Recovery from heat shock injury by activation of Na+-glucose cotransporter in renal epithelial cells

AbstractExposure of cells or organs to sublethal physical or chemical stresses induces disruption of cellular structures and functions. Here, we examined whether Na+-glucose cotransporter (SGLT1) is involved in the recovery from heat shock (HS) injury in porcine renal epithelial LLC-PK1 cells. Recovery from HS (42 °C for 3 h, then 37 °C for 12 h) increased SGLT1 activity, assessed by [14C]α-methyl glucopyranoside uptake, and a maximal transport rate (Vmax) from 2.4 to 5.9 nmol/mg protein/30 min, but did not alter an apparent affinity constant (Km). Protein distribution of SGLT1 in apical membrane fraction was also increased after recovery from HS without changing in total membrane fraction. Membrane integrity assessed by calcein accumulation was decreased by HS, and then returned to basal level. This recovery was inhibited by phloridzin, a potent SGLT1 inhibitor, and nonmetabolizable glucose analogues. Anti-transforming growth factor-β1 (TGF-β1) antibody inhibited both elevation of SGLT1 distribution in apical membrane and recovery of calcein accumulation induced by HS. Taken together, HS increases in the number of SGLT1 protein in apical membrane mediated via TGF-β1 signaling pathway. The increase of glucose uptake is necessary to repair plasma membrane integrity

Suppression and Regression of Choroidal Neovascularization in Mice by a Novel CCR2 Antagonist, INCB3344

PURPOSE: To investigate the effect of an intravitreally administered CCR2 antagonist, INCB3344, on a mouse model of choroidal neovascularization (CNV). METHODS: CNV was induced by laser photocoagulation on Day 0 in wild type mice. INCB3344 or vehicle was administered intravitreally immediately after laser application. On Day 14, CNV areas were measured on retinal pigment epithelium (RPE)-choroid flat mounts and histopathologic examination was performed on 7 µm-thick sections. Macrophage infiltration was evaluated by immunohistochemistry on RPE-choroid flat mounts and quantified by flow cytometry on Day 3. Expression of vascular endothelial growth factor (VEGF) protein in RPE-choroid tissue was examined by immunohistochemistry and ELISA, VEGF mRNA in sorted macrophages in RPE-choroid tissue was examine by real-time PCR and expression of phosphorylated extracellular signal-regulated kinase (p-ERK 1/2) in RPE-choroid tissue was measured by Western blot analysis on Day 3. We also evaluated the efficacy of intravitreal INCB3344 to spontaneous CNV detected in Cu, Zn-superoxide dismutase (SOD1) deficient mice. Changes in CNV size were assessed between pre- and 1week post-INCB3344 or vehicle administration in fundus photography and fluorescence angiography (FA). RESULTS: The mean CNV area in INCB3344-treated mice decreased by 42.4% compared with the vehicle-treated control mice (p<0.001). INCB3344 treatment significantly inhibited macrophage infiltration into the laser-irradiated area (p<0.001), and suppressed the expression of VEGF protein (p = 0.012), VEGF mRNA in infiltrating macrophages (p<0.001) and the phosphorylation of ERK1/2 (p<0.001). The area of spontaneous CNV in Sod1⁻/⁻ mice regressed by 70.35% in INCB3344-treated animals while no change was detected in vehicle-treated control mice (p<0.001). CONCLUSIONS: INCB3344 both inhibits newly forming CNV and regresses established CNV. Controlling inflammation by suppressing macrophage infiltration and angiogenic ability via the CCR-2/MCP-1 signal may be a useful therapeutic strategy for treating CNV associated with age-related macular degeneration

Hypoxia-inducible factor 1 alpha in oral squamous cell carcinoma and its relation to prognosis

The aim of this study was to investigate the correlation between the expression of hypoxia-inducible factor 1 alpha (HIF-1 alpha) and proliferative activity in tumor cells, lymph node metastasis, as well as prognosis in patients with oral squamous cell carcinoma (OSCC). Fifty-seven biopsy specimens of OSCC were investigated for the expression of HIF-1 alpha and proliferating cell nuclear antigen (PCNA) by immunohistochemistry. None of the patients had received any prior treatments. The percentage of HIF-1 alpha immunopositive area (PHIA) was calculated using computer-assisted image analysis for quantitative assessment of HIF-1 alpha expression. The PCNA labeling index (LI) was evaluated as a proliferation marker. We found that the mean PHIA in all stages was 12.1% in the poor prognosis patients, and it was 6.4% in the good prognosis patients. There was a significant difference of PHIA between poor prognosis and good prognosis patients (P = 0.0065). Furthermore, the mean PHIA in the patients who had no metastatic lymph nodes was 7.5%, while it was 11.7% in the patients who had metastatic lymph nodes. There was also a significant difference of PHIA between patients who had no metastatic lymph nodes and those who had metastatic lymph nodes (P = 0.0487). On the other hand, significant correlation between PHIA and PCNA LI was not observed. These results provide the clinical data indicating that HIF-1 alpha may play an important role in lymph node metastasis and prognosis in patients with OSCC

Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes

Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis