Fig 2: Regulation of the activity of the p38 mitogen-activated protein kinase signaling pathway by the histamine H4 receptor in ARPE-19 cells cultivated under high glucose conditions. ARPE-19 cells pretreated with selective inhibitors of ERK (U0126), JNK (SP600125) and p38 (SB203580) for 60 min were incubated with high glucose (25 mM D-glucose) or osmotically controlled (25 mM mannitol) medium for 48 hours, and then treated with histamine (0.1 mM, 8 hours). (A–C) Levels of p-ERK, ERK, p-JNK, JNK, p-p38, p38 and HRH4 are shown, as determined using an immunoblot assay. ß-Actin is used as the internal control. (D) ARPE-19 cells transfected with either scrambled small interfering RNA (siRNA) or HRH4 siRNA were cultivated with high glucose (25 mM D-glucose) or osmotically controlled (25 mM mannitol) medium for 48 hours, and then treated with histamine (0.1 mM) for 8 hours. Levels of p-ERK, ERK, p-JNK, JNK, p-p38, p38 and HRH4 are shown, as evaluated by immunoblotting. (E–H) Densitometric analysis was performed using ImageJ. ERK, extracellular signal-regulated kinase; HG, high glucose; HRH4, histamine receptor H4; JNK, c-Jun N-terminal kinase; OC, osmotically controlled.

Fig 3: Schematic illustration showing the regulatory mechanism for pro-angiogenic and anti-angiogenic factor expression in the RPE of diabetic retina via H4 receptor/p38 MAPK axis. HRH4, histamine receptor H4; IL-6, interleukin-6; mRNA, messenger RNA; p38 MAPK, p38 mitogen-activated protein kinase; PEDF, pigment epithelium-derived factor; RPE, retinal pigment epithelium; VEGF, vascular endothelial growth factor.

Fig 4: Role of the HRH4/p38 mitogen-activated protein kinase signaling pathway in histamine-induced regulation of VEGF, IL-6 and PEDF expression in ARPE-19 cells. ARPE-19 cells transfected either with negative control small interfering RNA (siRNA) or with HRH4 siRNA were exposed to high glucose-containing (25 mM) medium for 48 hours. Then, ARPE-19 cells from each group were incubated with histamine-containing (0.1 mM) medium, in the presence and absence of SB203580, a specific inhibitor of p38. (A–C) Levels of VEGF, IL-6 and PEDF messenger RNA (mRNA) are shown, as determined by quantitative real-time PCR. All experiments are performed in triplicate, and error bars represent the mean±SEM. *P<0.05. GAPDH; glyceraldehyde 3-phosphate dehydrogenase, HG, high glucose; HRH4, histamine receptor H4; IL-6, interleukin 6; NS, not significant; PEDF, pigment epithelium-derived factor; VEGF, vascular endothelial growth factor.

Fig 5: Increased histaminergic activity in the eyes of patients with diabetic retinopathy and upregulation of HRH4 expression in the retinal pigment epithelium of experimental diabetic conditions. (A–H) Levels of VEGF, IL-6, histamine and HDC in the serum and vitreous of patients with diabetic retinopathy (9 without VH and 11 with VH) are shown, as determined using ELISA and compared with those of non-diabetic controls (n=20). (I) Representative images of the RPE/choroid/scleral complex flat mounts from sham or streptozotocin-injected C57BL/6 mice (13 weeks after injection), db/m and db/db mice (25 weeks old) immunostained using the HRH4 antibody. Scale bar, 25 µm. (J, K) Protein and messenger RNA (mRNA) transcript levels of HRH4 in ARPE-19 cells cultivated in high glucose medium (D-glucose, 25 mM) or under osmotically controlled medium (mannitol, 25 mM) are shown, as determined using immunoblot assay and quantitative real-ime PCR, respectively. **P<0.01, ***p<0.001. Error bars represent the mean±SEM. Con, control; DAPI, 4',6-diamidino-2-phenylindole; DM, diabetes mellitus; HDC, histidine decarboxylase; HG, high glucose; HRH4, histamine receptor H4; IL-6, interleukin 6; NS, not significant; OC, osmotically controlled; STZ, streptozotocin-induced mice wth diabetes; VEGF, vascular endothelial growth factor; VH, vitreous hemorrhage.