matrix constituents, angiogenesis, and cell migration as the main biological processes enhanced in stromata, while the tumor compartment was characterized by an overexpression of many ribosomal protein genes. A further gene set enrichment analysis against a comprehensive ribosomal protein gene set finally revealed that only cytosolic ribosomes (80S) were affected by such upregulation, while mitochondrial ribosomes were virtually unaltered.Because of its high incidence and poor prognosis, colorectal cancer (CRC) represents an important health issue in several countries. As with other carcinomas, the so-called tumour microenvironment (TME) has been shown to play key roles in CRC progression and related therapeutical outcomes, even though a deeper understanding of the underlying molecular mechanisms is needed to devise new treatment strategies. For some years now, omics technologies and consolidated bioinformatics pipelines have allowed scientists to access large amounts of biologically relevant information, even when starting from small tissue samples; thus, in order to shed new light upon the role of the TME in CRC, we compared the gene expression profiles of 6 independent tumour tissues  to the expression profile of the surrounding stromata. To do this, paraffin-embedded whole tissues were first microdissected to obtain samples enriched with tumour and stromal cells, respectively. Afterwards, RNA was extracted and analysed using a microarray-based approach. A thorough bioinformatics analysis was then carried out to identify transcripts differentially expressed between the two groups and possibly enriched functional terms. Overall, 193 genes were found to be significantly downregulated in tumours compared to the paired stromata. The functional analysis of the downregulated gene list revealed three principal macro areas of interest: the extracellular matrix, cell migration, and angiogenesis. Conversely, among the upregulated genes, the main alterations detected by the functional annotation were related to the ribosomal proteins (rProteins) of both the large (60S) and small (40S) subunits of the cytosolic ribosomes. Subsequent gene set enrichment analysis (GSEA) confirmed the massive overexpression of most cytosolic—but not mitochondrial—ribosome rProteins. Solid tumours are not only composed of neoplastic cells but also of the stromal components surrounding the tumour mass and interacting with it—the so-called tumour microenvironment (TME). The TME is a complex network of cells and an extracellular matrix, which is physically part of the tumour itself and can play a central role in tumour progression, both helping neoplastic cells to survive and proliferate in hostile conditions and dictating therapy resistance ,2,3.HOX2D and RHOB, involved in apoptosis; SQSTM1, which mediates NFkB activation; RRAD, a member of the Ras/GTPase superfamily) were identified in tumour compared to stromal cells, which were separated using fluorescence-activated cell sorting [The cellular component of the TME is represented by mesenchymal cells, such as myofibroblasts or specialised cells  and tumour-associated fibroblasts. In addition, endothelial and muscle cells, as well as cells of both native and adaptive immune systems, contribute to make the TME a highly heterogenous tissue (reviewed in ). In mor sorting . Through sorting . More re sorting . OverallKRAS mutations and with wild-type BRAF  through a paired sample design carried out using microarray analysis.The aim of this study is to compare the transcriptomic profiles of microdissected tumour tissue and the surrounding tumour stromata in metastatic CRC patients harbouring KRAS mutations and not BRAF-mutated were enrolled for the study between April 2016 and October 2018 at the Medical Oncology Unit, Azienda Ospedaliero-Universitaria Careggi (Florence). All patients provided informed written consent and the study was approved by the local Ethical Committee of Azienda Ospedaliero-Universitaria Careggi . Paraffin-embedded samples of the primary tumours were retrieved from the archives of the Department of Experimental and Clinical Medicine (University of Florence). The clinical and pathological features of the patients were defined by experienced medical oncologists and pathologists according to the relevant guidelines. Overall, 6 patients were included in the study, since 4 out of 10 were not analysed due to array hybridisation failure  suffering from metastatic colorectal cancer harbouring In order to compare tumour tissue with the surrounding stroma, paraffin-embedded samples were manually microdissected. Briefly, 20 μm thick sections were put onto uncharged glass slides and counterstained with haematoxylin. Using a syringe needle, the tumour and stromal tissues were separated under a light microscope, collected, and transferred to a sterile tube.® DNA/RNA FFPE kit  from all paraffinized tissues for transcriptomics analyses. The RNA concentration was evaluated using a Nanodrop ND-1000 instrument , while the quality and integrity were evaluated using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano kit .From the enriched samples, total RNA extraction was carried out with the AllPrepGene expression analysis of RNA was carried out through a One-Color Microarray-Based Gene Expression Analysis using the Agilent-026652 Whole Human Genome Microarray 4 × 44 K v2 platform (Agilent Technologies) and following the manufacturer’s protocols. An Agilent G49000 DA SureScan Microarray scanner was used to scan the microarrays. Data were then extracted by Agilent Feature Extraction and stored for further analyses.normexp algorithm, log2-transformed, and inter-array-normalized through the quantile–quantile procedure. Based on the average value of the Agilent Negative Control probes (namely (−)3xSLv1), the gene expression matrix was then subjected to filtering in order to discard unexpressed genes. In particular, only probes featuring log2 expression above 6.8 in at least 75% of the samples of at least one group were retained for subsequent analysis. Overall, 25,605 probes out of 34,128 (75.03%) survived the filtering procedure. Upon sample pairing, expression values were used for log2 fold change (FC) computation and differential expression assessment according to their rank product statistics  [p-values were adjusted for multiple comparisons and statistical significance was assessed using the Benjamini–Hochberg procedure to control the false discovery rate (BH-FDR) to 5%  [2FC| < 0.5.After array scanning, raw data were processed using a custom script based on R/Bioconductor packages. Specifically, fluorescence intensity signals were background-subtracted using the  design) ,22,23,24https://toppgene.cchmc.org/, accessed on 12 March 2021) was used for hypergeometric test-based functional enrichment analysis [q-value < 0.05 were considered statistically significant.The ToppFun web tool  enrichment statistic was chosen. Normalized enriched score (NES) and BH-FDR q-values are reported in the text for each gene set of interest.To evaluate the global transcriptional alterations involving ribosomal protein, a comprehensive list of rProtein gene symbols was compiled by sourcing gene symbols from the HGNC database, then a gene set enrichment analysis was performed using GSEA v4.1.0 . To copeKRAS mutations and none carrying BRAF mutations  and stromal tissues  belonging to the same lesion, we performed manual microdissection  are shown in 2FC values observed in each patient—is represented as a heatmap in Afterwards, the two tissue types were statistically tested for differential gene expression using a paired-sample design. Collectively, 193 genes turned out to be significantly deregulated in tumour samples compared to the associated stroma. The top 50 differentially expressed genes (DEGs) sorted by average loghttps://toppgene.cchmc.org/, accessed on 15 April 2021) for functional enrichment analysis. To this end, ToppFun sources annotations from many different databases, including Gene Ontology (GO), KEGG, BioCarta, Reactome, and HUGO Gene Nomenclature Committee (HGNC). The full lists of the statistically significant terms  resulting from the enrichment analysis of both up- and downregulated DEGs are provided as The two lists of statistically significant up- and downregulated genes were separately fed to the ToppFun web tool , further confirming the relevance and the consistency of these data.The main alterations revealed by the functional annotation of the up-DEG list were related to ribosomal proteins (rProteins), ribosome structural constituents, and protein targeting. In particular, 11 different rProtein gene transcripts—corresponding to 14 unique Agilent probes—related to both the large (60S) and the small (40S) cytosolic ribosome subunits shown to be significantly overexpressed in tumour samples compared to the adjacent stroma , ECM glycoproteins , proteoglycans , and various integrin-related genes . The angiogenesis cluster—besides containing some of the same previous ECM proteins—comprised the angiopoietin ANGPTL4 and other genes that are known to act in tumour-induced angiogenesis , for a total of 21 entries. Finally, the set related to cell migration accounted for 29 genes, some of which (such as ARHGAP4) specifically related to cell motility or migration. For the precise gene content of each cluster, see On the other hand, from the functional enrichment analysis of the down-DEG list, three main clusters of functional terms emerged: (i) core extracellular matrix (ECM) structure and organization; (ii) angiogenesis, blood vessel development, and morphogenesis; (iii) cell migration and motility  ribosomes, with 58 out of 75 rProtein genes belonging the positive leading edge subset  nor those related to the small one (28S) showed any significant enrichment .Based on these observations, GSEA allowed us to conclude that the increased transcription of ribosomal genes in colorectal cancer cells compared to stromal cells mainly relates to the 80S ribosomes, while the mitochondrial ribosomes are left substantially unaffected.KRAS mutations and none with BRAF mutations. We provided evidence that: (i) RNA extracted from enriched tumour and stromal samples, obtained by manual microdissection, can be efficiently used for transcriptomic analysis; (ii) the comparison of the gene expression profiles of tumour and stroma pairs from CRC samples highlighted the presence of several differentially expressed genes (DEGs); (iii) the main overexpressed genes emerging from enriched stroma samples compared to tumour samples belonged to angiogenesis, cell migration, and extracellular matrix functions; (iv) the main result of our analysis is that transcripts related to ribosomes underwent a general overexpression in tumour tissue with respect to stromata, and this increased transcription of ribosomal genes mainly affects the 80S ribosomes, while the mitochondrial ribosomes are not affected.In recent years, several pieces of evidence have pointed out the relevance of the cross-talk between the tumour and the associated stromal components in determining the biological features of a cancer and its progression. Furthermore, omics technologies applied to the analysis of cancer tissues uncovered the possibility that a different molecular profile of differentially expressed genes characterises the tumour tissue and the surrounding stromata. In the present paper, we analysed the gene expression profiles of microdissected tumour and stromal tissues obtained from CRC samples. To avoid conflicting results due to the different clinicopathological characteristics of the patients, we studied a homogeneous cohort of CRC patients, all carrying From a technical point of view, our results regarding the separation (and cell component enrichment) of tumour tissue vs. stroma tissue are similar to those reported by Carlomagno’s group in terms of showing the efficacy of manual microdissection as a separation technique, although they applied a slightly different protocol to obtain enriched samples from paraffin-embedded specimens to be used for transcriptomic analysis . Our appKRAS mutations and none carrying BRAF mutations, 193 genes turned out to be significantly deregulated in the tumour compared to the paired stroma. Although we applied a conservative analysis after proper filtering and cleaning of the background noise, the number of DEGs we found was higher than reported in other papers presenting omics data, such as the study by Sugiyama et al., 2005 [ARHGAP4, GNAI2, AQP1, HSPB1) and others being implicated in the secretion of promigratory proteins , possibly acting as a chemoattractant source for the tumour cells in the neighbouring compartment. In this regard, SPARC (secreted protein acidic and cysteine-rich) has already been reported in different types of metastatic cancer—including CRC—and has attracted attention as a possible biomarker [Overall, in our cohort of 6 metastatic CRC patients all harbouring l., 2005 ; howeverl., 2005 . Likewisiomarker ,30.Different reports have addressed the DEG profile in CRC, although most of them have been focused on comparisons between CRC and healthy colon samples. Nevertheless, the relevance of separately evaluating the two components was pointed out several years ago by Smith and colleagues, who showed that some DE genes were detected when comparing laser-microdissected tumours with the whole lesion , althoughttp://xena.ucsc.edu/, accessed on 5 August 2021) [https://gtexportal.org/home/, accessed on 5 August 2021) [KRAS mutations and with wild-type BRAF. This led to a final cohort of n = 118 tumour samples and n = 304 normal tissues   for the st 2021) . Xena alRPS20, encoding a component of the small ribosomal subunit, increases the predisposition to develop a particular form of hereditary CRC named familial colorectal cancer type X (FCCX).A distinctive feature of cancer cells is the constitutive activation of growth factor signalling pathways that alter the activity of transcription factors ,34 respoIn general, the remodelling of the ribosomal machinery is likely to exert relevant biological effects in terms of protein synthesis and targeting, and could be integrated with the investigation of the translational and post-translational modifications in CRC. It is generally accepted that tumour cells, being characterised by enhanced growth, might also have increased ribosome production associated with altered biogenesis and nucleolar modifications  . MoreoveOverall, our data highlight the relevance of 80S ribosomes in cancer, which are in line with the recently proposed use of these organelles for innovative therapies (reviewed in ).KRAS mutations, our results are in accordance with a recent report [KRAS mutations in codon 13 are characterised by a significant upregulation of several genes involved in ribosome genesis, metabolism, and mRNA translation paralleled by increased proliferation and protein synthesis.Since all of the patients carried t report  showing Another interesting finding obtained in this study was the observation that the affected ribosomes belong to the cytosolic compartment, while mitochondrial ribosomes are not significantly altered. The link between cancer and metabolism was discovered many years ago, thanks to pivotal studies on aerobic glycolysis in cancer , and hasThe Warburg effect was shown to be a finely regulated metabolic state that can represent an advantage for cancer cells in the context of increased biosynthetic demand . It is wKRAS mutated–BRAF wild-type CRC agree with Warburg’s hypothesis, suggesting that the scenario might be different to that observed in other tumours, as in the case of the relevant role of OXPHOS in lymphomas [The mitochondrial remodelling in ymphomas .It is interesting to notice that the cohort analysed in this paper was composed of surgically resected CRC belonging to TNM stages II–IV, although all of the patients were enrolled when the disease became metastatic; therefore, it could be argued that the molecular signature we described could identify more aggressive cancers that are likely to progress towards a systemic disease.The data provided in this paper are promising but deserve further investigation. One option for further study could be a comparison between tumour tissue and healthy colorectal mucosa, as well as between a tumour stroma and normal stroma. It would also be useful to extend the analysis to different tumours in order to understand whether this DEG profile is unique to CRC or could represent a more general pattern. Moreover, proteomic screening could provide different and complementary information compared to the transcriptomic analysis presented herein.Tumour tissue and the surrounding stroma tissue are characterised by the differential expression of several genes. Omics approaches could help in identifying molecular patterns useful for the management of cancer patients. Data reported in this study represent a first piece of evidence pointing to the overexpression of cytosolic ribosome transcripts in CRC tumours vs. stromata. Further analyses performed in larger cohorts and different neoplasias are warranted.


Bone morphogenetic proteins (BMPs) play an important role in bone formation and repair. Recent studies underscored their essential role in the normal development of several organs and vascular homeostasis in health and diseases. Elevated levels of BMPs have been linked to the development of cardiovascular complications of diabetes mellitus. However, their particular role in the pathogenesis of microvascular dysfunction associated with diabetic retinopathy (DR) is still under-investigated. Accumulated evidence from our and others’ studies suggests the involvement of BMP signaling in retinal inflammation, hyperpermeability and pathological neovascularization in DR and age-related macular degeneration (AMD). Therefore, targeting BMP signaling in diabetes is proposed as a potential therapeutic strategy to halt the development of microvascular dysfunction in retinal diseases, particularly in DR. The goal of this review article is to discuss the biological functions of BMPs, their underlying mechanisms and their potential role in the pathogenesis of DR in particular. Diabetic retinopathy (DR) is one of the most common vascular complications of diabetes mellitus. The diagnosis of DR relies on the identification of microvascular abnormalities which are characterized by the breakdown of the blood retinal barrier (BRB), microaneurysms and pathological retinal neovascularization (RNV). The early stage of DR is called non-proliferative DR (NPDR), in which patients suffer from diabetic macular edema (DME) due to vascular hyperpermeability. DME is the most common cause of vision loss in diabetic patients due to the swelling and thickening of the macula that results from vascular leakage. The late stage of the disease is called proliferative DR (PDR) due to the development of RNV ,2. The gIntravitreal sustained release corticosteroid devices such as triamcinolone acetonide, dexamethasone and fluocinolone acetonide are beneficial, especially in patients with insufficient response to anti-VEGF, serving as anti-inflammatory agents and VEGF inhibitors ,5. They The retina is a highly specialized neuronal tissue which has the ability to convert visible light into an electrochemical signal. This signal is carried to the brain, which interprets it as vision. BRB plays a fundamental role in maintaining the privilege of the eye by the regulation of the fluid and molecular movement between ocular vascular beds and retinal tissues. The BRB includes the inner blood retinal barrier (iBRB) and the outer blood retinal barrier (oBRB). iBRB consists of tight junctions between retinal endothelial cells, pericytes and glial cells. On the other hand, retinal pigment epithelium (RPE) cells linked by tight junctions create the oBRB and rest on the underlying Bruch’s membrane. oBRB plays an essential role in maintaining the microenvironment of the outer retina, while iBRB maintains the microenvironment in the inner neural retina, and any dysfunction in iBRB or oBRB contributes to the pathophysiology of a number of retinal diseases, such as DR and AMD ,12,13.The goal of the current review article is to present BMPs and their downstream signaling systems as potential players and novel therapeutic targets to prevent, or at least minimize, the microvascular dysfunction associated with DR.BMPs are a subgroup of the transforming growth factor β (TGFβ) superfamily. There are ~20 BMPs which share structural similarities and were first described as key players in the formation and repair of bones . BMP family members are classified according to their structural homology into numerous subgroups, including the BMP2/4 group, the BMP5/6/7/8 group, the BMP9/10 group and the BMP12/13/14 group. Different BMPs are produced as inactive large pre-pro-polypeptides . The inactively synthesized BMPs contain mature polypeptides at their carboxyl terminals, with pro-domains separating them from signal peptides at their amino terminals . Mature BMPs include seven cysteines, where one of them is included in the process of dimerization by a covalent disulfide bond with another BMP monomer, generating a BMP-dimer capable of binding to, and activating, a BMP receptor . BMPs have other biological functions beyond their role in bone formation and repair. For example, BMP2 is crucial for retinal development, and both BMP2 and BMP4 are implicated in the formation of cardiac septa, and their deficiency may cause heart defects in mice. Moreover, BMP7 plays a role in the development of the kidney and the heart ,16. ReceBMP signaling involves a canonical pathway and a non-canonical one . The canStudies showed that BMPs are capable of binding to two types of serine-threonine kinase BMP receptors (BMPR1 and BMPR2) ,22. BMPsIt was believed that the extracellular matrix (ECM) represented an inert mechanical barrier getting rid of BMPs. However, recent work demonstrated that ECM may have a role in regulating BMP signaling . BMP sigBMPs promote angiogenesis by facilitating endothelial motility and invasion, as well as cell proliferation . During The interplay between BMPs and immune response should be taken into consideration. Studies on macrophages and endothelial cells have suggested both pro-inflammatory and anti-inflammatory roles ,38. TumoMonocytes from type 2 diabetic patients express higher levels of BMP2. Additionally, human macrophages shift to the M1 inflammatory phenotype when exposed to high glucose. Accordingly, higher levels of BMP2 in type 2 diabetes may contribute to the activation of the inflammatory response ,42.Several studies have highlighted the role of BMP2 in endothelial cell inflammation ,44,45. PBmper knockout mice were used where there was a significantly higher vascular leakage into interstitial lung tissue when compared with wild-type mice. Moreover, Bmper knockdown in endothelial cells increased endothelial permeability and reduced the VE-cadherin expression. These effects were rescued by the use of the recombinant human BMPER protein. Interestingly, enhanced BMP activity induced the effects of Bmper knockdown on both VE-cadherin expression and endothelial permeability. Increased levels of BMPER antagonized BMP4 signaling and prevented BMP4-induced endothelial barrier dysfunction and VE-cadherin downregulation, proposing BMPER as a BMP antagonist capable of restoring the endothelial barrier function [Bmper+/− mice showed increased vascular branching and sprouting. Although BMP levels did not increase in the retinas of those mice, BMP signaling was activated, as demonstrated by the increased endothelial phosphorylated SMAD protein levels and the increased expression of BMP target genes [Agonists and antagonists of the TGF-β family, including BMPs, were extensively reviewed previously . Among tfunction . Retinaset genes . Howeveret genes  postulatet genes . Taken tBMPs are essentially known for their salient role in embryogenesis and development. “BMPs are essentially known for their critical role in embryogenesis and development. Therefore, genetic elimination of BMPs, their downstream effectors or their receptors is embryonically lethal or causes major developmental anomalies”, their downstream effectors or their receptors were either embryonically lethal or suffered from major developmental anomalies. This reflects their striking function during the process of development. For instance, knocking out BMP 2 in mouse models showed that it is embryonically lethal, with defects in the formation of the amnion and chorion, impaired limb and cartilage development and impaired cardiac development. Animals lacking BMP 4 died in utero with a deficient development of the mesoderm and multiple organ anomalies, including cardiovascular and gonadal anomalies ,56,57,58Akita/+ and db/db mice, also demonstrated an increased vascular expression of BMP ligands, inhibitors and receptors [Akita/+ and db/db mice [kita/+ that lacked the BMP inhibitor matrix Gla protein . Taken together, these data suggest the emerging role of BMPs in vascular changes during diabetes. They also suggest that the BMP signaling system could be a therapeutic target to halt the development of the vascular complications of diabetes.BMPs are multifunctional growth factors that contribute to the pathogenesis of vascular complications of diabetes. BMPs play an important role in glucose homeostasis, being implicated in the development of the pancreas and insulin secretion ,71. Thereceptors . Recentl/db mice . MoreoveEarlier studies in our lab on human pre-osteoblasts drew our attention to the important role of BMPs as regulatory players in the process of inflammation and angiogenesis via upregulating VEGF and IL-6 . These pWe published the first report showing increased levels of retinal BMP2 in human and experimental DR . In thisMicrovascular endothelial cells challenged with hypoxia or treated with VEGF showed upregulated BMP2 at both RNA and protein levels , suggestRecently, our group reported that circulating levels of BMP2 significantly increased in plasma derived from different experimental diabetic animal models . The samCollectively, we are suggesting the presence of a differential impact of BMPs affecting retinal-blood barriers, with BMP2 dysregulation affecting mostly the inner barrier ,72, and Interestingly, Xu et al.  have shoRemarkably, human ocular samples from neovascular age-related macular degeneration (AMD) donors were rich in BMP2 and BMP4, particularly in RPE and choroid rather than in retina. However, only BMP4 was upregulated in blood samples from those donors. Therefore, our group suggested that circulating BMP4 but not BMP2 or local retinal BMP4 is implicated in the pathogenesis of neovascular AMD. This conclusion was confirmed by demonstrating that BMP4 but not BMP2 interrupted the RPE barrier function, induced RPE migration and increased MMPs’ activity in RPE .In summary, although the role of the BMP signaling system in the pathogenesis of diabetic retinopathy is under-investigated, accumulated evidence from our lab and other studies underscored the potential involvement of BMPs in inducing microvascular dysfunction in DR. Thus, targeting BMP signaling may represent a future therapeutic intervention to treat DR.


PMP22 gene overexpression in the most frequent CMT1A type are under investigation. PXT3003 is the compound in the most advanced phase for CMT1A, as a second-phase III trial is ongoing. Gene therapy to substitute defective genes or insert novel ones and compounds acting on pathways important for different CMT types are being developed and tested in animal models. Modulation of the Neuregulin pathway determining myelin thickness is promising for both hypo-demyelinating and hypermyelinating neuropathies; intervention on Unfolded Protein Response seems effective for rescuing misfolded myelin proteins such as P0 in CMT1B. HDAC6 inhibitors improved axonal transport and ameliorated phenotypes in different CMT models. Other potential therapeutic strategies include targeting macrophages, lipid metabolism, and Nav1.8 sodium channel in demyelinating CMT and the P2X7 receptor, which regulates calcium influx into Schwann cells, in CMT1A. Further approaches are aimed at correcting metabolic abnormalities, including the accumulation of sorbitol caused by biallelic mutations in the sorbitol dehydrogenase (SORD) gene and of neurotoxic glycosphingolipids in HSN1.There is still no effective drug treatment available for Charcot-Marie-Tooth neuropathies (CMT). Current management relies on rehabilitation therapy, surgery for skeletal deformities, and symptomatic treatment of pain; fatigue and cramps are frequent complaints that are difficult to treat. The challenge is to find disease-modifying therapies. Several approaches, including gene silencing, to counteract the  GJB1 gene on chromosome Xq13.1; less common autosomal dominant or recessive forms are on the record. Pure motor forms of CMT are characterised by the preservation of sensory nerves and are labelled distal Hereditary Motor Neuropathies (dHMNs), which can have autosomal dominant, autosomal recessive, or X-linked transmission. Pure or predominantly sensory neuropathies are grouped under the term of Hereditary Sensory (and Autonomic) Neuropathies (HS(A)Ns) and can have autosomal dominant or recessive inheritance patterns. Eventually, Hereditary Neuropathy with liability to Pressure Palsies is an autosomal dominant disorder characterised by recurrent focal neuropathies, which are typically painless and transient, and it is associated with the deletion of the PMP22 gene, whereas the duplication of the same gene with PMP22 overexpression is associated with the most frequent CMT subtype, CMT1A. Overall, this group of inherited peripheral neuropathies is also labelled as CMT and related neuropathies and is further classified according to the mutated gene. Indeed, about 100 genes have been associated with CMT and related disorders, which code for proteins involved in many different functions relevant for the nerves, including myelin formation and maintenance, axonal transport, vesicular trafficking, mitochondrial homeostasis, Endoplasmic Reticulum (ER) stress, channels formation, etc. . Ni. Ni62]. UPR is an adaptive, protective cellular reaction that is able to relieve stress from misfolded proteins and is activated when mutated proteins are retained in the ER. This is what has convincingly been demonstrated to occur in CMT1B for many MPZ mutations . HoweverBoth primary demyelinating and axonal CMT cause clinical disability through axonal degeneration. Sterile alpha and toll/interleukin 1 receptor motif-containing 1 (SARM1) is a key molecule in the axonal degeneration process, and loss of SARM1 is able to prevent axonal degeneration and improve recovery after nerve injury and in different neuropathic models . TherefoC201R/+ mice (model of CMT2D/dHMN type VA) and the MFN2R94Q mouse model of CMT2A, with effective correction or prevention of electrophysiological and clinical dysfunction [Disturbances of axonal transport are common to many hereditary neuropathies, and another active field of research is the development of selective inhibitors of Histon De-ACetylase type 6 (HDAC6), which by increasing acetylated alpha-tubulin were able to improve axonal transport and ameliorate the phenotype of mutant HSPB1 mice, which is a model of CMT2/dHMN . Thereaffunction ,76. An ifunction ,75 and iThere are several compounds that have been tested or are under consideration for different CMT models and types, and the most interesting are listed below.ClinicalTrials.gov NCT03124459) has been recently completed that employed ACE-083, which is a molecule that is able to generate muscle hypertrophy by inhibiting myostatin and other muscle regulators [A phase II trial , which activates macrophages, decreased their presence in the nerves and improved clinical and pathological features in CMT1B and CMTX1 mice .Some CMT types represent true metabolic neuropathies resulting from enzymatic deficits leading to product shortage or neurotoxic accumulation; in such cases, correction of the metabolic anomalies can potentially rescue the neuropathic phenotype. This is the case for CMT4B1 and CMT4B2, the hypermyelinating neuropathies already mentioned above, which are caused by mutations in myotubularin-related proteins  involved in phosphoinositides’ metabolism and for which inhibitors of the PIKfyve kinase, the enzyme counteracting the activity of MTMR2, are under investigation as potential treatments .SPTCL1/2) coding for serine palmitoyltransferase (SPT), which is the first enzyme in the sphingolipid synthesis pathway that conjugates L-serine with palmitoyl-CoA. Such SPTCL1/2 mutations lead to an accumulation of neurotoxic 1-deoxysphingolipids where serine is substituted by alanine or glycine and causes Hereditary Sensory Neuropathy type 1 (HSN1); L-serine supplementation was metabolically and phenotypically effective in the mouse model and improved the metabolic abnormalities also in the patients, although a pilot trial did not result in significant clinical benefit, which was likely because it was underpowered [A second example is provided by mutations in the two genes . Two patients with Arts syndrome appeared to benefit from S-adenosylmethionine (SAM) supplementation administered with the aim to refill the purine nucleotides pool ; whetherThe most recent and interesting instance is constituted by the recessive mutations in the gene encoding the sorbitol dehydrogenase (SORD) enzyme, associated with CMT2/dHMN, which catalyses the first step of the polyol pathway by oxidising sorbitol into fructose. SORD-defective patients have very high levels of sorbitol in their blood; studies on diabetic neuropathies have shown that sorbitol may be toxic for the nerves and inhibitors of aldose reductase, which transforms glucose into sorbitol in the step preceding SORD action, can reduce sorbitol accumulation and were effective in the SORD-defective cellular models .Nowadays, the main challenge for CMT is to find disease-modifying treatments. Although no drug treatment for any CMT type is still available, in the last few years, great advances have been made: the first clinical trials in patients have been conducted, others are ongoing or planned, and several therapeutic approaches are being tested in experimental models. There is a cautious optimism among researchers that it will not take a long time to see the first effective therapies for CMT. In the meanwhile, there is a lot we can do for CMT patients, including proper rehabilitation therapies, the use of appropriate orthotics, surgery for skeletal deformities in selected cases, management of pain, cramps, and fatigue, adequate general care, avoidance of neurotoxic drugs, and genetic counselling.


Neurodevelopmental disorders (NDDs) are a group of heterogeneous conditions, which include mainly intellectual disability, developmental delay (DD) and autism spectrum disorder (ASD), among others. These diseases are highly heterogeneous and both genetic and environmental factors play an important role in many of them. The introduction of next generation sequencing (NGS) has lead to the detection of genetic variants in several genetic diseases. The main aim of this report is to discuss the impact and advantages of the implementation of NGS in the diagnosis of NDDs. Herein, we report diagnostic yields of applying whole exome sequencing in 87 families affected by NDDs and additional data of whole genome sequencing (WGS) from 12 of these families.THOC2, SETD1B and CHD9 genes. Finally the use of WGS only allowed the identification of disease causing variants in 8% (1/12) of the patients in which previous WES failed to identify a genetic aetiology.The use of NGS technologies allowed identifying the causative gene alteration in approximately 36% (31/87) of the families. Among them, de novo mutation represented the most common cause of genetic alteration found in 48% (15/31) of the patients with diagnostic mutations. The majority of variants were located in known neurodevelopmental disorders genes. Nevertheless, some of the diagnoses were made after the use of GeneMatcher tools which allow the identification of additional patients carrying mutations in NGS is more powerful in identifying causative pathogenic variant than conventional algorithms based on chromosomal microarray as first-tier test. Our results reinforce the implementation of NGS as a first-test in genetic diagnosis of NDDs. Neurodevelopmental disorders (NDDs) are a group of heterogeneous conditions that result from an abnormal brain development that may give rise to impaired cognition, communication, adaptive behaviour, and psychomotor skills . NDDs inNext-generation sequencing (NGS) technologies have improved diagnostics of rare genetic diseases. Specifically, whole exome sequencing (WES) has led to enormous progress in deciphering monogenic forms of NDDs , 6. If cCurrent recommendations of genetic studies for diagnosis NDDs are based in the guidelines reported by Miller and collaborators reported more than 10 years ago in which chromosomal microarray (CMA) is considered the first-tier clinical diagnosis test for individuals with DD or congenital anomalies . The diaA total of 87 families with one or several members affected by affected by NDDs were referred to the Biochemistry and Molecular Genetics department for genetic testing. This cohort reflects a heterogeneous collection of clinical presentations, making it representative of a typical genetic clinic. Before enrolment, all patients underwent an extensive diagnostic workup, including clinical evaluation and genomic profiling  in subtelomeric regions by MLPA and CMA . The institutional review board approved the collection and use of these samples for research purposes . Written informed consent was obtained from all subjects prior to their participation.versus a trio-based analysis or solo-based analysis (sporadic case). As shown in Table We used four different family series in order to test the impact of the testing strategy of an extended family analysis (more than one generation affected) Analysis of cases that underwent NGS has been previously described –15. BrieOur patient cohort consisted in 87 families with one or more individuals diagnosed with NDDs in which Fragile X syndrome and pathogenic CNVs were previously discarded. According to the high clinical heterogeneity of NDDs, approximately 75% (67/87) of our patients referred heterogeneous co-occurring conditions, where DD/ID was present in 90% of the patients (79/87). Other clinical co-occurring conditions included ADHD, speech disorders, epilepsy, micro/macrocephaly, and the presence of dysmorphic features/ malformations. Clinical features and genetic results of the diagnosed patients are shown in Table The use of NGS technologies allowed identifying the causative genetic alteration in 36% of the cases, which is a significant increase over conventional CMA testing. The initial cohort included 97 cases, with a diagnostic value of 8.2% and 32% for CMA and NGS, respectively. Using NGS we achieved genetic diagnosis in 31 families identifying all patterns of inheritance . We also found three patients with previously reported variants, two of them with de novo variants in NIPBL gene and one with a de novo variant in the KCNT2 gene ; patient 21 carried the splicing variant c.5329-15A > G in NIPBL reported in the literature to result the skipping of exon 28 without altering the reading frame [KCNT2 gene previously reported in the literature [Most of the de novo pathogenic variants were found to be novel and detected in different genes previously associated with NDDs , which is currently not associated with ID but some patients with autistic features have been already reported in HGMD database.On the other hand, we identified a de novo missense variant in the ACSL4, CLCN4, DDX3X, GRIA3, IQSEC2, MID1, OCRL, SMC1A and THOC2), representing 32% of the solved cases. We identified two families (patient 8 and patient 16) with pathogenic variants in the THOC2 recently associated with the Mental retardation, X-linked 12/35 syndrome .Our results have identified 10 families with pathogenic variants in 9 genes located in the X-chromosome  was found to carry a de novo missense pathogenic variant in VPS13B (patient 2), CSPP (patient 23) and EMC1 (patient 24) and one consanguineous family with a homozygous variant in the PYCR1 (patient 7) gene (Table TRAPPC9 (patient 31) gene  of the families were found to carry genetic alterations associated with an AR pattern of inheritance  of the patients. On the other hand, no differences were found in the diagnosis rate between the other cohort structures . Nevertheless, trio-based analysis reduced the turnaround time of genetic diagnosis (data not shown) and despite not having WES data from parents, segregation analysis were in most cases essential in order to definitively classify variants as disease causing.De novo variation has been proposed as a major pathomechanism in NDDs representing approximately 20–30% of ASD and ID cases , 23. OurKCNT2 gene, usually associated with Developmental and epileptic encephalopathy type 57 (OMIM # 617771). This syndrome is characterized by epileptic encephalopathy, global DD with hypotonia, variably impaired intellectual development and poor or absent language. The KCNT2-associated developmental and epileptic encephalopathy comprises West syndrome, Lennox-Gastaut syndrome as well as epilepsy of infancy with migrating focal seizures. Currently, there are 4 patients with de novo mutations in the Arg190 residue described in the literature [KCNT2 gene present ID, neonatal hypotonia, hirsutism, thick hair, prominent eyebrows, long eyelashes, and diastema [Regardless the pattern of inheritance, most of the identified genes are implied in syndromic neurodevelopmental disorders, such as Cornelia de Lange syndrome (CdLS), Dent disease or Cohen syndrome, which are considered clinically recognizable syndromes. Nevertheless, several of the identified variants are rare and in some cases the clinical manifestations do not correspond to the classic syndromic forms or clinical features are not really specific. For example, patient 29 was referred to clinical consultation for presenting ID and dysmorphic features. Results from NGS identified a de novo previously reported variant in the terature , 21). OuNGS is also unveiling a large number of VUS, which effectively represent mutations where the pathogenicity and the function of the gene involved is unclear, hampering their endorsement to a clinical phenotype. The process of variant prioritization is performed based on several factors, including their possible presence in databases such as gnomAD, ClinVar and HGMD; the relevance to the reported clinical features, the impact of the variant or the associated mode of inheritance, among others. In order to determine whether a VUS may be associated with a patients’ phenotype, additional studies are needed including segregation analysis or functional studies. There are several possibilities of functional tests that can be performed to answer questions about the function of genes and the functional consequences of genetic variants. RNA studies are the easiest and fastest ways to determine if a variant of unknown significance could be pathogenic. Nevertheless it is only indicated in variants that produce changes in mRNA expression levels, for example, variants that cause alternative splicing events or that cause loss of function. Protein expression studies have already proven their value to demonstrate pathogenicity of genetic variants. Another strategy is to test the genetic variants in model systems  [reviewed in NIPBL gene  in case 20 clinically diagnosed with CdLS. This patient has global cognitive and growth retardation, microcephaly, and dysmorphia. Physical examination revealed obvious clinical signs of CdLs such as long philtrum, long eyelashes, high-arched palate and anteverted nares, hypospadias . Howevehttp://www.genematcher.org) to share phenotype and genotype data to broaden the exchange between scientific and medical teams have allowed the discovery of new genes responsible for the disease. In our cohort, we identified two families diagnosed with pathogenic variants in THOC2 gene  gene family consists of nine genes (CHD1–CHD9), and pathogenic variants in all CHD genes except CHD9 are implicated in NDDs [CHD9 variants in ASD patients [CHD9 is a candidate gene for ASD.The application of NGS technologies and the development of several web-based tools such as GeneMatcher . However, it should be taken into consideration that pathogenic CNVs were discarded by CMA in all patients presented in this report before performing NGS studies which might be a limitation of the study. On the other hand, new bioinformatic pipelines allow the detection not only of SNV but also reliable CNVs through WES data which further reinforces this change in diagnostic algorithm. The genetic architecture of NDDs is complex and interdisciplinary approaches combining genetics, functional genomics and biological models will be essential.


Premature birth results in an increased risk of respiratory distress and often requires oxygen therapy. While the supplemental oxygen has been implicated as a cause of bronchopulmonary dysplasia (BPD), in clinical practice this supplementation usually only occurs after the patient’s oxygen saturation levels have dropped. The effect of hyperoxia on neonates has been extensively studied. However, there is an unanswered fundamental question: which has the most impact-hyperoxia, hypoxia or fluctuating oxygen levels? In this review, we will summarize the reported effect of hypoxia, hyperoxia or a fluctuation of oxygen levels (hypoxia/hyperoxia cycling) in preterm neonates, with special emphasis on the lungs. These abnormalities have been attributed to ventilator induced injury as well as to oxygen therapy.15Bronchopulmonary dysplasia (BPD) is a form of chronic lung disease that affects premature newborns and infants and is the result of damage to the lungs caused by mechanical ventilation and long-term use of oxygen.16 High rates of in utero and perinatal exposure to infection may be causally related to preterm delivery and subsequent lung injury.17 Over the past three decades, the histological presentation of BPD has changed from heterogeneous pulmonary inflammation and fibrosis (“Old BPD”) to uniform arrest of alveolar development and variable interstitial cellularity and/or fibroproliferation (“New BPD”).19BPD is recognized as a chronic lung disease of infancy that presents as a systemic syndrome and can be associated with neurodevelopmental deficits, cognitive impairments, failure to thrive, pulmonary hypertension and cor pulmonale.24 The developing lung of the neonate is a perfect example of those vulnerable tissues; the endothelial cells and the alveolar type II cells are especially susceptible to oxidative injury. Oxidative stress in these cells activates transcription factors and pathways leading to cellular dysfunction, inactivation of surfactant, and impaired cell survival.27The fetus normally lives in a physiologically hypoxic environment, and relative hyperoxic exposure can cause an increase in the generation of “reactive oxygen species” (ROS). Preterm newborns are particularly vulnerable to oxygen toxicity due to inadequate levels of antioxidant enzymes, and hence to decreased protection from oxidative injury of rapidly growing tissues.28 Excess levels of oxygen (hyperoxia), as occur in supplemental oxygen administration, generate accumulation of ROS1. Hypoxia/hypoxemia, as in episodes of oxygen desaturations, also results in generation of ROS via a superoxide burst that occurs rapidly with hypoxic exposure.31 This ROS generation has been shown to be necessary for stabilization of Hypoxia Inducible Factor 1α (HIF-1α) and activation of the HIF system.30Several pre- and post-natal adverse events generate ROS exposure and contribute to BPD pathogenesis; these include hypoxic, hyperoxic, and mechanical stimuli.32 Altered regulation of these transcription factors and pathways can disrupt postnatal alveolar development, cause inflammation, and potentially lead to fibrosis in preterm infants.34 ROS generation activates the HIF system.27 Nrf2 deficiency was shown to augment lung injury and arrest of alveolarization caused by hyperoxia during the newborn period.32 Both NF-κB and AP-1 are activated in multiple cell types, as well as in lung, following hyperoxia.2 A summary of ROS generation and neonatal lung injury is summarized in The ROS generated by both hyperoxia and hypoxia can result in alterations in cellular proliferation and apoptosis. Mechanisms may involve dysregulation of key transcription factors involved in ROS signaling such as HIF, nuclear factor E2 related factor 2 (Nrf2), NF-κB, and activator protein-1 (AP-1).36 Placental hypoxia also induces an imbalance between pro-angiogenic and anti-angiogenic factors inhibiting VEGF signaling.38 This reduction in VEGF signaling in the developing lung was shown to result in impaired pulmonary vascular growth and alveolarization in neonatal rats.39 Maternal exposure to hypobaric hypoxia at high-altitudes can alter placental function, influence oxygen delivery to the fetus, result in lower birth weight, and increase the risk for pre-eclampsia.41Tissue injury due to oxidative stress is also noted in placental hypoxia, as is seen in preterm born neonates of pre-eclamptic mothers, where ROS are elevated and antioxidant levels are decreased in the maternal circulation.42 Hypoxia of the infant leads to generalized pulmonary vasoconstriction and increases the pulmonary vascular resistance, causing pulmonary hypertension. Sustained hypoxic pulmonary vasoconstriction causes vascular remodeling of the pulmonary vascular bed leading ultimately to right heart failure.43 Postnatal hypoxia causes impaired alveolarization and alveolar simplification with fewer and larger alveoli. The impaired alveolarization also impairs the vascular maturation in the alveolar wall via mechanisms involving. altered signaling of HIF-1α, VEGF, and TGF-β,45Premature babies are exposed post-natally to chronic or intermittent hypoxia as a result of immature lungs, apnea of prematurity, inadequate ventilation, or persistence of intrapulmonary arteriovenous shunts causing hypoxemia.28 systemically circulating oxygen saturation levels (SPO2) is generally targeted to reach above 85%. However, the optimal systemic oxygen saturation in preterm infants is currently not clear. To compensate for the premature lung’s simplified alveolar structure and thick alveolar septae, higher levels of supplemental oxygen are often required to achieve the targeted intravascular oxygen levels, further augmenting the already existing (relative) postnatal hyperoxic state.27 Hyperoxia leads to impaired VEGF expression and disrupted angiogenesis and alveolarization due to rapid proteasomal degradation of HIF-1α.48 This induces vascular arrest, leading to pulmonary vascular diseases. Relative hyperoxia also increases generation of ROS and induces oxidative stress, an important contributor to the development of neonatal BPD. In addition, lower levels of antioxidants including vitamin E, transferrin, and superoxide dismutase, and higher levels of free iron further predispose preterm infants to oxidative injury.50Preterm birth leads to premature transition of the pulmonary circulation from the physiologic hypoxic fetal environment to a relative hyperoxic postnatal environment (room air). In addition, oxygen supplementation is often required to fulfill the oxygen demands for adequate functioning of the body’s tissues and organs, in particular the brain, intestines, and kidneys.55 The development of that injury is dependent on the developmental stage of the lung, and the type, duration, volume and pressure of the mechanical ventilation.54 Mechanical ventilation also results in down regulation of VEGF-1 and its receptor flt-1 and up-regulation of the TGF-β co-receptor endoglin. This imbalance in mechanically ventilated lungs likely contributes to altered alveolarization and angiogenesis.56Although mechanical ventilation is often essential and life-saving, it can provoke ventilator-induced lung injury in severely premature infants mainly by over-stretching of the distal epithelium and capillary endothelium.1 and AT2; both belong to a super family of seven transmembrane G-protein coupled receptors. The AT1 receptor mediates all of the classical actions of Ang II , while the AT2 receptor promotes vasodilatation, cell differentiation, inhibition of cell growth and apoptosis and may play a counterbalancing role to the effects of Ang II on the AT1 receptor.57 ACE-2 and its product angiotensin 1–7 (Ang 1–7) acting on its receptor Mas were shown to have counteracting effects against the adverse actions of the other RAS components. Findings from numerous experimental studies have suggested notable protective effects of ANG1–7/Mas activation in the cardiovascular system.58 Local tissue effects of RAS have been identified in a variety of tissues such as heart, kidney, liver, lung, brain, pancreas and adipose tissue, where RAS component expression has been detected.60 Local RAS is involved in injury and inflammatory and fibrogenic diseases of many organs including heart,62 lung,65 liver,66 pancreas,67 and kidneys,69 by mechanisms independent of the blood-derived RAS.The RAS  is tradi70 In addition, neonatal rat ACE inhibition lowered the surface tension of bronchoalveolar lavage fluid and caused widening of respiratory airspaces and thinning of alveolar septa.71 Autopsy of human BPD patients showed decreased ACE expression in lungs as compared to controls without lung disease.72 Wagenaar et al.4 reported that Mas receptor and angiotensin receptor 2 (AT2) agonists reduced inflammation of oxygen-induced lung injury in rats. In this study, mRNA levels of the RAS component genes were measured during normal lung development. The mRNA levels of AT1, AT2, and ACE-2 decreased gradually, whereas expression of angiotensinogen and ACE-1 increased gradually as the neonatal rat lung develops. Exposure to 100% oxygen for 10 days resulted in an increase in expression of AT2 and a decrease in expression of AT1, angiotensinogen, and ACE.4 These studies suggest a critical role for angiotensin-angiotensin receptor signaling involving pulmonary alveolarization in the normal physiology of the neonatal lung and in the pathophysiology of BPD,. Angiotensinogen and ACE-1 play a role in alveolar development and septation, while the reported lower expression levels of ACE-2 in the neonatal lung could be facilitating the lung injury in the neonatal period.72RAS is believed to play a role in neonatal lung development and BPD pathogenesis . Perinat73 Previous studies from our lab and other groups suggest that ACE-2 is down-regulated in fibrotic conditions of the adult and neonatal human lung,73 via Mas receptor mechanisms.74 We have also demonstrated that ACE-2 regulates alveolar epithelial cell survival by balancing the proapoptotic Ang II and its antiapoptotic degradation product Ang 1–7, through Ang 1–7 action on its receptor Mas.74 Furthermore, we showed that ACE-2 is expressed in fetal human lung fibroblasts but is significantly decreased by hyperoxic lung injury in a cell culture model.3 Importantly, this effect was reversed by hypoxia preceding hyperoxia.65 Recombinant human ACE-2 has been tested in healthy individuals in clinical trials to determine medication pharmacokinetics and pharmacodynamics,75 and has been investigated as pipeline drug “GSK2586881” in a pilot clinical trial to treat adult acute lung injury.76 ACE-targeted therapies might be future beneficial treatments for BPD.77ACE-2 has been shown to play a protective role in lung disease through effects mediated by the receptor Mas, the receptor for the ACE-2 peptide product ANG1–7.78. Postnatal exposure to intermittent hypoxia followed by interventional hyperoxia induces oxidative stress and free radicals, which leads to direct cellular injury, oxidation of DNA, induction of cytokines, and recruitment of neutrophils and macrophages to the lung, manifested as pulmonary inflammation.21 Furthermore, ROS are released by immune cells resulting in epithelial and endothelial cell injury.26 Clinically unsuspected oxygen desaturation occurs frequently in preterm infants with and without bronchopulmonary dysplasia, and profound hypoxemia is claimed responsible for sudden unexplained deaths in these infants.79 Furthermore, infants who develop BPD experience more frequent episodes of oxygen desaturations than infants who recover from respiratory distress syndrome without developing BPD.80Premature infants are known to experience intermittent episodes of hypoxemia lasting from a few seconds to several minutes19 demonstrated that cycling hypoxia with hyperoxia episodes exacerbated lung injury in neonatal mice.19 In their study they tested the effects of hypoxic episodes on a normoxia background, continuous hyperoxia, and hypoxic episodes combined with a hyperoxia background on neonatal mice and compared the results with the normoxia group. Compared with the normoxia control, the hypoxic episodes on normoxia background had no significantchanges on radial alveolar count (RAC) as a marker of lung injury, on oxidized glutathione, or on protein carbonyls as markers of oxidative stress, while continuous hyperoxia significantly reduced RAC and increased oxidative stress markers compared with normoxia. Interestingly, the intermittent hypoxia on hyperoxia background group of mice showed significantly lower RAC and higher oxidative stress markers compared with continuous hyperoxia. Their study suggested that the combination of two oxidative stress mechanisms, hypoxia and hyperoxia, cause a more profound lung injury than either one alone. Furthermore, their study even suggests that hypoxic mice did not have any BPD-like changes.19Very few studies in literature directly compare the effects of hyperoxia, hypoxia, or their fluctuation on the neonatal lung development or injury. 82 on animal models of BPD compared the different effects of prenatal hypoxia and postnatal hyperoxia in neonatal mouse lung. Compared with normoxia controls, the lung volume, total air space volume and total septal surface were significantly reduced in the postnatal hyperoxia groups compared with either prenatal normoxia or prenatal hypoxia. The volume weighted mean volume of the parenchymal airspaces and the wall thickness of septa was significantly higher, and the volume density and the volume weighted mean volume of lamellar bodies in alveolar epithelial cells type II (AEII) were significantly lower in the prenatal hypoxia-postnatal hyperoxia group compared with normoxia controls, while prenatal normoxia-postnatal hyperoxia did not cause these changes. The study suggested that the prenatal hypoxia and postnatal hyperoxia model was found to best reflect morphological changes in lung development comparable with alterations found in BPD.81A study by Schmiedl et al.82 examined the effects of intravitreal bevacizumab, a treatment for retinopathy of prematurity (ROP), on the lungs of a newborn rat pup model. The study showed long term effects of exposure to intermittent hypoxia and hyperoxia during the first 2 weeks of life. At postnatal days P23 and P45, both pO2 and SaO2 were lower in intermittent hypoxia exposure, while hyperoxia alone increased pO2 compared to the room air group. Interestingly, no significant difference was found in SaO2 in the hyperoxia group compared to room air.82 The results of this study suggest long-term effects of hypoxemia-hyperoxemia fluctuation on blood oxygenation and indicate the presence of a discrepancy between pO2 and SaO2 measures. In a 3-dimiensional cell culture organoid model of BPD, Sucre et al.83 showed that markers of fibroblast activation were increased by hypoxia-hyperoxia cycling as seen in BPD; α-SMA, Col1A1, TGFβ1, and PDE5a, and downstream targets of Wnt signaling  were increased in hypoxia-hyperoxia relative to normoxia cultured organoids.83 A study by Chang et al.84 investigated the effects of hyperoxia with intermittent hypoxia on neonatal rats and found that repeated intermittent hypoxia during hyperoxia can alter biomarkers responsible for normal microvascular and alveolar development.84 However, both the Sucre et al.83 and Chang et al.84 studies have not compared the effects of hyperoxia alone to the effects of intermittent hypoxia during hyperoxia.A recent study by Valencia et al.91 Many of these studies have been discussed in detail in other reviews,93 we summarize these in 93 These guides are based on the NeOProM meta-analysis study of the three largest and most recent RCTs94; the Surfactant Positive Pressure and Pulse Oximetry Randomized Trial (SUPPORT),89 the Benefits of Oxygen Saturation Targeting II (BOOST II),90 and the Canadian Oxygen Trial (COT).91 In these trials oxygen saturation monitoring by pulse oximetry was the method used to monitor oxygen levels. Pulse oximetry is currently the prevalent monitoring technology to detect blood oxygenation.95 However, invasive blood gas analysis is needed in order to monitor the oxygenation status when SpO2 is close to saturation (≥97%). A new technological measure using multiwavelength pulse co-oximetry called oxygen reserve index (ORI) can be used to monitor oxygenation in the moderate hyperoxemic range (PaO2 100–200mmHg). The ORI is an index with a unit-less scale between 0.00 and 1.00, which is a relative indicator of changes in PaO2 in the moderate hyperoxemic range, and is used as a companion to pulse oximetry monitoring in patients receiving supplemental oxygen.96Several clinical studies and randomized controlled trials (RCTs) were performed to determine the range of optimal saturation by pulse oximetry in preterm infants receiving supplemental oxygen.98 An increase in arterial oxygen saturation, as when the preterm infant is resuscitated with high oxygen concentrations, is believed to be damaging to the newly developed retinal capillaries. After a preterm infant is no longer in supplemental oxygen, the avascular retina becomes hypoxic, leading to overexpression of angiogenic factors and vasoproliferation of intravitreal blood vessels.101 Penn et al.102 reported that exposure to variable hyperoxia (hypoxia/hyperoxia fluctuations) has been shown to be much more effective at producing proliferative retinopathy in neonatal rat than exposure to constant hyperoxia.102 Numerous animal studies established that intermittent hypox(em)ia cycling with hyperox(em)ia produces severe oxygen induced retinopathy.107 These studies emphasize the critical role of oxygen level fluctuations.ROP is a disease affecting the development of the retinal vasculature characterized by abnormal growth of retinal blood vessels in the incompletely vascularized retina in preterm infants receiving supplemental oxygen therapy. ROP was first reported by Theodore L. Terry in 1942 and referred to as “retrolental fibroplasia”.115 A transcriptomic study of neonatal ventricular chamber growth and development during perinatal circulatory transition identified Wnt11 as a prominent regulator of chamber-specific proliferation. Perinatal hypoxia treatment in mice suppressed Wnt11 expression and was associated with cyanotic congenital heart defect (CHD) phenotypes and correlated with O2 saturation levels in hypoxemic infants with Tetralogy of Fallot (TOF).116 Several animal studies have shown cardiovascular adverse effects of neonatal hyperoxia with long-term consequences into adulthood. Mouse model studies on perinatal or maternal inflammation combined with neonatal hyperoxia exposure showed altered fetal development affecting cardiac structure and function, resulting in early cardiac dysfunction leading to cardiac failure in adulthood.118 In newborn pigs, hyperoxia was found to trigger oxygen free radical-mediated membrane injury together with an inability of the newborn heart to up-regulate its antioxidant enzyme defenses while impairing myocardial function and hemodynamics.119 In addition, neonatal hyperoxia exposure was shown to increase systemic blood pressure and impair vasoreactivity in adult rats,120 possibly due to developmental programming of endothelial nitric oxide synthase uncoupling and enhanced vascular oxidative stress.121 Neonatal hyperoxia exposure also increased adult airway reactivity and was associated with left ventricular (LV) dysfunction in adult mice.44 Cardiac effects of hyperoxia also affect adults. In adult patients with and without congestive heart failure (CHF), hyperoxia was associated with impairment of cardiac relaxation and increased LV filling pressures. These studies indicate that caution should be used in the administration of high inspired O2 fractions.122Several independent studies in humans and animal models have reported that chronic fetal hypoxia can trigger a fetal origin of cardiac dysfunction and increase the risk of cardiovascular disease in later life.123 Neonatal hyperoxia exposure also resulted in impaired nephrogenesis causing reduction in both nephrogenic zone width and glomerular diameter and increased apoptotic cell count.124 Since the kidneys are highly vascular organs, the relative hyperoxia and oxygen supplementation in preterm infants could be responsible for the renal developmental abnormalities and for affecting glomerular vascularization, and should be examined in future studies.Oxygen supplementation and hyperoxic exposure of newborn animal models resulted in enlarged renal corpuscles and decreased number of nephrons in the kidneys in early adulthood.Various groups are shedding light on the importance of events that are commonly seen in neonatal ICUs, particularly with extremely low birth weight preterm babies, and with how prematurity affects the lung as well as other organs. Hyperoxia might not be the only risk factor for worsening BPD. However, uncontrollable fluctuation/cycling between hypoxemia, normoxemia and hyperoxemia might be a higher risk than hyperoxemia alone. There are trials of simulations to those cycling effects in different animal models. In the neonatal ICU there is no clear cut-off to delineate the “sweet-spot” in the oxygen saturation zone; how much is too much has not yet been clearly defined.19These results suggest that the aggressive prevention of hypoxemic episodes in human neonates at risk for the development of BPD needs to be further investigated. Our group, along with other groups, has studied the role of RAS in local tissue injury,130 and showed that ACE-2 might be beneficial to attenuate lung injury.73 A future study by our group is planned to look at the effect of cycling of hypoxemia/normoxemia/hyperoxemia and the effect of ACE-2 in a small animal model of BPD. Measurement of partial pressure of oxygen and/or SPO2 is planned to better assess and quantify the effects of hypoxemia with that of hyperoxemia/hypoxemia.In mice, the episodic “fluctuation” of hypoxia and hyperoxia during the induction of BPD potentiated the oxidative stress in lung tissue and exacerbated the alveolar developmental arrest.


Bladder cancer (BC) in dogs is often lethal at the time of diagnosis. Therefore, there is a constant need for novel research on improvements of its characterization and treatment. Due to high cost and limited number of available dog patients, in vitro models of canine BC have been increasingly used for the last 25 years. In the present article, we present existing in vitro models of canine BC, including available simple  and more complex  models.Bladder cancer (BC) constitutes approximately 2% of all spontaneously occurring cancers in dogs. It is characterized by a devastating clinical course in most cases, which emphasizes a constant need for the development of novel methods of disease characterization and treatment. Over the past years, advances in cell engineering have resulted in the development of various canine in vitro models of BC, emerging as complements for in vivo research. In this article, we aimed to review the available data on existing in vitro models of canine BC, focusing primarily on their characteristics, applications in veterinary medicine, as well as advantages and disadvantages. The most commonly used in vitro models of canine BC comprise immortalized cell lines grown as adherent monolayers. They provide an unlimited supply of research material, however, they do not faithfully reflect the conditions prevailing in vivo, since the spatial cellular interactions are lost. The importance of the three-dimensional (3D) features of solid tumors in relation to carcinogenesis or drug response process has resulted in the development of the first canine 3D models of BC available for in vitro research. So far, results obtained with in vitro and in vivo research should be interpreted together. With the constantly growing complexity of in vitro models of BC cancer, animal-based research might be reduced in the future. Bladder cancer (BC) constitutes approximately 2% of all spontaneously occurring cancers in dogs . With esThough obvious benefits that could be gained from the research of canine BC in vivo, there are some inevitable limitations , making extensive in vivo research difficult ,2. CurreIn the present article, we aimed to review the available data on existing in vitro models of canine BC as a tool in veterinary research, focusing primarily on their characteristics, potential applications in veterinary medicine, and advantages and disadvantages. To the best of our knowledge, this is the first review summarizing the current evidence regarding this topic. We conducted a literature search using two electronic databases, namely, Pubmed and Scopus. The most recent search was performed on 28 January 2022. Screening of the literature was conducted using the following search string:  AND (“cancer” OR “carcinoma” OR “neoplasm”) AND (“in vitro” OR “model” OR “cell*” OR “culture”) AND (“dog” OR “canine”). Auto-alerts in Medline were run, as well as reference lists of original articles and review articles for further eligible data. We exclusively included data regarding in vitro models of canine bladder TCC. Only papers in English were considered eligible without restrictions on publication year. The flow diagram of the study selection process is presented in The most commonly used two dimensional (2D) models of canine BC comprise immortalized cell lines and primary cell cultures grown as adherent monolayers in appropriate media. The first immortalized canine BC cell line, called K9TCC, was established by Knapp et al. in 1995 . Since tThe majority of canine BC cell lines express specific cancer-related markers, resembling those presented by the primary tumors in vivo. Due to differences in the development process (mainly related to primary tumor characteristics), the available canine TCC cell lines differ in terms of expressed biomarkers . This alTo date, only a few studies have provided data regarding high-throughput molecular characterization of existing canine BC cell lines. Initial molecular characterization (including genotypic data) of several canine BC cell lines was performed by the Flint Animal Cancer Center (FACC) . SubsequIn vitro studies using BC cell lines play a significant role in the novel drug discovery and development process, providing crucial data on drug effects in the early preclinical stages. Such information is of paramount importance in the decision-making process for drugs moving forward into more expensive and time-consuming in vivo clinical trials. Initial studies using canine TCC cell lines were extensively focused on non-selective cyclooxygenase inhibitors  and various chemotherapeutic agents . FollowiCanine BC cell lines can be also used for investigation of other forms of anti-cancer therapy. As an example, Parfitt et al. investigated and characterized the radiosensitivity and capacity for cellular damage repair of canine BC cell lines . AuthorsConventional 2D cultures have several advantages supporting their important role in preclinical research. Research using cell lines is significantly less expensive than in vivo animal studies and provide an unlimited supply of material, which is widely available and easy to propagate under completely controlled and reproducible environmental conditions . NeverthThe importance of the three-dimensional (3D) features of solid tumors in relation to carcinogenesis or drug response process has prompted efforts to develop in vitro models mimicking in vivo tumor growth more precisely. Examples of 3D culture systems include multicellular aggregates grown as spheroids, scaffold-based models grown within polymer networks, and organoids defined as stem cell-containing self-organizing structures possessing multiple features of the original tumor ,32. DespThe first 3D models of canine BC were established by Elbadawy et al. . They ge3D cell culture approaches hold great potential and offer complex systems for various purposes, such as disease modeling and investigation of anticancer drug efficacy. The similarities in the drug responsiveness among the 3D in vitro models and the in vivo models might largely be due to their similarities in enhanced cellular interactions via adhesion and secretion of soluble factors of tumors ,36. ThesAlthough significant advances have been made over the past years, modeling the complexity of canine BC in in vitro models has not been completely successful. There are still various challenges, including the need for extensive molecular characterization of existing cell lines or creation of new reliable 3D models, incorporating multicellular cultures and diverse cellular microenvironments. So far, results obtained by in vitro and in vivo research should be interpreted together. With the constantly growing complexity of in vitro models of BC cancer, animal-based research might be reduced in the future