Catalpol Prevents Glomerular Angiogenesis Induced by Advanced Glycation End Products via Inhibiting Galectin-3

Abstract

Objective

The main characteristics of diabetic nephropathy (DN) at the early stage are abnormal angiogenesis of glomerular endothelial cells (GECs) and macrophage infiltration. Galectin-3 plays a pivotal role in the pathogenesis of DN via binding with its ligand, advanced glycation end products (AGEs). Catalpol, an iridoid glucoside extracted from Rehmannia glutinosa, has been found to ameliorate vascular inflammation, reduce endothelial permeability, and protect against endothelial damage in diabetic milieu. However, little is known about whether catalpol could exert an anti-angiogenesis and anti-inflammation effect induced by AGEs.

Methods

Mouse GECs (mGECs) and RAW 264.7 macrophages were treated with different concentrations of AGEs (0, 50, 100, 200 and 400 µg/mL) for different time (0, 6, 12, 24 and 48 h) to determine the optimal concentration of AGEs and treatment time. Cells were treated with catalpol (10 µmol/L), GB1107 (1 µmol/L, galectin-3 inhibitor), PX-478 (50 µmol/L, HIF-1α inhibitor), adenovirus-green fluorescent protein (Ad-GFP) [3×10⁷ plaque-forming unit (PFU)/mL] or Ad-galectin-3-GFP (2×10⁸ PFU/mL), which was followed by incubation with 50 µg/mL AGEs. The levels of galectin-3, vascular endothelial growth factor A (VEGFA) and pro-angiogenic factors angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), tunica interna endothelial cell kinase-2 (Tie-2) were detected by enzymelinked immunosorbent assay (ELISA). Cell counting kit-8 (CCK-8) assay was used to evaluate the proliferation of these cells. The expression levels of galectin-3, vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and hypoxia-inducible factor-1α (HIF-1α) in mGECs and those of galectin-3 and HIF-1α in RAW 264.7 macrophages were detected by Western blotting and immunofluorescence (IF) staining. The rat DN model was established. Catalpol (100 mg/kg) or GB1107 (10 mg/kg) was administered intragastrically once a day for 12 weeks. Ad-galectin-3-GFP (6×10⁷ PFU/mL, 0.5 mL) or Ad-GFP (6×10⁶ PFU/mL, 0.5 mL) was injected into the tail vein of rats 48 h before the sacrifice of the animals. The expression of galectin-3, VEGFR1, VEGFR2, and HIF-1α in renal cortices was analyzed by Western blotting. The expression of galectin-3, F4/80 (a macrophage biomarker), and CD34 (an endothelium biomarker) in renal cortices was detected by IF staining, and collagen accumulation by Masson staining.

Results

The expression levels of galectin-3 and VEGFA were significantly higher in mGECs and RAW 264.7 macrophages treated with 50 µg/mL AGEs for 48 h than those in untreated cells. Catalpol and GB1107 could block the AGEs-induced proliferation of mGECs and RAW 264.7 macrophages. Over-expression of galectin-3 was found to reduce the inhibitory effect of catalpol on the proliferation of cells. Catalpol could significantly decrease the levels of Ang-1, Ang-2 and Tie-2 released by AGEs-treated mGECs, which could be reversed by over-expression of galectin-3. Catalpol could significantly inhibit AGEs-induced expression of galectin-3, HIF-1α, VEGFR1, and VEGFR2 in mGECs. The inhibitory effect of catalpol on galectin-3 in AGEs-treated mGECs was impaired by PX-478. Moreover, catalpol attenuated the AGEs-activated HIF-1α/galectin-3 pathway in RAW 264.7 macrophages, which was weakened by PX-478. Additionally, catalpol significantly inhibited the expression of galectin-3, macrophage infiltration, collagen accumulation, and angiogenesis in the kidney of diabetic rats. Over-expression of galectin-3 could antagonize these inhibitory effects of catalpol.

Conclusion

Catalpol prevented the angiogenesis of mGECs and macrophage proliferation via inhibiting galectin-3. It could prevent the progression of diabetes-induced renal damage.

Keywords

catalpol / glomerular angiogenesis / advanced glycation end products / galectin-3

References

[1] Vasanth RaoA B V, TanSH, CandasamyM, et al.. Diabetic nephropathy: An update on pathogenesis and drug development. Diabetes Metab Syndr, 2019, 13(1): 754-762

[2] HohensteinB, HausknechtB, BoehmerK, et al.. Local VEGF activity but not VEGF expression is tightly regulated during diabetic nephropathy in man. Kidney Int, 2006, 69(9): 1654-1661

[3] KanesakiY, SuzukiD, UeharaG, et al.. Vascular endothelial growth factor gene expression is correlated with glomerular neovascularization in human diabetic nephropathy. Am J Kidney Dis, 2005, 45(2): 288-294

[4] RichterA, AlexdottirMS, MagnusSH, et al.. EGFL7 Mediates BMP9-Induced Sprouting Angiogenesis of Endothelial Cells Derived from Human Embryonic Stem Cells. Stem Cell Reports, 2019, 12(6): 1250-1259

[5] ElshabrawyHA, ChenZ, VolinMV, et al.. The pathogenic role of angiogenesis in rheumatoid arthritis. Angiogenesis, 2015, 18(4): 433-448

[6] TanabeK, MaeshimaY, SatoY, et al.. Antiangiogenic Therapy for Diabetic Nephropathy. Biomed Res Int, 2017, 2017: 5724069

[7] StevensM, NealCR, CraciunEC, et al.. The natural drug DIAVIT is protective in a type II mouse model of diabetic nephropathy. PLoS One, 2019, 14(3): e0212910

[8] FunasakaT, RazA, Nangia-MakkerP. Galectin-3 in angiogenesis and metastasis. Glycobiology, 2014, 24(10): 886-891

[9] AboulhagagNA, El-DeekHEM, SherifMF. Expression of galectin-1 and galectin-3 in renal cell carcinoma; immunohistochemical study. Ann Diagn Pathol, 2018, 36: 31-37

[10] SinghR, BardenA, MoriT, et al.. Advanced glycation end-products: a review. Diabetologia, 2001, 44(2): 129-146

[11] PuglieseG, IacobiniC, PesceCM, et al.. Galectin-3: an emerging all-out player in metabolic disorders and their complications. Glycobiology, 2015, 25(2): 136-150

[12] PuglieseG, PricciF, LetoG, et al.. The diabetic milieu modulates the advanced glycation end product-receptor complex in the mesangium by inducing or upregulating galectin-3 expression. Diabetes, 2000, 49(7): 1249-1257

[13] KikuchiY, KobayashiS, HemmiN, et al.. Galectin-3-positive cell infiltration in human diabetic nephropathy. Nephrol Dial Transplant, 2004, 19(3): 602-607

[14] LogueOC, McGowanJW, GeorgeEM, et al.. Therapeutic angiogenesis by vascular endothelial growth factor supplementation for treatment of renal disease. Curr Opin Nephrol Hypertens, 2016, 25(5): 404-409

[15] MarkowskaAI, JefferiesKC, PanjwaniN. Galectin-3 protein modulates cell surface expression and activation of vascular endothelial growth factor receptor 2 in human endothelial cells. J Biol Chem, 2011, 286(34): 29913-29921

[16] ViscontiRP, RichardsonCD, SatoTN. Orchestration of angiogenesis and arteriovenous contribution by angiopoietins and vascular endothelial growth factor (VEGF). Proc Natl Acad Sci USA, 2002, 99(12): 8219-8224

[17] NayakBK, ShanmugasundaramK, FriedrichsWE, et al.. HIF-1 Mediates Renal Fibrosis in OVE26 Type 1 Diabetic Mice. Diabetes, 2016, 65(5): 1387-1397

[18] ZimnaA, KurpiszM. Hypoxia-Inducible Factor-1 in Physiological and Pathophysiological Angiogenesis: Applications and Therapies. Biomed Res Int, 2015, 2015: 549412

[19] LiQ, WenY, WangL, et al.. Hyperglycemia-induced accumulation of advanced glycosylation end products in fibroblast-like synoviocytes promotes knee osteoarthritis. Exp Mol Med, 2021, 53(11): 1735-1747

[20] ZhuY, MaWQ, HanXQ, et al.. Advanced glycation end products accelerate calcification in VSMCs through HIF-1α/PDK4 activation and suppress glucose metabolism. Sci Rep, 2018, 8(1): 13730

[21] NakagawaT, KosugiT, HanedaM, et al.. Abnormal angiogenesis in diabetic nephropathy. Diabetes, 2009, 58(7): 1471-1478

[22] SunW, GaoY, DingY, et al.. Catalpol ameliorates advanced glycation end product-induced dysfunction of glomerular endothelial cells via regulating nitric oxide synthesis by inducible nitric oxide synthase and endothelial nitric oxide synthase. IUBMB Life, 2019, 71(9): 1268-1283

[23] LiuK, XuH, LvG, et al.. Loganin attenuates diabetic nephropathy in C57BL/6J mice with diabetes induced by streptozotocin and fed with diets containing high level of advanced glycation end products. Life Sci, 2015, 123: 78-85

[24] GoligorskyMS. Vascular endothelium in diabetes. Am J Physiol Renal Physiol, 2017, 312(2): F266-F275

[25] KimuraK, ItoM, AmanoM, et al.. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rhokinase). Science, 1996, 273(5272): 245-248

[26] SunL, HuangT, XuW, et al.. Advanced glycation end products promote VEGF expression and thus choroidal neovascularization via Cyr61-PI3K/AKT signaling pathway. Sci Rep, 2017, 7(1): 14925

[27] ZhangJ, BiR, MengQ, et al.. Catalpol alleviates adriamycin-induced nephropathy by activating the SIRT1 signalling pathway in vivo and in vitro. Br J Pharmacol, 2019, 176(23): 4558-4573

[28] WangL, XueGB. Catalpol suppresses osteosarcoma cell proliferation through blocking epithelial-mesenchymal transition (EMT) and inducing apoptosis. Biochem Biophys Res Commun, 2018, 495(1): 27-34

[29] LinC, LuY, YanX, et al.. Catalpol protects glucose-deprived rat embryonic cardiac cells by inducing mitophagy and modulating estrogen receptor. Biomed Pharmacother, 2017, 89: 973-982

[30] HanY, ShenM, TangLY, et al.. Antiangiogenic effects of catalpol on rat corneal neovascularization. Mol Med Rep, 2018, 17(2): 2187-2194

[31] YaoY, ZhouL, LiaoW, et al.. HH1-1, a novel Galectin-3 inhibitor, exerts anti-pancreatic cancer activity by blocking Galectin-3/EGFR/AKT/FOXO3 signaling pathway. Carbohydr Polym, 2019, 204: 111-123

[32] McLeodK, WalkerJT, HamiltonDW. Galectin-3 regulation of wound healing and fibrotic processes: insights for chronic skin wound therapeutics. J Cell Commun Signal, 2018, 12(1): 281-287

[33] ChenWS, CaoZ, LefflerH, et al.. Galectin-3 Inhibition by a Small-Molecule Inhibitor Reduces Both Pathological Corneal Neovascularization and Fibrosis. Invest Ophthalmol Vis Sci, 2017, 58(1): 9-20

[34] PiyushT, ChackoAR, SindrewiczP, et al.. Interaction of Galectin-3 with MUC1 on cell surface promotes EGFR dimerization and activation in human epithelial cancer cells. Cell Death Differ, 2017, 24(11): 1937-1947

[35] LobryT, MillerR, NevoN, et al.. Interaction between Galectin-3 and cystinosin uncovers a pathogenic role of inflammation in kidney involvement of cystinosis. Kidney Int, 2019, 96(2): 350-362

[36] ZhangZ, ZhengY, WangH, et al.. CD146 interacts with Galectin-3 to mediate endothelial cell migration. FEBS Lett, 2018, 592(11): 1817-1828

[37] NatarajamurthySH, SistlaS, DharmeshSM. Disruption of Galectin-3 and Galectin-3 binding protein (G3BP) interaction by dietary pectic polysaccharides (DPP) - Arrest of metastasis, inhibition of proliferation and induction of apoptosis. Int J Biol Macromol, 2019, 139: 486-499

[38] ZhangP, SunY, PengR, et al.. Long non-coding RNA Rpph1 promotes inflammation and proliferation of mesangial cells in diabetic nephropathy via an interaction with Gal-3. Cell Death Dis, 2019, 10(7): 526