Histochemistry and Cell Biology最新文献

Diabetic wounds pose significant clinical challenges owing to delayed healing associated with chronic inflammation, impaired angiogenesis, and poor extracellular matrix (ECM) remodeling. Bioengineered scaffolds incorporating natural bioactives offer promising strategies for enhancing skin regeneration. In this study, the author developed and evaluated a collagen-hyaluronic acid (Col-HA) scaffold loaded with epigallocatechin gallate (EGCG), a polyphenolic compound known for its antioxidant, anti-inflammatory, and proangiogenic properties. EGCG was incorporated into porous Col-HA scaffolds, and their physicochemical properties, degradation rate, and drug release profile were characterized. In vitro cell viability assays were performed using mesenchymal stem cells to assess biocompatibility. A full-thickness excisional wound model was established in streptozotocin-induced diabetic rats, which were treated with control (no scaffold), Col-HA scaffold, EGCG alone, or Col-HA + EGCG scaffolds. Wound healing was evaluated on days 7 and 14 via macroscopic closure, histological stereology (epidermal/dermal volume and fibroblast and vascular density), cytokine profiling (transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α), hydroxyproline quantification, and tensile strength testing. The EGCG-loaded Col-HA scaffold exhibited a porous microstructure (~ 120 µm pore size) and a biphasic release profile, with sustained EGCG release up to 14 days. In vivo, the Col-HA + EGCG group demonstrated significantly accelerated wound closure compared with other groups (p < 0.05). Histological analysis revealed enhanced regeneration of epidermis and dermis, increased fibroblast proliferation and angiogenesis, and reduced inflammatory cell infiltration. ELISA results showed upregulated TGF-β and VEGF levels and downregulated IL-1β and TNF-α in the Col-HA + EGCG group. Moreover, collagen content and tensile strength were highest in this group, indicating superior ECM remodeling and mechanical restoration. The multifunctional Col-HA scaffold incorporated with EGCG effectively promotes diabetic wound healing by modulating inflammation, enhancing angiogenesis, and supporting tissue regeneration. This combinatorial strategy holds significant potential for advanced wound care therapies.

Actin is a pivotal cytoskeletal protein that also regulates chromatin remodeling, transcription, and RNA processing within the nucleus. These nuclear functions are regulated by post-translational modifications (PTMs), but the roles of specific PTMs of nuclear actin remain poorly understood. Of these, the O-GlcNAcylation of Ser199 (gS199) is of particular interest, because this residue can also be phosphorylated (pS199) and is adjacent to the Thr201-203 cluster, a known promoter of filament elongation. In this study, we aimed to elucidate the role of Ser199 O-GlcNAcylation in nuclear actin organization and function. We demonstrate that O-GlcNAcylation at Ser199 actin is associated with actin localization to nuclear speckles and suppresses filament formation. In vivo and in vitro assays revealed that gS199- and pS199-actin have a punctate distribution within the nucleus and colocalize with the speckle marker SRSF2 (SC35). Immunoelectron microscopy showed that this localization was markedly enhanced under diabetic conditions. Furthermore, the introduction of an anti-gS199-actin antibody induced nuclear filament formation, directly linking Ser199 O-GlcNAcylation to the inhibition of actin polymerization. Immunoprecipitation and mass spectrometry identified glyceraldehyde 3-phosphate dehydrogenase and histone H1.4 as nuclear binding partners of modified Ser199-actin. These findings suggest a mechanism by which Ser199 O-GlcNAcylation restricts actin polymerization, anchors actin to nuclear speckles, and thereby influences RNA processing. Dysregulation of this pathway in diabetes may destabilize nuclear speckle organization and contribute to the transcriptional defects that underlie diabetic complications.

Histopathological diagnosis relies on careful and expert assessment of tissue as guided by multiple criteria relevant to specific immunohistochemical (IHC) markers. Computer-aided detection or diagnosis systems have recently been deployed to detect abnormalities in histological samples, transforming many areas of research and medicine such as pathology. These software packages can provide a helpful decision support tool for accelerating analysis, but they would need to capture information from the sample in a manner that facilitates the multicriteria assessment/interpretation demanded by the IHC markers and other histochemical stains. As a result of this potential, and the limited assessment of the performance of software utilized for automated analysis of histological samples, we conducted this study. We aimed to provide a technical assessment of two analysis approaches that are utilized in two commercially available image analysis software platforms, namely positive pixel count analysis approach and cell-by-cell analysis approach. These two approaches are used in many digital histopathological slide analysis software packages including ImageScope (Leica Biosystems) and HALO (Indica Labs), which respectively deploy the aforementioned algorithms and thus were used as proxies for the comparison in this study. Thirty-seven whole slide images of immunohistochemically stained tumor samples from breast, colon, and endometrium were analyzed using three different sampling methods recording percentage of antibody marker positivity. The pixel-based software was better able to identify color intensity, offering the option for grading the IHC marker. However, the object-based software outperformed pixel-based software, having more consistent positivity estimates across the three sampling methods. These results are limited by the small number of clinical samples, IHC marker heterogeneity, and the lack of ground-truth data. Nonetheless, neither of the software packages' metrics performed in a manner required for comprehensive assessment of the IHC markers in this study, yet they can be used to address specific questions related to quantitative expression of tumor diagnostic markers.

Fetal sex influences gene expression in the healthy feto-placental endothelium, potentially contributing to sex-dependent developmental programming and disease risk. Gestational diabetes mellitus (GDM) alters maternal-fetal homeostasis and placental vascular function. Building on previous findings of sex-biased gene expression in healthy feto-placental endothelial cells (fpEC), we investigated whether these biases persist or change following GDM exposure. We first identified sex-biased gene expression in fpEC from GDM pregnancies, then analyzed GDM-induced changes separately in male and female fpEC. Gene ontology enrichment was performed using the PANTHER database. Proliferation and network formation were assessed by BrdU incorporation assay and Matrigel assay, respectively. Female fpEC exhibited a greater transcriptional response to GDM, with more differentially expressed genes than male cells. Functionally, GDM reduced proliferation and increased network formation in female fpEC, while male cells were comparatively unaltered. In healthy conditions, male and female fpEC showed clear transcriptomic and functional dimorphism, which was abolished by GDM. Interestingly, GDM amplified sex-biased gene expression despite convergence in cellular behavior. These findings highlight fetal sex as a key modifier of the placental endothelial response to GDM and support its relevance in sex-specific pregnancy outcomes.

Intervertebral disc degeneration (IDD) is known as a primary contributor to low back pain, a debilitating condition which is the leading cause of disability worldwide. Traditionally, its assessment is based on clinical parameters, including magnetic resonance imaging (MRI). However, patients with similar radiological findings may have significantly different prognoses suggesting the involvement of patient-specific biomarkers and little-investigated molecules supporting the complexity of the pathophysiological microenvironment of the intervertebral disc (IVD). We conducted a study on IVD biopsies from 40 patients with mild IDD (Pfirrmann III), to identify a potential molecular signature that correlates with clinical and behavioral parameters including sex, age, smoking, body mass index (BMI), duration of symptoms prior to surgery, inflammatory cell density, or surgical site. Immunohistological analysis focused on the expression of proteins involved in the defense against oxidative stress, in the maintenance of IVD homeostasis, and energy metabolism: the transcription factors FOXO3a, HIF1α, Bry, the enzyme SOD2, and the glucose transporter GLUT1. Significant differences in protein expression were observed only in relation to Pfirrmann grade. Within the grade III subgroup, expression levels did not vary with patient-specific parameters or clinical outcomes such as complete healing, recurrence, or persistent pain after surgery. This highlights the importance of broadening the scope of assessment in pathological conditions such as IDD. Rather than limiting the evaluation to the expression level of a single protein marker, it is crucial to collect comprehensive data on the various factors that may influence individual patient responses to disc degeneration.

Skin aging is associated with fibroblast senescence, impaired wound healing, and dysregulation of markers such as sirtuin 1 (SIRT1) and fibroblast activation protein-α (FAP-α). It is known that proliferation and migration decrease in aging fibroblasts, which delays the repair process. Metformin, a widely used anti-diabetic drug, can regulate cellular senescence pathways. This study evaluated the effects of metformin on wound healing and SIRT1 and FAP-α expression in senescent fibroblasts. Cellular senescence was induced in primary human dermal fibroblasts using 100 µM hydrogen peroxide (H₂O₂), as validated by a WST-8 assay and SA-β-gal staining. Wound healing assay and immunocytochemistry were performed on control, senescent, and metformin-treated groups (2.5, 5, and 10 mM). Wound closure was significantly impaired in senescent fibroblasts (38% at 72 h versus 89% in the control group). Metformin restored wound healing in a dose-dependent manner; the 10 mM group achieved 94% closure at 72 h, which was comparable to the control group. SIRT1 expression decreased in senescent fibroblasts (90.17 ± 4.67 vs. 124.83 ± 4.31 in controls, p < 0.001) and increased progressively with metformin treatment, reaching control levels at 10 mM. FAP-α expression increased in senescent fibroblasts (91.83 ± 4.36 vs. 78.17 ± 2.56 in controls, p < 0.05) and declined towards baseline with metformin treatment, being significantly reduced at 5 and 10 mM. Metformin improved wound healing capacity and normalized age-related alterations in SIRT1 and FAP-α expression in senescent fibroblasts. These results imply that metformin alleviates senescence-associated dysfunction, suggesting its potential as a therapeutic agent to enhance wound repair in aging skin.

Histological changes in skeletal muscle after heart failure have been widely investigated in the lower limbs, including slow-to-fast fiber type transition, fast fiber-predominant atrophy, reduced capillary number, and increased collagen content. However, histological changes in the upper limb muscles remain largely unexplored. Given the prognostic significance of grip strength and upper limb muscle mass in heart failure, elucidating these changes is essential. We aimed to investigate histological changes in forelimb muscles in a rat model of monocrotaline (MCT)-induced heart failure. Rats were assigned to control (vehicle-injected) or heart failure (MCT-injected) groups. Grip strength was measured on day 27, and histological analyses of the biceps brachii (BiB) and flexor digitorum profundus (FDP) were performed on day 28. Grip strength significantly reduced in the heart failure group. Both BiB and FDP exhibited significant atrophy of fast fibers without changes in slow fiber size. The BiB showed a reduced capillary-to-muscle fiber ratio and no change in fiber type, whereas the FDP showed a shift toward a faster fiber phenotype and no change in capillary number. Collagen content remained unchanged in both muscles. MCT-induced heart failure leads to fast fiber-specific atrophy in forelimb muscles, with muscle-specific differences in capillary and fiber type adaptations. These changes may underlie the impaired upper limb muscle functions in heart failure. While some findings are consistent with those in lower limb muscles, others differ, suggesting region- and muscle-specific responses. Thus, findings from a single muscle cannot necessarily be extrapolated to all skeletal muscles.