Although many advances have been made during the last six decades, the controlled processes by which vertebrate hard tissues acquire, organize, and maintain their mineral component remain only partially understood. Mineralized tissues can be classified into two categories based on their developmental origin. Enamel is the only hard tissue of epithelial origin; all other hard tissues are of connective tissue origin and therefore possess a collagen-rich extracellular matrix as their main organic component. The cells responsible for the formation of collagen-based hard tissues regulate the synthesis and degradation of non-collagenous components, among which proteoglycans (PGs) play a key role in biomineralization. This review focuses on the mineralization process of bone and dentin, highlighting the highly coordinated sequential events leading to the acquisition of the mineral phase. Initially, the forming cells release numerous small spherical membrane-limited bodies known as matrix vesicles (MVs), which constitute the nanocompartments necessary for the onset of mineral nucleation (vesicular stage). At this stage, extracellular matrix PGs bind to the MV membrane and subsequently to calcium ions, thereby allowing the accumulation of high concentrations of calcium around the MVs. Following PG degradation, calcium ions enter the MVs through annexin channels, leading to the nucleation and growth of the first mineral crystals. Subsequently, cells orchestrate the conditions required for mineral propagation into the surrounding collagenous matrix (fibrillar stage). During this phase, PGs and other non-collagenous matrix components play a crucial role in promoting mineralization progression as well as in limiting and interrupting the process.
Equine asthma (EA) is the most prevalent chronic respiratory disease in horses, but molecules thought to be relevant in its pathogenesis are not yet fully understood. Recently, increased Vanin-1 (VNN1) levels were found in bronchoalveolar lavage fluid from horses with severe EA. Human VNN1 is known as a pantetheinase, which is associated with respiratory diseases such as asthma, but the equine vanins (eVNNs) are uncharacterized. We identified the domain architecture of eVNN in silico and analyzed its tissue expression in healthy horses using RT-PCR (n=5) and in situ hybridization (n=3). Recombinant eVNNs were expressed in HEK293 cells to assess cellular transport and glycosylation via Endo H and PNGase F treatment. Three putative functional eVNNs were identified on chromosome 10, each containing canonical Nitrilase superfamily domains. All genes showed a broad tissue expression, but distinct airway localization: eVNN1 and eVNN2 were predominantly expressed in respiratory epithelium, whereas eVNN3 was localized in submucosal glands. eVNN1 and eVNN3 were secreted, while eVNN2 remained cell associated. Pantetheinase activity of recombinant eVNN1 was confirmed by mass spectrometry. The three eVNNs appear to cover different functional niches in equine airways, enabling future research into their putative role in EA.
Keratan sulfate (KS) is a glycosaminoglycan consisting of repeating N-acetyllactosamine disaccharides, in which both galactose and N-acetylglucosamine are often 6-O-sulfated. It has been reported that KS recognized by the monoclonal antibodies 5D4 and 373E1 is preferentially expressed in papillary thyroid carcinoma (PTC) and only minimally in other thyroid tumors/lesions or normal thyroid tissue. However, the precise epitopes recognized by these antibodies remain incompletely characterized, and the expression and extent of low-sulfated KS in PTC have not been systematically evaluated. To better understand the nature of KS expressed in PTC, we generated a novel anti-KS monoclonal antibody, 299-1C1, and performed immunohistochemical analyses using 299-1C1 together with two existing anti-KS monoclonal antibodies, 5D4 and R-10G, in combination with keratanase II and endo-β-galactosidase. The results showed that both highly sulfated and low-sulfated KS are preferentially expressed in PTC, including lymph node metastases, accompanied by upregulation of genes encoding key KS biosynthetic enzymes (B3GNT7, B4GALT4, CHST2, and CHST6) in integrated TCGA/GTEx transcriptomic datasets. Expression of B3GNT7, B4GALT4, and CHST2, but not CHST6, was further increased in BRAF-mutant PTCs. These findings indicate that anti-KS monoclonal antibodies are useful for the pathological diagnosis of PTC, particularly for distinguishing lymph node metastases from intranodal thyroid inclusions.
Human cytomegalovirus (HCMV) has been linked to tumor progression in several cancers, but its role in breast cancer remains uncertain. This study investigated HCMV long non-coding RNA4.9 (lncRNA4.9) in three breast cancer subtypes (ER+/PR+, n=25; HER2-positive n=24; triple-negative, n=26) and normal breast tissue (n=5) using RNAscope. Serum HCMV IgG and IgM levels were measured, and immunohistochemistry was used to assess the pan-macrophage marker CD68 in breast tissue. HCMV IgG seropositivity was more common in ER+ patients, while IgM positivity was more frequent in HER2-positive and triple-negative cases. HCMV lncRNA4.9 signals were detected in over half of tumor samples, localizing to invasive tumor cells, stromal cells, adipose tissue, and ductal carcinoma in situ (DCIS). Although detection rates did not differ significantly between breast cancer subtypes, triple-negative tumors exhibited mid- to high-level lncRNA4.9 expression. Immunohistochemical analysis demonstrated CD68+ cells present in most tumors and were more abundant in HCMV lncRNA4.9-positive samples. These cells co-localized with lncRNA4.9-positive cells at invasive margins, while in associated foci of DCIS, CD68+ cells were present in surrounding stroma. This study provides the first demonstration of HCMV lncRNA4.9 in breast tissue and supports HCMV's potential role in the biology of a subset of breast cancers.
Sickle cell disease (SCD) is an inherited hemoglobinopathy leading to the deformability of red blood cells, enhanced cellular adhesion, and blockage in microcirculation. "Sickle Neutrophils" cause oxidative stress and decreased bioavailability of nitric oxide (NO) due to enhanced generation of reactive oxygen species (ROS). The mechanism of action of the common medication of SCD, hydroxyurea (HU), has not yet been thoroughly examined. This investigation examines the subcellular localization of neuronal nitric oxide synthase (nNOS) using immunoelectron microscopy, and confocal microscopy, as well as NO production and ROS generation by flow cytometry in neutrophils from steady-state SCD patients, and SCD patients on HU therapy. nNOS was increased and prominently distributed in the cytoplasmic compartment, with no nNOS staining in the nucleus of neutrophils in SCD-HU patients. ROS was found to be increased in SCD patients and was significantly reduced after HU treatment. An increase in NO production and decrease in ROS are linked to nNOS's nucleo-cytoplasmic shuttling in SCD neutrophils after HU therapy that provides a novel cellular mechanism through which HU regulates neutrophil function. In conclusion, this study establishes for the first time that HU therapy induces a favorable nucleo-cytoplasmic translocation of nNOS in SCD neutrophils, indicating a key mechanism behind the therapeutic effectiveness of HU in SCD.
Both macrophages and neutrophils are recognized as innate immune cells; the former engulf cholesterol crystals (CCs) while secreting NLRP3, which also promote inflammatory responses and drive the latter into atherosclerotic plaques. Moreover, recent studies have implied a critical role of NLRP3 in neutrophil recruitment into atherosclerotic lesions as neutrophil extracellular traps (NETs), which have not been visualized in vivo adhering on CCs so far. We have observed that neutrophils simultaneously attracted by NLRP3 cause NETs formation in situ from spontaneously ruptured atherosclerotic plaques by using touch-imprints method. This study aimed to confirm by imaging that NETs react to CCs, release NLRP3-related inflammatory factor, and degenerate on the surfaces that might result in breakages. These findings raise the possibility that NET-forming neutrophils not only respond to CCs but may also contribute to their structural modification or fragmentation. Such interactions may represent a previously underappreciated bidirectional relationship between CCs and NETs in human atherothrombosis.
Sulphated glycosaminoglycan (sGAG) is a major component of cartilage and known to be involved in cell signaling processes, but little is known about expression and localization in different cartilage types. Using common histochemical staining methods to assess regional sGAG distribution in bovine articular, meniscal, auricular, and nasal cartilage, different staining patterns were found for alcian blue, Safranin O and thionine. Immunohistochemistry to identify expression and location of different epitopes of chondroitin sulfate (C-0-S, C-4-S and C-6-S), dermatan sulfate, keratan sulfate, and heparan sulfate, also demonstrated regional differences which could not explain the differences in histochemical staining. Chondroitin sulfate and keratan sulfate were expressed in all cartilage types investigated, however keratan sulfate was not expressed in the superficial zone of articular cartilage. Dermatan sulfate was only present in tensile load-bearing zones of articular and meniscal cartilage, and in the perichondrium of auricular and nasal cartilage. Despite being hyaline, nasal cartilage and articular cartilage showed dissimilar sGAG expressions. In conclusion, sGAG localization in different types of cartilage is distinctly different, suggesting diverse structural and mechanical functions governed by GAGs. These differences are important for our understanding of the role of sGAG in cartilage function and in cartilage tissue engineering strategies.
Aquaporin-4 (AQP4), the predominant water channel in the central nervous system, has been extensively characterized in astroglia, but its presence in peripheral mechanoreceptive end-organs remains unknown. We investigated AQP4 localization in human mechanosensory corpuscles using formalin-fixed, paraffin-embedded penile tissues from 11 adult cadavers and 12 surgical prepuces. Single and double immunohistochemistry was performed in serial sections with a comprehensive battery of antibodies targeting axons (neurofilaments, neuron-specific enolase [NSE], protein gene product 9.5 [PGP9.5], synaptophysin), glial cells (S100, nestin, nerve growth factor receptor [NGFR], vimentin, Wilms' tumor 1 [WT1], growth-associated protein 43 [GAP43]), perineurial and endoneurial cells (α-smooth muscle actin [α-SMA], epithelial membrane antigen [EMA], glucose transporter 1 [Glut-1], CD34), and extracellular matrix components (collagen type IV [COLIV]). AQP4 immunoreactivity was consistently observed in the terminal glial cells (TGCs) of all types of mechanosensory corpuscles across all penile regions examined, including Meissner, genital, Krause, and Pacinian corpuscles. Outer-core lamellar cells of Pacinian corpuscles also exhibited AQP4 expression. The AQP4+ mechanosensory corpuscles exhibited immunohistochemical profiles consistent with the established marker patterns of sensory corpuscles (S100+/WT1+/vimentin+/nestin+/Gap-43+/NGFR+ TGCs; synaptophysin+/neurofilaments+/PGP9.5+/NSE+ axons; CD34+ intermediate layer; EMA+/Glut-1+/vimentin+/COLIV+/NGFR+/Gap-43+ outer core; COLIV+ extracellular matrix). Beyond sensory corpuscles, AQP4 was present in Schwann cells of nerve bundles and inconsistently in vascular endothelium. These findings provide the first evidence that AQP4 is a molecular constituent of TGCs in human mechanosensory corpuscles, suggesting roles in local water-ion homeostasis or other non-transporting functions of AQP4, and warranting further investigation of its biological significance in low-threshold mechanoreceptor end-organs.
Artificial intelligence (AI) is transforming how cell biologists generate, analyze, and interpret visual data. Automated pipelines and large-scale image analysis increase throughput and reproducibility, yet they also modify how visual evidence is validated and trusted. This Perspective examines the conceptual implications of this shift, arguing that the integration of AI into imaging demands rather than replaces human morphological expertise. We distinguish between three conceptually distinct phenomena: (1) automation of acquisition, (2) AI-driven interpretation, and (3) the emergence of synthetic images generated without experimental basis. Through examples ranging from classical morphological misinterpretations to the rise of generative models, we show how both human and algorithmic systems can distort meaning when interpretation is detached from context. While AI can match or surpass human precision, metavisual competence remains essential to distinguish signals from artifacts. The current challenge is not technological but scientific: to ensure that computational power and human insight co-evolve toward reliable visual knowledge. We call for a renewed education in critical visual literacy, formal recognition of imaging specialists, and transparent standards of image provenance, such as the publication of raw instrument metadata. Preserving interpretative competence is crucial for the integrity of visual evidence in biomedical science.
Salivary gland cancer is a rare heterogeneous group of neoplasms with complex histopathologic patterns, including carcinoma with squamous differentiation, for which surgery is the standard treatment. However, further research is essential for developing new therapies. Head and neck squamous cell carcinoma is positive for p63 and negative for the bicellular tight junction protein cingulin (CGN). p63 plays a key role in cancer progression such as cell proliferation, migration, apoptosis, and squamous differentiation. To understand the roles of p63 in salivary duct adenocarcinoma, we investigated the malignancy by using overexpression of deltaNp63 in p63-negative salivary duct adenocarcinoma cells PGC2E derived from parotid gland duct adenocarcinoma. By transfection with deltaNp63, the PGC2E cells exhibited increased nuclear p63 expression and reduced CGN levels at the membrane. Overexpression of deltaNp63 disrupted epithelial polarity and epithelial permeability barriers, promoted cell proliferation and migration, and enhanced cellular metabolism. Treatment with inhibitors of histone deacetylase and nuclear factor kappa B, and antibodies to tumor necrosis factor-α and tricellular tight junction protein lipolysis-stimulated lipoprotein receptor induced apoptosis in PGC2E cells. However, overexpression of deltaNp63 prevented the induced apoptosis. These findings suggest that p63 contributes to cancer malignancy and the complex phenotype, and it may be possible to develop a novel treatment via p63.
Microvascular dysfunction due to hypoxia is a key contributor in the pathogenesis of many disorders including cancer and retinal and cardiovascular diseases, but relevant human models are missing. Here, we present a robust 3D in vitro method with the use of human induced pluripotent stem cell-derived blood vessel organoids to analyze in vitro microvascular remodeling. We present a detailed practical pipeline combining optical tissue clearing, high-resolution immunofluorescence, and surface marker analysis to quantitatively assess hypoxia-driven changes in endothelial cells, pericytes, and the basal lamina. Exposure of these blood vessel organoids to chronic hypoxia (1% O-2) for 1 week recapitulated key pathological features, including structural remodeling and a dysregulated secretome with altered vascular endothelial growth factor signaling. This approach establishes a versatile and human-relevant platform to study microvascular remodeling induced by chronic hypoxia and other pathological stimuli and their contribution to microvascular-related diseases:
Quantitative assessment of collagen fibril diameter is essential for understanding ultrastructural changes in aging, connective tissue disorders, and extracellular matrix remodeling. Although transmission electron microscopy (TEM) is widely used for this purpose, existing methods for fibril measurement are predominantly manual, operator-dependent, and prone to inconsistent reporting. To address this, we developed a semi-automated Fiji/ImageJ (IJ1) macro that standardizes fibril diameter measurements from two-dimensional TEMs. The macro uses a wand-based region-of-interest (ROI) detection strategy with integrated geometric validation. It calculates multiple metrics, including area-equivalent diameters, ellipse-derived major and minor axes, shape consistency indices, and spatial localization across the image field. Built-in quality assurance thresholds exclude oblique or irregular profiles, ensuring accurate identification of true fibril cross-sections. Real-time visual overlays support live validation and user feedback during analysis. We detail the implementation, analytical workflow, and validation approach, provide practical guidance for reproducible use across operators and datasets, and show proof-of-concept utility for analysis of collagen fibrils in vascular Ehlers-Danlos syndrome (VEDS) subject dermal samples. This open-source, extensible tool enhances standardization and reproducibility in collagen fibril morphometry for ultrastructural research.
ADAMTS1 (a disintegrin-like and metalloproteinase domain with thrombospondin type 1 repeats) is a secreted metalloproteinase with a known role in extracellular matrix remodeling in cardiovascular development and female fertility. Because of conflicting report of embryonic lethality and survival in Adamts1 mutant mice, we report generation and characterization of a new knockout (KO) allele, which led to detection of neonatal lethal omphalocele (persistent umbilical hernia) as a new Adamts1-dependent birth defect. This phenotype was also detected in another mutants with an in-frame lacZ insertion. β-galactosidase staining in the latter showed that Adamts1 was strongly expressed in the undifferentiated cells in the developing ventral abdominal wall preceding midline fusion at the umbilical cord attachment site. The omphalocele was characterized by the accumulation of the proteoglycan versican along with reduced proteolysis and impaired development of the superficial muscle layer, the panniculus carnosus, around the site of umbilical cord attachment. Furthermore, we observed sustained expression of the transcription factor, paired-like homeodomain transcription factor 2 in KO embryos, whereas it was downregulated in wild-type embryos during late embryogenesis. ADAMTS1 is thus a new matrisome component required for body wall closure.
Forebrain organoids (FOs) closely replicate key features of human brain, but often develop necrotic cores due to oxygen diffusion limits, resulting in disassembly. While dynamic culture devices can mitigate this, they add variability and diverge from adult cerebral static environment. Inspired by allometric scaling principles of brain growth, we developed a uniform, static culture protocol applying a 1-day transient high-dose Fibroblast Growth Factor 2 (100 ng/ml) treatment before neural induction. This acted as a proliferative stimulus, promoting long-term viability and structural integrity. Using human embryonic stem cells, early-FOs with diameters of 500-1000 µm achieved an 83.33% survival rate at 20 days in vitro (DIV). Area and volume increased significantly during 60 DIV culture period, but between 30 and 60 DIV they plateaued, indicating a transition from neural development to maturation. Weight increased until 30 DIV, but it significantly dropped between 30 and 60 DIV, possibly reflecting the formation of lumen-like structures. At 60 DIV, immunofluorescence revealed organized PAX6+ ventricle-like structures, where SOX2 marked neural progenitors, TUJ1 and MAP2 indicated mature neurons, GFAP identified astrocytes, and SYN1 highlighted emerging synaptic networks. This scalable protocol supports robust FOs generation, providing optimization and practical improvement within established frameworks to advance precision medicine.
SummaryMyogenic precursor cells within skeletal muscles are responsible for the maintenance of skeletal muscle over a lifetime. Neurotrophic and growth factors play critical roles in this maintenance and in responses of myogenic precursor cells. Both glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) play roles in the maintenance and/or development of strabismus, yet few studies have examined their roles in the control of myogenic precursor cell proliferation and differentiation. Two populations of myogenic precursor cells were isolated from extraocular and leg muscle by fluorescence-activated cell sorting: EECD34 cells, largely PITX2-positive, and PAX7-positive cells. Cultures were treated with GDNF or CNTF and processed immunohistochemically to determine proliferation and differentiation rates. Neither GDNF nor CNTF affected cell proliferation rates for either muscle. Both treatments impacted cell differentiation by increasing multinucleated cell number, with TA-derived precursor cells producing cells containing large numbers of nuclei and EOM-derived precursor cells producing shorter multinucleated fibers with fewer nuclei. These differences may explain the presence of extremely short myofibers within normal adult EOM compared with limb muscle. As GDNF and CNTF are downregulated in strabismic muscles, data suggest that myofiber length homeostasis may be disrupted in strabismic EOM and suggest possible approaches for strabismus treatment.
This study investigated the spatiotemporal dynamics of cardiac CD34⁺ stromal cells (SCs) during the reparative/proliferative phase of post-myocardial infarction (MI) healing. A transmural, non-reperfused MI was induced in middle-aged male Sprague-Dawley rats via left anterior coronary artery ligation, and proliferating cells were labeled with 5-bromo-2'-deoxyuridine. Hearts were collected at days 3, 7, and 14 after MI and analyzed using histology and immunohistochemistry. We found that the myocardial interstitium and coronary vessel adventitia harbored a population of cardiac CD34⁺ SCs. Following MI, activated CD34⁺ SCs expanded from the peri-infarct region across the healing wound through proliferation and migration, often alongside activated fibroblasts/myofibroblasts. While α-SMA⁺ myofibroblasts accumulated at pro-fibrotic granulation tissue sites, CD34⁺ SCs preferentially repopulated residual endomysial scaffolds spared by phagocytic macrophages. Over time, expanding fibrotic tissue progressively overtook these regions, leading to disappearance of CD34⁺ SCs. Importantly, clusters of CD34⁺ SCs accumulated at the scar border around the stumps of surviving cardiac myocytes, seemingly facilitating integration of endomysial connective tissue from non-infarcted myocardium into the developing fibrotic scar matrix. Collectively, these findings suggest that, unlike α-SMA⁺ myofibroblasts, cardiac CD34⁺ SCs seemed to support regenerative rather than fibrotic repair during post-MI wound healing by contributing to the preservation of myocardial stromal architecture.
This study investigated the contribution of macrophage migration inhibitory factor (MIF) for the development of ventral prostate in pubertal and adult mice. Mice aged 30 or 60 days from C57BL/6 WT (wild-type) and MIF-/-(knockout) strains were studied. Histological analysis, immunohistochemistry (smooth muscle alpha-actin, vimentin, PCNA, WNT5a), serum testosterone, and western blotting for ERK1/2 were performed. Thirty-day-old MIF-/- mice exhibited higher testosterone serum levels, and the ventral prostate presented enlarged luminal area as well as decreased collagen and smooth muscle cell content. Regarding cell proliferation, there was an important reduction in MIF-/- mice of both ages. In addition, MIF-/- 30 mice presented elevated ERK activation and WNT5a scores in the prostate. This study showed that developmental change expected only at adulthood of prostate is already very evident at 30 days of age in MIF-/- mice, anticipating the pubertal development. Thus, MIF has a stimulative role in proliferation and also modulates androgenic stimuli in the prostate, which can contribute to gland development from puberty to adulthood.
The functions of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor, depend on its subcellular localization. At the plasma membrane, PTEN dephosphorylates phosphatidylinositol-3,4,5-triphosphate to inhibit AKT signaling, whereas nuclear PTEN contributes to the maintenance of genomic stability. Fluorescent proteins (FPs) are widely used to assess PTEN's subcellular localization; however, both the intrinsic properties of FPs (e.g., molecular size) and the choice of FP can influence subcellular localization. This study aimed to determine whether FP fusion affects the subcellular localization of PTEN and its mutant forms under conditions involving DNA damage. mCherry typically promotes cytosolic localization of FP-fused PTEN, indicating that FP selection may affect the interpretation of localization data. Furthermore, FP fusion increases the molecular size of the truncated PTEN fragment, which may impede its nuclear import. In comparison, PTEN mutants such as PTENK13R or PTENA4, which predominantly localize to the cytoplasm or nucleus, respectively, show a minimal dependence on the type of FP. Similarly, DNA damage-induced nuclear accumulation of PTEN appears to be independent of the FP type. These findings underscore the importance of carefully considering the effects of FP fusion when investigating the mechanisms regulating the nuclear translocation of PTEN.
Maintaining the native morphology and characteristic staining patterns of dissected tissues is critical for histological analysis. Reliable preservation enables accurate assessment of structural integrity and cellular components, which is fundamental to identifying viable avenues for tissue regeneration and improving clinical outcomes. Acknowledging the constraints of conventional paraffin embedding, we propose an alternative method employing gelatin as an embedding medium. Gelatin is a natural, water-soluble protein derived from collagen, known for its biocompatibility. As a hydrogel, it provides a supportive matrix that closely mimics the natural extracellular matrix of soft tissues facilitating excellent preservation of structural and molecular features during the histological process. Here we demonstrate that this strategy offers superior support for fragile tissues, including fibrocartilage and hyaline cartilage across various anatomical sites, such as the temporomandibular joint, the knee joint, the long bone growth plate, and the intervertebral disc. To assess the maintenance of tissue morphology and matrix composition, histological sections were subjected to various staining techniques, including hematoxylin and eosin, Masson's trichome, safranin O, immunofluorescence, and von Kossa staining. Gelatin embedding resulted in superior maintenance of fibrocartilage architecture, as evidenced by histological evaluation. In addition, quantification of safranin O staining showed significantly greater glycosaminoglycan content in gelatin-embedded samples.
