Novel Architecture of Costal Cartilage and Implications in Chest Wall Deformities

Costal cartilage is a type of hyaline cartilage that forms rod-like structures that connect the ribs to the sternum. Deformation of costal cartilage is observed in the chest wall deformities, pectus excavatum and pectus carinatum. Pectus excavatum involves a sternal displacement causing a depression of the chest while pectus carinatum causes a protrusion of the chest. As costal cartilage is not a widely studied tissue, this leaves little knowledge into possible factors involved in the pathogenesis of pectus deformities. Costal cartilage in these deformities has been described as being weakened and may implicate proteoglycans which play an important role in the structure and maintenance of the extracellular matrix. This study focused on the major proteoglycans aggrecan, biglycan, and decorin. Immunohistochemistry and western blot analysis of these proteoglycans was performed on costal cartilage from patients with pectus deformities along with an age-matched control. Western blotting was also performed on normal costal cartilage from fetal, adolescent, and adult tissue to observe any changes in relation to age. We demonstrated that aggrecan forms distinct patterns of localization. Western blot analysis of biglycan and decorin indicated that these proteoglycans exist in a proform and mature form of different sizes likely based on varied N-glycanation. Both forms of biglycan and prodecorin were present in fetal. adolescent, and adult tissue while mature decorin was present in adolescent and adult tissue suggesting biglycan has a greater role in early costal cartilage development. Immunohistochemistry of biglycan and decorin showed both proteoglycans localized in the territorial matrix but not the interterritorial matrix and may be indicative of an alternative method of collagen organization or hindered visualization caused by the formation of large collagen nanostraws. Further studies will be performed to establish the functional differences of the different glycanated forms of biglycan and decorin as well as their inclusion in collagen fibrils

The Chondrocyte Channelome: A Novel Ion Channel Candidate in the Pathogenesis of Pectus Deformities

Costal cartilage is a type of rod-like hyaline cartilage connecting the ribs to the sternum. The chest wall deformities pectus excavatum (PE) and pectus carinatum (PC) involve displacement of the sternum causing a depression or protrusion of the chest. There is little knowledge about costal cartilage and pectus deformities with much of its understanding based on assumptions from articular cartilage. Chondrocytes are subjected to a constantly changing environment with fluctuations in pH and osmolarity. Ion channels detect these changes and in turn regulate proliferation, differentiation, and extracellular matrix production. Using ion channel qPCR arrays, we produced expression profiles for normal, fetal, PE-affected, and PC-affected costal chondrocytes as well as articular chondrocytes. Costal and articular chondrocytes had many commonly expressed ion channels with certain channels specific to each cartilage type. The discrepancy in ion channel expression is likely to be a reflection of the functional differences between the two cartilage types. Additionally, fetal costal chondrocytes had several other distinct ion channels possibly due to the differentiation status of the cells. In PC and PE chondrocytes, ACCN1 (ASIC2) and KCNN2 (SK2) were consistently down-regulated compared to normal costal chondrocytes. However, Western blot analysis found deceases only in ASIC2 protein levels. ASIC2 is a proton-gated ion channel involved in cell response to extracellular pH changes. Calcium monitoring revealed a delay in the formation calcium transients in PC cells when challenged with low pH which may be caused by aberrant signaling from ASIC channels. Immunofluorescent analysis of connexins found that Cx43 was present in chondrocytes with phosphorylated Cx43 localizing in and around the nucleus. Analysis of ATP release found that release is likely a connexin-mediated process, though external acidosis did not induce ATP release. Analysis of microRNAs found upregulation and down-regulation of several microRNAs in PC versus control cells, though further studies are needed to identify a possible microRNA signature for pectus deformities. Overall, we have generated a comprehensive ion channel profile for the costal chondrocytes, as well as identified a possible contributing factor for pectus deformities

Effects of Nanosecond Pulse Electric Fields on Cellular Elasticity

We investigated the effects of a single 60 nanosecond pulsed electric field (nsPEF) of low (15 kV/cm) and high (60 kV/cm) field strengths on cellular morphology and membrane elasticity in Jurkat cells using fluorescent microscopy and atomic force microscopy (AFM). We performed force displacement measurements on cells using AFM and calculated the Young\u27s modulus for membrane elasticity. Differential effects were observed depending upon pulsing conditions. We found that a single nsPEF of low field strength did not induce any apparent cytoskeletal breakdown and had minor morphological changes. Interestingly, force measurements and calculation of Young\u27s modulus showed a significant decrease in membrane elasticity. A single nsPEF of high field strength induced stark morphological changes due to disruption of the actin cytoskeleton and a marked decrease in elasticity likely caused by irreversible membrane damage. We suggest that the cellular morphology is mainly dependent on stabilization by the actin cytoskeleton, while the elasticity changes are partially dependent on the cytoskeletal integrity

Nanosecond Pulsed Electric Field Induced Changes in Cell Surface Charge Density

This study reports that the surface charge density changes in Jurkat cells with the application of single 60 nanosecond pulse electric fields, using atomic force microscopy. Using an atomic force microscope tip and Jurkat cells on silica in a 0.01 M KCl ionic concentration, we were able to measure the interfacial forces, while also predicting surface charge densities of both Jurkat cell and silica surfaces. The most important finding is that the pulsing conditions varyingly reduced the cells\u27 surface charge density. This offers a novel way in which to examine cellular effects of pulsed electric fields that may lead to the identification of unique mechanical responses. Compared to a single low field strength NsPEF (15 kV/cm) application, exposure of Jurkat cells to a single high field strength NsPEF (60 kV/cm) resulted in a further reduction in charge density and major morphological changes. The structural, physical, and chemical properties of biological cells immensely influence their electrostatic force; we were able to investigate this through the use of atomic force microscopy by measuring the surface forces between the AFM\u27s tip and the Jurkat cells under different pulsing conditions as well as the interfacial forces in ionic concentrations

Presence and Localization of Pro-and Mature Forms of Biglycan and Decorin in Human Costal Cartilage Derived from Chest Wall Deformities

Costal cartilage is a type of hyaline cartilage that forms rod-like structures that connect the ribs to the sternum. The most common chest wall deformities, pectus excavatum and pectus carinatum involved efective costal cartilage resulting in sternal displacement. Costal cartilage is not widely studied leaving little insight into possible factors involved in the pathogenesis of these pectus deformities. This study focused on the presence and distribution of two important regulators of collagen fibrillogenesis and organization, biglycan and decorin. Immunohistochemical analysis of transverse cross sections of normal and deformed costal cartilage revealed that biglycan and decorin mainly localized in the territorial matrix except for prodecorin which was only found within chondrocytes. Western blot analysis of whole protein extracts demonstrated the presence of both pro and mature forms of biglycan and mature decorin in patients and controls. In normal costal cartilage of different ages, the mature form of decorin was absent in a fetal sample whereas mature biglycan was weakly expressed, suggestive that mature biglycan may play a role in early costal cartilage development. Further studies are needed to determine the functional differences between the pro- and mature forms of biglycan and decorin both in age and disease

Atomic Force Microscopy Characterization of Collagen ‘Nanostraws’ in Human Costal Cartilage

Costal cartilage, a type of hyaline cartilage that bridges the bony ribs and sternum, is relatively understudied compared to the load bearing cartilages. Deformities of costal cartilage can result in deformation of the chest wall, where the sternum is largely pushed toward or away from the spine, pectus excavatum and pectus carinatum, respectively, with each condition having significant clinical impact. In the absence of extensive literature describing morphological features of costal cartilage, we characterized a sample from the costal margin immunohistologically and through atomic force microscopy. We had previously observed the presence of collagen ‘nanostraws’ running the length of costal cartilage. Hypothesizing that these structures may be responsible for fluid flow within this thick, avascular tissue, and prior to microfluidic analysis, we estimated the diameters and measured Young\u27s modulus of elasticity of the collagen nanostraws. We found significant differences in results between treatment type and fixation. Significant differences in nanostraw elasticity and diameter obviously affect nano-fluidic transport calculations, and therefore, we consider these results of importance to the scientific community relying upon measurements in the nanoscale

Transient ALT Activation Protects Human Primary Cells From Chromosome Instability Induced by Low Chronic Oxidative Stress

Cells are often subjected to the effect of reactive oxygen species (ROS) as a result of both intracellular metabolism and exposure to exogenous factors. ROS-dependent oxidative stress can induce 8-oxodG within the GGG triplet found in the G-rich human telomeric sequence (TTAGGG), making telomeres highly susceptible to ROS-induced oxidative damage. Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes and their dysfunction is believed to affect a wide range of cellular and/or organismal processes. Acute oxidative stress was shown to affect telomere integrity, but how prolonged low level oxidative stress, which may be more physiologically relevant, affects telomeres is still poorly investigated. Here, we explored this issue by chronically exposing human primary fibroblasts to a low dose of hydrogen peroxide. We observed fluctuating changes in telomere length and fluctuations in the rates of chromosome instability phenotypes, such that when telomeres shortened, chromosome instability increased and when telomeres lengthened, chromosome instability decreased. We found that telomere length fluctuation is associated with transient activation of an alternative lengthening of telomere (ALT) pathway, but found no evidence of cell death, impaired proliferation, or cell cycle arrest, suggesting that ALT activation may prevent oxidative damage from reaching levels that threaten cell survival

A ubiquitin-based effector-to-inhibitor switch coordinates early brain, craniofacial, and skin development

The molecular mechanisms that coordinate patterning of the embryonic ectoderm into spatially distinct lineages to form the nervous system, epidermis, and neural crest-derived craniofacial structures are unclear. Here, biochemical disease-variant profiling reveals a posttranslational pathway that drives early ectodermal differentiation in the vertebrate head. The anteriorly expressed ubiquitin ligase CRL3-KLHL4 restricts signaling of the ubiquitous cytoskeletal regulator CDC42. This regulation relies on the CDC42-activating complex GIT1-βPIX, which CRL3-KLHL4 exploits as a substrate-specific co-adaptor to recognize and monoubiquitylate PAK1. Surprisingly, we find that ubiquitylation converts the canonical CDC42 effector PAK1 into a CDC42 inhibitor. Loss of CRL3-KLHL4 or a disease-associated KLHL4 variant reduce PAK1 ubiquitylation causing overactivation of CDC42 signaling and defective ectodermal patterning and neurulation. Thus, tissue-specific restriction of CDC42 signaling by a ubiquitin-based effector-to-inhibitor is essential for early face, brain, and skin formation, revealing how cell-fate and morphometric changes are coordinated to ensure faithful organ development