{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,11]],"date-time":"2026-10-11T08:06:12Z","timestamp":1791705972304,"version":"4.3.4"},"reference-count":39,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,1,6]],"date-time":"2026-01-06T00:00:00Z","timestamp":1767657600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Pharmacol."],"abstract":"<jats:sec>\n                    <jats:title>Introduction<\/jats:title>\n                    <jats:p>Anti-vascular endothelial growth factor (VEGF) therapy fails in approximately 30% of diabetic retinopathy (DR) patients, indicating the limitations of solely targeting VEGF-A. This study aimed to design a bispecific nanobody, Nb-TV, targeting both tumor necrosis factor-\u03b1 (TNF-\u03b1) and VEGF-A to achieve dual anti-inflammatory and anti-angiogenic effects for potential DR treatment.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      The inhibitory effects of Nb-TV were evaluated\n                      <jats:italic>in vitro<\/jats:italic>\n                      using adult retinal pigment epithelium-19 (ARPE-19) cells and human umbilical vein endothelial cells (HUVECs). Key assays included analysis of nuclear translocation (p65, p38\u03b1), phosphorylation (Erk1\/2), expression of inflammatory factors (IL-6, IL-8, MCP-1) and inducible nitric oxide synthase (iNOS), Caspase-1 activation, and assessments of HUVEC proliferation, migration, and tube formation. The\n                      <jats:italic>in vivo<\/jats:italic>\n                      therapeutic efficacy was investigated in a DR mouse model, where Nb-TV was administered via eye drops. Retinal levels of TNF-\u03b1 and VEGF-A, neovascularization, and structural lesions were examined.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      <jats:italic>In vitro<\/jats:italic>\n                      , Nb-TV inhibited over 60% of TNF-\u03b1-induced p65 nuclear translocation in ARPE-19 cells, significantly reducing IL-6, IL-8, and MCP-1 expression by 24.6%, 26.6%, and 37.8%, respectively, and suppressing Caspase-1 activation. In HUVECs, it effectively blocked VEGF-A-driven proliferation, migration, and tube formation by inhibiting p38\u03b1 nuclear translocation (28.5% reduction) and Erk1\/2 phosphorylation (98.6% inhibition), while downregulating iNOS expression.\n                      <jats:italic>In vivo<\/jats:italic>\n                      , topical Nb-TV application decreased TNF-\u03b1 and VEGF-A levels in retinal tissues of DR mice, controlled pathological neovascularization, and improved retinal structural damage.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Discussion<\/jats:title>\n                    <jats:p>Our findings demonstrate that Nb-TV mechanistically dual-targets the TNF-\u03b1\/NF-\u03baB and VEGF-A\/MAPK pathways, mitigating both inflammation and angiogenesis synergistically. This validates Nb-TV as a novel bispecific nanobody with promising therapeutic potential for DR by simultaneously modulating key pathological pathways. This study not only proposes a strategic approach for refractory DR but also provides a framework for targeting other multifactorial ocular and systemic diseases.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fphar.2025.1729606","type":"journal-article","created":{"date-parts":[[2026,1,6]],"date-time":"2026-01-06T06:34:17Z","timestamp":1767681257000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":4,"title":["Synergistic therapy for diabetic retinopathy via dual blockade of VEGF-A and TNF-\u03b1 with a bispecific nanobody"],"prefix":"10.3389","volume":"16","author":[{"given":"Chenlu","family":"Wu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yue","family":"Shen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chengnan","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chong","family":"Bi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenyue","family":"Yan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinping","family":"Niu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenyun","family":"Zheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xingyuan","family":"Ma","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,1,6]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"240","DOI":"10.3109\/08820539909069543","article-title":"Molecular mechanisms of vascular permeability in diabetic retinopathy","volume":"14","author":"Antonetti","year":"1999","journal-title":"Semin. 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