Search Results (160)

Background: Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), remain major causes of morbidity and mortality, yet no targeted pharmacological therapy is available. Excessive neutrophil and macrophage infiltration drives reactive oxygen species (ROS) production and cytokine release, leading to alveolar–capillary barrier disruption and fatal respiratory failure. Methods: We applied an integrative multi-omics strategy combining single-cell transcriptomics, peripheral blood proteomics, and lung tissue proteomics in a lipopolysaccharide (LPS, 10 mg/kg)-induced mouse ALI model to identify key signaling pathways. Harpagide, an iridoid glycoside identified from our natural compound screen, was evaluated in vivo (40 and 80 mg/kg) and in vitro (0.1–1 mg/mL). Histopathology, oxidative stress markers (SOD, GSH, and MDA), cytokine levels (IL-6 and IL-1β), and signaling proteins (HIF-1α, p-PI3K, p-AKT, Nrf2, and HO-1) were quantitatively assessed. Direct target engagement was probed using surface plasmon resonance (SPR), the cellular thermal shift assay (CETSA), and 100 ns molecular dynamics (MD) simulations. Results: Multi-omics profiling revealed robust activation of HIF-1, PI3K/AKT, and glutathione-metabolism pathways following the LPS challenge, with HIF-1α, VEGFA, and AKT as core regulators. Harpagide treatment significantly reduced lung injury scores by ~45% (p < 0.01), collagen deposition by ~50%, and ROS accumulation by >60% relative to LPS (n = 6). The pro-inflammatory cytokines IL-6 and IL-1β were reduced by 55–70% at the protein level (p < 0.01). Harpagide dose-dependently suppressed HIF-1α and p-AKT expression while enhancing Nrf2 and HO-1 levels (p < 0.05). SPR confirmed direct binding of Harpagide to HIF-1α (KD = 8.73 µM), and the CETSA demonstrated enhanced thermal stability of HIF-1α. MD simulations revealed a stable binding conformation within the inhibitory/C-TAD region after 50 ns. Conclusions: This study reveals convergent immune–microenvironmental regulatory mechanisms across cellular and tissue levels in ALI and demonstrates the protective effects of Harpagide through multi-pathway modulation. These findings offer new insights into the pathogenesis of ALI and support the development of “one-drug, multilayer co-regulation” strategies for systemic inflammatory diseases. Full article

(1) Background: The extracellular matrix (ECM) is a natural scaffold for cells, creating a three-dimensional architecture composed of fibrous proteins (mainly collagen) and proteoglycans, which are synthesized by resident cells. In this study, a physiological hypoxic environment was utilized to enhance ECM production by human mesenchymal stem cells (hMSCs), a process relevant to tissue engineering and regenerative medicine. (2) Methods: hMSCs were treated with deferoxamine (DFO), a pharmaceutical hypoxia-mimetic agent that induces cellular responses similar to low-oxygen conditions through stabilization of hypoxia inducible factor-1α (HIF-1α). The time points 0 h 24 h, 3 h 24 h, and 24 h 24 h refer to DFO being added immediately after cell seeding (before cells adhesion), 3 h after cell seeding (during initial cells attachment), and 24 h after cell seeding (after focal adhesions formation and actin organization), respectively, to evaluate the influence of cell adhesion on ECM deposition. hMSCs incubated in culture media were subsequently exposed to DFO for 24 h. Samples were then subjected to cell viability tests, scanning electron microscopy (SEM), atomic force microscopy (AFM) and laser scanning confocal microscopy (CLSM) assessments. (3) Results: Viability tests indicated that DFO concentrations in the range of 0–300 µM were non-toxic over 24 h. The presence of collagen fibers in the DFO-derived ECM was confirmed with anti-collagen antibodies under CLSM. Increased ECM secretion was observed under the following conditions: 3 μM DFO (24 h 24 h), 100 μM DFO (0 h 24 h) and 300 μM DFO (3 h 24 h). SEM and AFM images revealed the morphology of various stages of collagen formation with both collagen fibrils and fibers identified. (4) Conclusions: Our preliminary study demonstrated enhanced ECM secretion by hMSC treated with DFO at concentrations of 3, 100, and 300 µM within a short cultivation period of 24–48 h without significant affecting cell viability. By mimicking physiological processes, it may be possible to stimulate endogenous tissue regeneration, for example, at an injury site. Full article

Background: Hypoxia is a hallmark of solid tumors, including hepatocellular carcinoma (HCC), where it drives oxidative stress and extracellular matrix (ECM) remodeling, promoting tumor invasion and metastasis. Investigating these mechanisms in patients remains challenging due to the complexity of the tumor microenvironment. Methods: We developed a scaffold-free three-dimensional (3D) spheroid model of HCC using human hepatocellular carcinoma HepG2 cells (ATCC HB-8065). To characterize hypoxia-driven processes, a multiparametric approach combining MTT assays for metabolic activity, confocal microscopy for viability and ECM organization, flow cytometry for apoptosis and ROS detection, qRT-PCR for gene expression, and FTIR spectroscopy for biochemical profiling were performed. Results: The 3D model exhibited progressive ROS accumulation, stabilization of HIF-1α, and metabolic reprogramming toward aerobic glycolysis. In parallel, ECM remodeling was evident, with increased expression of SPARC and FN1 and collagen fiber alignment, reflecting an invasive tumor phenotype. Conclusions: This scaffold-free 3D HCC model recapitulates key physiopathological features of tumor progression, providing a robust and physiologically relevant platform to investigate the hypoxia–ROS–ECM relationship and to support preclinical evaluation of targeted therapeutic strategies. Full article

Hypoxia-induced oxidative stress and inflammation in the brain are critical contributors to neurological disorders. Hydrogen gas has emerged as a therapeutic agent with potent antioxidant and anti-inflammatory properties. In this study, we evaluated the protective effects of hydrogen against acute hypoxia-induced brain injuries in medaka. Fish were exposed to hypoxia and then recovered in water bubbled with air, hydrogen, or ozone. LOX-1 hypoxia probe imaging and HIF-1α immunostaining showed persistent tissue hypoxia in the air and ozone groups, which was significantly reduced by hydrogen treatment. Histological analysis revealed extensive vascular congestion in the midbrain after hypoxia, which was markedly alleviated by hydrogen. TUNEL assay demonstrated that hydrogen suppressed hypoxia-induced neuronal apoptosis. Immunohistochemistry and ELISA showed elevated levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) and proinflammatory markers (COX-2, IL-6, TNF-α) in the brains of air- and ozone-treated fish; these increases were significantly attenuated by hydrogen. ORAC assay confirmed that hydrogen restored brain antioxidant capacity. Behavioral analysis further demonstrated that hydrogen treatment improved locomotor activity and stabilized respiratory function. These results indicate that hydrogen protects medaka against hypoxia-induced oxidative and inflammatory injuries and may represent a promising therapeutic strategy for hypoxia-related neurological disorders. Full article

Background/Objectives: Propolis is a complex natural product with long-standing traditional use as an antimicrobial remedy. Several studies suggest that Brazilian varieties of propolis may promote wound healing and protect the skin from UV damage, most likely due to antioxidant and anti-inflammatory mechanisms. However, the literature provides limited support for this topic. The present work aimed at characterizing the polyphenolic profile of two Brazilian propolis samples, investigating their biological activity. Methods: Biological experiments were conducted in human keratinocytes (HaCaT) and fibroblasts (HDF) stimulated by cytokines involved in skin inflammation and remodeling (TNF-α and IL-1β), while phytochemical analyses were conducted by LC-MS techniques. Results: Our findings indicate that artepillin C and drupanin were the principal phytochemicals of green propolis, while vestitol, medicarpin, and neovestitol were the most abundant in red propolis. The presence of phenolic compounds was correlated with the antioxidant activity demonstrated by ORAC and intracellular ROS assays. Accordingly, both Brazilian propolis samples impaired NF-κB activity, while only red propolis hindered IL-8 release in both cell lines with an IC₅₀ lower than 25 μg/mL. Surprisingly, both propolis samples at the same concentrations enhanced the production of IL-6 and VEGF, thus suggesting the coexistence of anti-inflammatory, antioxidant, and trophic mechanisms contributing to skin repair. In line with this hypothesis, propolis also induced the stabilization of HIF-1α, paralleling the biological effect of a well-known synthetic HIF stabilizer (DMOG). Conclusions: This work supports the investigation of Brazilian red and green propolis as potential modulators of the inflammatory phase in wound healing. Full article

Diabetes mellitus, as a metabolic disorder, has received growing attention for its detrimental effects on the male reproductive system (particularly the testes) manifesting as increased oxidative stress, reduced blood perfusion, heightened inflammation, and germ cell apoptosis under hyperglycemic conditions. Hypoxia-inducible factor (HIF)-1α, a pivotal transcription factor in cellular hypoxia responses, plays a crucial role in regulating metabolism, angiogenesis, and apoptosis. Emerging evidence underscores its significant physiological and pathological roles in diabetic testicular injury. This review outlines the structural domains, activation mechanisms, and key target genes of HIF-1α, and further examines its involvement in diabetes-induced oxidative stress, impaired perfusion, endocrine dysregulation, and the imbalance of apoptosis and autophagy in testicular tissue. Notably, HIF-1α exerts protective effects by activating canonical signaling pathways such as phosphoinositide-3 kinase (PI-3K)/protein kinase B (Akt), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and nuclear factor (NF)-κB, thereby enhancing antioxidant gene expression, promoting angiogenesis, and upregulating anti-apoptotic proteins. Furthermore, HIF-1α may help stabilize androgen levels by preserving Leydig cell function, potentially alleviating diabetes-associated gonadal dysfunction. This review also discusses the feasibility of targeting HIF-1α as a novel therapeutic strategy. In conclusion, a comprehensive understanding of HIF-1α’s mechanistic role in diabetic testicular damage provides valuable insights into the pathogenesis of diabetes-related reproductive disorders and offers new avenues for therapeutic intervention. Full article

Angiogenesis, the formation of new blood vessels from pre-existing ones, is crucial for various physiological and pathological conditions, including embryonic development, wound healing, tissue regeneration and tumor progression. While traditionally attributed to the actions of growth factors and their receptors, emerging evidence highlights the crucial regulatory roles of mitochondria in angiogenesis. This narrative review explores the multifaceted functions of mitochondria in endothelial cells, which are central to blood vessel formation. Beyond their classical role in ATP production, mitochondria contribute to angiogenesis through redox signaling, calcium homeostasis, biosynthetic activity, and reactive oxygen species (ROS) generation. These organelles help regulate key endothelial behaviors such as proliferation, migration, and tube formation through mechanisms that include mitochondrial calcium signaling and ROS-mediated stabilization of hypoxia-inducible factor-1α (HIF-1α), leading to increased vascular endothelial growth factor (VEGF) expression. Additionally, mitochondrial dynamics, dysfunction, and genetic factors are discussed for their influence on angiogenic outcomes. Understanding these complex mitochondrial functions opens new therapeutic avenues for modulating angiogenesis in diseases such as cancer and cardiovascular disorders. Full article

Cobalt is an essential trace element involved in key biological processes. It serves most notably as a component of vitamin B₁₂ (cobalamin) and a regulator of erythropoiesis. While cobalt deficiency can lead to disorders such as megaloblastic anemia, excess cobalt poses toxicological risks to the thyroid, cardiovascular, and hematopoietic systems. In recent years, cobalt ions (Co²⁺) have gained attention for their ability to mimic hypoxia and promote angiogenesis. This represents a crucial mechanism for tissue regeneration. Cobalt mediates this effect mainly by stabilizing hypoxia-inducible factor 1α (HIF-1α) under normoxic conditions, thereby upregulating angiogenic genes, including VEGF, FGF, and EPO. Experimental studies—from cell culture to animal models—have demonstrated cobalt-induced enhancement of endothelial proliferation, migration, and microvascular formation. Emerging evidence also indicates that Co²⁺-stimulated macrophages secrete integrin-β1-rich exosomes. These exosomes enhance endothelial motility and tubulogenesis independently of VEGF. Furthermore, cobalt-modified biomaterials have been developed to deliver cobalt ions in a controlled manner. Examples include cobalt-doped β-tricalcium phosphate or bioactive glasses. These materials support both angiogenesis and osteogenesis.This review summarizes current findings on cobalt’s role in angiogenesis. The emphasis is on its potential in cobalt-based biomaterials for tissue engineering and regenerative medicine. Full article

Chronic kidney disease (CKD)-associated anemia is a global health concern and is linked to vascular and ocular complications. Hypoxia-inducible factor (HIF) stabilizers, or HIF prolyl hydroxylase inhibitors (PHIs), are promising candidates for the treatment of CKD-associated anemia. Since hypoxia and angiogenesis are involved in eye diseases, this study examined the effects of HIF-PHIs on metabolism and gene expression in retinal pigment epithelium (RPE) cells. Results revealed that PHIs differentially induced angiogenic (VEGFA, ANG) and glycolytic (PDK1, GLUT1) gene expression, with Roxadustat causing the strongest transcriptional changes. However, Roxadustat-induced angiogenic signals did not promote endothelial tube formation. Moreover, it did not induce oxidative stress, inflammation, or significant antioxidant gene responses in ARPE-19 cells. Roxadustat also reduced the inflammatory cytokine response to tumor necrosis factor-α, including IL-6, IL-8, and MCP-1, and did not exacerbate VEGF expression under high-glucose conditions. Overall, Roxadustat triggered complex gene expression changes without promoting inflammation or oxidative stress in RPE cells. Despite these findings, ophthalmologic monitoring is advised during PHI treatment in CKD patients receiving HIF-PHIs. Full article

The mitochondrial regulator MNRR1 is reduced in several pathologies, including the mitochondrial heteroplasmic disease MELAS, and genetic restoration of its level normalizes the pathological phenotype. Here, we investigate the upstream mechanism that reduces MNRR1 levels. We have identified the hypoxic regulator HIF2α to bind the MNRR1 promoter and inhibit transcription by competing with RBPJκ. In MELAS cells, there is a pseudohypoxic state that transcriptionally induces HIF2α and stabilizes HIF2α protein. MELAS cybrids harboring the m.3243A > G mutation display reduced levels of prolyl hydroxylase 3 (PHD3), which contributes to the HIF2α stabilization. These results prompted a search for compounds that could increase MNRR1 levels pharmacologically. The screening of a 2400-compound library uncovered the antifungal drug nitazoxanide and its metabolite tizoxanide as enhancers of MNRR1 transcription. We show that treating MELAS cybrids with tizoxanide restores cellular respiration, enhances mitophagy, and, importantly, shifts heteroplasmy toward wild-type mtDNA. Furthermore, in fibroblasts from MELAS patients, the compound improves mitochondrial biogenesis, enhances autophagy, and protects from LPS-induced inflammation. Mechanistically, nitazoxanide reduces HIF2α levels by increasing PHD3. Chemical activation of MNRR1 is thus a potential strategy to improve mitochondrial deficits seen in MELAS. Finally, our data suggests a broader physiological pathway wherein two proteins, induced under severe (1% O2; HIF2α) and moderate (4% O2; MNRR1) hypoxic conditions, regulate each other inversely. Full article

Transcription factors (TFs) are proteins that control gene expression by binding to specific DNA sequences and are essential for cell development, differentiation, and homeostasis. Dysregulation of TFs is implicated in numerous diseases, including cancer, autoimmune disorders, and neurodegeneration. While TFs were traditionally considered “undruggable” due to their lack of well-defined binding pockets, recent advances have made it possible to modulate their activity using diverse pharmacological strategies. Major TF families include NF-κB, p53, STATs, HIF-1α, AP-1, Nrf2, and nuclear hormone receptors, which take part in the regulation of inflammation, tumor suppression, cytokine signaling, hypoxia and stress response, oxidative stress, and hormonal response, respectively. TFs can perform multiple functions, participating in the regulation of opposing processes depending on the context. NF-κB, for instance, plays dual roles in immunity and cancer, and is targeted by proteasome and IKKβ inhibitors. p53, often mutated in cancer, is reactivated using MDM2 antagonist Nutlin-3, refunctionalizing compound APR-246, or stapled peptides. HIF-1α, which regulates hypoxic responses and angiogenesis, is inhibited by agents like acriflavine or stabilized in anemia therapies by HIF-PHD inhibitor roxadustat. STATs, especially STAT3 and STAT5, are oncogenic and targeted via JAK inhibitors or novel PROTAC degraders, for instance SD-36. AP-1, implicated in cancer and arthritis, can be inhibited by T-5224 or kinase inhibitors JNK and p38 MAPK. Nrf2, a key antioxidant regulator, can be activated by agents like DMF or inhibited in chemoresistant tumors. Pharmacological strategies include direct inhibitors, activators, PROTACs, molecular glues, and epigenetic modulators. Challenges remain, including the structural inaccessibility of TFs, functional redundancy, off-target effects, and delivery barriers. Despite these challenges, transcription factor modulation is emerging as a viable and promising therapeutic approach, with ongoing research focusing on specificity, safety, and efficient delivery methods to realize its full clinical potential. Full article

Hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitors continually stabilize hypoxia-inducible factor-1α (HIF-1α). These inhibitors are effective in the clinical treatment of renal anemia. However, the effects of continued HIF-1α stabilization on skeletal muscle differentiation remain unclear. This study aimed to investigate the effects of continued HIF-1α stabilization on skeletal muscle differentiation using a HIF-PH inhibitor in both in vitro and in vivo models. We cultured mouse C2C12 myoblasts to differentiate into myotubes with or without FG-4592, a HIF-PH inhibitor. Additionally, we treated nine-week-old male C57BL/6 mice with either FG-4592 or vehicle via intraperitoneal injections three times a week for four weeks. In vitro, FG-4592 treatment stabilized HIF-1α continually. Morphological analysis revealed that 72 h FG-4592 treatment suppressed differentiation of C2C12 myoblasts into myotubes. This treatment decreased the gene and protein expression of MyoD and myogenin, reduced the protein expression of myosin heavy chain (MHC), and increased the gene and protein expression of myostatin. HIF-1α knockdown mitigated the decrease in MHC protein expression induced by FG-4592. In vivo, FG-4592 treatment increased HIF-1α protein expression and decreased MyoD, myogenin, and MHC protein expression in gastrocnemius muscle. These findings suggest that pharmacological HIF-PH inhibition suppresses myoblast differentiation through continued HIF-1α stabilization. Full article

This study developed electrosprayed deferoxamine (DFO)-loaded poly(lactic-co-glycolic acid) microspheres (DFO-MS) combined with a sucrose acetate isobutyrate (SAIB) depot (DFO-MS@SAIB) for bone-defect repair, targeting the coordinated regulation of angiogenesis and osteogenesis in vascularized bone regeneration—where new blood vessels support functional bone integration. In vitro/in vivo evaluations confirmed its dual pro-angiogenic and pro-osteogenic effects via HIF-1α pathway activation. Background/Objectives: Emerging evidence underscores the indispensability of vascularization in bone-defect repair, a clinical challenge exacerbated by limited intrinsic healing capacity. While autologous grafts and growth-factor-based strategies remain mainstream, their utility is constrained by donor-site morbidity, transient bioactivity, and poor spatiotemporal control over angiogenic–osteogenic coupling. Here, we leveraged DFO, a hypoxia-mimetic HIF-1α stabilizer with angiogenic potential, to engineer an injectable DFO-MS@SAIB depot. This system was designed to achieve sustained DFO release, thereby synchronizing vascular network formation with mineralized tissue regeneration in critical-sized defects. Methods: DFO-MS were fabricated via electrospraying and combined with SAIB (DFO-MS@S) to form an injectable sustained-release depot. Their physicochemical properties, including morphology, encapsulation efficiency, degradation, release kinetics, and rheology, were systematically characterized. In vitro, the angiogenic capacity of HUVECs co-cultured with DFO-MS was evaluated; conditioned HUVECs were then co-cultured with BMSCs to assess the BMSCs’ cytocompatibility and osteogenic differentiation. In vivo bone regeneration in a rat calvarial defect model was evaluated using micro-CT, histology, and immunohistochemistry. Results: The DFO-MS@SAIB system achieved sustained DFO release, stimulating HUVEC proliferation, migration, and tubulogenesis. In a Transwell co-culture model, pretreated HUVECs promoted BMSC migration and osteogenic differentiation via paracrine signaling involving endothelial-secreted factors (e.g., VEGF). HIF-1α pathway activation upregulated osteogenic markers (ALP, Col1a1, OCN), while in vivo experiments demonstrated enhanced vascularized bone regeneration, with significantly increased bone volume/total volume (BV/TV) and new bone area compared with controls. Conclusion: The DFO-MS@SAIB system promotes bone regeneration via sustained deferoxamine release and HIF-1α-mediated signaling. Its angiogenesis–osteogenesis coupling effect facilitates vascularized bone regeneration, thereby offering a translatable strategy for critical-sized bone-defect repair. Full article

Background: Von Hippel-Lindau (VHL) disease, a hereditary cancer syndrome, is characterized by mutations in the VHL gene, which result in the stabilization of hypoxia-inducible factors (HIF)-1α and -2α, ultimately leading to the development of highly vascularized tumors, such as hemangioblastomas of the central nervous system (CNS-HBs). The standard treatment for these brain tumors is neurosurgical resection. However, multiple surgeries are often necessary due to tumor recurrence, which increases the risk of neurological sequelae. Thus, elucidation of the proliferative behavior of hemangioblastomas (with the aim of identifying biomarkers associated with tumor progression) and the development of pharmacological therapies could reduce the need for repeated surgical interventions and provide alternative treatment options for unresectable CNS-HBs. Belzutifan (Welireg™), a selective HIF-2α inhibitor and the only FDA-approved non-surgical option, has shown limited efficacy in CNS-HBs, highlighting the need for alternative therapeutic strategies. Results: In this study, primary cell cultures were successfully established from CNS-HB tissue samples of VHL patients, achieving a 75% success rate. These cultures were predominantly composed of stromal cells and pericytes. The proliferative patterns of patient-derived HB cell cultures significantly correlated with tumor burden and recurrence in VHL patients. Furthermore, flow cytometry, reverse transcription-PCR, and Western blot analyses revealed marked overexpression of both HIF-1α and HIF-2α isoforms in primary HB cells. In addition, evaluation of the therapeutic potential of acriflavine, a dual HIF-1α/HIF-2α inhibitor, demonstrated reduced HB cells viability, induced G2/M cell cycle arrest, and predominantly triggered necrotic cell death in patient-derived HB cultures. Conclusions: These results suggest that the in vitro proliferative dynamics of HB cell cultures may reflect clinical characteristics associated with CNS-HB progression, potentially serving as indicators to predict tumor development in patients with VHL. Furthermore, our findings support the simultaneous targeting of both HIF-1α and HIF-2α isoforms as a promising non-invasive therapeutic strategy. Full article

The Tibetan goat (Capra hircus) exhibits remarkable adaptations to high-altitude hypoxia, yet the molecular mechanisms remain unclear. This study integrates RNA-seq, WGCNA, and machine learning to explore gene-environment interactions (G × E) in hypoxia adaptation. Fibroblasts from the Tibetan goat and Yunling goat were cultured under hypoxic (1% O₂) and normoxic (21% O₂) conditions, respectively. This identified 68 breed-specific (G), 100 oxygen-responsive (E), and 620 interaction-driven (I) Differentially Expressed Genes (DEGs). The notably higher number of interaction-driven DEGs compared to other effects highlights transcriptional plasticity. We defined two gene sets: Environmental Stress Genes (n = 632, E ∪ I) and Genetic Adaptation Genes (n = 659, G ∪ I). The former were significantly enriched in pathways related to oxidative stress defense and metabolic adaptation, while the latter showed prominent enrichment in pathways associated with vascular remodeling and transcriptional regulation. CTNNB1 emerged as a key regulatory factor in both gene sets, interacting with CASP3 and MMP2 to form the core of the protein–protein interaction (PPI) network. Machine learning identified MAP3K5, TGFBR2, RSPO1 and ITGB5 as critical genes. WGCNA identified key modules in hypoxia adaptation, where FOXO3, HEXIM1, and PPARD promote the stabilization of HIF-1α and metabolic adaptation through the HIF-1 signaling pathway and glycolysis. These findings underscore the pivotal role of gene–environment interactions in hypoxic adaptation, offering novel perspectives for both livestock breeding programs and biomedical research initiatives. Full article