Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Immunohistochemical analysis of epiretinal fibrovascular membranes that were obtained from 14 patients with proliferative diabetic retinopathy during pars plana vitrectomy. Significant positive correlations between the numbers of blood vessels expressing CD31, reflecting angiogenic activity, and the numbers of blood vessels expressing hyaluronan synthase (HAS)2 (A) , the numbers of stromal cells expressing HAS2 (B) , the numbers of blood vessels expressing hyaluronidase (Hyal)-2 (C) and the numbers of blood vessels expressing CD44 (D) (Pearson’s correlation coefficient). Significant positive correlations between vitreous fluid levels of hyaluronan and the levels of hyaluronidase (Hyal)-1 (E) , heparan sulphate (F) and soluble syndecan-1 (G) (Pearson’s correlation coefficient). Samples from patients with proliferative diabetic retinopathy (PDR) are indicated by open circles, whereas samples from non-diabetic patients with rhegmatogenous retinal detachment (RD) are indicated by closed circles, illustrating sample clustering according to disease entity.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for human Hyal-1 (Cat No DY7358), human Hyaluronan (Cat No DY3614-05), and human syndecan-1 (Cat No DY2780), human vascular endothelial growth factor (VEGF) (Cat No DY293B), human monocyte chemoattractant protein-1 (MCP-1/CCL2) (Cat No DY279), human matrix metalloproteinase-9 (MMP-9) (Cat No DY911) and human angiopoietin-2 (Cat No DY623) were purchased from R&D Systems.

Techniques: Immunohistochemical staining, Expressing, Activity Assay

Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Determination of hyaluronan synthase (HAS)2 (A) , hyaluronidase (Hyal)-1 (B) , Hyal-2 (C) , soluble CD44 (D) , syndecan-1 (E) and heparan sulphate (F) levels in vitreous fluid samples. Equal volumes (15 µL) of vitreous fluid from patients with proliferative diabetic retinopathy (PDR; n=10) and from non-diabetic patients with rhegmatogenous retinal detachment (RD; n=10) were subjected to gel electrophoresis and the presence of HAS2, Hyal-1, Hyal-2, soluble CD44, syndecan-1, and heparan sulphate were detected by Western blot analysis. Representative sets of samples are shown. The intensity of the protein bands was determined in all samples and band intensities were compared between RD and PDR patients. Results are expressed as mean ± standard deviation or standard error of mean (*p < 0.05, independent t-test).

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for human Hyal-1 (Cat No DY7358), human Hyaluronan (Cat No DY3614-05), and human syndecan-1 (Cat No DY2780), human vascular endothelial growth factor (VEGF) (Cat No DY293B), human monocyte chemoattractant protein-1 (MCP-1/CCL2) (Cat No DY279), human matrix metalloproteinase-9 (MMP-9) (Cat No DY911) and human angiopoietin-2 (Cat No DY623) were purchased from R&D Systems.

Techniques: Nucleic Acid Electrophoresis, Western Blot, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Expression levels of hyaluronidase (Hyal)-1 (A) , CD44 (B) and receptor for hyaluronan-mediated motility (RHAMM) (C) in the retinal lysates of non-diabetic control rats (C) (n=12) and diabetic rats (D) (n=12) were determined by Western blot analysis. After determination of the intensity of the protein bands, intensities were adjusted to those of β-actin in the samples. Oxidative stress was monitored with the use of 2’,7’-Dichlorofluorescein (DCF) fluorescence intensity analysis (D) . Results are expressed as mean ± standard deviation. Ultra-Low molecular weight hyaluronan (ULMW-HA) induces breakdown of blood-retinal barrier (E) . ULMW-HA was injected intravitreally at the dose of 50 ng in 5 µL in one eye and the same volume of phosphate-buffered saline (PBS) was injected in the contralateral eye of normal rats. The BRB was quantified with the fluorescein isothiocyanate-conjugated dextran technique. Results are expressed as mean ± standard deviation of 12 rats. *p < 0.05 compared to the values obtained from PBS-injected eyes. (independent t-test). Western blot analysis of retinas demonstrated that intravitreal injection of ULMW-HA induced significant upregulation of the expression of phospho-NF-κB (F) , phospho-ERK1/2 (G) , vascular endothelial growth factor (VEGF) (H) , intercellular adhesion molecule-1 (ICAM-1) (I) , vascular cell adhesion molecule-1 (VCAM-1) (J) and high-mobility group box-1 (HMGB1) (K) . Results are expressed as mean ± standard deviation or standard error of mean of 8–10 rats in each group (*p < 0.05; independent t-test).

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for human Hyal-1 (Cat No DY7358), human Hyaluronan (Cat No DY3614-05), and human syndecan-1 (Cat No DY2780), human vascular endothelial growth factor (VEGF) (Cat No DY293B), human monocyte chemoattractant protein-1 (MCP-1/CCL2) (Cat No DY279), human matrix metalloproteinase-9 (MMP-9) (Cat No DY911) and human angiopoietin-2 (Cat No DY623) were purchased from R&D Systems.

Techniques: Expressing, Control, Western Blot, Fluorescence, Standard Deviation, Molecular Weight, Injection, Saline

Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Human retinal Müller glial cells were left untreated or treated with high-glucose (HG) (25 mM) for 24 (h). For HG treatment, cultures treated with mannitol (25 mM) were used as a control. Protein expression of hyaluronan synthase (HAS)2 (A) , hyaluronidase (Hyal)-1 (B) , Hyal-2 (C) , CD44 (D) and receptor for hyaluronan-mediated motility (RHAMM) (E) in cell lysate was determined by Western blot analysis. Human retinal Müller glial cells were left untreated or treated with HG (25 mM), cobalt chloride (CoCl 2 ) (300 µM), tumor necrosis factor-α (TNFα) (5 ng/mL) or hydrogen peroxide (H 2 O 2 ) (10 mM) for 24 (h) Levels of hyaluronidase (Hyal)-1 were quantified in the culture media by ELISA. (F) Results are expressed as mean ± standard deviation or standard error of mean from three different experiments each performed in triplicate (*p < 0.05; independent t-test).

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for human Hyal-1 (Cat No DY7358), human Hyaluronan (Cat No DY3614-05), and human syndecan-1 (Cat No DY2780), human vascular endothelial growth factor (VEGF) (Cat No DY293B), human monocyte chemoattractant protein-1 (MCP-1/CCL2) (Cat No DY279), human matrix metalloproteinase-9 (MMP-9) (Cat No DY911) and human angiopoietin-2 (Cat No DY623) were purchased from R&D Systems.

Techniques: Control, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Human retinal microvascular endothelial cells (HRMECs) were left untreated or treated with high-glucose (HG) (25 mM) for 24h. For HG treatment, cultures treated with mannitol (25 mM) were used as a control. Protein expression of hyaluronan synthase (HAS)2 (A) , hyaluronidase (Hyal)-1 (B) , Hyal-2 (C) and CD44 (D) in cell lysate was determined by Western blot analysis. (E) HRMECs were left untreated or treated with tumor necrosis factor–α (TNF-α) (5ng/ml) or hydrogen peroxide (H 2 O 2 ) (10mM) for 24h. Levels of Hyal-1 were quantified in the culture media by ELISA. (F) HRMECs and human retinal Müller glial cells were compared after treatment with mannitol (25 mM) or HG (25 mM) for 24h. Levels of hyaluronan (HA) were quantified in the culture media by ELISA. Results are expressed as mean ± standard deviation from three different experiments each performed in triplicate (*p < 0.05; independent t-test).

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for human Hyal-1 (Cat No DY7358), human Hyaluronan (Cat No DY3614-05), and human syndecan-1 (Cat No DY2780), human vascular endothelial growth factor (VEGF) (Cat No DY293B), human monocyte chemoattractant protein-1 (MCP-1/CCL2) (Cat No DY279), human matrix metalloproteinase-9 (MMP-9) (Cat No DY911) and human angiopoietin-2 (Cat No DY623) were purchased from R&D Systems.

Techniques: Control, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Immunohistochemical analysis of epiretinal fibrovascular membranes that were obtained from 14 patients with proliferative diabetic retinopathy during pars plana vitrectomy. Significant positive correlations between the numbers of blood vessels expressing CD31, reflecting angiogenic activity, and the numbers of blood vessels expressing hyaluronan synthase (HAS)2 (A) , the numbers of stromal cells expressing HAS2 (B) , the numbers of blood vessels expressing hyaluronidase (Hyal)-2 (C) and the numbers of blood vessels expressing CD44 (D) (Pearson’s correlation coefficient). Significant positive correlations between vitreous fluid levels of hyaluronan and the levels of hyaluronidase (Hyal)-1 (E) , heparan sulphate (F) and soluble syndecan-1 (G) (Pearson’s correlation coefficient). Samples from patients with proliferative diabetic retinopathy (PDR) are indicated by open circles, whereas samples from non-diabetic patients with rhegmatogenous retinal detachment (RD) are indicated by closed circles, illustrating sample clustering according to disease entity.

Article Snippet: To determine the presence of Hyal-1, Hyal-2, HAS2, CD44, syndecan-1, heparan sulphate and RHAMM in the vitreous samples, equal volumes (10 μL) of vitreous samples were boiled in Laemmli’s sample buffer (1:1, v/v) under reducing condition for 10 min. Immunodetection was performed with the use of rabbit polyclonal anti-Hyal-1 antibody (1:1000, NBP2-16906, Novus Biologicals), mouse polyclonal anti-Hyal-2 antibody (1:1000, H00008692-B02P, Novus Biologicals), mouse monoclonal anti-HAS2 antibody (1:1000, ab140671, Abcam), rabbit monoclonal anti-CD44 antibody (1:1000, ab189524, Abcam), rabbit monoclonal anti-RHAMM antibody (1:1000, ab124729, Abcam), rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1000, MAB1018, R&D Systems), rabbit polyclonal anti-p65 subunit of nuclear factor-kappa B (phospho-NF-κB) (1:1000, NB100-82086, Novus Biologicals), rabbit polyclonal anti-high-mobility group box1 (HMGB1) (1:1000, Cat. no. ab18256, Abcam), mouse monoclonal anti-VEGF antibody (1:750, MAB293, R&D Systems), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:100, sc-8439, Santa Cruz Biotechnology Inc.), and mouse monoclonal anti-vascular cell adhesion molecule-1 (VCAM-1) antibody (1:100, sc-13160, Santa Cruz Biotechnology Inc.).

Techniques: Immunohistochemical staining, Expressing, Activity Assay

Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Determination of hyaluronan synthase (HAS)2 (A) , hyaluronidase (Hyal)-1 (B) , Hyal-2 (C) , soluble CD44 (D) , syndecan-1 (E) and heparan sulphate (F) levels in vitreous fluid samples. Equal volumes (15 µL) of vitreous fluid from patients with proliferative diabetic retinopathy (PDR; n=10) and from non-diabetic patients with rhegmatogenous retinal detachment (RD; n=10) were subjected to gel electrophoresis and the presence of HAS2, Hyal-1, Hyal-2, soluble CD44, syndecan-1, and heparan sulphate were detected by Western blot analysis. Representative sets of samples are shown. The intensity of the protein bands was determined in all samples and band intensities were compared between RD and PDR patients. Results are expressed as mean ± standard deviation or standard error of mean (*p < 0.05, independent t-test).

Article Snippet: To determine the presence of Hyal-1, Hyal-2, HAS2, CD44, syndecan-1, heparan sulphate and RHAMM in the vitreous samples, equal volumes (10 μL) of vitreous samples were boiled in Laemmli’s sample buffer (1:1, v/v) under reducing condition for 10 min. Immunodetection was performed with the use of rabbit polyclonal anti-Hyal-1 antibody (1:1000, NBP2-16906, Novus Biologicals), mouse polyclonal anti-Hyal-2 antibody (1:1000, H00008692-B02P, Novus Biologicals), mouse monoclonal anti-HAS2 antibody (1:1000, ab140671, Abcam), rabbit monoclonal anti-CD44 antibody (1:1000, ab189524, Abcam), rabbit monoclonal anti-RHAMM antibody (1:1000, ab124729, Abcam), rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1000, MAB1018, R&D Systems), rabbit polyclonal anti-p65 subunit of nuclear factor-kappa B (phospho-NF-κB) (1:1000, NB100-82086, Novus Biologicals), rabbit polyclonal anti-high-mobility group box1 (HMGB1) (1:1000, Cat. no. ab18256, Abcam), mouse monoclonal anti-VEGF antibody (1:750, MAB293, R&D Systems), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:100, sc-8439, Santa Cruz Biotechnology Inc.), and mouse monoclonal anti-vascular cell adhesion molecule-1 (VCAM-1) antibody (1:100, sc-13160, Santa Cruz Biotechnology Inc.).

Techniques: Nucleic Acid Electrophoresis, Western Blot, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Expression levels of hyaluronidase (Hyal)-1 (A) , CD44 (B) and receptor for hyaluronan-mediated motility (RHAMM) (C) in the retinal lysates of non-diabetic control rats (C) (n=12) and diabetic rats (D) (n=12) were determined by Western blot analysis. After determination of the intensity of the protein bands, intensities were adjusted to those of β-actin in the samples. Oxidative stress was monitored with the use of 2’,7’-Dichlorofluorescein (DCF) fluorescence intensity analysis (D) . Results are expressed as mean ± standard deviation. Ultra-Low molecular weight hyaluronan (ULMW-HA) induces breakdown of blood-retinal barrier (E) . ULMW-HA was injected intravitreally at the dose of 50 ng in 5 µL in one eye and the same volume of phosphate-buffered saline (PBS) was injected in the contralateral eye of normal rats. The BRB was quantified with the fluorescein isothiocyanate-conjugated dextran technique. Results are expressed as mean ± standard deviation of 12 rats. *p < 0.05 compared to the values obtained from PBS-injected eyes. (independent t-test). Western blot analysis of retinas demonstrated that intravitreal injection of ULMW-HA induced significant upregulation of the expression of phospho-NF-κB (F) , phospho-ERK1/2 (G) , vascular endothelial growth factor (VEGF) (H) , intercellular adhesion molecule-1 (ICAM-1) (I) , vascular cell adhesion molecule-1 (VCAM-1) (J) and high-mobility group box-1 (HMGB1) (K) . Results are expressed as mean ± standard deviation or standard error of mean of 8–10 rats in each group (*p < 0.05; independent t-test).

Article Snippet: To determine the presence of Hyal-1, Hyal-2, HAS2, CD44, syndecan-1, heparan sulphate and RHAMM in the vitreous samples, equal volumes (10 μL) of vitreous samples were boiled in Laemmli’s sample buffer (1:1, v/v) under reducing condition for 10 min. Immunodetection was performed with the use of rabbit polyclonal anti-Hyal-1 antibody (1:1000, NBP2-16906, Novus Biologicals), mouse polyclonal anti-Hyal-2 antibody (1:1000, H00008692-B02P, Novus Biologicals), mouse monoclonal anti-HAS2 antibody (1:1000, ab140671, Abcam), rabbit monoclonal anti-CD44 antibody (1:1000, ab189524, Abcam), rabbit monoclonal anti-RHAMM antibody (1:1000, ab124729, Abcam), rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1000, MAB1018, R&D Systems), rabbit polyclonal anti-p65 subunit of nuclear factor-kappa B (phospho-NF-κB) (1:1000, NB100-82086, Novus Biologicals), rabbit polyclonal anti-high-mobility group box1 (HMGB1) (1:1000, Cat. no. ab18256, Abcam), mouse monoclonal anti-VEGF antibody (1:750, MAB293, R&D Systems), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:100, sc-8439, Santa Cruz Biotechnology Inc.), and mouse monoclonal anti-vascular cell adhesion molecule-1 (VCAM-1) antibody (1:100, sc-13160, Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Control, Western Blot, Fluorescence, Standard Deviation, Molecular Weight, Injection, Saline

Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Human retinal Müller glial cells were left untreated or treated with high-glucose (HG) (25 mM) for 24 (h). For HG treatment, cultures treated with mannitol (25 mM) were used as a control. Protein expression of hyaluronan synthase (HAS)2 (A) , hyaluronidase (Hyal)-1 (B) , Hyal-2 (C) , CD44 (D) and receptor for hyaluronan-mediated motility (RHAMM) (E) in cell lysate was determined by Western blot analysis. Human retinal Müller glial cells were left untreated or treated with HG (25 mM), cobalt chloride (CoCl 2 ) (300 µM), tumor necrosis factor-α (TNFα) (5 ng/mL) or hydrogen peroxide (H 2 O 2 ) (10 mM) for 24 (h) Levels of hyaluronidase (Hyal)-1 were quantified in the culture media by ELISA. (F) Results are expressed as mean ± standard deviation or standard error of mean from three different experiments each performed in triplicate (*p < 0.05; independent t-test).

Article Snippet: To determine the presence of Hyal-1, Hyal-2, HAS2, CD44, syndecan-1, heparan sulphate and RHAMM in the vitreous samples, equal volumes (10 μL) of vitreous samples were boiled in Laemmli’s sample buffer (1:1, v/v) under reducing condition for 10 min. Immunodetection was performed with the use of rabbit polyclonal anti-Hyal-1 antibody (1:1000, NBP2-16906, Novus Biologicals), mouse polyclonal anti-Hyal-2 antibody (1:1000, H00008692-B02P, Novus Biologicals), mouse monoclonal anti-HAS2 antibody (1:1000, ab140671, Abcam), rabbit monoclonal anti-CD44 antibody (1:1000, ab189524, Abcam), rabbit monoclonal anti-RHAMM antibody (1:1000, ab124729, Abcam), rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1000, MAB1018, R&D Systems), rabbit polyclonal anti-p65 subunit of nuclear factor-kappa B (phospho-NF-κB) (1:1000, NB100-82086, Novus Biologicals), rabbit polyclonal anti-high-mobility group box1 (HMGB1) (1:1000, Cat. no. ab18256, Abcam), mouse monoclonal anti-VEGF antibody (1:750, MAB293, R&D Systems), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:100, sc-8439, Santa Cruz Biotechnology Inc.), and mouse monoclonal anti-vascular cell adhesion molecule-1 (VCAM-1) antibody (1:100, sc-13160, Santa Cruz Biotechnology Inc.).

Techniques: Control, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Human retinal microvascular endothelial cells (HRMECs) were left untreated or treated with high-glucose (HG) (25 mM) for 24h. For HG treatment, cultures treated with mannitol (25 mM) were used as a control. Protein expression of hyaluronan synthase (HAS)2 (A) , hyaluronidase (Hyal)-1 (B) , Hyal-2 (C) and CD44 (D) in cell lysate was determined by Western blot analysis. (E) HRMECs were left untreated or treated with tumor necrosis factor–α (TNF-α) (5ng/ml) or hydrogen peroxide (H 2 O 2 ) (10mM) for 24h. Levels of Hyal-1 were quantified in the culture media by ELISA. (F) HRMECs and human retinal Müller glial cells were compared after treatment with mannitol (25 mM) or HG (25 mM) for 24h. Levels of hyaluronan (HA) were quantified in the culture media by ELISA. Results are expressed as mean ± standard deviation from three different experiments each performed in triplicate (*p < 0.05; independent t-test).

Article Snippet: To determine the presence of Hyal-1, Hyal-2, HAS2, CD44, syndecan-1, heparan sulphate and RHAMM in the vitreous samples, equal volumes (10 μL) of vitreous samples were boiled in Laemmli’s sample buffer (1:1, v/v) under reducing condition for 10 min. Immunodetection was performed with the use of rabbit polyclonal anti-Hyal-1 antibody (1:1000, NBP2-16906, Novus Biologicals), mouse polyclonal anti-Hyal-2 antibody (1:1000, H00008692-B02P, Novus Biologicals), mouse monoclonal anti-HAS2 antibody (1:1000, ab140671, Abcam), rabbit monoclonal anti-CD44 antibody (1:1000, ab189524, Abcam), rabbit monoclonal anti-RHAMM antibody (1:1000, ab124729, Abcam), rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1000, MAB1018, R&D Systems), rabbit polyclonal anti-p65 subunit of nuclear factor-kappa B (phospho-NF-κB) (1:1000, NB100-82086, Novus Biologicals), rabbit polyclonal anti-high-mobility group box1 (HMGB1) (1:1000, Cat. no. ab18256, Abcam), mouse monoclonal anti-VEGF antibody (1:750, MAB293, R&D Systems), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:100, sc-8439, Santa Cruz Biotechnology Inc.), and mouse monoclonal anti-vascular cell adhesion molecule-1 (VCAM-1) antibody (1:100, sc-13160, Santa Cruz Biotechnology Inc.).

Techniques: Control, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation