Skull base infiltration by GBM cells in an orthotopic xenograft mouse model of GBM.Subsequent hydrocephalus and precocious death.In vivo models are essential for the study of glioblastoma (GBM). Patient-Derived Orthotopic Xenograft (PDOX) using GBM cells in immunodeficient mice classically results into 2 main scenarios of tumor development, either intracerebral or intraventricular. In the present brief report, we have used brain clarification and lightsheet imaging to highlight the occurrence of a communicating hydrocephalus associated to the ventral leptomeningeal invasion by cancer cells, leading to a decreased survival. We believe that this uncommon event should be taken into account for interpretation of forthcoming survival manipulations in preclinical studies.1 In this context, multiple novel therapeutic strategies are tested in preclinical studies, in order to increase survival in vivo. Currently available mouse models include human GBM cell line orthotopic xenografts, Patient-Derived Orthotopic Xenograft (PDOX), Genetically Engineered Mouse Models (GEM) and syngeneic mouse models.2 For PDOX, tumor-associated immune response is impeded due to the use of immunodeficient mice, but the tumor development seems to recapitulate more accurately the genetic and phenotypic features of the disease in comparison to the other mouse models.2 In xenograft models, Irtenkauf et al. reported the possible development of a tumor either in the brain parenchyma or in the ventricles, depending on the site of injection.3 Using brain clarification and lightsheet imaging after PDOX, we report a third possible scenario.Glioblastoma (GBM) is the most frequent malignant primary brain tumor in adults, well-known for its poor prognosis despite multimodal therapies.IDH wild-type, p53 wild-type, MGMT non-methylated) obtained after informed patient and cultivated as neurospheres.4 GB1 cells were transduced to express RFP before being grafted orthotopically in nude mice.4 Clarification of each brain hemisphere was performed separately, as described by Tomer et al.5 Images of each hemisphere were obtained using a dual illumination lightsheet fluorescence microscope (Zeiss®). Images were stitched, reconstructed, and analyzed using Imaris® software (version 9.5.0). This study has been approved by the ethical committee of the University of Liège (IRB 2197).The human primary GBM culture GB1 was established from a resected newly-diagnosed tumor , a single mouse (specimen I) presented severe signs of suffering (curved back and weight loss of more than 15% in 3 days) 1 month after being grafted, while the remaining 9 mice developed similar symptoms only 3 months after the engraftment. This symptomatic mouse was hence precociously sacrificed. After brain clarification , images The tumor cells observed at the ventral part of the forebrain are forming a symmetrical pseudo-mass of 300–500 μm thickness, adjacent to the ventral part of not-invaded brain and forebrain, which extend anteriorly along the ventral walls of the optic tract and the olfactive bulb (In a second study (N2) using the same GBM cells, we highlighted the occurrence of tumoral leptomeningeal dissemination in 2 additional specimens. The second specimen (II) presented identical suffering symptoms and we observed the same pattern of ventral dissemination with subsequent severe hydrocephalus and precocious death at 6 weeks, although tumor cells engraftment in the striatum was not detected . The thi6 The accumulation of tumor cells in the ventral subarachnoid spaces could, therefore, explain the development of communicating hydrocephalus. In GBM patients, the onset of communicating hydrocephalus is a late event, usually reported after surgery and irradiation, occurring in less than 10% of patients.7 Its association with leptomeningeal dissemination remains controversial.8The mechanism of ventral leptomeningeal infiltration in PDOX remains uncertain. Two hypotheses could be considered and might not be exclusive. (1) Due to reflux during cell injection, tumor cells might not graft in the brain and might drift in the declivitous ventral part of the brain. In this condition, the presented observation would rather result from a technical issue at the engraftment and would therefore not be expected in GEM GBM models. (2) It is possible that tumor cells follow the cerebro-spinal fluid flow to the ventral skull base as recently reported by Ahn et al.3 Ventral leptomeningeal infiltration and intraventricular tumors both seem to be associated with shorter survival. In case of early onset of suffering symptoms, we would recommend MRI realization, looking either for the development of communicating hydrocephalus associated to the restricted development of a tumor mass in the striatum or for the formation of an intraventricular tumor mass. Taken together, those findings suggest that preclinical survival studies using PDOX should confirm the localization of the tumor in order to adequately compare mice survival and to determine more accurately if tested therapies display significant improvement.In conclusion, communicant hydrocephalus associated to ventral leptomeningeal invasion leading to precocious death after PDOX is a rare event, as shown by the 3 out of 35 mice  affected in the present report. Irtenkauf et al. reported the occurrence of intraventricular tumor in around 10% of mice, using the same coordinates as us.


In this perspective review, we discuss the limitations of current murine models and outline how the combination of microphysiological systems (MPSs) and targeted computational models can lead to breakthroughs in this field. In particular, while MPSs enable the experimentation on human cells in controlled and physiological environments, in silico models can provide a versatile tool to translate the in vitro findings to the more complex in vivo setting. As a showcase example, we focus on Notch-related cardiovascular diseases, such as Alagille syndrome, Adams–Oliver syndrome, and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL).Notch is an evolutionary, conserved, cell–cell signaling pathway that is central to several biological processes, from tissue morphogenesis to homeostasis. It is therefore not surprising that several genetic mutations of Notch components cause inherited human diseases, especially cardiovascular disorders. Despite numerous efforts, current in vivo models of genetic Notch cardiovascular diseases is provided, followed by a discussion over the potential of microphysiological systems and computational models in overcoming these limitations and in potentiating drug testing and modeling of these pathologies.In this review, a comprehensive overview of the limitations of current  Notch is a signaling pathway that relies on direct cell–cell interaction for its activation. Notch ligands  presented at the surface of a cell interact with membrane-bound Notch receptors (Notch1–4) of another adjoining cell. The ligand–receptor interaction results in proteolytic processing of the Notch receptor, releasing the active signaling unit of the receptor, the Notch intracellular domain (NICD). Upon its release into the cytoplasm, the NICD translocates into the nucleus where it binds RBPJk, thereby inducing transcription of target genes and activation of transcriptional programs. This contributes to the determination of cell fate and regulation of tissue development and homeostasis.1–3 and homeostasis.5 Concurrently, mutations in different Notch ligands or receptors can cause several pathologies such as congenital heart defects (CHDs) and diseases such as Alagille syndrome (ALGS), Adams–Oliver syndrome (AOS), calcific aortic valve disease (AoVD), and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL).6In the cardiovascular system, Notch controls heart morphogenesis as well as blood vessel formation6 Each of these diseases is associated with mutations in genes coding for different Notch components  and white matter, together with ischemic strokes.16 Unfortunately, current treatments cannot cure these diseases and only aim at mitigating the symptoms. To overcome this limitation and develop new regenerative therapeutic strategies, further understanding of the underlying molecular mechanisms is required.Mutations in in vivo disease models fall short of replicating the actual human disease characteristics, which suggests that fundamental differences between species are present in this context. At the same time, the inherent complexity of in vivo models complicates the identification of the exact molecular mechanisms involved, thereby hindering the development of new therapies. In this perspective review, after summarizing current in vivo models of Notch-related cardiovascular diseases, we propose that their limitations could be overcome by combining engineered in vitro models, providing a controlled environment to investigate specific aspects of the pathologies, together with in silico models, providing a digital twin for the in vivo setting.Despite several efforts, current In vivo animal models are generally the most reliable in mimicking the complexity of human organisms. As such, they have been widely adopted to screen for the effects of Notch mutations. However, although ALGS, AOS, AoVD, and CADASIL are caused by autosomal dominant mutations, cardiovascular phenotypes are not obvious in mice presenting targeted disruption of one allele in the associated Notch genes,17–19 except for Dll4 in AOS.20–22 This limitation suggests that murine models are not fully reliable in this case as mice might have differences or compensatory mechanisms masking the effects of Notch mutations.23 Furthermore, mouse models have a short life span and different hallmarks of aging, which affect the possibility to mimic the temporal features of Notch-related diseases, including the fact that the first symptoms of CADASIL start to become evident in young adults16 despite the Notch-related mutation being present since birth. Nevertheless, a few animal models replicate some details of cardiovascular Notch-related diseases and thus provide valuable information on the disease etiology.For example, mice most likely exhibit a different hemodynamic environment compared to humans, which can influence Notch responses to mechanics.24–29 While mice heterozygous for either Jag1 or Notch2 cannot mimic the disease,19 double heterozygous mice (Jag1dDSL/+Notch2del1/+) can recapitulate several ALGS symptoms, including cardiac defects.24 However, ALGS is known to arise from single mutations in JAG1 and, more rarely, NOTCH2. Recently, Andersson et al.27 have described a novel mouse (Jag1Ndr/Ndr) with a phenotype mimicking ALGS in humans. The proposed mutation renders JAG1 hypomorphic, both inhibiting signaling through NOTCH1 and hindering activation of NOTCH2 and NOTCH3.27 This new model suggests that ALGS may result from Notch mutations causing partial loss of function, leading to reduced Notch activity. Overall, not only do these findings highlight the importance of understanding the specific roles of different Notch components in human physiology but they also point to the necessity of further analyzing Notch dose sensitivity.Mice carrying an array of different Notch mutations have been proposed as ALGS models.31 . Differently, introduction and overexpression of NOTCH3R169C into mice in a heterozygous state successfully created a phenotype also displaying white matter degeneration.33 This model has recently led to new findings suggesting that CADASIL is caused by Notch3 gain-of-function mutations that cause increased NAPDH oxidase 5 activation and consequential endoplasmic reticulum stress.34 Similar hypermorphic associations have been made with mutant Notch3 and NF-κB signaling, aggravating vascular remodeling and inflammation.35 However, this NOTCH3R169C model required excessive overexpression of the mutant protein, above endogenous levels,33 and whether CADASIL is caused by loss- or gain-of-function mutations is still strongly debated.37 Future studies should aim at clarifying this point, in addition to identifying the mechanisms causing CADASIL symptoms to start appearing in young adults and mainly in the brain region. Characterizing the temporal progression and localization of the disease might lead to development of strategies for early diagnosis and treatments.Several mouse models of CADASIL, expressing mutant variants of NOTCH3 with altered numbers of cysteines, partially recapitulate the CADASIL phenotype. These mice exhibit the characteristic deposition of granular osmiophilic material and accumulation of NOTCH3, but no evident brain lesionsNotch1 or RBPJk can mimic the human phenotype of AoVD to some extent. In particular, Notch1+/− mice show enhanced valve calcification and activation of alkaline phosphatase.38RBPJk+/− mice display greater cardiac impairment, including thickened, calcified valve leaflets, macrophage infiltration, and collagen deposition, as well as activation of procalcific and profibrotic signaling pathways.39 Moreover, RBPJk+/− mice also have upregulated levels of Runx2,39 a transcription factor promoting osteoblast fate and heavily upregulated in human AoVD.40 The seemingly important role of RBPJk39 suggests that AoVD is a result of generally disrupted Notch transcriptional activation, which, given the results with the Notch1 mice,38 mainly occurs through Notch1, although other receptors must be involved. Unfortunately, Notch1+/−, RBPJk+/−, and Dll4+/− mice are, however, not representative models of AOS as they do not display the terminal transverse limb defects and scalp aplasia cutis characteristic of AOS. It has been proposed that vascular defects would be the main drivers of AOS,42 and mice with induced inhibition of Notch activity display loss of VSMC coverage and hemorrhage in different regions of the developing brain and limbs.41 In conclusion, animal models of Notch-associated cardiovascular diseases are currently not completely satisfactory; therefore, alternative and complementary strategies are necessary.Mice heterozygous for either 43 MPSs can also aid in elucidating molecular mechanisms underlying human pathologies and the identification of therapeutic targets. As described above, this can be difficult in animal models, which may not reflect human pathophysiology and where dynamic molecular signaling events and complex cell–microenvironment interactions are difficult to capture. This is especially true for the cardiovascular system, where changes in hemodynamics can critically affect Notch signaling.23 Furthermore, many of the Notch-associated cardiovascular disorders listed above are rare disorders.44Failure to meet the expectations of drug development and drug screening incentives has driven the development of microphysiological systems (MPSs) or organs-on-chips that recapitulate the function of human organs.45 and modeling rare disease has high potential to overcome this issue. Complex and rare diseases can be modeled in MPSs46 using cells from patient donors, either from primary or induced stem cell sources, or using genetic tools to induce a disease phenotype that can be studied in vitro against an isogenic background.Identifying molecular therapeutic targets and treating rare disease patients are huge challenges due to the limited amount of data available and the lack of relevant model systems. Fewer than around 5% of around 7000 currently identified rare diseases have effective drug therapies,47 Other rare diseases that have been modeled using iPSCs and organ-on-chip technologies include hereditary hemorrhagic telangiectasia, Rett syndrome, Alpers–Huttenlocher syndrome (as reviewed by Low and Tagle46), and Barth syndrome, where a heart-on-chip technology provided new insights into the pathogenesis.48 For all these reasons, we expect that the MPS will be a game changer also in the investigation of genetic Notch disorders as it could be adopted to study disease-mimicking cells and test drugs under physiologically relevant conditions (The Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic condition that is caused by a mutation in the Lamin A gene. Recently, a tissue-engineered blood vessel model of HGPS using induced pluripotent stem cell (iPSC)-derived endothelial cells identified the role of the endothelium in HGPS.nditions .27 mediated by the interactions between multiple Notch ligands and receptors,39 each having a specific role. To elucidate these mechanisms, therefore, techniques able to engineer cells expressing different (ratios of) Notch ligands and receptors in a controlled manner should be adopted, and MPSs should enable long-term culture of cells in environments mimicking the structure, components, and stimuli of blood vessels.As described in the sections above, most of the genetic Notch cardiovascular disorders seem to arise from defects in blood vessels, which are in turn caused by incorrect temporal changes in Notch dosage,50 Notch signaling dynamics is important for tissue development and morphogenesis,52 and Notch signaling dose and specific receptor–ligand interactions affect cell fate54 and physiology.20 Advances in synthetic biology, stem cell, and organoid technologies, as well as gene editing, combined with advanced microengineering of humanized model systems, will drive cutting-edge translational research.Possible disease-mimicking cells might be obtained through synthetic biology and cellular engineering, which have already helped in elucidating Notch signaling networks and the impact of receptor–ligand specificity on signaling dynamics.ex vivo vascular networks have been useful to study the impact of Notch signaling dynamics on tissue morphogenesis and malformation,55 but these systems lack the impact of mechanics. Coupling dynamic molecular activity and mechanics to tissue pathology, to understand the interrelationship between biochemical regulation and mechanical impact on cardiovascular disease, requires real-time monitoring of molecular and cellular activities in humanized physiological systems and control over hemodynamic parameters. In vitro models with simplified mechanical characteristics or reduced tissue complexity, ex vivo microfluidic channels, and cellular strain platforms have all been used to study the mechanosensitivity of Notch in vascular cells.56–59 The complexity of these systems should be gradually increased to more closely mimic the physiological environment.Within vascular biology, three-dimensional (3D), in vivo. Perfused, self-organized vascular systems in 3D gels have helped in elucidating novel mechanisms of Notch in vascular barrier functions.60The hemodynamic environment of blood vessels consists of both shear stress (due to blood flow) and circumferential stretch (due to blood pressure). The tissue is layered, multicellular, and 3D. While simple microfluidic or strain devices can be employed to assess the impact of mechanics on Notch, more complex models and tissue-engineered constructs are a step closer toward mimicking the complexity 61 A bioreactor that independently combined shear stress and stretch was developed to study the development of vascular grafts,62 and another bioreactor with a controlled, pressurized tissue system was developed to study the long-term effect of constant strain versus constant stress on tissue growth.63We recently developed a vessel wall on a chip, mimicking the physiological cell composition, organization, and hemodynamic environment of the arterial vessel wall, to study the impact of simultaneous shear stress and strain on endothelial-VSMC signaling in tissue remodeling.In sum, several bioreactors and MPSs have already been developed and hold great promise in mimicking several aspects of genetic Notch cardiovascular disorders. The modeling of disease states on MPSs opens new avenues for understanding molecular mechanisms of pathologies and potential treatments of Notch-linked vascular disease. These models will provide insight into complexity, dose sensitivity, and receptor–ligand specificity of Notch signaling in cardiovascular tissue and especially in relation to changes in tissue mechanics and hemodynamics.in vitro findings remain challenging to translate to in vivo settings, where several biological processes interact, creating feedback loops with counterintuitive results. This conceptual gap between in vitro and in vivo models may be bridged by in silico models, providing digital twins of in vivo conditions and analytic tools for understanding the systemic consequences of single biological mechanisms.Although MPSs enable the investigation of human diseases with human donor cells and the investigation of isolated stimuli acting on them, In silico models have a wide range of applications within the investigation of vascular diseases and associated treatments. For example, computational fluid dynamics models can potentiate the optimization of in vitro systems64–66 and analysis of the effects of vascular network geometry on hemodynamics.67–69 Not only can these models compute the shear stress experienced by endothelial cells and other cell types in vitro70–72 or in human pathologic conditions73 but they can also be adopted to optimize surgical strategies for Notch-related diseases such as ALGS.74–7677–79 both for idealized and patient-specific geometries.81 Coupled with this, an increasing number of modeling approaches include the possibility to simulate cell behavior in response to chemo-mechanical cues within cardiovascular tissues.82 Given the growing evidence of Notch mechanosensitivity, the capability of computational models to analyze cellular mechanical stimuli will be increasingly crucial for the study of Notch-related diseases. To this aim, these biomechanical models have to be coupled with in silico Notch models.Similarly, computational models are widely adopted to compute the local strain experienced by cardiovascular cells resulting from blood pressure-induced tissue deformation,in silico models of this signaling pathway have been proposed,83 usually to investigate the effects of different Notch ligand–receptor relationships on the resulting cellular phenotypic patterns.84–89 For example, modeling studies have uncovered that spatial patterns of cells with two alternating phenotypes can arise both from Notch lateral inhibition,84 defined as downregulation of Notch ligands in receiving cells after Notch activation, and from cis-inhibition,85 that is, the mutual exclusion of Notch receptor–ligand pairs within a single cell without Notch activation. Similarly, simulations have been crucial to understand the effects of filopodia,91 cell area and size,88 mechanics,93 and cell connectivity89 on Notch signaling dynamics and consequential tissue patterning. To our knowledge, there are currently no computational studies simulating Notch signaling in genetic Notch cardiovascular disorders. Nevertheless, we envision that combining and extending the features of the existing Notch in silico models will provide great versatility and opportunities for investigation of Notch-related diseases and for acceleration of targeted drug development and approval as it is also suggested and currently occurs in other fields.95Due to the importance of Notch in numerous contexts, many 4 By accounting for Notch3-Jag1 signaling and their sensitivity to cyclic strain, this model identified Notch mechanosensitivity as a possible key factor of blood vessel homeostasis4  they have helped in understanding whether these pathologies result from loss- or gain-of-function Notch mutations, this information is still not enough for development of drugs targeting the disease instead of the symptoms. To this aim, fully understanding the molecular pathways involved is necessary. Achieving such understanding solely with animal models is challenging, if not impossible, not only because of their incapability to fully recapitulate Notch-related diseases but also because of their complexity. A further challenge stems from potential species differences. In general, the insufficient contribution of animal models is also highlighted by the disproportionate number of drugs that positively pass animal trials, but fail to reach the clinical setting due to negative results in humans.in vivo, where multiple phenomena interact over a long time period. Therefore, these two approaches complement each other and could lead to breakthroughs in the field of Notch-related cardiovascular diseases.We expect that the combination between MPSs and computational models will significantly aid our understanding of Notch signaling mechanisms in cardiovascular disorders and will potentiate the development of targeted treatments . MPSs prFuture MPSs should aim at more closely mimicking the complexity of human tissues and conditions while keeping their advantages in terms of control. Further complexity and physiological relevance may be achieved by including more features of the systemic environment, such as gradients of nutrients, gasses, cytokines, hormones, circulating cells, and other relevant cell types, and by tuning the composition and organization of the extracellular matrix. Ideally, this should be performed following a modular design so that complexity can be increased or decreased depending on the research question. Such an approach should also be followed by computational models to increase their versatility and enable their application in different contexts.Importantly, given the long timescale features of Notch-related diseases and tissue morphogenesis, growth, and remodeling more in general, both MPSs and computational models should reach the potential to recapitulate long-term organ function, enabling long-term cell culture on the one hand and long-term simulations with contained computational costs on the other hand. Reaching these goals remains challenging and requires engineering skills that are not yet widely accessible. To fill this gap, we need to develop new tools and a new framework where expertise in cell biology, molecular biomedicine, engineering, and computational biology is combined. To facilitate communication and the exchange of information and tools between these different disciplines, common hybrid international conferences should be organized and strategies should be optimized to increase the accessibility of tissue chip technology and computational models. For this latter aim, these technologies should be accurately validated and commercialized for community-wide access.In conclusion, the coupling and further development of MPSs and computational models may accelerate the investigation of genetic Notch cardiovascular disorders and could provide new, personalized medicine approaches as drugs may be tested directly on patient-derived cells within MPSs mimicking the human physiology and disease. The potential of this coupling is currently largely unexplored in this field and therefore, despite the challenges ahead, we envision that the interplay between these two approaches will lead to accelerated drug discovery and an increased approval rate, in addition to facilitating the transition from care, where only symptoms are treated, to cure, where disease causes are targeted.


The development of cardiovascular disease during renal impairment involves striking multi-tiered, multi-dimensional complex alterations encompassing the entire oxygen transport system. Complex interactions between target organ systems involving alterations of the heart, vascular, musculoskeletal and respiratory systems occur in Chronic Kidney Disease (CKD) and collectively contribute to impairment of cardiovascular function. These systemic changes have challenged our diagnostic and therapeutic efforts, particularly given that imaging cardiac structure at rest, rather than ascertainment under the stress of exercise, may not accurately reflect the risk of premature death in CKD. The multi-systemic nature of cardiovascular disease in CKD patients provides strong rationale for an integrated approach to the assessment of cardiovascular alterations in this population. State-of-the-art cardiopulmonary exercise testing (CPET) is a powerful, dynamic technology that enables the global assessment of cardiovascular functional alterations and reflects the integrative exercise response and complex machinery that form the oxygen transport system. CPET provides a wealth of data from a single assessment with mechanistic, physiological and prognostic utility. It is an underutilized technology in the care of patients with kidney disease with the potential to help advance the field of cardio-nephrology. This article reviews the integrative physiology and pathophysiology of cardio-renal impairment, critical new insights derived from CPET technology, and contemporary evidence for potential applications of CPET technology in patients with kidney disease. The patAs members of the scientific and health care community, we are challenged with an ethical and moral obligation to help reduce premature mortality from CVD in our growing CKD population. However, the challenge of helping to improve patient outcomes in this population is compounded by the highly complex processes involved in CVD development as kidney failure ensues. As renal function declines, the development of CVD involves both traditional and non-traditional risk factors such as uremia, pro-inflammatory cytokines, volume overload, mineral disorders, electrolyte disturbances, anemia, sympathetic nerve activation, renin-angiotensin-aldosterone (RAAS) activation and vitamin D deficiency. Additionally, emerging risk factors such as elevated fibroblast growth factor (FGF)-23, low Klotho levels, post-translational protein modifications, and gut derived uremic toxins have now been tightly linked with CVD development in CKD  Figure . CentralThe striking multi-tiered, multi-dimensional complexities of CVD development in CKD have challenged our diagnostic and therapeutic efforts. For example, imaging of single organs such as the heart by echocardiography or MRI does not adequately reflect the complex systemic processes that lead to cardiovascular impairment in CKD and is therefore insufficiently sensitive to accurately reflect cardiovascular health in this population. Despite huge scientific, economic and financial investment, many of our cardio-renal outcome trials in nephrology have yielded neutral results and have not demonstrated a treatment benefit . This maThe emergence of state-of-the-art Cardiopulmonary Exercise Testing (CPET) technology and its application to cardiovascular disease research in nephrology is one such alternative approach gaining significant traction. While only a handful of studies have applied CPET technology to study patients with CKD, these studies have already revealed significant mechanistic and physiological insights, and have provided evidence to support the potential use of CPET-derived indices in a variety of applications in nephrology. This article considers integrative physiological and pathophysiological insights into cardiovascular impairment in CKD derived from CPET technology, and appraises contemporary evidence for the potential application of CPET to help advance the field of cardio-nephrology.As CKD progresses, the integrated metabolic machinery required for the cardiovascular system to function and enable optimal exercise performance is impaired. At the level of the heart and vasculature, kidney failure leads to a uremic phenotype that recapitulates many features of cardiovascular aging, including myocyte hypertrophy, reduced myocardial capillarization and non-vascularized myocardial interstitial fibrosis and calcification as well as vascular calcification, arteriosclerosis and arterial stiffening of systemic vasculature . The molCritically, CKD results in the failure of multiple organ systems beyond alterations to the heart and vasculature  Walsh, : The mus2) equals cardiac output multiplied by the arterial minus venous oxygen content, as illustrated in These diverse systemic alterations contribute to the impairment of the cardiovascular system to fulfill its primary function, that is, to be an effective oxygen transport system . The rol–1/kg–1 or one metabolic equivalent (MET). However, it has now become clear that resting cardiac and pulmonary function testing cannot reliably predict exercise performance and functional capacity, and that overall health status is more strongly associated with exercise tolerance than with resting measurements (2) at maximal or peak exercise (VO2Peak) as a robust, objective and reproducible index of cardiovascular functional capacity. While the definition of VO2Max and VO2Peak are different and will be discussed in further detail below, for the purposes of this article we will use the term VO2Peak . VO2Peak reflects the maximal ability of an individual to take in, transport and use oxygen and defines the individual’s functional aerobic capacity  is highly prevalent in the dialysis population and is associated with high rates of all-cause and cardiovascular mortality . However2 peak can occur in CKD. As an example, in advanced CKD patients, a reduction in maximal heart rate or a blunted chronotropic response occurs, leading to reduced cardiac output (2 – Cvo2max) , or conditions that affect the musculoskeletal system  and are too numerous to completely list here, but have also been described in bed-rest and deconditioning  . All patitioning .2uptake (VO2), carbon dioxide output (VCO2) and minute ventilation (VE) and some CPET systems, can provide breath-by-breath analysis of these variables. These measures are used to derive various other gas exchange patterns and can provide organ-specific information on the dysregulated responses to exercise. In the general heart failure population, CPET-derived indices have been recognized as robust markers for the assessment of cardiovascular disease compared to conventional resting imaging for several reasons. Firstly, the coupling of morphological cardiac alterations to cardiovascular performance is largely unknown; secondly, resting cardiopulmonary imaging tests cannot reliably predict functional performance; and thirdly, there is increasing appreciation that overall health status correlates better with exercise tolerance  is defined as the highest rate of oxygen uptake during intense, maximal exercise whereby no further increases in work rate can cause additional rises in VO2  (2 (VO2Peak) is directly reflective of VO2Max and is defined as the highest value of VO2 obtained upon an incremental or other high-intensity exercise test that brings the individual to the limit of tolerance (2Peak considers the integrative exercise response involving the degree of ventricular function (pumping capacity), oxygen transport in blood (O2 carrying capacity), pulmonary and vascular function (O2 delivery), skeletal muscle metabolic capacity (O2 utilization), and as well as ultrastructural and molecular changes of organ systems involved, and their interactions  at peak or incremental exercise (Maximum oxygen uptake (VOplateau) . Peak VOolerance . VO2Peakractions . During l effort . The perexercise . It is t2Peak declines by 10% per decade after the age of 30 and this has been attributed to decreasing maximal heart rate, stroke volume, blood flow to skeletal muscle and skeletal muscle aerobic potential  increasing linearly with VO2 and VCO2. This is followed by a second phase during which VE increases disproportionately relative to first VO2 and then VCO2  causes of exercise limitation . Above VT, however, progressive increases in VE result in the liberation of “excess” CO2 from bicarbonate stores. This helps to buffer the protons being produced as a result of high rates of glycolysis/glycogenolysis, but invalidates the use of RER as a measure of substrate oxidation. Nonetheless, RER still provides an objective descriptor of subject motivation. An RER greater than approximately 1.0 implies that an individual has attained a maximal volitional effort, whereas an RER less than this value suggests that they have not. Measurement of RER during CPET testing and is therefore of crucial significance to assist in the attainment of reliable and clinically meaningful VO2Peak values O2 (Oxygen pulse is the ratio between VOC(a-v)O2 . FlattenC(a-v)O2 . Under cC(a-v)O2 .2Peak (18 ± 4.1 ml/min–1/kg–1) compared to controls   . The stufunction . It is p2Peak) . These results become even more revealing considering that the study found that LV mass index did not change (p = 0.20). This highlights the limitations of using LV mass or hypertrophy as a surrogate endpoint for tracking disease progression in advanced CKD patients, and that other processes beyond LV geometric indices are likely involved in driving overall cardiovascular functional decline in this population.The CAPER  study by p = 0.003). Among the variables that were correlated with VO2Peak changes from baseline to 12 months in the non-transplanted dialysis group, were LV mass index, LV ejection fraction, and maximal heart rate as well as hemoglobin level (anemia). It is currently unknown how cardiovascular functional capacity alters with increasing dialysis vintage  beyond 1 year and this data will be critical to help inform cardiovascular risk. Additionally, it is unknown how processes such as myocardial remodeling through fibrosis, myocardial arteriole calcification, capillary rarefaction, myocardial stunning and complex circulating factors such as FGF23, Klotho and metabolites modulate VO2Peak decline in dialysis. We postulate that these factors may contribute to reduced cardiac compliance and ventricular contractility, loss of arterial elasticity, as well as end-organ damage of the lungs and skeletal muscle system. Further studies are needed to evaluate this.CAPER found that LV ejection fraction declined after 1 year . However, cardiovascular functional capacity was not restored to the level observed in the control group without CKD, and this was consistent with the incomplete normalization of kidney function with transplantation . These findings become all the more meaningful considering that LVMI did not change after kidney transplantation, despite a significant improvement in VO2Peak and when contrasted with the inverse direction of change in non-transplanted dialysis patients. Despite unaltered LVMI, the study reported an improvement in LVEF in the transplanted group at 12 months. These latter findings are consistent with a recent study using volume-independent cardiac magnetic imaging that failed to show significant change in LVMI and LVEF after transplantation  as well as corrected calcium level , lower peak heart rate  and impaired heart rate recovery  compared to patients with a GFR > 60. These findings are suggestive of subclinical cardiovascular disease that can be objectively assessed in pre-dialytic CKD patients. Given that microvascular dysfunction has been shown to be involved in cardiovascular disease in CKD and subclinical disease  functional class, worse symptoms, poor quality of life and decreased patient survival. Exercise capacity is reduced even in mild heart failure and is reflective of an inability of cardiac output to increase adequately with mild exertion . VO2Peakc output . In hearc output . Becausec output . Measurec output . The ide2Peak ≤ 14 mL.min–1.kg–1 as a criterion for which 1 year survival was significantly lower than that achieved through transplantation. Individuals with a VO2Peak > 14 mL.min–1.kg–1 had a 6% 1 year mortality and this suggests that heart transplantation can be safely deferred in this subgroup of symptomatic heart failure patients. VO2Peak potently risk stratifies heart failure patients (with reduced ejection fraction and preserved ejection fraction) into Weber classes A, B, C, and D corresponding to VO2Peak > 20, 16–20, 10–16, <10 mL.min–1.kg–1 which associate with 3 years transplant and mechanical circulatory support-free survival of 97, 93, 83, and 64% respectively . Significantly, among the patients who had VT < 40%, those that underwent kidney transplantation had a significantly better survival compared with the non-transplanted patients. Among the patients with VT ≥ 40%, survival did not differ significantly between those who were transplanted and those who were not. These results suggest that assessment of VT using CPET can provide a high level of discrimination to risk stratify patients for timely kidney transplantation, and that those with a VT < 40% may benefit from early transplantation. These findings are critical given the growing body of evidence that single surrogate markers from the most established clinicopathologic factors, such as age and LV mass index, have limited prognostic value for cardiovascular outcomes in CKD.CPET-derived indices of cardiovascular functional capacity have recently been shown to be robust predictors of cardiovascular morbidity and premature death among patients with advanced CKD, independent of LV measures , 2014. I2 uptake patterns and mechanical or musculoskeletal parameters. In the general heart failure population, a number of indices have been associated with important measures of cardiovascular physiology such as circulatory power (an index of cardiac systolic function), VE/VCO2 (an index of ventilatory efficiency and pulmonary vascular resistance) and mean response time  that can be easily attainable using CPET non-invasively (A significant strength of CPET is that the technology provides a wealth of data from a single assessment, including hemodynamics, ventilatory efficiency, stability and Ovasively . CPET as2 and arterial, as well as mixed venous blood gasses during linear ramp exercise. These data will allow Fick cardiac output derivation and evaluation of the component variables of the Fick equation. Patients with identical VO2Peak values with a diagnosis of heart failure, may have significantly different levels of impairment in the reserve capacity of each Fick variable (CPET that is coupled with invasive hemodynamic assessment, for example using radial or pulmonary arterial catheters enables highly detailed patient phenotyping. This permits accurate assessment of blood pressure and oxyhemoglobin measurement, as well as simultaneous measurement of VOvariable . No studThe significant public health need for better diagnostic and therapeutic approaches to help improve cardiovascular outcomes in CKD requires the implementation of effective, holistic and forward-thinking strategies in our research efforts. Given the multi-systemic nature of CVD development in CKD, an integrative approach at all levels to study mechanisms of disease, to track disease progression or improvement, for risk stratification, and for implementation of appropriate clinical trial endpoints maybe warranted. CPET provides a global test of cardiovascular functional capacity that reflects the entire oxygen transport system and may provide a potential solution for an integrative approach to assessing the cardiovascular system in CKD. The technology provides a wealth of data per single assessment with mechanistic, physiological and prognostic utility. Because a significant advantage of CPET is the ability to couple the technology with other investigative modalities such as invasive CPET and advanced imaging techniques, this can provide comprehensive phenotyping and characterization of multisystem changes that determine overall cardiovascular functional capacity with a wide array of applications. While CPET is a powerful technology, there are also important limitations to the technology that warrants a brief discussion. Firstly, CPET assessment does select for patients who retain some functional ability and therefore, patients who are unable to complete a cycle or treadmill test would be inherently excluded from assessment; Secondly, like all types of exercise testing, patients are required to present to a CPET lab and tests must be conducted by a trained exercise physiologist or clinician. Development of CPET technology has now enabled measurement of ventilatory parameters using handheld devices that can be used in an office-based setting, making the technology increasingly more accessible.Although CPET is a tool that is now widely available and supported by sound scientific evidence in several clinical fields, only a handful of studies have utilized CPET to study kidney patients. Despite this, CPET has already provided a wealth of hypothesis-generating data and has opened up many other exciting questions in the cardio-renal field. The potential to capitalize on next-generation CPET technology to help move the cardio-renal field forward has provided strong rationale for the urgent need for further CPET-related research in nephrology. Here in the Division of Nephrology at Indiana University School of Medicine, we have developed the first nephrology-based CPET laboratory in the United States dedicated to studying kidney patients. These efforts are in parallel with increasing interest among nephrologists worldwide in exercise impairment and musculoskeletal disorders in CKD patients. CPET technology has the potential to drive actionable strategies in our collective efforts to help reduce the burden of CVD in CKD. Strong efforts to recognize these promising advances and to place them as a primary research agenda in cardio-nephrology are needed.KL wrote the manuscript. GM generated all the figures. AC, GL, and SM provided the intellectual guidance and edited the manuscript. All the authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The human spinal column is a dynamic, segmented, bony, and cartilaginous structure that protects the neurologic system and simultaneously provides balance and flexibility. Children with developmental disorders that affect the patterning or shape of the spine can be at risk of neurologic and other physiologic dysfunctions. The most common developmental disorder of the spine is scoliosis, a lateral deformity in the shape of the spinal column. Scoliosis may be part of the clinical spectrum that is observed in many developmental disorders, but typically presents as an isolated symptom in otherwise healthy adolescent children. Adolescent idiopathic scoliosis (AIS) has defied understanding in part due to its genetic complexity. Breakthroughs have come from recent genome-wide association studies (GWAS) and next generation sequencing (NGS) of human AIS cohorts, as well as investigations of animal models. These studies have identified genetic associations with determinants of cartilage biogenesis and development of the intervertebral disc (IVD). Current evidence suggests that a fraction of AIS cases may arise from variation in factors involved in the structural integrity and homeostasis of the cartilaginous extracellular matrix (ECM). Here, we review the development of the spine and spinal cartilages, the composition of the cartilage ECM, the so-called “matrisome” and its functions, and the players involved in the genetic architecture of AIS. We also propose a molecular model by which the cartilage matrisome of the IVD contributes to AIS susceptibility. For the rest of this review, we focus on the biologic factors that control IVD development and their genetic contributions to AIS susceptibility. We also discuss the implications of these discoveries for new hypothesis-driven research and therapeutic interventions.19 Briefly, in early development, presomitic mesodermal cells proliferate and move from the tail bud at a specific rhythmic pace, creating somites that emerge at the anterior tip and progress along the embryonic axis , Notch, and Wnt/β-catenin—that set up waves of gene expression through “circuits” controlling the timing of oscillations, spatial expression, and synchronicity of somitogenesis.21 The subsequent formation of the sclerotome, i.e., future spine and associated structures, requires that a subset of mesodermal cells in the ventral somite region undergo an epithelial-to-mesenchymal transition and then migrate toward the midline to surround the notochord . This two-step transition is regulated by Wnt and Fgf signaling pathways.22 The vertebrate spine also derives from the axial mesoderm in addition to the paraxial mesoderm (somites)  Fig. .Fig. 3Ca23 During embryonic spine development, the notochord regresses from regions of developing vertebral bodies and expands in regions of the future IVD , the gel-like inner core of the IVD, and Pax1 expressing cells contribute to the region of the outer annulus fibrosus (AF) IVD.26 The spontaneous mutant mouse strains “undulated” and “scoli” display varying spinal phenotypes resulting from mutations in the Pax1 locus.30 Two undulated strains  exhibit mild kink tail phenotypes due to hypomorphic Pax1 mutations. By comparison, the homozygous Uns/Uns strain harbors a 120 kb deletion encompassing Pax1 and completely lacks lower thoracic and lumbar vertebral units. Of note, Uns/Uns displays a more severe phenotype than a homozygous targeted knockout of Pax1, suggesting that noncoding sequences outside of Pax1 may drive expression of additional loci related to spine development. Indeed, analysis of Pax1 enhancers using transgenic assays in mouse identified a strong extra-genic enhancer “Xe1” which drives expression in the sclerotome and whose loss presumably contributes to the Uns/Uns phenotype.31 More comprehensive analysis of enhancers, using transcriptomic and chromatin immunoprecipitation sequencing (ChIP-seq) studies of Pax1-positive flow-sorted E12.5 vertebral column cells, found enrichment of signals at loci related to ECM, cartilage development, and collagen fibrillogenesis biological pathways. Moreover, Pax1-negative cells display a general downregulation of ECM genes, e.g., Col11A1, suggesting Pax1 is a critical transcriptional regulator of ECM matrix biogenesis in the outer AF. Proteoglycan accumulation  is also significantly reduced in animals lacking Pax1.33 It is clear from these and other studies that Pax1, together with its paralog Pax9, act synergistically to drive development of IVD of the entire vertebral spine.34In early mouse embryonic development, the sclerotome migrates out from the somites to surround the notochord in a pattern of condensed and less condensed segments corresponding to future IVD and vertebral bodies, respectively, along the anterior/posterior axis. The paired box 1 (Pax1) transcription factor is a well-described marker of sclerotome development which is induced via factors secreted from the notochord including: Sonic Hedgehog (Shh) and through noggin-dependent inhibition of bone morphogenetic protein (BMP) signaling.38Sox5/6 is expressed in the notochord and the sclerotome, where they share partially redundant roles and cooperate with Sox9 to drive the expression of chondrogenesis genes in the growth plate.44 Sox5 and 6 are clearly important in IVD development as well. Both are expressed in the notochord and the sclerotome, and the notochordal sheath fails to form in Sox5−/−;Sox6−/− mice. The consequence of this is failure of the NP to form. In addition to severe chondrodysplasia, mice lacking Sox6 alone develop a kinked tail that occurs in segments where the NP is small and misplaced,45 while Sox5−/−;Sox6−/− double mutant mice display a virtual absence of cartilage and chondrogenic tissues of the spine.46 Other striking features of Sox5−/−;Sox6−/− double mutant mice are the downregulation of genes encoding major ECM components including collagen II, aggrecan, and cartilage link protein, and a general delay in differentiation of the inner AF (IAF).47 Sox9 controls Sox5/6 expression and is required for the normal patterning and development of the spine, failing to generate obvious patterned vertebral bodies or IVD tissues when ablated in embryonic mice.48 Sox9 is continuously required for homeostasis of the spine in adult mice, where its ablation in skeletally mature mice leads to kyphoscoliosis, disc compression, and degeneration of the IVD.37 Interestingly, Pax1 transcripts are upregulated when Sox5 and 6 together are removed genetically from the IVD anlagen, suggesting regulatory feedback between Sox5/6 and Pax1 signaling, which may help to define the regulatory programs driving development of the inner and outer AF. Indeed, Pax1, as well as its paralog Pax9, also assists in the robust gene expression of Sox5. Thus, Pax1/9 and Sox9 function in concert with BMP and transforming growth factor (TGF) signaling and are critical to initiate expression of chondrogenic genes during early IVD differentiation. Later, the expression of Pax1 is restricted to the outer annulus by the Sox5/Sox6 negative feedback mechanism49  that connects to cartilage fibers extending from the AF.In humans, the spine grows most rapidly in the first 5 years of life, with an average T1 to S1 segment length increase of 10 cm during this time (2 cm per year). There is a slower T1 to S1 growth from age 5 to 10 years of ~5 cm until adolescence (1 cm per year). From age 10 years to adulthood, T1 to S1 grows an additional 10 cm; this includes the adolescent growth spurt (2 cm per year).ies Fig. . Cells w3 in the AF and only 4 000 “chondrocyte-like” cells per mm3 in the NP.52 In the latter case, notochordal-like cells that begin as large vacuolated structures are slowly replaced by distinctly smaller and less metabolic cells in postnatal development, up to ~10 years of age in humans  production via BMP2 receptor SMAD1/5 signaling.63The proteoglycan content is highest in the NP and declines outward toward the AF. In the NP, chondroitin sulfate-linked proteoglycans interact with collagens to retain hydration and form a gel-like matrix that resists compression and provides height to the disc. The predominant proteoglycan of the IVD matrisome is aggrecan, present in both NP and AF.56 Other proteins effectively bridge the structural components of the ECM, re-enforcing and strengthening the network and also connecting it to cells and to other soluble factors. The multidomain structures of these connectors facilitate direct binding to cell surface receptors, other ECM connectors, and soluble growth factors. Example glycoproteins in this class include the fibronectins and laminins. Like collagens, laminins are triple-helical proteins made up of α, β, and γ chains that combine via a central coiled-coil domain. Multifunctional domains of the laminins can interact with cell surface receptors such as integrins and also other components of the ECM, thereby mediating cell–cell interactions. Fibronectin, a large secreted glycoprotein, dimerizes through C-terminal disulfide bonds where it binds to collagens, gelatins, integrins, and other ECM molecules. In particular, fibronectin binding to integrin receptors promotes their clustering in the cell membrane, which in turn leads to further assembly of fibronectin into fibrils around the cells.58 In the adult disc, fibronectin proteolysis into fragments is correlated with the grade of disc degeneration, and has been shown to stimulate catabolic events that lead to the same.52Other proteoglycans in the disc function as accessory proteins that may, for example, participate in collagen assembly and help to regulate the appropriate width and length of fibrils. For example, small leucine-rich repeat proteins (SLRPs) are proteoglycans such as versican, decorin, biglycan, fibromodulin, lumican, and perlecan that contain characteristic domains including carboxy-terminal leucine-rich repeats Fig. . These p57 The primary structural component of microfibrils in the IVD are the large, structurally distinctive fibrillin glycoproteins . Associated with the fibrillins are a number of ancillary proteins, such as microfibril-associated glycoprotein 1, that provide specialized functions. In the OAF and IAF microfibrils co-localize with elastin fibers. While in the OAF microfibrils closely align with collagen lamellae of the ECM, in the IAF fibrillin-positive microfibrils are also seen in an elastin-negative region around cells. In the NP microfibrils are found almost exclusively in the pericellular region, away from elastin fibers in the ECM.51 The distribution of microfibrils may reflect differing roles in the OAF, which must absorb the brunt of compression and torsional forces, compared with the IAF and NP that are subjected to relatively minor, isotropic forces. It is also now appreciated that the microfibrillar niche is a command center for regulating cellular behavior. This is apparently achieved through direct interactions with cell surface receptors as well as by serving as a reservoir for various growth factors, particularly in the TGF family.65Within the disc, the AF is a collagen-rich fibrillar ECM constructed to withstand tensile and torsional forces transmitted outward from the gelatinous NP. Microfibrils are an important structure distinct from collagen and elastin fibrils.66 By definition, SNPs are common in the population and consequently can be informative in statistical tests such as logistic regression that measure frequency differences between affected cases and matched controls. Because of the many tests performed in a typical GWAS , a significance level of P < 5 ×10−8 is generally required for any association to be declared significant (GWAS-level significance). Replication of GWAS-significant loci is necessary to independently validate candidate SNPs and provide realistic estimates of effect size and fraction of risk conferred across populations. It is important to note that while GWAS are proven to be powerful for mapping disease loci, further fine-mapping approaches are needed to define the underlying causal mutations. Multiple AIS risk loci that were originally identified in GWAS have been further validated by replication and meta-analysis studies, as given in Table 67The majority of genetic risk in AIS has been defined thus far by hypothesis-free genome-wide association studies (GWAS) that map the location of genetic risk factors in the genome relative to fixed markers, usually single nucleotide polymorphisms (SNPs).PAX1 gene was originally discovered by a GWAS in 3 102 individuals of European descent (North American) and replicated in independent North American, Japanese, and East Asian female cohorts.68 Further investigation revealed that the association was driven almost entirely by females  but not males , identifying it as an apparently sex-specific AIS locus. Resequencing highly conserved sequence elements within the associated interval in AIS subjects refined the signal to a putative enhancer near the Xe1 enhancer described earlier. The new enhancer, called PEC7, was shown to drive reporter gene expression in zebrafish somites, an activity that was disrupted by AIS-associated SNPs.68 These data suggest that alterations in the spatial and/or temporal control of PAX1 expression can increase risk of spinal deformity (AIS). The association signal within an intron of the SOX6 gene implicates its transcriptional regulation in AIS. SOX6 is expressed in several tissues including testis, muscle, and spinal cord in addition to chondrocytes.45 However, it is tempting to speculate that AIS-associated variation in the SOX6 enhancer could specifically diminish its expression in cartilaginous tissues and thus dysregulate SOX5/6/9, PAX1, and possibly even GPR126 (see below) in these tissues. GPR126  is a member of the adhesion G protein-coupled receptor family with multiple cellular functions ranging from cell adhesion to migration, to name a few.69 In humans, GPR126 is highly expressed in cartilages and the IVD. In mouse embryo, Gpr126 is highly expressed in the proliferating cartilage of the spine, suggesting its role in spinal development could be exerted through effects on chondrogenesis.70 As outlined below, further experiments in conditional mutants have supported this hypothesis, and in particular, have shown that Gpr126 is a positive regulator of cartilage differentiation and possibly its homeostasis. In the chondrogenic ATDC5 cell line, Gpr126 mRNA expression increases with early chondrogenic differentiation.71Gpr126 overexpression in this cell line increases expression of the cartilage marker genes Col2a1 (encoding type II collagen) and Acan (encoding aggrecan), while Gpr126 knockdown decreases their expression.72 Gpr126 and Sox9 are also positive regulators of each other, as knockdown or knockout of either reduces expression of the other in ATDC5 cells, chondrocytic N1511 cells, or adult IVD.72 In zebrafish, Gpr126 is required for Schwann cell myelination, an effect that could be overcome by elevating cAMP levels with forskolin, suggesting a classic signaling mechanism through G proteins.73 Gpr126 receptor signaling can be self-activated through autoproteolytic processing.74 Gpr126 additionally mediates certain activities such as Schwann cell development through interaction with a laminin-211 ligand.75 However, the mechanisms by which Gpr126 signals in chondrocyte cells are yet to be characterized.An association distal to the CDH13 encodes the cadherin 13, or “T-cadherin” protein, a unique member of the cadherin family that lacks transmembrane and cytoplasmic domains, attaching to the plasma membrane via a glycosylphosphatidylinositol anchor.76 During development T-cadherin is highly expressed throughout the early developing sclerotome, with later expression restricted to the caudal sclerotome and migrating neural crest cells.77 Strong CDH13 expression is also reported in a chondrocytic cell line and rib cartilage, but its role in these tissues is unknown.67 ABO encodes glycosyltransferase isoforms that catalyze the transfer of carbohydrates to the H antigen, forming the antigenic structures of the ABO blood group [MIM:616093]. ABO is expressed at varying levels in a variety of tissues and is reportedly undetected in cartilage. BNC2 (OMIM 608669) encoding Basonuclin 2, is a highly conserved transcription factor belonging to the C2H2 group of zinc finger proteins. In humans, BNC2 is produced in a variety of tissues including bone and cartilage. The strongest and most reproduced association with AIS in multiple studies is with SNP rs11190870 that is located near the end of the LBX1 gene. LBX1 is encoded head-to-head with another gene FLJ41350 ). LBX1 (OMIM 604255) encodes the ladybird late homeobox transcription factor. Lbx1 is specifically expressed early in embryogenesis in the developing dermamyotome and is suggested to control the expression of genes that guide lateral migration of muscle precursors and maintain their migratory potential.78 Lbx1 is also critical for specification of dorsal interneuron fates and somatosensory function.79 Thus, LBX1 may be implicated in myogenic and neurogenic etiology of human AIS.Whether genetic variation of other associated loci contributes to mechanisms of cartilage biogenesis awaits investigation. For example, 80 rs12946942 and the LD block containing it are in a gene desert between SOX9 and KCNJ2. The block contains six enhancer elements, one of which forms a long-range chromatin loop to SOX9 in a prostate cancer cell line, and SNPs within the enhancer appear to direct allele-specific gene expression. It is posited that variants in the region may confer significant risk of progressive AIS by controlling scoliosis-related spatio-temporal SOX9 expression.Identifying patients with a high likelihood of curve progression is a top priority of AIS clinical management. Consequently, a comprehensive search for severity-correlated SNPs was conducted in a GWAS by limiting cases to those with spinal deformity above 40 degrees after skeletal maturity or surgical correction. Through a two-stage association study using a total of 12 000 Japanese subjects, a novel locus, rs12946942 on chromosome 17q24.3, showed significant association and was replicated in a Han Chinese population.81 Although MIR4300 expression has been noted in bone, cartilage, IVD, and spinal cord among other tissues, its function and potential role in AIS is not yet known.82A later study in the Japanese cohort identified association with SNPs in MIR4300HG, the gene encoding MIR4300 microRNA. Correlated SNPs were mapped within a putative enhancer in a conserved toplogically associated domain (TAD). So-called TADs are large, self-interacting genomic regions that are implicated in gene regulation.r2) estimate that <7% of the total genetic variance underlying AIS has been discovered.67 More recently, DNA sequence-based approaches have been applied to both AIS case/control cohorts and to individual AIS families, with the goal of identifying rare disease-causing variants that would be missed by standard GWAS and that could account for “missing” heritability.83 The discovery of rare disease alleles has been bolstered by large-scale publicly available reference sets such as the Genome Aggregated Database (gNOMAD) that provide summary data for sequenced alleles.84 The search space for rare genetic risk factors is often limited to the exons of the genome, the so-called “exome” for practical reasons, e.g., cost, computation time, and biologic interpretability. As the costs of sequencing decrease, the field is moving beyond exome sequencing (ES) toward genome sequencing (GS). Simultaneously, the tools for identifying and interpreting noncoding rare disease variants are rapidly improving.85 Single rare variants may be tested for enrichment in cases versus controls or co-segregation with disease in families. Alternatively, a potentially more informative approach is to statistically measure the burden of rare alleles per gene  in cases versus controls. The first ES burden study of 91 AIS cases and 337 controls showed FBN1 and FBN2, encoding fibrillins-1 and -2, respectively, as the most significantly associated genes. Dominantly inherited mutations in FBN1 are known to cause Marfan syndrome (OMIM 154700). Marfan syndrome patients typically present with distinctive skeletal features including increased height, disproportionately long limbs and digits, anterior chest deformity, mild-to-moderate joint laxity, scoliosis, dural ectasia , and a narrow, highly arched palate with crowding of the teeth. Cardiovascular features can be life-threatening and include mitral valve prolapse, mitral regurgitation, dilatation of the aortic root, and aortic regurgitation.86 Mutations in FBN2 are associated with the related disorder Congenital Contractural Arachnodactyly (CCA) (OMIM 121050). The mutations identified in AIS tended to be associated with tall stature and larger spinal curves, but without the cardiac manifestations or other complications that are typical of MFS or CCA,87 suggesting they exert hypomorphic effects on the functions of these proteins.Heritability studies . The Stickler/Marshall syndromes are hereditary conditions characterized by distinctive, flattened facial features, abnormalities of the vitreous, conductive hearing loss, scoliosis or kyphosis, and joint problems including joint hypermobility and early onset arthritis. Many Stickler patients exhibit Pierre Robin sequence, a combination of features  that can lead to feeding problems and difficulty breathing.90 On average, significantly more novel variants in ECM genes were observed in the AIS cohort compared with controls, suggesting that the polygenic accumulation of novel variants within potentially many ECM genes contributes to AIS risk. More recently, an exome-wide association study that was restricted to severe AIS identified a variant in the SLC39A8 gene. SLC39A8 is a ubiquitously expressed divalent metal cation transporter that, not surprisingly, has been genetically linked to disorders of numerous organ systems in humans including blood pressure, body mass index (BMI), circulating manganese levels, and circulating cholesterol levels.91 The AIS-associated variant altered alanine at position 391 to threonine. This single amino acid change was also correlated with greater spinal curvature, decreased height, increased BMI, and lower plasma manganese in AIS cases. Mutations in the zebrafish homolog caused vertebral anomalies and mild spinal curvatures that were noted to be reminiscent of zebrafish knockouts of the gene encoding alpha-1 chain of type VIII collagen described below.92 This observation suggests the possibility of a functional interaction between Slc39a8 and the extracellular matrix in zebrafish, and possibly other vertebrates including humans. Taken together, rare variant-based studies are interpreted to suggest that rare, hypomorphic variants in genes responsible for Mendelian connective tissue disorders may be contributing factors in AIS.Other sequence-based studies have highlighted rare mutations in ECM genes. In a pathway-based analysis of severe (i.e. >40 degree curves) AIS, restricting the analysis to novel (not found in dbSNP at that time) coding variants revealed association with musculoskeletal collagen genes, particularly 93 In a separate hypothesis-driven study, the HSPG2 gene encoding the SLRP perlecan was selected as a candidate per ES of an extended AIS family. One particular HSPG2 variant, Asn786Ser, was marginally overrepresented in a larger case-control cohort.94 Recessively inherited mutations in the HSPG2 gene cause Schwartz–Jampel syndrome type I (OMIM 255800), a rare disorder characterized by joint contractures, myotonic myopathy (muscle stiffness), dystrophy of epiphyseal cartilages, eye abnormalities, short stature, distinct facial features and propensity to malignant hyperthermia.95 In a separate study, linkage analysis and ES of a Swedish pedigree with an autosomal dominant inheritance of AIS identified a rare, nonsynonymous variant (V2287I) in the CELSR2 gene encoding cadherin EGF LAG seven-pass G-type receptor 2. Furthermore, analysis of tagging SNPs revealed significant association (P = 0.000 1) to this variant in a set of 1 739 Swedish-Danish AIS cases and 1 812 controls but not with case-control cohorts from Japan and the US. It is interesting to note that CELSR2 is a planar cell polarity cadherin, like GPR126, that encodes an adhesion-class G protein receptor. Mice that lack Celsr2 develop progressive hydrocephalus and display dysgenesis of ependymal cilia. Further family-based studies hold promise for discovering higher-impact AIS disease alleles, although reduced penetrance of the phenotype is an acknowledged challenge. Continued genome sequencing in both populations and families is warranted to fully define the polygenic architecture of the disease.Families with multiple affected members provide the opportunity to map and identify disease variants by their co-segregation with AIS. In one study, filtering for rare variants ) identified by ES in five multigenerational families followed by gene ontology term enrichment analyses produced top results for actin- and microtubule-based projections and ECM.96 Furthermore, MRI studies of AIS patients found that progressive scoliosis was associated with displacement of the NP toward the convexity of the curve.96 Whether the relatively subtle hypomorphic genetic changes in components of the IVD ECM identified with AIS so far are necessary or sufficient to cause AIS is not clear at present. Consequently, the ability to affect spatio-temporal expression of specific genes with precision, and to create knock-ins of disease-associated mutations in vertebrate models will be key for addressing these questions.Taken together, evidence from human genetic studies and animal models supports a role for vertebral cartilages, particularly the cartilage matrisome, in AIS pathogenesis. This model focuses attention on the IVD due to its role in withstanding and dissipating the forces of spinal growth. Radiographic studies of the spine in patients with progressive scoliosis are consistent with this model. In AIS patients that were followed longitudinally, wedging of the involved discs preceded wedging of the involved vertebrae until the end of the growth spurt, at which point vertebral wedging progressed somewhat more.Gpr126 using a Col2Cre driver produced thoracic scoliosis beginning at age P20 and becoming more prevalent with age. From this study it was posited that AIS may arise from deficiencies in a specific osteoprogenitor cell type expressing type II collagen.97 The study also suggested that scoliosis may have its beginnings in early development. While cell proliferation was unaffected in rib and spinal cartilage (IVD) cells, a slight increase in apoptotic cells was noted in both locations at P1 before the expected onset of spinal curvature. This and additional experiments defined a critical role for Gpr126 on cartilage development, including effects on the morphogenesis of the annulus fibrosis, chondrocyte survival, and the expression of Gal3st4 encoding galactose-3-O-sulfotranserase 4.98 These results also support the hypothesis that the dysregulation, and possibly loss, of specific cell populations early in postnatal development leads to a “weakened” IVD, perhaps through deficiencies in the ECM, that is vulnerable to the increasing axial and torsional forces exerted during rapid growth.The specific tissues in which AIS originates has been difficult to discern, as bone and soft tissue components of the spine in human patients appear to develop and function normally, at least superficially. Likewise, although AIS manifests around the adolescent growth spurt, it has been unclear whether its origins trace back to deficiencies in early development. Research addressing these questions has been severely limited by a lack of appropriate animal models that recapitulate a true AIS phenotype. This issue is likely a reflection of several factors, including reduced penetrance and the relatively late onset that may be missed in typical forward genetic screens. Moreover, the genetic factors identified in humans thus far point to hypomorphic mutations and variations in regulatory elements that may manifest in tissue- and temporal-specific ways. Fortunately, breakthroughs in this regard have come from use of the Cre/loxP system to create tissue-specific and inducible knockouts of AIS candidate genes. For example, removing 99 It has been conjectured that the cranial to caudal spinal loads that fish experience while swimming may recapitulate, to some extent, the same directional forces on the human spine due to walking upright.100 Zebrafish (Danio rerio) in particular is a workhorse laboratory fish that has become a staple in the armamentarium of tools to dissect human developmental disorders. Attractive features of the zebrafish include its relatively rapid generation time of 6–12 weeks, external and near-transparent development, and genetic and genomic tractability. Early models of spinal deformity in zebrafish were mapped to mutations in the Col8a1a gene encoding alpha-1 chain of collagen type VIII. These mutants exhibited vertebral anomalies, reminiscent of human “congenital” scoliosis, that were correlated with early defects of notochord development.101 Similar morphologic changes were later noted in Slc39a8 knockout zebrafish as described above, raising the question of a possible pathogenic overlap between congenital scoliosis that involves frank vertebral anomalies, and idiopathic scoliosis in which the vertebrae appear to be morphologically normal. Evidence in support of this came from another gene-targeting study in zebrafish that produced the first genetically defined developmental model of IS. In that study, genetic removal of an atypical protein tyrosine kinase called Ptk7 evoked a rotational spinal deformity beginning at late larval stages and progressing until sexual maturity. This model also recapitulates human IS in that there was a strong bias toward severe curves in females. Further genetic manipulations that removed maternal copies of Ptk7 in early development led to loss-of-function mutants with vertebral anomalies reminiscent of congenital scoliosis, suggesting that the same genetic mutations could result in idiopathic versus congenital scoliosis phenotypes in a context-dependent manner.102 This study of ptk7 mutants implicated abnormal spinal fluid flow. A more recent study further implicated neuroinflammation as an underlying cause of spine curve formation in ptk7 mutants.103 A correlation between inflammation and onset of scoliosis was also supported by independent studies in which the transcription factor Stat3 was removed from maternal and zygotic zebrafish. During later development, stat3 mutant zebrafish exhibit stunted growth, scoliosis, excessive inflammation, and fail to thrive. In humans, dominant-negative mutations in STAT3 cause Hyper IgE syndrome marked by dysregulation of immunity as well as late-onset scoliosis reminiscent of AIS.104Fish (teleosts) are emerging as powerful tools for understanding developmental mechanisms of spinal deformities. Scoliotic curves have been observed in various fish species, including early studies in “curveback” guppy that demonstrated a heritable phenotype reminiscent of human idiopathic scoliosis.Xe1/PEC7, using genome editing tools will be critical for hypothesis-testing in vertebrate systems. It remains to be seen whether single variants in individual genes will be sufficient to create AIS-like phenotypes in mouse. More likely, in keeping with the complex heritability of AIS, it may be combinations of genetic changes affecting multiple components of the cartilage matrisome that will be required to evoke progressive deformity. The introduction of multiple genetic changes into individual animals by breeding strategies or sequential genome editing may be required to model AIS appropriately.The ability to create knock-ins of specific human mutations, whether in exons or in disease-associated enhancers such as 14 Bracing addresses both biologic and biomechanical susceptibility in AIS by relieving asymmetric loading in the spine and thereby allowing symmetric growth. Although bracing has shown efficacy in preventing progression in clinical trials,8 it is typically prescribed to be worn 12–16 h each day over a course of years, and compliance is notoriously poor.8,105 Surgical correction is required when bracing fails to halt progression, not only to address the appearance of the deformity but also to prevent future pulmonary complications. Lung volume normally increases dramatically during the juvenile and adolescent years, approximately doubling from the age of 10 years to skeletal maturity. Progressive AIS is associated with diminished lung volumes, particularly for curves ≥70 degrees. Patients with curves reaching 100 degrees are at significant risk of symptomatic restrictive pulmonary disease. Given all of these issues, the first goal of surgical approaches to AIS is to halt progression beyond an acceptable limit of ~50 degrees Cobb angle, while a secondary goal is to achieve some correction of the existing deformity.1Natural-history studies have shown AIS measuring less than 30 degrees at the cessation of growth rarely increases in adulthood. Fortunately, the majority of patients are in this category but nevertheless require careful monitoring with radiography to monitor the curve. Curves greater than 50 degrees, however, will slowly progress throughout adulthood at a rate of 0.75–1.00 degrees per year. Therefore, the AIS patient with an immature skeleton and scoliosis of greater than about 20 degrees is at risk for progression and may be treated by bracing.109 Therapies proposed to stimulate ECM production in IVDD include local delivery of growth factors or viral-based gene therapy, or adoptive transfer of adult mesenchymal stem cells. In this regard, the avascular nature of the IVD and its limited systemic connection could prove advantageous for direct in vivo therapeutic delivery, similar to the eye. Direct injection of therapeutic viral vectors has shown such promise for correcting several inherited eye disorders and it is now being pursued in clinical trials.108 In IVDD, increased proteoglycan synthesis has been observed after direct injection of adenovirus, adeno-associated virus, or lentivirus expressing Tgf-β, latent membrane protein 1 (Lmp1), Sox9, and other factors in rabbit models.106 Cell-based therapies would aim to replace or regenerate resident IVD cells, likely using endogenous progenitor cells or MSCs to increase ECM production. However, harvesting endogenous cells would require surgery, and the cells would be predicted to harbor the same genetic susceptibilities as resident IVD cells. The number of resident NP cells in the IVD is expected to be at its peak in individuals younger than 20 years of age.107 Although the cellular status in AIS patients is not well understood, delivering additional progenitors into the IVD environment might not prove beneficial. Modern genome editing technologies provide another promising line of gene therapy. Specifically, clustered regularly interspaced short palindromic repeat (CRISPR)-associated nucleases can be used to cleave specific sites, with subsequent removal or replacement of targeted DNA sequences.108 The benefits of genome editing are its simplicity and precision in removing or correcting an offending mutation. This is particularly relevant where simply stimulating endogenous gene expression would not apply. For example, genome editing could be envisioned to correct aberrant amino changes in type XI collagen in the AIS IVD. Another advantage of CRISPR-based genome editing is the efficiency of creating multiple genetic changes within each targeted cell, which may be necessary for more complex disorders such as AIS. It should be noted that while genome editing holds promise, its clinical application to any acquired or inherited human disorder is in its infancy until potential side effects (off-target changes and possible leakage to germline) are fully understood and controlled. Considering AIS, biologic therapies are particularly attractive given the “window of opportunity” in children with mild AIS who are apparently otherwise healthy.The fact that bracing can be successful suggests that future biologic therapies aimed at preventing progression should target the same “window of opportunity”. As outlined in this review, genomic studies of human AIS point to the biogenesis and/or maintenance of the ECM of the IVD in disease susceptibility. Future therapies consequently could be aimed at stimulating proper ECM production in cells of the IVD in patients with mild AIS, with the goal of providing better biomechanical and/or biologic resistance to asymmetric growth forces. Approaches to IVD-targeted therapy could perhaps build upon the data armamentarium developed by the intervertebral disc degeneration (IVDD) research community, as well as on other successful gene therapy efforts.110 In this scenario, the late onset and ostensibly healthy metabolism in AIS could prove advantageous, analogous to preventing osteoporosis by early calcium and vitamin D intake. It is notable however that in the IVD, the ECM is key in governing the rate of molecular transport through the long pathway from the cartilaginous endplate to the relatively sparse NP and/or AF cells. Aggrecan in particular intercalates with the collagen framework and thereby largely determines solute distribution and mobility.110 Clearly, appropriate in vitro and in vivo model systems and more fundamental understanding of the genetic, cellular, and developmental mechanisms of AIS are needed to advance any biologic therapeutic strategies.Prevention of AIS altogether has been a futuristic goal but not inconceivable given the emerging understanding of underlying mechanisms. For some patients, improving mechanical function in the IVD by boosting glycoprotein content and hydration early in life could be envisioned as a preventative strategy, perhaps by small molecule or peptide therapies, or by better nutritional delivery to the disc.The embryologic development of the spine is an exquisitely controlled process both spatially and temporally. Deleterious mutations in single genes controlling somitogenesis can lead to rare Mendelian disorders affecting spinal integrity. Idiopathic scoliosis, usually occurring at adolescence, is the most common developmental disorder of the spine in children. AIS is genetically complex, but emerging data both from GWAS and next-generation sequencing of human AIS cohorts have highlighted a role for determinants of cartilage biogenesis and development of the IVD, particularly pathways controlling extracellular matrix, or matrisome production. DNA sequence variants have been identified that potentially alter the transcriptional regulation of matrisome genes, or that create hypomorphic changes in protein-coding sequences. Combinations of these variations may effectively create tissue-specific alterations, with major effects on the composition and/or organization of the IVD matrisome in cells of both NP and AF. Cellular maturation in the NP occurs in the first decade of life in humans and also may be a contributor to AIS. In either scenario, impairments in the disc may cause asymmetric responses to the strong torsional forces along the spine during adolescent growth, leading the spine to twist away from these forces. In contrast to the disc in adult IVD degeneration, the IVD in AIS does not exhibit overt signs of degeneration. Given this and the fact that current bracing therapies during the adolescent growth spurt can be effective suggests that there is a window of opportunity for providing biologic therapies to control or prevent AIS and subsequent curve progression. The IVD of AIS patients may be amenable to direct in vivo delivery of molecular or nutritional therapies aimed at preserving its structural integrity. These factors encourage optimism and justify continued research to understand underlying disease mechanisms and to develop robust model systems for in vivo testing.


PYGL gene. Patients with GSD VI usually present with hepatomegaly, recurrent hypoglycemia, and short stature. Results: We report on two non-related Turkish patients with a novel homozygous splice site variant, c.345G>A, which was shown to lead to exon 2 skipping of the PYGL-mRNA by exome and transcriptome analysis. According to an in silico analysis, deletion Arg82_Gln115del is predicted to impair protein stability and possibly AMP binding. Conclusion: GSD VI is a possibly underdiagnosed disorder, and in the era of next generation sequencing, more and more patients with variants of unknown significance in the PYGL-gene will be identified. Techniques, such as transcriptome analysis, are important tools to confirm the pathogenicity and to determine therapeutic measures based on genetic results.Introduction: Glycogen storage disease type VI (GSD VI) is a disorder of glycogen metabolism due to mutations in the  PYGL gene resulting in deficiency of hepatic glycogen phosphorylase (PYGL). Different isoforms of glycogen phosphorylase are expressed in various tissues including brain (PYGB), heart (PYGB), muscle (PYGM), and liver (PYGL). In contrast to the intra-organ activity of brain and muscle isoforms of glycogen phosphorylase, liver PYGL allows rapid release of free glucose into circulation, thus providing a constant supply of energy to extrahepatic tissues ; [244_356del], p.[(Arg82_Gln115del)]; [(Arg82_Gln115del)].Trio exome analysis of patient 2 and both parents yielded a novel homozygous splice site variant in horylase . AccordiM of the enzyme for its substrate glucose-1-phosphate [Human liver glycogen phosphorylase (PYGL) is a homodimer formed by monomers of 846 amino acid residues. The activity of the enzyme is controlled by phosphorylation of a highly conserved serine residue Ser14) [4 [1]. ThGSD type VI is a rare inborn error of glycogen metabolism with only about 50 cases reported so far. The severity of clinical symptoms varies significantly. While most patients present with hepatomegaly ,3,6, theWhile hyperlactatemia is a typical feature of GSD type I and is associated with impaired growth, it is no common finding in patients with ketotic forms of hepatic GSDs . InteresThe genetic background of GSD VI is heterogeneous, and apart from the Mennonite pathogenic variant c.1620+1G>A that generates a transcript lacking all or part of exon 13 while maintaining some residual enzyme activity, no common mutations have been identified so far . TherefoPYGL-gene will be identified by these diagnostic approaches. Therefore, techniques such as transcriptome analysis are important tools to confirm the pathogenicity and to determine therapeutic measures based on the genetic results.As GSD VI is often associated with rather mild symptoms, it is well conceivable that this disorder is underdiagnosed. In the era of next generation sequencing, it can be assumed that more and more patients with variants of unknown significance in the